PMID19074876	Coordinated regulation of cell cycle transcripts by p53-Inducible microRNAs, miR-192 and miR-215. Cell cycle arrest in response to DNA damage is an important antitumorigenic mechanism. MicroRNAs (miRNAs) were recently shown to play key regulatory roles in cell cycle progression. For example, miR-34a is induced in response to p53 activation and mediates G(1) arrest by down-regulating multiple cell cycle-related transcripts. Here we show that genotoxic stress promotes the p53-dependent up-regulation of the homologous miRNAs miR-192 and miR-215. Like miR-34a, activation of miR-192/215 induces cell cycle arrest, suggesting that multiple miRNA families operate in the p53 network. Furthermore, we define a downstream gene expression signature for miR-192/215 expression, which includes a number of transcripts that regulate G(1) and G(2) checkpoints. Of these transcripts, 18 transcripts are direct targets of miR-192/215, and the observed cell cycle arrest likely results from a cooperative effect among the modulations of these genes by the miRNAs. Our results showing a role for miR-192/215 in cell proliferation combined with recent observations that these miRNAs are underexpressed in primary cancers support the idea that miR-192 and miR-215 function as tumor suppressors.
PMID19699171	The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs. MicroRNAs regulated by lipopolysaccharide (LPS) target genes that contribute to the inflammatory phenotype. Here, we showed that the protein kinase Akt1, which is activated by LPS, positively regulated miRNAs let-7e and miR-181c but negatively regulated miR-155 and miR-125b. In silico analyses and transfection studies revealed that let-7e repressed Toll-like receptor 4 (TLR4), whereas miR-155 repressed SOCS1, two proteins critical for LPS-driven TLR signaling, which regulate endotoxin sensitivity and tolerance. As a result, Akt1(-/-) macrophages exhibited increased responsiveness to LPS in culture and Akt1(-/-) mice did not develop endotoxin tolerance in vivo. Overexpression of let-7e and suppression of miR-155 in Akt1(-/-) macrophages restored sensitivity and tolerance to LPS in culture and in animals. These results indicate that Akt1 regulates the response of macrophages to LPS by controlling miRNA expression.
PMID19074875	p53-Responsive micrornas 192 and 215 are capable of inducing cell cycle arrest. microRNAs provide a novel layer of regulation for gene expression by interfering with the stability and/or translation of specific target mRNAs. Overall levels of microRNAs are frequently down-regulated in cancer cells, and reducing general microRNA processing increases cancerogenesis in transgenic models, suggesting that at least some microRNAs might act as effectors in tumor suppression. Accordingly, the tumor suppressor p53 up-regulates miR-34a, a microRNA that contributes to apoptosis and acute senescence. Here, we used array hybridization to find that p53 induces two additional, mutually related clusters of microRNAs, leading to the up-regulation of miR-192, miR-194, and miR-215. The same microRNAs were detected at high levels in normal colon tissue but were severely reduced in many colon cancer samples. On the other hand, miR-192 and its cousin miR-215 can each contribute to enhanced CDKN1A/p21 levels, colony suppression, cell cycle arrest, and cell detachment from a solid support. These effects were partially dependent on the presence of wild-type p53. Antagonizing endogenous miR-192 attenuated 5-fluorouracil-induced accumulation of p21. Hence, miR-192 and miR-215 can act as effectors as well as regulators of p53; they seem to suppress cancerogenesis through p21 accumulation and cell cycle arrest.
PMID20388878	MicroRNA cluster 221-222 and estrogen receptor alpha interactions in breast cancer. Several lines of evidence have suggested that estrogen receptor alpha (ERalpha)-negative breast tumors, which are highly aggressive and nonresponsive to hormonal therapy, arise from ERalpha-positive precursors through different molecular pathways. Because microRNAs (miRNAs) modulate gene expression, we hypothesized that they may have a role in ER-negative tumor formation. Gene expression profiles were used to highlight the global changes induced by miRNA modulation of ERalpha protein. miRNA transfection and luciferase assays enabled us to identify new targets of miRNA 206 (miR-206) and miRNA cluster 221-222 (miR-221-222). Northern blot, luciferase assays, estradiol treatment, and chromatin immunoprecipitation were performed to identify the miR-221-222 transcription unit and the mechanism implicated in its regulation. Different global changes in gene expression were induced by overexpression of miR-221-222 and miR-206 in ER-positive cells. miR-221 and -222 increased proliferation of ERalpha-positive cells, whereas miR-206 had an inhibitory effect (mean absorbance units [AU]: miR-206: 500 AU, 95% confidence interval [CI]) = 480 to 520; miR-221: 850 AU, 95% CI = 810 to 873; miR-222: 879 AU, 95% CI = 850 to 893; P < .05). We identified hepatocyte growth factor receptor and forkhead box O3 as new targets of miR-206 and miR-221-222, respectively. We demonstrated that ERalpha negatively modulates miR-221 and -222 through the recruitment of transcriptional corepressor partners: nuclear receptor corepressor and silencing mediator of retinoic acid and thyroid hormone receptor. These findings suggest that the negative regulatory loop involving miR-221-222 and ERalpha may confer proliferative advantage and migratory activity to breast cancer cells and promote the transition from ER-positive to ER-negative tumors.
PMID18834855	Effects of miR-34a on cell growth and chemoresistance in prostate cancer PC3 cells. Tumor suppressor p53 transcriptionally regulates expression of microRNA-34a, which confers translational inhibition and mRNA degradation of genes involved in cell cycle control and apoptosis. In various cancers, miR-34a expression is lost or reduced. Here, we investigated the role of miR-34a in prostate cancer cell lines. MiR-34a expression was markedly reduced in p53-null PC3 cells and p53-mutated DU145 cells compared with LNCaP cells expressing wild-type p53. In PC3 cell, ectopic expression of miR-34a decreased the SIRT1 mRNA and protein levels as well as protein levels of known direct target genes. Reporter assays revealed that miR-34a-induced SIRT1 inhibition occurred at the transcriptional but not post-transcriptional level despite the presence of a potential miR-34a binding site within its 3'-UTR. Ectopic miR-34a expression resulted in cell cycle arrest and growth inhibition and attenuated chemoresistance to anticancer drug camptothecin by inducing apoptosis, suggesting a potential role of miR-34a for the treatment of p53-defective prostate cancer.
PMID20624818	A minicircuitry involving REST and CREB controls miR-9-2 expression during human neuronal differentiation. miRNAs play key roles in the nervous system, where they mark distinct developmental stages. Accordingly, dysregulation of miRNA expression may have profound effects on neuronal physiology and pathology, including cancer. Among the neuronal miRNAs, miR-9 was shown to be upregulated during in vitro neuronal differentiation and downregulated in 50% of primary neuroblastoma tumors, suggesting a potential function as an oncosuppressor gene. In this study we characterized the promoter and the transcriptional regulation of the miR-9-2 gene during neuronal differentiation. We found that, despite its localization inside an exon of a putative host-gene, miR-9-2 is expressed as an independent unit with the promoter located in the upstream intron. By promoter fusion and mutational analyses, together with RNAi and Chromatin immunoprecipitation assays, we demonstrated that the concerted action of the master transcriptional factors RE1-silencing transcription factor (REST) and cAMP-response element binding protein (CREB) on miR-9-2 promoter induces miRNA expression during differentiation. We showed that the repressor REST inhibits the activity of the miR-9-2 promoter in undifferentiated neuroblastoma cells, whereas REST dismissal and phosphorylation of CREB trigger transcription in differentiating cells. Finally, a regulatory feed-back mechanism, in which the reciprocal action of miR-9 and REST may be relevant for the maintenance of the neuronal differentiation program, is shown.
PMID22020437	miR-34 miRNAs provide a barrier for somatic cell reprogramming. Somatic reprogramming induced by defined transcription factors is a low-efficiency process that is enhanced by p53 deficiency. So far, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation, indicating that additional p53 targets may regulate this process. Here, we demonstrate that miR-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. Mir34a deficiency in mice significantly increased reprogramming efficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of Mir34a promoted iPSC generation without compromising self-renewal or differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (also known as N-Myc). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three miR-34 miRNAs acted cooperatively in this process. Taken together, our findings identified miR-34 miRNAs as p53 targets that play an essential role in restraining somatic reprogramming.
PMID22170100	Interferon regulatory factor 3 inhibits astrocyte inflammatory gene expression through suppression of the proinflammatory miR-155 and miR-155*. Astrocytes, together with microglia and macrophages, participate in innate inflammatory responses in the CNS. Although inflammatory mediators such as interferons generated by astrocytes may be critical in the defense of the CNS, sustained unopposed cytokine signaling could result in harmful consequences. Interferon regulatory factor 3 (IRF3) is a transcription factor required for IFNβ production and antiviral immunity. Most cells express low levels of IRF3 protein, and the transcriptional mechanism that upregulates IRF3 expression is not known. In this study, we explored the consequence of adenovirus-mediated IRF3 gene transfer (Ad-IRF3) in primary human astrocytes. We show that IRF3 transgene expression suppresses proinflammatory cytokine gene expression upon challenge with IL-1/IFNγ and alters astrocyte activation phenotype from a proinflammatory to an anti-inflammatory one, akin to an M1-M2 switch in macrophages. This was accompanied by the rescue of neurons from cytokine-induced death in glial-neuronal co-cultures. Furthermore, Ad-IRF3 suppressed the expression of microRNA-155 and its star-form partner miR-155*, immunoregulatory miRNAs highly expressed in multiple sclerosis lesions. Astrocyte miR-155/miR155* were induced by cytokines and TLR ligands with a distinct hierarchy and involved in proinflammatory cytokine gene induction by targeting suppressor of cytokine signaling 1, a negative regulator of cytokine signaling and potentially other factors. Our results demonstrate a novel proinflammatory role for miR-155/miR-155* in human astrocytes and suggest that IRF3 can suppress neuroinflammation through regulating immunomodulatory miRNA expression. © 2011 Wiley-Liss, Inc.
PMID20980664	A network connecting Runx2, SATB2, and the miR-23a~27a~24-2 cluster regulates the osteoblast differentiation program. Induced osteogenesis includes a program of microRNAs (miRs) to repress the translation of genes that act as inhibitors of bone formation. How expression of bone-related miRs is regulated remains a compelling question. Here we report that Runx2, a transcription factor essential for osteoblastogenesis, negatively regulates expression of the miR cluster 23a∼27a∼24-2. Overexpression, reporter, and chromatin immunoprecipitation assays established the presence of a functional Runx binding element that represses expression of these miRs. Consistent with this finding, exogenous expression of each of the miRs suppressed osteoblast differentiation, whereas antagomirs increased bone marker expression. The biological significance of Runx2 repression of this miR cluster is that each miR directly targets the 3' UTR of SATB2, which is known to synergize with Runx2 to facilitate bone formation. The findings suggest Runx2-negative regulation of multiple miRs by a feed-forward mechanism to cause derepression of SATB2 to promote differentiation. We find also that miR-23a represses Runx2 in the terminally differentiated osteocyte, representing a feedback mechanism to attenuate osteoblast maturation. We provide direct evidence for an interdependent relationship among transcriptional inhibition of the miR cluster by Runx2, translational repression of Runx2 and of SATB2 by the cluster miRs during progression of osteoblast differentiation. Furthermore, miR cluster gain of function (i.e., inhibition of osteogenesis) is rescued by the exogenous expression of SATB2. Taken together, we have established a regulatory network with a central role for the miR cluster 23a∼27a∼24-2 in both progression and maintenance of the osteocyte phenotype.
PMID20209161	Helicobacter pylori induces miR-155 in T cells in a cAMP-Foxp3-dependent manner. Amongst the most severe clinical outcomes of life-long infections with Helicobacter pylori is the development of peptic ulcers and gastric adenocarcinoma--diseases often associated with an increase of regulatory T cells. Understanding H. pylori-driven regulation of T cells is therefore of crucial clinical importance. Several studies have defined mammalian microRNAs as key regulators of the immune system and of carcinogenic processes. Hence, we aimed here to identify H. pylori-regulated miRNAs, mainly in human T cells. MicroRNA profiling of non-infected and infected human T cells revealed H. pylori infection triggers miR-155 expression in vitro and in vivo. By using single and double H. pylori mutants and the corresponding purified enzymes, the bacterial vacuolating toxin A (VacA) and gamma-glutamyl transpeptidase (GGT) plus lipopolysaccharide (LPS) tested positive for their ability to regulate miR-155 and Foxp3 expression in human lymphocytes; the latter being considered as the master regulator and marker of regulatory T cells. RNAi-mediated knockdown (KD) of the Foxp3 transcription factor in T cells abolished miR-155 expression. Using adenylate cyclase inhibitors, the miR-155 induction cascade was shown to be dependent on the second messenger cyclic adenosine monophosphate (cAMP). Furthermore, we found that miR-155 directly targets the protein kinase A inhibitor alpha (PKIalpha) mRNA in its 3'UTR, indicative of a positive feedback mechanism on the cAMP pathway. Taken together, our study describes, in the context of an H. pylori infection, a direct link between Foxp3 and miR-155 in human T cells and highlights the significance of cAMP in this miR-155 induction cascade.
PMID20871480	Human SMG-1 is involved in gemcitabine-induced primary microRNA-155/BIC up-regulation in human pancreatic cancer PANC-1 cells. Human primary microRNA-155/B-cell integration cluster (BIC) transcript is the precursor of microRNA-155. The overexpression of them has been widely observed in the progression of various types of tumors. Our objective was to investigate the effect of anticancer agents on the expression of BIC and possible signal pathways that involved in. Quantitative real-time reverse transcriptase polymerase chain reaction was used to measure the expression of BIC. Chemical inhibitors against c-Jun N-terminal kinase 1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2, protein kinase C, checkpoint kinase 1, and phosphatidylinositol 3 kinase (PI3K) were used for the evaluation of involved signal pathways. RNA interference was used to knock down the expression of ataxia-telangiectasia mutated, ataxia-telangiectasia and Rad3 related, and suppressor of morphogenesis in genitalia-1 (SMG-1), and Western blot was carried out to evaluate the knockdown effect. B-cell integration cluster expression was induced by a representative anti-pancreatic cancer drug, gemcitabine, in human pancreatic cancer PANC-1 cells. The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 and c-Jun N-terminal kinase inhibitors, but not the checkpoint kinase 1 and protein kinase C inhibitors, suppressed the up-regulation of BIC. B-cell integration cluster up-regulation was also significantly inhibited by the PI3K inhibitor wortmannin. RNA interference studies showed that wortmannin-sensitive SMG-1 but not ataxia-telangiectasia mutated or ataxia-telangiectasia and Rad3 related was involved in the up-regulation. Our results show that multiple pathways can be involved in the up-regulation of BIC. Furthermore, we demonstrate for the first time that PI3K SMG-1 is required for gemcitabine-induced up-regulation of BIC transcript.
PMID22198213	MicroRNA-34a inhibits migration and invasion of colon cancer cells via targeting to Fra-1. MicroRNA-34a (miR-34a), a transcriptional target of p53, is a well-known tumor suppressor gene. Here, we identified Fra-1 as a new target of miR-34a and demonstrated that miR-34a inhibits Fra-1 expression at both protein and messenger RNA levels. In addition, we found that p53 indirectly regulates Fra-1 expression via a miR-34a-dependant manner in colon cancer cells. Overexpression of miR-34a strongly inhibited colon cancer cell migration and invasion, which can be partially rescued by forced expression of the Fra-1 transcript lacking the 3'-untranslated region. The expression of matrix metalloproteinase (MMP)-1 and MMP-9, two enzymes involved in cell migration and invasion, was decreased in miR-34a-transfected cells, and this can be rescued by Fra-1 overexpression. Moreover, we found that miR-34a was downregulated in 25 of 40 (62.5%) colon cancer tissues, as compared with the adjacent normal colon tissues and that the expression of miR-34a was correlated with the DNA-binding activity of p53. Unexpectedly, the DNA-binding activity of p53 was not inversely correlated with Fra-1 expression, and a significant statistical inverse correlation between miR-34a and Fra-1 expression was only observed in 14 of 40 (35%) colon cancer tissues. Taken together, our in vitro data suggest that p53 regulates Fra-1 expression, and eventually cell migration/invasion, via a miR-34a-dependent manner. However, in vivo data indicate that the p53-miR-34a pathway is not the major regulator of Fra-1 expression in human colon cancer tissues.
PMID22020335	β-Catenin/LEF1 transactivates the microRNA-371-373 cluster that modulates the Wnt/β-catenin-signaling pathway. The microRNA-371-373 (miR-371-373) cluster is specifically expressed in human embryonic stem cells (ESCs) and is thought to be involved in stem cell maintenance. Recently, microRNAs (miRNAs) of this cluster were shown to be frequently upregulated in several human tumors. However, the regulatory mechanism for the involvement of the miR-371-373 cluster in human ESCs or cancer cells remains unclear. In this study, we explored the relationship between this miRNA cluster and the Wnt/β-catenin-signaling pathway, which has been shown to be involved in both stem cell maintenance and tumorigenesis. We show that miR-371-373 expression is induced by lithium chloride and is positively correlated with Wnt/β-catenin-signaling activity in several human cancer cell lines. Mechanistically, three TCF/LEF1-binding elements (TBEs) were identified in the promoter region and shown to be required for Wnt-dependent activation of miR-371-373. Interestingly, we also found that miR-372&373, in turn, activate Wnt/β-catenin signaling. In addition, four protein genes related to the Wnt/β-catenin-signaling pathway were identified as direct targets of miR-372&373, including Dickkopf-1 (DKK1), a well-known inhibitor of Wnt/β-catenin signaling. Using a lentiviral system, we showed that overexpression of miR-372 or miR-373 promotes cell growth and the invasive activity of tumor cells as knockdown of DKK1. Taken together, our study demonstrates a novel β-catenin/LEF1-miR-372&373-DKK1 regulatory feedback loop, which may have a critical role in regulating the activity of Wnt/β-catenin signaling in human cancer cells.
PMID18776219	Transcriptional mechanism for the paired miR-433 and miR-127 genes by nuclear receptors SHP and ERRgamma. MicroRNAs (miRNAs, miRs) are genomically encoded small approximately 22 nt RNA molecules that have been shown to mediate translational repression of target mRNAs involved in cellular proliferation, differentiation and death. Despite intensive studies on their physiological and pathological functions, the molecular mechanism of how miRNA gene transcription is regulated remains largely unknown. Microarray profiling revealed 21 miRNAs clustered on chromosome 12, including miR-433 and miR-127, that were co-upregulated in small heterodimer partner (SHP, NR0B2) SHP knockouts (SHP(-/-)) liver. Gene cloning revealed that the 3'-coding region of pri-miR-433 served as the promoter region of pri-miR-127. Estrogen related receptor (ERRgamma, NR3B3) robustly activated miR-433 and miR-127 promoter reporters through ERRE, which was transrepressed by SHP. The strong elevation of miR-433 and miR-127 in Hepa-1 cells correlated with the down-regulation of SHP and up-regulation of ERRgamma. Ectopic expression of ERRgamma induced miR-433 and miR-127 expression, which was repressed by SHP coexpression. In contrast, knockdown ERRgamma decreased miR-433 and miR-127 expression. In addition, the ERRgamma agonist GSK4716 induced miR-433 and miR-127 expression both in vitro and in vivo, respectively. In summary, the coupled miR-433 and miR-127 genes were transcribed from independent promoters regulated by nuclear receptors ERRgamma/SHP in a compact space by using overlapping genomic regions.
PMID20395557	Inhibition and role of let-7d in idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation. To determine changes in expression and role of microRNAs in IPF. RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry. Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells. Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).
PMID19351822	The miR-17/92 polycistron is up-regulated in sonic hedgehog-driven medulloblastomas and induced by N-myc in sonic hedgehog-treated cerebellar neural precursors. Medulloblastoma is the most common malignant pediatric brain tumor, and mechanisms underlying its development are poorly understood. We identified recurrent amplification of the miR-17/92 polycistron proto-oncogene in 6% of pediatric medulloblastomas by high-resolution single-nucleotide polymorphism genotyping arrays and subsequent interphase fluorescence in situ hybridization on a human medulloblastoma tissue microarray. Profiling the expression of 427 mature microRNAs (miRNA) in a series of 90 primary human medulloblastomas revealed that components of the miR-17/92 polycistron are the most highly up-regulated miRNAs in medulloblastoma. Expression of miR-17/92 was highest in the subgroup of medulloblastomas associated with activation of the sonic hedgehog (Shh) signaling pathway compared with other subgroups of medulloblastoma. Medulloblastomas in which miR-17/92 was up-regulated also had elevated levels of MYC/MYCN expression. Consistent with its regulation by Shh, we observed that Shh treatment of primary cerebellar granule neuron precursors (CGNP), proposed cells of origin for the Shh-associated medulloblastomas, resulted in increased miR-17/92 expression. In CGNPs, the Shh effector N-myc, but not Gli1, induced miR-17/92 expression. Ectopic miR-17/92 expression in CGNPs synergized with exogenous Shh to increase proliferation and also enabled them to proliferate in the absence of Shh. We conclude that miR-17/92 is a positive effector of Shh-mediated proliferation and that aberrant expression/amplification of this miR confers a growth advantage to medulloblastomas.
PMID20233879	MicroRNA-519c suppresses hypoxia-inducible factor-1alpha expression and tumor angiogenesis. Hypoxia-inducible factor-1alpha (HIF-1alpha) is widely considered to be one of the key regulators of tumor angiogenesis. The upstream regulation is complex and involves several growth factors, cytokines, and hypoxia. Herein, we have identified miR-519c as a hypoxia-independent regulator of HIF-1alpha, acting through direct binding to the HIF-1alpha 3' untranslated region and leading to reduced tumor angiogenesis. Overexpression of miR-519c resulted in a significant decrease of HIF-1alpha protein levels and reduced the tube formation of human umbilical vein endothelial cells; similarly, antagomir inhibition of miR-519c increased the level of HIF-1alpha protein and enhanced angiogenic activity, suggesting an important role of miR-519c in HIF-1alpha-mediated angiogenesis. Consistent with the overexpression of miR-519c in cancer patients with better prognosis, mice injected with miR-519c-overexpressing cells exhibited dramatically reduced HIF-1alpha levels, followed by suppressed tumor angiogenesis, growth, and metastasis. In addition, we found that hepatocyte growth factor (HGF), a known HIF-1alpha inducer, reduced the miR-519c levels through an Akt-dependent pathway. This regulation was posttranscriptional and may be mediated by suppression of miR-519c maturation. Taken together, our findings provide the first evidence that miR-519c is a pivotal regulator of tumor angiogenesis and that microenvironmental HGF contributes to regulating miR-519c biogenesis in cancer cells.
PMID19351827	Thrombin induces tumor cell cycle activation and spontaneous growth by down-regulation of p27Kip1, in association with the up-regulation of Skp2 and MiR-222. The effect of thrombin on tumor cell cycle activation and spontaneous growth was examined in synchronized serum-starved tumor cell lines and a model of spontaneous prostate cancer development in TRAMP mice. BrdUrd incorporation and propidium iodide staining of prostate LNCaP cells arrested in G(0) and treated with thrombin or serum revealed a 48- and 29-fold increase in S phase cells, respectively, at 8 hours. Similar results were obtained with TRAMP cells and a glioblastoma cell line, T98G. Cell cycle kinases and inhibitors in synchronized tumor cells revealed high levels of p27(Kip1) and low levels of Skp2 and cyclins D1 and A. Addition of thrombin, TFLLRN, or serum down-regulated p27(Kip1) with concomitant induction of Skp2, Cyclin D1, and Cyclin A with similar kinetics. LNCaP p27(Kip1)-transfected cells or Skp2 knockdown cells were refractory to thrombin-induced cell cycle activation. MicroRNA 222, an inhibitor of p27(Kip1), was robustly up-regulated by thrombin. The in vitro observations were tested in vivo with transgenic TRAMP mice. Repetitive thrombin injection enhanced prostate tumor volume 6- to 8-fold (P < 0.04). Repetitive hirudin, a specific potent antithrombin, decreased tumor volume 13- to 24-fold (P < 0.04). Thus, thrombin stimulates tumor cell growth in vivo by down-regulation of p27(Kip1).
PMID20201077	MicroRNA-29, a key regulator of collagen expression in systemic sclerosis. To investigate the role of microRNA (miRNA) as posttranscriptional regulators of profibrotic genes in systemic sclerosis (SSc). MicroRNA, which target collagens, were identified by in silico analysis. Expression of miRNA-29 (miR-29) was determined by TaqMan real-time polymerase chain reaction analysis of skin biopsy and fibroblast samples from SSc patients and healthy controls as well as in the mouse model of bleomycin-induced skin fibrosis. Cells were transfected with precursor miRNA (pre-miRNA)/anti-miRNA of miR-29 using Lipofectamine. Collagen gene expression was also studied in luciferase reporter gene assays. For stimulation, recombinant transforming growth factor beta (TGFbeta), platelet-derived growth factor B (PDGF-B), or interleukin-4 (IL-4) was used. The effects of inhibiting PDGF-B and TGFbeta signaling on the levels of miR-29 were studied in vitro and in the bleomycin model. We found that miR-29a was strongly down-regulated in SSc fibroblasts and skin sections as compared with the healthy controls. Overexpression in SSc fibroblasts significantly decreased, and accordingly, knockdown in normal fibroblasts increased, the levels of messenger RNA and protein for type I and type III collagen. In the reporter gene assay, cotransfection with pre-miR-29a significantly decreased the relative luciferase activity, which suggests a direct regulation of collagen by miR-29a. TGFbeta, PDGF-B, or IL-4 reduced the levels of miR-29a in normal fibroblasts to those seen in SSc fibroblasts. Similar to human SSc, the expression of miR-29a was reduced in the bleomycin model of skin fibrosis. Inhibition of PDGF-B and TGFbeta pathways by treatment with imatinib restored the levels of miR-29a in vitro and in the bleomycin model in vivo. These data add the posttranscriptional regulation of collagens by miR-29a as a novel aspect to the fibrogenesis of SSc and suggest miR-29a as a potential therapeutic target.
PMID19333945	Expression of MicroRNA-146a in osteoarthritis cartilage. A role of microRNA, which are approximately 22-nucleotide noncoding RNAs, has recently been recognized in human diseases. The objective of this study was to identify the expression pattern of microRNA-146a (miR-146a) in cartilage from patients with osteoarthritis (OA). The expression of miR-146a in cartilage from 15 patients with OA was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Induction of the expression of miR-146a by cultures of normal human articular chondrocytes following stimulation with interleukin-1beta (IL-1beta) was examined by quantitative RT-PCR. All cartilage samples were divided into 3 groups according to a modification of the Mankin score (grade I = mild OA scored 0-5, grade II = moderate OA scored 6-10, and grade III = severe OA scored 11-14). In grade I OA cartilage samples, the expression of miR-146a and COL2A1 was significantly higher than that in the other groups (P < 0.05). In grades II and III OA cartilage, the expression of miR-146a and COL2A1 was decreased, whereas the expression of matrix metalloproteinase 13 (MMP-13) was elevated in grade II OA cartilage. These data showed that miR-146a is expressed intensely in cartilage with a low Mankin grade and that miR-146a expression decreases in parallel with the level of MMP-13 expression. Tissue section in situ hybridization of primary miR-146a (pri-miR-146a) revealed that pri-miR-146a was expressed in chondrocytes residing in all tissue layers, especially in the superficial layer, where it was intensely expressed. The expression of miR-146 was markedly elevated by IL-1beta stimulation in human chondrocytes in vitro. This study shows that miR-146 is intensely expressed in low-grade OA cartilage and that its expression is induced by stimulation of IL-1beta. Thus, miR-146 might play a role in OA cartilage pathogenesis.
PMID22052544	Sox9 is upstream of microRNA-140 in cartilage. MicroRNA-140 (miR-140) is specifically expressed in developing cartilage tissues. We have previously reported that miR-140 plays an important role during palatal cartilage development by modulating platelet-derived growth factor receptor alpha (pdgfra) in zebrafish. However, the regulatory mechanism of miR-140 in cartilage is still unknown. Using developing zebrafish, sox9a mutant (sox9a-/-) and sox9b mutant (sox9b-/-) zebrafish and SOX9 small interfering RNA in human chondrocytes, T/C-28 cells, we found that miR-140 is regulated by the cartilage master transcription regulator Sox9 in zebrafish and mammalian cells.
PMID21673106	down-regulation of Kruppel-like factor-4 (KLF4) by microRNA-143/145 is critical for modulation of vascular smooth muscle cell phenotype by transforming growth factor-beta and bone morphogenetic protein 4. In the postnatal vasculature, fully differentiated and quiescent vascular smooth muscle cells (VSMCs) in a "contractile" phenotype are required for the normal regulation of vascular tone. The transforming growth factor-β (TGF-β) superfamily of growth factors (TGF-βs and bone morphogenetic proteins (BMPs)) are potent inducers of contractile phenotype and mediate (i) induction of contractile genes, and (ii) inhibition of VSMC growth and migration. Transcription of contractile genes is positively regulated by a regulatory DNA element called a CArG box. The CArG box is activated by the binding of serum response factor and its coactivators, myocardin (Myocd) or Myocd-related transcription factors (MRTFs). Krüppel-like factor-4 (KLF4) is known to inhibit activation of the CArG box. However, the potential role of KLF4 in the contractile activities of TGF-β or BMP has not been explored. Here, we demonstrate that TGF-β and BMP4 rapidly down-regulate KLF4 through induction of microRNA-143 (miR-143) and miR-145, which leads to a reduction of KLF4 transcripts and decreased KLF4 protein expression. Inhibition of miR-145 prevents down-regulation of KLF4 and activation of contractile genes by TGF-β or BMP4, suggesting that modulation of KLF4 is a prerequisite for induction of contractile genes by TGF-β and BMP4. Interestingly, both TGF-β and BMP4 activate transcription of the miR-143/145 gene cluster through the CArG box, however, TGF-β mediates this effect through induction of Myocd expression, whereas BMP4 utilizes nuclear translocation of MRTF-A. Thus, this study sheds light on both the similarities and the differences of TGF-β and BMP4 signaling in the regulation of KLF4 and contractile genes.
PMID20368621	Cross talk between microRNA and epigenetic regulation in adult neurogenesis. Both microRNAs (miRNAs) and epigenetic regulation have important functions in stem cell biology, although the interactions between these two pathways are not well understood. Here, we show that MeCP2, a DNA methyl-CpG-binding protein, can epigenetically regulate specific miRNAs in adult neural stem cells (aNSCs). MeCP2-mediated epigenetic regulation of one such miRNA, miR-137, involves coregulation by Sox2, a core transcription factor in stem cells. miR-137 modulates the proliferation and differentiation of aNSCs in vitro and in vivo. Overexpression of miR-137 promotes the proliferation of aNSCs, whereas a reduction of miR-137 enhances aNSC differentiation. We further show that miR-137 post-transcriptionally represses the expression of Ezh2, a histone methyltransferase and Polycomb group (PcG) protein. The miR-137-mediated repression of Ezh2 feeds back to chromatin, resulting in a global decrease in histone H3 trimethyl lysine 27. Coexpression of Ezh2 can rescue phenotypes associated with miR-137 overexpression. These results demonstrate that cross talk between miRNA and epigenetic regulation contributes to the modulation of adult neurogenesis.
PMID18619954	Specific requirements of MRFs for the expression of muscle specific microRNAs, miR-1, miR-206 and miR-133. The expression of three microRNAs, miR-1, miR-206 and miR-133 is restricted to skeletal myoblasts and cardiac tissue during embryo development and muscle cell differentiation, which suggests a regulation by muscle regulatory factors (MRFs). Here we show that inhibition of C2C12 muscle cell differentiation by FGFs, which interferes with the activity of MRFs, suppressed the expression of miR-1, miR-206 and miR-133. To further investigate the role of myogenic regulators (MRFs), Myf5, MyoD, Myogenin and MRF4 in the regulation of muscle specific microRNAs we performed gain and loss-of-function experiments in vivo, in chicken and mouse embryos. We found that directed expression of MRFs in the neural tube of chicken embryos induced ectopic expression of miR-1 and miR-206. Conversely, the lack of Myf5 but not of MyoD resulted in a loss of miR-1 and miR-206 expression. Taken together our results demonstrate differential requirements of distinct MRFs for the induction of microRNA gene expression during skeletal myogenesis.
PMID20048743	Downregulation of miR-21 inhibits EGFR pathway and suppresses the growth of human glioblastoma cells independent of PTEN status. MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that regulate gene expression after transcription. Aberrant expression of miRNAs has been shown to be involved in tumorigenesis. We showed that miR-21 was one of the most frequently overexpressed miRNA in human glioblastoma (GBM) cell lines. To explore whether miR-21 can serve as a therapeutic target for glioblastoma, we downregulated miR-21 with a specific antisense oligonucleotide and found that apoptosis was induced and cell-cycle progression was inhibited in vitro in U251 (PTEN mutant) and LN229 (PTEN wild-type) GBM cells; xenograft tumors from antisense-treated U251 cells were suppressed in vivo. Antisense-miR-21-treated cells showed a decreased expression of EGFR, activated Akt, cyclin D, and Bcl-2. Although miR-21 is known to regulate PTEN and downregulation of miR-21 led to increased PTEN expression both endogenously and in a reporter gene assay, the GBM suppressor effect of antisense-miR-21 is most likely independent of PTEN regulation because U251 has mutant PTEN. Microarray analysis showed that the knockdown of miR-21 significantly altered expression of 169 genes involved in nine cell-cycle and signaling pathways. Taken together, our studies provide evidence that miR-21 may serve as a novel therapeutic target for malignant gliomas independent of PTEN status.
PMID21853155	Let-7a is a direct EWS-FLI-1 target implicated in Ewing's sarcoma development. Ewing's sarcoma family tumors (ESFT) are the second most common bone malignancy in children and young adults, characterized by unique chromosomal translocations that in 85% of cases lead to expression of the EWS-FLI-1 fusion protein. EWS-FLI-1 functions as an aberrant transcription factor that can both induce and suppress members of its target gene repertoire. We have recently demonstrated that EWS-FLI-1 can alter microRNA (miRNA) expression and that miRNA145 is a direct EWS-FLI-1 target whose suppression is implicated in ESFT development. Here, we use miRNA arrays to compare the global miRNA expression profile of human mesenchymal stem cells (MSC) and ESFT cell lines, and show that ESFT display a distinct miRNA signature that includes induction of the oncogenic miRNA 17-92 cluster and repression of the tumor suppressor let-7 family. We demonstrate that direct repression of let-7a by EWS-FLI-1 participates in the tumorigenic potential of ESFT cells in vivo. The mechanism whereby let-7a expression regulates ESFT growth is shown to be mediated by its target gene HMGA2, as let-7a overexpression and HMGA2 repression both block ESFT cell tumorigenicity. Consistent with these observations, systemic delivery of synthetic let-7a into ESFT-bearing mice restored its expression in tumor cells, decreased HMGA2 expression levels and resulted in ESFT growth inhibition in vivo. Our observations provide evidence that deregulation of let-7a target gene expression participates in ESFT development and identify let-7a as promising new therapeutic target for one of the most aggressive pediatric malignancies.
PMID22384255	Cooperation of p300 and PCAF in the control of microRNA 200c/141 transcription and epithelial characteristics. Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype.
PMID21522133	Aging and chronic DNA damage response activate a regulatory pathway involving miR-29 and p53. Aging is a multifactorial process that affects most of the biological functions of the organism and increases susceptibility to disease and death. Recent studies with animal models of accelerated aging have unveiled some mechanisms that also operate in physiological aging. However, little is known about the role of microRNAs (miRNAs) in this process. To address this question, we have analysed miRNA levels in Zmpste24-deficient mice, a model of Hutchinson-Gilford progeria syndrome. We have found that expression of the miR-29 family of miRNAs is markedly upregulated in Zmpste24(-/-) progeroid mice as well as during normal aging in mouse. Functional analysis revealed that this transcriptional activation of miR-29 is triggered in response to DNA damage and occurs in a p53-dependent manner since p53(-/-) murine fibroblasts do not increase miR-29 expression upon doxorubicin treatment. We have also found that miR-29 represses Ppm1d phosphatase, which in turn enhances p53 activity. Based on these results, we propose the existence of a novel regulatory circuitry involving miR-29, Ppm1d and p53, which is activated in aging and in response to DNA damage.
PMID18940871	Epstein-Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-kappaB pathway. The Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, substantially contributes to EBV's oncogenic potential by activating nuclear factor-kappaB (NF-kappaB). miR-155 is an oncogenic miRNA critical for B-cell maturation and immunoglobulin production in response to antigen. We report that miR-155 expression is much higher in EBV-immortalized B cells than in EBV-negative B cells. LMP1, but not LMP2, up-regulated the expression of miR-155, when transfected in EBV-negative B cells. We analyzed two putative NF-kappaB binding sites in the miR-155 promoter; both sites recruited NF-kappaB complex, in nuclear extract from EBV-immortalized cells. The exogenous expression of LMP1, in EBV-negative background, is temporally correlated to induction of p65 with binding on both NF-kappaB sites and with miR-155 overexpression. The induction of p65 binding together with increased RNA polymerase II binding, confirms that LMP1-mediated activation of miR-155 occurs transcriptionally. In reporter assays, miR-155 promoter lacking NF-kappaB binding sites was no longer activated by LMP1 expression and an intact AP1 site is needed to attain maximum activation. Finally, we demonstrate that LMP1-mediated activation of miR-155 in an EBV-negative background correlates with reduction of protein PU.1, which is a possible miR target.
PMID20118412	MicroRNA-224 is involved in transforming growth factor-beta-mediated mouse granulosa cell proliferation and granulosa cell function by targeting Smad4. Many members of the TGF-beta superfamily are indicated to play important roles in ovarian follicular development, such as affecting granulosa cell function and oocyte maturation. Abnormalities associated with TGF-beta1 signaling transduction could result in female infertility. MicroRNAs (miRNAs), as small noncoding RNAs, were recently found to regulate gene expression at posttranscriptional levels. However, little is known about the role of miRNAs in TGF-beta-mediated granulosa cell proliferation and granulosa cell function. In this study, the miRNA expression profiling was identified from TGF-beta1-treated mouse preantral granulosa cells (GCs), and three miRNAs were found to be significantly up-regulated and 13 miRNAs were down-regulated. Among up-regulated miRNAs, miR-224 was the second most significantly elevated miRNA. This up-regulation was attenuated by treatment of GCs with SB431542 (an inhibitor of TGFbeta superfamily type I receptors, thus blocking phosphorylation of the downstream effectors Smad2/3), indicating that miR-224 expression was regulated by TGF-beta1/Smads pathway. The ectopic expression of miR-224 can enhance TGF-beta1-induced GC proliferation through targeting Smad4. Inhibition of endogenous miR-224 partially suppressed GC proliferation induced by TGF-beta1. In addition, both miR-224 and TGF-beta1 can promote estradiol release from GC, at least in part, through increasing CYP19A1 mRNA levels. This is the first demonstration that miRNAs can control reproductive functions resulting in promoting TGF-beta1-induced GC proliferation and ovarian estrogen release. Such miRNA-mediated effects could be potentially used for regulation of reproductive processes or for treatment of reproductive disorders.
PMID20098732	Genomic profiling of messenger RNAs and microRNAs reveals potential mechanisms of TWEAK-induced skeletal muscle wasting in mice. Skeletal muscle wasting is a devastating complication of several physiological and pathophysiological conditions. Inflammatory cytokines play an important role in the loss of skeletal muscle mass in various chronic diseases. We have recently reported that proinflammatory cytokine TWEAK is a major muscle-wasting cytokine. Emerging evidence suggests that gene expression is regulated not only at transcriptional level but also at post-transcriptional level through the expression of specific non-coding microRNAs (miRs) which can affect the stability and/or translation of target mRNA. However, the role of miRs in skeletal muscle wasting is unknown. To understand the mechanism of action of TWEAK in skeletal muscle, we performed mRNA and miRs expression profile of control and TWEAK-treated myotubes. TWEAK increased the expression of a number of genes involved in inflammatory response and fibrosis and reduced the expression of few cytoskeletal gene (e.g. Myh4, Ankrd2, and TCap) and metabolic enzymes (e.g. Pgam2). Low density miR array demonstrated that TWEAK inhibits the expression of several miRs including muscle-specific miR-1-1, miR-1-2, miR-133a, miR-133b and miR-206. The expression of a few miRs including miR-146a and miR-455 was found to be significantly increased in response to TWEAK treatment. Ingenuity pathway analysis showed that several genes affected by TWEAK are known/putative targets of miRs. Our cDNA microarray data are consistent with miRs profiling. The levels of specific mRNAs and miRs were also found to be similarly regulated in atrophying skeletal muscle of transgenic mice (Tg) mice expressing TWEAK. Our results suggest that TWEAK affects the expression of several genes and microRNAs involved in inflammatory response, fibrosis, extracellular matrix remodeling, and proteolytic degradation which might be responsible for TWEAK-induced skeletal muscle loss.
PMID21516127	Regulation of transcription factor E2F3a and its clinical relevance in ovarian cancer. Recently we showed an integral epidermal growth factor receptor (EGFR)-E2F3a signaling path, in which E2F3a was found to be essential in EGFR-mediated proliferation in ovarian cancer cells. The present work evaluates the clinical relevance of this novel axis and of E2F3a itself in a large set of 130 ovarian cancer specimens. For this purpose E2F3a and its counterpart, E2F3b, were measured by RT-PCR and activated EGFR was assessed by immunohistochemistry. When compared with healthy control tissue, both E2F3 isoforms were overexpressed in the cancers, but only E2F3a expression correlated with tumor stage (ρ=0.349, P=0.0001) and residual disease (ρ=0.254, P=0.004). Univariate survival analyses showed E2F3a and activated EGFR to be associated with poor PFS and OS. Furthermore, a strong, positive correlation between activated EGFR and E2F3a expression was shown (P=0.0001). We further identified two EGFR-independent mechanisms that regulate E2F3a expression, namely one, acting by promoter methylation of miR-34a, which by its physical interaction with E2F3a transcripts causes their degradation, and the second based on 6p22 gene locus amplification. MiRIDIAN-based knockdown and induction of miR-34a evidenced a direct regulatory link between miR-34a and E2F3a, and the tumor-suppressive character of miR-34a was documented by its association with improved survival. Although, 6p22 gene locus amplification was detected in a significant number of ovarian cancer specimens, 6p22 ploidy was not relevant in predicting survival. In Cox regression analysis, E2F3a, but not activated EGFR or miR-34a expression, retained independent prognostic significance (PFS: hazards ratio 3.785 (1.326-9.840), P=0.013; OS: hazards ratio 4.651 (1.189-15.572), P=0.013). These clinical findings highlight the relevance of E2F3a in the biology of ovarian cancer. Moreover, identification of EGFR-independent mechanisms in E2F3a control can be helpful in explaining the non-responsiveness of therapeutic EGFR targeting in ovarian cancer.
PMID19221490	MiR-34, SIRT1 and p53: the feedback loop. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Several studies have linked dysregulation of miRNA with tumorigenesis. The TP53 is one of the most commonly mutated genes in human cancers, and its gene product p53 activates transcription of a set of miRNA including the miR-34 family of miRNA. The miR-34 family regulates cell cycle progression, cellular senescence and apoptosis, but the targets of miR-34 are not completely defined. We recently found that miR-34a inhibits SIRT1, a gene that regulates cellular senescence and limits longevity. SIRT1 also regulates p53 dependent apoptosis through deacetylating and stabilizing p53. We also discovered that SIRT1 mediates miR-34a activation of apoptosis by regulating p53 activity. Based on this observation, we propose a positive feedback loop, in which p53 induces expression of miR-34a which suppresses SIRT1, increasing p53 activity.
PMID20351064	MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro. Smooth muscle cell (SMC) plasticity plays an important role during development and in vascular pathologies such as atherosclerosis and restenosis. It was recently shown that down-regulation of microRNA (miR)-143 and -145, which are coexpressed from a single promoter, regulates the switch from contractile to synthetic phenotype, allowing SMCs to migrate and proliferate. We show in this study that loss of miR-143/145 in vitro and in vivo results in the formation of podosomes, which are actin-rich membrane protrusions involved in the migration of several cell types, including SMCs. We further show that platelet-derived growth factor (PDGF) mediates podosome formation in SMCs through the regulation of miR-143/145 expression via a pathway involving Src and p53. Moreover, we identify key podosome regulators as targets of miR-143 (PDGF receptor alpha and protein kinase C epsilon) and miR-145 (fascin). Thus, dysregulation of the miR-143 and -145 genes is causally involved in the aberrant SMC plasticity encountered during vascular disease, in part through the up-regulation of an autoregulatory loop that promotes podosome formation.
PMID18818206	Down-regulation of micro-RNA-1 (miR-1) in lung cancer. Suppression of tumorigenic property of lung cancer cells and their sensitization to doxorubicin-induced apoptosis by miR-1. Micro-RNAs are approximately 21-25-nucleotide-long noncoding RNAs that regulate gene expression primarily at the post-transcriptional level in animals. Here, we report that micro-RNA-1 (miR-1), abundant in the cardiac and smooth muscles, is expressed in the lung and is down-regulated in human primary lung cancer tissues and cell lines. In situ hybridization demonstrated localization of miR-1 in bronchial epithelial cells. The tumor suppressor C/EBPalpha, frequently suppressed in lung cancer, reactivated miR-1 expression in the lung cancer cells. Repressed miR-1 was also activated in lung cancer cells upon treatment with a histone deacetylase inhibitor. These observations led us to examine the antitumorigenic potential of miR-1 in lung cancer cells. Expression of miR-1 in nonexpressing A549 and H1299 cells reversed their tumorigenic properties, such as growth, replication potential, motility/migration, clonogenic survival, and tumor formation in nude mice. Exogenous miR-1 significantly reduced expression of oncogenic targets, such as MET, a receptor tyrosine kinase, and Pim-1, a Ser/Thr kinase, frequently up-regulated in lung cancer. Similarly, the levels of two additional targets, FoxP1, a transcription factor with oncogeneic property, and HDAC4 that represses differentiation-promoting genes, were reduced in miR-1-expressing cells. Conversely, depletion of miR-1 facilitated N417 cell growth with concomitant elevation of these targets. Further, ectopic miR-1 induced apoptosis in A549 cells in response to the potent anticancer drug doxorubicin. Enhanced activation of caspases 3 and 7, cleavage of their substrate PARP-1, and depletion of anti-apoptotic Mcl-1 contributed to the sensitivity of miR-1-expressing cells to doxorubicin. Thus, miR-1 has potential therapeutic application against lung cancers.
PMID18710938	Oct4/Sox2-regulated miR-302 targets cyclin D1 in human embryonic stem cells. Oct4 and Sox2 are transcription factors required for pluripotency during early embryogenesis and for the maintenance of embryonic stem cell (ESC) identity. Functional mechanisms contributing to pluripotency are expected to be associated with genes transcriptionally activated by these factors. Here, we show that Oct4 and Sox2 bind to a conserved promoter region of miR-302, a cluster of eight microRNAs expressed specifically in ESCs and pluripotent cells. The expression of miR-302a is dependent on Oct4/Sox2 in human ESCs (hESCs), and miR-302a is expressed at the same developmental stages and in the same tissues as Oct4 during embryogenesis. miR-302a is predicted to target many cell cycle regulators, and the expression of miR-302a in primary and transformed cell lines promotes an increase in S-phase and a decrease in G(1)-phase cells, reminiscent of an ESC-like cell cycle profile. Correspondingly, the inhibition of miR-302 causes hESCs to accumulate in G(1) phase. Moreover, we show that miR-302a represses the productive translation of an important G(1) regulator, cyclin D1, in hESCs. The transcriptional activation of miR-302 and the translational repression of its targets, such as cyclin D1, may provide a link between Oct4/Sox2 and cell cycle regulation in pluripotent cells.
PMID21059906	microRNA-132 regulates dendritic growth and arborization of newborn neurons in the adult hippocampus. Newborn neurons in the dentate gyrus of the adult hippocampus rely upon cAMP response element binding protein (CREB) signaling for their differentiation into mature granule cells and their integration into the dentate network. Among its many targets, the transcription factor CREB activates expression of a gene locus that produces two microRNAs, miR-132 and miR-212. In cultured cortical and hippocampal neurons, miR-132 functions downstream from CREB to mediate activity-dependent dendritic growth and spine formation in response to a variety of signaling pathways. To investigate whether miR-132 and/or miR-212 contribute to the maturation of dendrites in newborn neurons in the adult hippocampus, we inserted LoxP sites surrounding the miR-212/132 locus and specifically targeted its deletion by stereotactically injecting a retrovirus expressing Cre recombinase. Deletion of the miR-212/132 locus caused a dramatic decrease in dendrite length, arborization, and spine density. The miR-212/132 locus may express up to four distinct microRNAs, miR-132 and -212 and their reverse strands miR-132* and -212*. Using ratiometric microRNA sensors, we determined that miR-132 is the predominantly active product in hippocampal neurons. We conclude that miR-132 is required for normal dendrite maturation in newborn neurons in the adult hippocampus and suggest that this microRNA also may participate in other examples of CREB-mediated signaling.
PMID18066065	Widespread microRNA repression by Myc contributes to tumorigenesis. The c-Myc oncogenic transcription factor (Myc) is pathologically activated in many human malignancies. Myc is known to directly upregulate a pro-tumorigenic group of microRNAs (miRNAs) known as the miR-17-92 cluster. Through the analysis of human and mouse models of B cell lymphoma, we show here that Myc regulates a much broader set of miRNAs than previously anticipated. Unexpectedly, the predominant consequence of activation of Myc is widespread repression of miRNA expression. Chromatin immunoprecipitation reveals that much of this repression is likely to be a direct result of Myc binding to miRNA promoters. We further show that enforced expression of repressed miRNAs diminishes the tumorigenic potential of lymphoma cells. These results demonstrate that extensive reprogramming of the miRNA transcriptome by Myc contributes to tumorigenesis.
PMID21868695	Transplantation of human pericyte progenitor cells improves the repair of infarcted heart through activation of an angiogenic program involving micro-RNA-132. Pericytes are key regulators of vascular maturation, but their value for cardiac repair remains unknown. We investigated the therapeutic activity and mechanistic targets of saphenous vein-derived pericyte progenitor cells (SVPs) in a mouse myocardial infarction (MI) model. SVPs have a low immunogenic profile and are resistant to hypoxia/starvation (H/S). Transplantation of SVPs into the peri-infarct zone of immunodeficient CD1/Foxn-1(nu/nu) or immunocompetent CD1 mice attenuated left ventricular dilatation and improved ejection fraction compared to vehicle. Moreover, SVPs reduced myocardial scar, cardiomyocyte apoptosis and interstitial fibrosis, improved myocardial blood flow and neovascularization, and attenuated vascular permeability. SVPs secrete vascular endothelial growth factor A, angiopoietin-1, and chemokines and induce an endogenous angiocrine response by the host, through recruitment of vascular endothelial growth factor B expressing monocytes. The association of donor- and recipient-derived stimuli activates the proangiogenic and prosurvival Akt/eNOS/Bcl-2 signaling pathway. Moreover, microRNA-132 (miR-132) was constitutively expressed and secreted by SVPs and remarkably upregulated, together with its transcriptional activator cyclic AMP response element-binding protein, on stimulation by H/S or vascular endothelial growth factor B. We next investigated if SVP-secreted miR-132 acts as a paracrine activator of cardiac healing. In vitro studies showed that SVP conditioned medium stimulates endothelial tube formation and reduces myofibroblast differentiation, through inhibition of Ras-GTPase activating protein and methyl-CpG-binding protein 2, which are validated miR-132 targets. Furthermore, miR-132 inhibition by antimiR-132 decreased SVP capacity to improve contractility, reparative angiogenesis, and interstitial fibrosis in infarcted hearts. SVP transplantation produces long-term improvement of cardiac function through a novel paracrine mechanism involving the secretion of miR-132 and inhibition of its target genes.
PMID20798686	Involvement of NF-κB/miR-448 regulatory feedback loop in chemotherapy-induced epithelial-mesenchymal transition of breast cancer cells. The epithelial-mesenchymal transition (EMT) induced by chemotherapeutic agents promotes malignant tumor progression; however, the mechanism underlying the drug-induced EMT remains unclear. In this study, we reported that miR-448 is the most downregulated microRNA following chemotherapy. Suppression of miR-448 correlated with EMT induction in breast cancer in vitro and in vivo. With the use of chromatin immunoprecipitation-seq analysis, we demonstrated that miR-448 suppression induces EMT by directly targeting special AT-rich sequence-binding protein-1 (SATB1) mRNA, leading to elevated levels of amphiregulin and thereby, increasing epidermal growth factor receptor (EGFR)-mediated Twist1 expression, as well as nuclear factor κB (NF-κB) activation. On the other hand, we also found that the adriamycin-activated NF-κB directly binds the promoter of miR-448 suppressing its transcription, suggesting a positive feedback loop between NF-κB and miR-448. Furthermore, all patients who received cyclophosphamide (CP), epirubicin plus taxotere/CP, epirubicin plus 5-fluorouracil chemotherapy showed miR-448 suppression, an increased SATB1, Twist1 expression and acquisition of mesenchymal phenotypes. These findings reveal an underlying regulatory pathway, in which the autoregulation between NF-κB and miR-448 is important for restrain miR-448 suppression upon chemotherapy and may have a role in the regulation of chemotherapy-induced EMT. Disruption of the NF-κB-miR-448 feedback loop during clinical treatment may improve the chemotherapy response of human breast cancers in which EMT is a critical component.
PMID19064005	Stable Argonaute2 overexpression differentially regulates microRNA production. microRNAs are a class of small RNA molecules that associate with Argonaute proteins to regulate gene expression. Argonaute proteins not only mediate the functions of microRNAs, but also play an essential role in the biogenesis of microRNAs. Here, we report that stable, long-term overexpression of Argonaute2, an Argonaute isoform, induces the production of a number of microRNAs, such as the let-7 family of microRNAs, in 293T cells. On the other hand, the expression of many microRNAs is insensitive to elevated Argonaute levels, and microRNAs in the miR-17-92 and homologous clusters are even down-regulated. The down-regulation may result from the let-7-mediated inhibition of the expression of Myc, which positively controls the transcription of the microRNAs clusters. Our data suggest that human cells have a mechanism for microRNA homeostasis, and that overexpression of a general microRNA processing factor can differentially regulate the expression of specific microRNAs.
PMID20093556	MicroRNA-21 is induced early in pancreatic ductal adenocarcinoma precursor lesions. Pancreatic ductal adenocarcinoma (PDAC) has the poorest overall prognosis among gastrointestinal cancers; however, curative resection in early-stage PDAC greatly improves survival rates, indicating the importance of early detection. Because abnormal microRNA production is commonly detected in cancer, we investigated noninvasive precursor pancreatic intraepithelial neoplasia (PanIN) lesions for microRNA production as a potential early biomarker of PDAC. Pathologists identified and classified ductal lesions. We extracted total RNA from laser-capture microdissected PanIN tissue samples from a conditional KRAS(G12D) mouse model (n = 29) or of human origin (n = 38) (KRAS is v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). MicroRNA production was quantified by quantitative real-time PCR. Internal controls included 5S and U6 RNAs. Production of microRNAs miR-21, miR-205, and miR-200 paralleled PanIN progression in the KRAS(G12D) mouse model, compared with microRNA production in samples of nonpathologic ducts. miR-21 demonstrated the highest relative concentrations in the precursor lesions. Interestingly, miR-205 and miR-21 up-regulation preceded phenotypic changes in the ducts. The production of microRNAs miR-21, miR-221, miR-222, and let-7a increased with human PanIN grade, with peak production occurring in hyperplastic PanIN-2/3 lesions. In situ hybridization analysis indicated miR-21 production to be concentrated in pathologic ductal cells. miR-21 production was regulated by KRAS(G12D) and epidermal growth factor receptor in PDAC-derived cell lines. Aberrant microRNA production is an early event in the development of PanIN. Our findings indicate that miR-21 warrants further investigation as a marker for early detection of PDAC.
PMID21656127	High-mobility group A1 proteins enhance the expression of the oncogenic miR-222 in lung cancer cells. High-mobility group A1 (HMGA1) is a non-histone chromatin protein that has the ability to regulate the transcriptional activity of many genes. Overexpression of HMGA1 is associated with malignant cellular behavior in a range of human cancers but the underlying mechanism is largely unknown. Here we showed that in a cohort of non-small cell lung cancer (NSCLC) tumors, HMGA1 overexpression was immediately associated with enhanced expression of an oncogenic miRNA, namely, miR-222. Chromatin immunoprecipitation (CHIP) assay revealed that HMGA1 directly binds to the proximal promoter of miR-222 in NSCLC cells. We further showed that HMGA1 silencing reduced miR-222 transcriptional activity, whereas forced HMGA1 expression increased it, indicating that miR-222 is directly regulated by HMGA1. Based on in silico prediction, one of the putative targets of miR-222 is phosphatase 2A subunit B (PPP2R2A) which inhibits Akt phosphorylation (p-Akt). We demonstrated that miR-222 inhibited protein expression of PPP2R2A in NSCLC cells by directly interacting with its 3'-UTR region, leading to an obvious increase of p-Akt. HMGA1 silencing augmented PPP2R2A protein expression and inhibited Akt signaling, resulting in significantly retarded cell growth response to IGF-I. These results suggested that HMGA1 is a positive regulator of miR-222, and HMGA1 overexpression might contribute to dysregulation of Akt signaling in NSCLC.
PMID22292433	Transcriptional activation of microRNA-34a by NF-kappa B in human esophageal cancer cells. miR-34a functions as an important tumor suppressor during the process of carcinogenesis. However, the mechanism of miR-34a dysregulation in human malignancies has not been well elucidated. Our study aimed to further investigate the regulation mechanism of miR-34a. We found that overexpression of NF-kappa B p65 subunit could increase miR-34a levels in EC109, an esophageal squamous cancer cell line, while ectopic expression of DN IkappaB leaded to a significant reduction of miR-34a expression. Bioinformatics analysis suggested three putative KB sites in promoter region of miR-34a gene. Mutation two of these KB sites impaired p65 induced miR-34a transcriptional activity. Chromatin immunoprecipitation and electrophoretic mobility shift assays both showed that NF-kappaB could specifically bind to the third KB site located in miR-34a promoter. In addition, we found that overexpression of NF-kappaB p65 could not successfully induce miR-34a expression in esophageal cancer cell lines with mutant p53 or decreased p53. Reporter assay further showed that NF-kappaB-induced miR-34a transcriptional activity was reduced by p53 impairment. Nevertheless, CHIP analysis suggested binding of NF-kappaB to miR-34a promoter was not affected in cells with mutant p53. Our work indicates a novel mechanism of miR-34a regulation that NF-kappaB could elevate miR-34a expression levels through directly binding to its promoter. And wildtype p53 is responsible for NF-kappaB-mediated miR-34a transcriptional activity but not for NF-kappaB binding. These findings might be helpful in understanding miR-34a abnormality in human malignancies and open new perspectives for the roles of miR-34a and NF-kappaB in tumor progression.
PMID22195016	Insulin-like growth factor-1 receptor is regulated by microRNA-133 during skeletal myogenesis. The insulin-like growth factor (IGF) signaling pathway has long been established as playing critical roles in skeletal muscle development. However, the underlying regulatory mechanism is poorly understood. Recently, a large family of small RNAs, named microRNAs (miRNAs), has been identified as key regulators for many developmental processes. Because miRNAs participate in the regulation of various signaling pathways, we hypothesized that miRNAs may be involved in the regulation of IGF signaling in skeletal myogenesis. In the present study, we determined that the cell-surface receptor IGF-1R is directly regulated by a muscle-specific miRNA, microRNA-133 (miR-133). A conserved and functional binding site for miR-133 was identified in the 3'untranslated region (3'UTR) of IGF-1R. During differentiation of C2C12 myoblasts, IGF-1R protein, but not messenger RNA (mRNA) expression, was gradually reduced, concurrent with the upregulation of miR-133. Overexpression of miR-133 in C2C12 cells significantly suppressed IGF-1R expression at the posttranscriptional level. We also demonstrated that both overexpression of miR-133 and knockdown of IGF-1R downregulated the phosphorylation of Akt, the central mediator of the PI3K/Akt signaling pathway. Furthermore, upregulation of miR-133 during C2C12 differentiation was significantly accelerated by the addition of IGF-1. Mechanistically, we found that the expression of myogenin, a myogenic transcription factor reported to transactivate miR-133, was increased by IGF-1 stimulation. Our results elucidate a negative feedback circuit in which IGF-1-stimulated miR-133 in turn represses IGF-1R expression to modulate the IGF-1R signaling pathway during skeletal myogenesis. These findings also suggest that miR-133 may be a potential therapeutic target in muscle diseases.
PMID20177001	Reciprocal repression between microRNA-133 and calcineurin regulates cardiac hypertrophy: a novel mechanism for progressive cardiac hypertrophy. Cardiac hypertrophy involves a remodeling process of the heart in response to diverse pathological stimuli. Both calcineurin/nuclear factor of activated T cells pathway and microRNA-133 (miR-133) have been shown to play a critical role in cardiac hypertrophy. It has been recognized that the expression and activity of calcineurin increases and miR-133 expression decreases in the hypertrophic heart, and inhibition of calcineurin or increase of miR-133 expression protects against cardiac hypertrophy. Here we tested the interaction between miR-133 and calcineurin in cardiac hypertrophy. Cardiac hypertrophy in vivo and in vitro was induced by transverse aortic constriction and phenylephrine treatment. mRNA levels were measured by using real-time PCR methods. Luciferase assays showed that transfection of miR-133 in HEK293 cells downregulated calcineurin expression, which was reversed by cotransfection with the miR-133-specific 2'-O-methyl antisense inhibitory oligoribonucleotides. These results were confirmed in cultured primary cardiomyocytes. miR-133 expression was downregulated, and calcineurin activity was enhanced in both in vivo and in vitro cardiac hypertrophy models. Treatment of cells and animals with cyclosporin A, an inhibitor of calcineurin, prevented miR-133 downregulation. Moreover, the antisense oligodeoxynucleotides against the catalytic subunits of calcineurin Abeta and the decoy oligodeoxynucleotides targeting nuclear factor of activated T cells transcription factor, a calcineurin downstream effector, increased miR-133 expression in cultured primary cardiomyocytes. Our data show that reciprocal repression between miR-133 and calcineurin regulates cardiac hypertrophy.
PMID18755897	miR-34a repression of SIRT1 regulates apoptosis. MicroRNA 34a (miR-34a) is a tumor suppressor gene, but how it regulates cell proliferation is not completely understood. We now show that the microRNA miR-34a regulates silent information regulator 1 (SIRT1) expression. MiR-34a inhibits SIRT1 expression through a miR-34a-binding site within the 3' UTR of SIRT1. MiR-34 inhibition of SIRT1 leads to an increase in acetylated p53 and expression of p21 and PUMA, transcriptional targets of p53 that regulate the cell cycle and apoptosis, respectively. Furthermore, miR-34 suppression of SIRT1 ultimately leads to apoptosis in WT human colon cancer cells but not in human colon cancer cells lacking p53. Finally, miR-34a itself is a transcriptional target of p53, suggesting a positive feedback loop between p53 and miR-34a. Thus, miR-34a functions as a tumor suppressor, in part, through a SIRT1-p53 pathway.
PMID22114673	Listeria monocytogenes infection in macrophages induces vacuolar-dependent host miRNA response. Listeria monocytogenes is a gram-positive facultative intracellular pathogen, causing serious illness in immunocompromised individuals and pregnant women. Upon detection by macrophages, which are key players of the innate immune response against infection, L. monocytogenes induces specific host cell responses which need to be tightly controlled at transcriptional and post-transcriptional levels. Here, we ask whether and how host miRNAs, which represent an important mechanism of post-transcriptional regulation in a wide array of biological processes, are altered by a model pathogen upon live infection of murine bone marrow derived macrophages. We first report that L. monocytogenes subverts the host genome-wide miRNA profile of macrophages in vitro. Specifically, we show that miR-155, miR-146a, miR-125a-3p/5p and miR-149 were amongst the most significantly regulated miRNAs in infected macrophages. Strikingly, these miRNAs were highly upregulated upon infection with the Listeriolysin-deficient L. monocytogenes mutant Δhly, that cannot escape from the phagosome thus representing a vacuolar-contained infection. The vacuolar miRNA response was significantly reduced in macrophages deficient for MyD88. In addition, miR-146a and miR-125a-3p/5p were regulated at transcriptional levels upon infection, and miR-125a-3p/5p were found to be TLR2 responsive. Furthermore, miR-155 transactivation in infection was regulated by NF-κB p65, while miR-146a and miR-125a-3p/5p expression was unaffected in p65-deficient primary macrophages upon L. monocytogenes infection. Our results demonstrate that L. monocytogenes promotes significant changes in the miRNA expression profile in macrophages, and reveal a vacuolar-dependent miRNA signature, listeriolysin-independent and MyD88-dependent. These miRNAs are predicted to target immune genes and are therefore most likely involved in regulation of the macrophage innate immune response against infection at post-transcriptional levels.
PMID21471220	MicroRNA-378 targets the myogenic repressor MyoR during myoblast differentiation. MicroRNAs play important roles in many cell processes, including the differentiation process in several different lineages. For example, microRNAs can promote differentiation by repressing negative regulators of transcriptional activity. These regulated transcription factors can further up-regulate levels of the microRNA in a feed-forward mechanism. Here we show that MyoD up-regulates miR-378 during myogenic differentiation in C2C12 cells. ChIP and high throughput sequencing analysis shows that MyoD binds in close proximity to the miR-378 gene and causes both transactivation and chromatin remodeling. Overexpression of miR-378 increases the transcriptional activity of MyoD, in part by repressing an antagonist, MyoR. The 3' untranslated region of MyoR contains a direct binding site for miR-378. The presence of this binding site significantly reduces the ability of MyoR to prevent the MyoD-driven transdifferentiation of fibroblasts. MyoR and miR-378 were anticorrelated during cardiotoxin-induced adult muscle regeneration in mice. Taken together, this shows a feed-forward loop where MyoD indirectly down-regulates MyoR via miR-378.
PMID21518799	p53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1 and ZEB2. p53 suppresses tumor progression and metastasis. Epithelial-mesenchymal transition (EMT) is a key process in tumor progression and metastasis. The transcription factors ZEB1 and ZEB2 promote EMT. Here, we show that p53 suppresses EMT by repressing expression of ZEB1 and ZEB2. By profiling 92 primary hepatocellular carcinomas (HCCs) and 9 HCC cell lines, we found that p53 up-regulates microRNAs (miRNAs), including miR-200 and miR-192 family members. The miR-200 family members transactivated by p53 then repress ZEB1/2 expression. p53-regulated miR-192 family members also repress ZEB2 expression. Inhibition or overexpression of the miRNAs affects p53-regulated EMT by altering ZEB1 and ZEB2 expression. Our findings indicate that p53 can regulate EMT, and that p53-regulated miRNAs are critical mediators of p53-regulated EMT.
PMID18492795	Inducible expression of microRNA-194 is regulated by HNF-1alpha during intestinal epithelial cell differentiation. Maintenance of the intestinal epithelium is based on well-balanced molecular mechanisms that confer the stable and continuous supply of specialized epithelial cell lineages from multipotent progenitors. Lineage commitment decisions in the intestinal epithelium system involve multiple regulatory systems that interplay with each other to establish the cellular identities. Here, we demonstrate that the microRNA system could be involved in intestinal epithelial cell differentiation, and that microRNA-194 (miR-194) is highly induced during this process. To investigate this inducible expression mechanism, we identified the genomic structure of the miR-194-2, -192 gene, one of the inducible class of miR-194 parental genes. Furthermore, we identified its transcriptional regulatory region that contains a consensus-binding motif for hepatocyte nuclear factor-1alpha (HNF-1alpha), which is well known as a transcription factor to regulate gene expression in intestinal epithelial cells. By chromatin immunoprecipitation assay and luciferase reporter analysis, we revealed that pri-miR-194-2 expression is controlled by HNF-1alpha, and its consensus binding region is required for the transcription of pri-miR-194-2 in vivo in an intestinal epithelial cell line, Caco-2. Our observations indicate that microRNA genes could be targets of lineage-specific transcription factors and that microRNAs are regulated by a tissue-specific manner in the intestinal epithelium. Therefore, our work suggests that induced expression of these microRNAs have important roles in intestinal epithelium maturation.
PMID22322403	miR-21 promotes migration and invasion by the miR-21-PDCD4-AP-1 feedback loop in human hepatocellular carcinoma. Distant migration and invasion is the main contributor to the death of cancer patients and miRNAs have been implicated in these processes. In the present study, we identified the role of microRNA-21 (miR-21) in hepatocellular carcinoma (HCC) migration and invasion and determined its underlying regulatory mechanism. miR-21 was significantly upregulated in HCC tissues and cell lines, compared with adjacent non-tumor tissues and normal hepatic cells. miR-21 upregulation was associated with the capacity of tumor migration and invasion in HCC. The expression level of miR-21 was inversely correlated with the protein expression level of a previously identified target gene, programmed cell death 4 (PDCD4). In HepG2 cells, inhibition of miR-21 expression repressed cell migration and invasion by upregulating both mRNA and protein levels of PDCD4 and downregulating key downstream signaling pathway molecules, including phospho-c-Jun, matrix metalloproteinases (MMP)-2 and MMP-9. Moreover, activation protein 1 (AP-1) could directly activate miR-21 transcription. Taken together, these results provide evidence that miR-21 promotes migration and invasion in HCC through the miR-21-PDCD4-AP-1 feedback loop, suggesting that targeting the miR-21-PDCD4-AP-1 loop may represent a promising strategy in the management of HCC.
PMID21909380	Targeting epigenetic regulation of miR-34a for treatment of pancreatic cancer by inhibition of pancreatic cancer stem cells. MicroRNA-34a (miR-34a) is a transcriptional target of p53 and is down-regulated in pancreatic cancer. This study aimed to investigate the functional significance of miR-34a in pancreatic cancer progression through its epigenetic restoration with chromatin modulators, demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC) and HDAC inhibitor Vorinostat (SAHA). Re-expression of miR-34a in human pancreatic cancer stem cells (CSCs) and in human pancreatic cancer cell lines upon treatment with 5-Aza-dC and SAHA strongly inhibited the cell proliferation, cell cycle progression, self-renewal, epithelial to mesenchymal transition (EMT) and invasion. In pancreatic CSCs, modulation of miR-34a induced apoptosis by activating caspase-3/7. Treatment of pancreatic CSCs with the chromatin-modulating agents resulted in the inhibition of Bcl-2, CDK6 and SIRT1, which are the putative targets of miR-34a. MiR-34a upregulation by these agents also induced acetylated p53, p21(WAF1), p27(KIP1) and PUMA in pancreatic CSCs. Inhibition of miR-34a by antagomiR abrogates the effects of 5-Aza-dC and SAHA, suggesting that 5-Aza-dC and SAHA regulate stem cell characteristics through miR-34a. In CSCs, SAHA inhibited Notch pathway, suggesting its suppression may contribute to inhibition of the self-renewal capacity and induction of apoptosis. Interestingly, treatment of pancreatic CSCs with SAHA resulted in the inhibition of EMT with the transcriptional up-regulation of E-Cadherin and down-regulation of N-Cadherin. Expression of EMT inducers (Zeb-1, Snail and Slug) was inhibited in CSCs upon treatment with SAHA. 5-Aza-dC and SAHA also retard in vitro migration and invasion of CSCs. The present study thus demonstrates the role of miR-34a as a critical regulator of pancreatic cancer progression by the regulating CSC characteristics. The restoration of its expression by 5-Aza-dC and SAHA in CSCs will not only provide mechanistic insight and therapeutic targets for pancreatic cancer but also promising reagents to boost patient response to existing chemotherapies or as a standalone cancer drug by eliminating the CSC characteristics.
PMID18695042	A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation. Decreased expression of specific microRNAs (miRNAs) occurs in human tumors, which suggests a function for miRNAs in tumor suppression. Herein, levels of the miR-17-5p/miR-20a miRNA cluster were inversely correlated to cyclin D1 abundance in human breast tumors and cell lines. MiR-17/20 suppressed breast cancer cell proliferation and tumor colony formation by negatively regulating cyclin D1 translation via a conserved 3' untranslated region miRNA-binding site, thereby inhibiting serum-induced S phase entry. The cell cycle effect of miR-17/20 was abrogated by cyclin D1 siRNA and in cyclin D1-deficient breast cancer cells. Mammary epithelial cell-targeted cyclin D1 expression induced miR-17-5p and miR-20a expression in vivo, and cyclin D1 bound the miR-17/20 cluster promoter regulatory region. In summary, these studies identify a novel cyclin D1/miR-17/20 regulatory feedback loop through which cyclin D1 induces miR-17-5p/miR-20a. In turn, miR-17/20 limits the proliferative function of cyclin D1, thus linking expression of a specific miRNA cluster to the regulation of oncogenesis.
PMID21892154	Experience-dependent expression of miR-132 regulates ocular dominance plasticity. miR-132 is a CREB-induced microRNA that is involved in dendritic spine plasticity. We found that visual experience regulated histone post-translational modifications at a CRE locus that is important for miR-212 and miR-132 cluster transcription, and regulated miR-132 expression in the visual cortex of juvenile mice. Monocular deprivation reduced miR-132 expression in the cortex contralateral to the deprived eye. Counteracting this miR-132 reduction with an infusion of chemically modified miR-132 mimic oligonucleotides completely blocked ocular dominance plasticity.
PMID20386864	MicroRNA-34a is induced via p53 during cisplatin nephrotoxicity and contributes to cell survival. MicroRNAs are small noncoding RNAs that are produced endogenously and have emerged as important regulators in pathophysiological conditions such as development and tumorigenesis. Very little is known about the regulation of microRNAs in renal diseases, including acute kidney injury (AKI). In this study, we examined the regulation of microRNA-34a (miR-34a) in experimental models of cisplatin-induced AKI and nephrotoxicity. By Northern blot and real-time polymerase chain reaction analyses, we detected an induction of miR-34a in vitro during cisplatin treatment of mouse proximal tubular cells and also in vivo during cisplatin nephrotoxicity in C57BL/6 mice. In cultured cells, miR-34a was induced within a few hours. In mice, miR-34a induction was detectable in renal tissues after 1 d of cisplatin treatment and increased to approximately four-fold of control at d 3. During cisplatin treatment, p53 was activated. Inhibition of p53 with pifithrin-α abrogated the induction of miR-34a during cisplatin treatment of proximal tubular cells. In vivo, miR-34a induction by cisplatin was abrogated in p53-deficient mice, a result that further confirms a role for p53 in miR-34a induction during cisplatin nephrotoxicity. Functionally, antagonism of miR-34a with specific antisense oligonucleotides increased cell death during cisplatin treatment. Collectively, the results suggest that miR-34a is induced via p53 during cisplatin nephrotoxicity and may play a cytoprotective role for cell survival.
PMID18977326	NF-kappaB-YY1-miR-29 regulatory circuitry in skeletal myogenesis and rhabdomyosarcoma. Studies support the importance of microRNAs in physiological and pathological processes. Here we describe the regulation and function of miR-29 in myogenesis and rhabdomyosarcoma (RMS). Results demonstrate that in myoblasts, miR-29 is repressed by NF-kappaB acting through YY1 and the Polycomb group. During myogenesis, NF-kappaB and YY1 downregulation causes derepression of miR-29, which in turn accelerates differentiation by targeting its repressor YY1. However, in RMS cells and primary tumors that possess impaired differentiation, miR-29 is epigenetically silenced by an activated NF-kappaB-YY1 pathway. Reconstitution of miR-29 in RMS in mice inhibits tumor growth and stimulates differentiation, suggesting that miR-29 acts as a tumor suppressor through its promyogenic function. Together, these results identify a NF-kappaB-YY1-miR-29 regulatory circuit whose disruption may contribute to RMS.
PMID21665894	Downregulation of miR-132 by promoter methylation contributes to pancreatic cancer development. MicroRNAs (miRNAs), which regulate gene expression by partial complementarity to the 3' untranslated region of their target genes, have been implicated in cancer initiation and progression. However, the molecular mechanism underlying the regulation of miRNA expression during pancreatic tumorigenesis has not been extensively reported. In this study, we first compared the miRNA expression in human pancreatic cancers and adjacent normal tissues by miRNA array and identified 12 differentially expressed miRNAs. miR-132, which is downregulated in tumors, was further studied in greater detail. Decreased expression of miR-132 was confirmed in 16 of 20 pancreatic carcinomas (P < 0.0001), compared with their respective benign tissues by TaqMan miRNA assays. miR-132 expression was remarkably influenced by promoter methylation in PANC1 and SW1990 cells. Promoter hypermethylation was observed in tumor samples but not in the normal counterparts, and the expression of miR-132 negatively correlated with its methylation status (P = 0.013). miR-132 was transcribed by RNA polymerase II, and Sp1 played a major role in miR-132 transcription. The expression of Sp1 correlated with that of miR-132 in tissues. Moreover, cancerous tissues showed significantly lower Sp1-binding affinity to the miR-132 promoter, relative to non-tumor samples. Proliferation and colony formation of pancreatic cancer cells were suppressed in cells transfected with miR-132 mimics and enhanced in cells transfected with miR-132 inhibitor by negatively regulating the Akt-signaling pathway. Our present findings illustrate the mechanism driving miR-132 downregulation and the important role of miR-132 in pancreatic cancer development.
PMID18203756	MeCP2-dependent repression of an imprinted miR-184 released by depolarization. Both fragile X syndrome and Rett syndrome are commonly associated with autism spectrum disorders and involve defects in synaptic plasticity. MicroRNA is implicated in synaptic plasticity because fragile X mental retardation protein was recently linked to the microRNA pathway. DNA methylation is also involved in synaptic plasticity since methyl CpG-binding protein 2 (MeCP2) is mutated in patients with Rett syndrome. Here we report that expression of miR-184, a brain-specific microRNA repressed by the binding of MeCP2 to its promoter, is upregulated by the release of MeCP2 after depolarization. The restricted release of MeCP2 from the paternal allele results in paternal allele-specific expression of miR-184. Our finding provides a clue to the link between the microRNA and DNA methylation pathways.
PMID18029763	Regulatory interaction of HNF1-alpha to microRNA-194 gene during intestinal epithelial cell differentiation. Maintenance of intestinal epithelium is based on well-balanced molecular mechanisms that confer the stable and continuous supply of specialized epithelial cell lineages from multipotent progenitors. Lineage commitment decisions in intestinal epithelium system involve multiple regulatory systems that interplay each other to establish the cellular identities. Here, we demonstrate that the microRNA system could be involved in intestinal epithelial cell differentiation and that microRNA-194 (miR194) is highly induced during this process and controlled by a transcription factor, HNF-alpha, that is well known to regulate gene expression in intestinal epithelial cells. Thus, the 5' conserved genomic region of miR-1942 gene, the inducible class of miR-194 parental gene, contains a binding motif for HNF1-alpha. This consensus region is required for the transcription of miR-1942 and active in intestinal epithelial cell line, Caco-2, in-vivo. Our observations indicate that microRNA genes could be targets of lineage specific transcription factors and that microRNAs are regulated in intestinal epithelial cells in a tissue specific manner. Given that role of microRNA in fine tuning of gene expression patterns, our results suggest that HNF1-alpha regulates the gene expression program by not only direct activation of genes but also modulation through induction of microRNAs such as miR-194, in intestinal epithelial cells. This represents a novel molecular machinery that might specify the fates of intestinal epithelial cell lineages during their differentiation.
PMID18794355	MicroRNA-155 is regulated by the transforming growth factor beta/Smad pathway and contributes to epithelial cell plasticity by targeting RhoA. Transforming growth factor beta (TGF-beta) signaling facilitates metastasis in advanced malignancy. While a number of protein-encoding genes are known to be involved in this process, information on the role of microRNAs (miRNAs) in TGF-beta-induced cell migration and invasion is still limited. By hybridizing a 515-miRNA oligonucleotide-based microarray library, a total of 28 miRNAs were found to be significantly deregulated in TGF-beta-treated normal murine mammary gland (NMuMG) epithelial cells but not Smad4 knockdown NMuMG cells. Among upregulated miRNAs, miR-155 was the most significantly elevated miRNA. TGF-beta induces miR-155 expression and promoter activity through Smad4. The knockdown of miR-155 suppressed TGF-beta-induced epithelial-mesenchymal transition (EMT) and tight junction dissolution, as well as cell migration and invasion. Further, the ectopic expression of miR-155 reduced RhoA protein and disrupted tight junction formation. Reintroducing RhoA cDNA without the 3' untranslated region largely reversed the phenotype induced by miR-155 and TGF-beta. In addition, elevated levels of miR-155 were frequently detected in invasive breast cancer tissues. These data suggest that miR-155 may play an important role in TGF-beta-induced EMT and cell migration and invasion by targeting RhoA and indicate that it is a potential therapeutic target for breast cancer intervention.
PMID22382496	MiRNA-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-β signalling pathway. Transforming growth factor-β (TGF-β) signaling regulates many diverse cellular activities through both canonical (SMAD-dependent) and non-canonical branches, which includes the mitogen-activated protein kinase (MAPK), Rho-like guanosine triphosphatase and phosphatidylinositol-3-kinase/AKT pathways. Here, we demonstrate that miR-335 directly targets and downregulates genes in the TGF-β non-canonical pathways, including the Rho-associated coiled-coil containing protein (ROCK1) and MAPK1, resulting in reduced phosphorylation of downstream pathway members. Specifically, inhibition of ROCK1 and MAPK1 reduces phosphorylation levels of the motor protein myosin light chain (MLC) leading to a significant inhibition of the invasive and migratory potential of neuroblastoma cells. Additionally, miR-335 targets the leucine-rich alpha-2-glycoprotein 1 (LRG1) messenger RNA, which similarly results in a significant reduction in the phosphorylation status of MLC and a decrease in neuroblastoma cell migration and invasion. Thus, we link LRG1 to the migratory machinery of the cell, altering its activity presumably by exerting its effect within the non-canonical TGF-β pathway. Moreover, we demonstrate that the MYCN transcription factor, whose coding sequence is highly amplified in a particularly clinically aggressive neuroblastoma tumor subtype, directly binds to a region immediately upstream of the miR-335 transcriptional start site, resulting in transcriptional repression. We conclude that MYCN contributes to neuroblastoma cell migration and invasion, by directly downregulating miR-335, resulting in the upregulation of the TGF-β signaling pathway members ROCK1, MAPK1 and putative member LRG1, which positively promote this process. Our results provide novel insight into the direct regulation of TGF-β non-canonical signaling by miR-335, which in turn is downregulated by MYCN.
PMID21324897	A Runx2/miR-3960/miR-2861 regulatory feedback loop during mouse osteoblast differentiation. Our recent study showed that miR-2861 promotes osteoblast differentiation by targeting histone deacetylase 5, resulting in increased runt-related transcription factor 2 (Runx2) protein production. Here we identified another new microRNA (miRNA) (miR-3960) that played a regulatory role in osteoblast differentiation through a regulatory feedback loop with miR-2861. miR-3960 and miR-2861 were found clustered at the same loci. miR-3960 was transcribed during bone morphogenic protein 2 (BMP2)-induced osteogenesis of ST2 stromal cells. Overexpression of miR-3960 promoted BMP2-induced osteoblastogenesis. However, the inhibition of miR-3960 expression attenuated the osteoblastogenesis. Homeobox A2 (Hoxa2), a repressor of Runx2 expression, was confirmed to be a target of miR-3960. Electrophoretic mobility shift assay and chromatin immunoprecipitation experiments confirmed that Runx2 bound to the promoter of the miR-3960/miR-2861 cluster. Furthermore, overexpression of Runx2 induced miR-3960/miR-2861 transcription, and block of Runx2 expression attenuated BMP2-induced miR-3960/miR-2861 transcription. Here we report that miR-3960 and miR-2861, transcribed together from the same miRNA polycistron, both function in osteoblast differentiation through a novel Runx2/miR-3960/miR-2861 regulatory feedback loop. Our findings provide new insights into the roles of miRNAs in osteoblast differentiation.
PMID17994015	Homeostatic regulation of MeCP2 expression by a CREB-induced microRNA. Both increases and decreases in methyl CpG-binding protein 2 (MeCP2) levels cause neurodevelopmental defects. We found that MeCP2 translation is regulated by microRNA 132 (miR132). Block of miR132-mediated repression increased MeCP2 and brain-derived neurotrophic factor (BDNF) levels in cultured rat neurons and the loss of MeCP2 reduced BDNF and miR132 levels in vivo. This feedback loop may provide a mechanism for homeostatic control of MeCP2 expression.
PMID19144316	Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein. Foxp3(+) regulatory T (Treg) cells limit pathogenic immune responses to self-antigens and foreign antigens. An essential role for microRNA (miRNA) in the maintenance and function of Treg cells, revealed by the Treg cell-specific Dicer ablation, raised a question as to a specific miRNA contribution. We found that Foxp3 controlled the elevated miR155 expression required for maintaining Treg cell proliferative activity and numbers under nonlymphopenic conditions. Moreover, miR155 deficiency in Treg cells resulted in increased suppressor of cytokine signaling 1 (SOCS1) expression accompanied by impaired activation of signal transducer and activator of transcription 5 (STAT5) transcription factor in response to limiting amounts of interleukin-2. Our studies suggest that Foxp3-dependent regulation of miR155 maintains competitive fitness of Treg cell subsets by targeting SOCS1, and they provide experimental support for a proposed role for miRNAs in ensuring the robustness of cellular phenotypes.
PMID20203269	MicroRNA and proliferation control in chronic lymphocytic leukemia: functional relationship between miR-221/222 cluster and p27. We investigated functional relationships between microRNA 221/222 (miR-221/222) cluster and p27, a key regulator of cell cycle, in chronic lymphocytic leukemia (CLL). The enforced expression of miR-221/222 in the CLL cell line MEC1 induced a significant down-regulation of p27 protein and conferred a proliferative advantage to the transduced cells that exhibited faster progression into the S phase of the cell cycle. Accordingly, expression of miR-221/miR-222 and p27 was found to be inversely related in leukemic cells obtained from peripheral blood (PB) of 38 patients with CLL. Interestingly, when miR-221/222 and p27 protein were evaluated in different anatomic compartments (lymph nodes or bone marrow) of the same patients, increased expression of the 2 miRNAs became apparent compared with PB. This finding was paralleled by a low expression of p27. In addition, when CLL cells were induced in vitro to enter cell cycle (eg, with cytosine phosphate guanine oligodeoxynucleotide), a significant increase of miR-221/222 expression and a marked down-regulation of p27 protein were evident. These data indicate that the miR-221/222 cluster modulates the expression of p27 protein in CLL cells and lead to suggest that miR-221/222 and p27 may represent a regulatory loop that helps maintaining CLL cells in a resting condition.
PMID19021527	Role of miRNA-146a in the regulation of the innate immune response and cancer. In mammalian cells, miRNAs (microRNAs) are the most abundant family of small non-coding RNAs that regulate mRNA translation through the RNA interference pathway. In general, it appears that the major function of miRNAs is in development, differentiation and homoeostasis, which is indicated by studies showing aberrant miRNA expression during the development of cancer. Interestingly, changes in the expression of miR-146a have been implicated in both the development of multiple cancers and in the negative regulation of inflammation induced via the innate immune response. Furthermore, miR-146a expression is driven by the transcription factor NF-kappaB (nuclear factor kappaB), which has been implicated as an important causal link between inflammation and carcinogenesis. In the present article, we review the evidence for a role of miR-146a in innate immunity and cancer and assess whether changes in miR-146a might link these two biological responses.
PMID21072171	The regulation of miRNA-211 expression and its role in melanoma cell invasiveness. The immediate molecular mechanisms behind invasive melanoma are poorly understood. Recent studies implicate microRNAs (miRNAs) as important agents in melanoma and other cancers. To investigate the role of miRNAs in melanoma, we subjected human melanoma cell lines to miRNA expression profiling, and report a range of variations in several miRNAs. Specifically, compared with expression levels in melanocytes, levels of miR-211 were consistently reduced in all eight non-pigmented melanoma cell lines we examined; they were also reduced in 21 out of 30 distinct melanoma samples from patients, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic. The levels of several predicted target mRNAs of miR-211 were reduced in melanoma cell lines that ectopically expressed miR-211. In vivo target cleavage assays confirmed one such target mRNA encoded by KCNMA1. Mutating the miR-211 binding site seed sequences at the KCNMA1 3'-UTR abolished target cleavage. KCNMA1 mRNA and protein expression levels varied inversely with miR-211 levels. Two different melanoma cell lines ectopically expressing miR-211 exhibited significant growth inhibition and reduced invasiveness compared with the respective parental melanoma cell lines. An shRNA against KCNMA1 mRNA also demonstrated similar effects on melanoma cells. miR-211 is encoded within the sixth intron of TRPM1, a candidate suppressor of melanoma metastasis. The transcription factor MITF, important for melanocyte development and function, is needed for high TRPM1 expression. MITF is also needed for miR-211 expression, suggesting that the tumor-suppressor activities of MITF and/or TRPM1 may at least partially be due to miR-211's negative post transcriptional effects on the KCNMA1 transcript. Given previous reports of high KCNMA1 levels in metastasizing melanoma, prostate cancer and glioma, our findings that miR-211 is a direct posttranscriptional regulator of KCNMA1 expression as well as the dependence of this miRNA's expression on MITF activity, establishes miR-211 as an important regulatory agent in human melanoma.
PMID19219026	c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism. Altered glucose metabolism in cancer cells is termed the Warburg effect, which describes the propensity of most cancer cells to take up glucose avidly and convert it primarily to lactate, despite available oxygen. Notwithstanding the renewed interest in the Warburg effect, cancer cells also depend on continued mitochondrial function for metabolism, specifically glutaminolysis that catabolizes glutamine to generate ATP and lactate. Glutamine, which is highly transported into proliferating cells, is a major source of energy and nitrogen for biosynthesis, and a carbon substrate for anabolic processes in cancer cells, but the regulation of glutamine metabolism is not well understood. Here we report that the c-Myc (hereafter referred to as Myc) oncogenic transcription factor, which is known to regulate microRNAs and stimulate cell proliferation, transcriptionally represses miR-23a and miR-23b, resulting in greater expression of their target protein, mitochondrial glutaminase, in human P-493 B lymphoma cells and PC3 prostate cancer cells. This leads to upregulation of glutamine catabolism. Glutaminase converts glutamine to glutamate, which is further catabolized through the tricarboxylic acid cycle for the production of ATP or serves as substrate for glutathione synthesis. The unique means by which Myc regulates glutaminase uncovers a previously unsuspected link between Myc regulation of miRNAs, glutamine metabolism, and energy and reactive oxygen species homeostasis.
PMID20388916	Identification of the miR-106b~25 microRNA cluster as a proto-oncogenic PTEN-targeting intron that cooperates with its host gene MCM7 in transformation. PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor that antagonizes signaling through the phosphatidylinositol 3-kinase-Akt pathway. We have demonstrated that subtle decreases in PTEN abundance can have critical consequences for tumorigenesis. Here, we used a computational approach to identify miR-22, miR-25, and miR-302 as three PTEN-targeting microRNA (miRNA) families found within nine genomic loci. We showed that miR-22 and the miR-106b~25 cluster are aberrantly overexpressed in human prostate cancer, correlate with abundance of the miRNA processing enzyme DICER, and potentiate cellular transformation both in vitro and in vivo. We demonstrated that the intronic miR-106b~25 cluster cooperates with its host gene MCM7 in cellular transformation both in vitro and in vivo, so that the concomitant overexpression of MCM7 and the miRNA cluster triggers prostatic intraepithelial neoplasia in transgenic mice. Therefore, the MCM7 gene locus delivers two simultaneous oncogenic insults when amplified or overexpressed in human cancer. Thus, we have uncovered a proto-oncogenic miRNA-dependent network for PTEN regulation and defined the MCM7 locus as a critical factor in initiating prostate tumorigenesis.
PMID20643754	microRNAome changes in bystander three-dimensional human tissue models suggest priming of apoptotic pathways. The radiation-induced bystander effect (RIBE) is a phenomenon whereby unexposed cells exhibit molecular symptoms of stress exposure when adjacent or nearby cells are traversed by ionizing radiation (IR). Recent data suggest that RIBE may be epigenetically mediated by microRNAs (miRNAs), which are small regulatory molecules that target messenger RNA transcripts for translational inhibition. Here, we analyzed microRNAome changes in bystander tissues after α-particle microbeam irradiation of three-dimensional artificial human tissues using miRNA microarrays. Our results indicate that IR leads to a deregulation of miRNA expression in bystander tissues. We report that major bystander end points, including apoptosis, cell cycle deregulation and DNA hypomethylation, may be mediated by altered expression of miRNAs. Specifically, c-MYC-mediated upregulation of the miR-17 family was associated with decreased levels of E2F1 and RB1, suggesting a switch to a proliferative state in bystander tissues, while priming these cells for impending death signals. Upregulation of the miR-29 family resulted in decreased levels of its targets DNMT3a and MCL1, consequently affecting DNA methylation and apoptosis. Altered expression of miR-16 led to changes in expression of BCL2, suggesting modulation of apoptosis. Thus, our data clearly show that miRNAs play a profound role in the manifestation of late RIBE end points. In summary, this study creates a roadmap for understanding the role of microRNAome in RIBE and for developing novel RIBE biomarkers.
PMID22298639	Transcriptional regulation of miR-196b by ETS2 in gastric cancer cells. E26 transformation-specific sequence (ETS)-2 is a transcriptional modulator located on chromosome 21, alterations in its expression have been implicated with a reduced incidence of solid tumors in Down syndrome patients. MicroRNAs (miRNAs) are thought to participate in diverse biological functions; however, the regulation of miRNAs is not well characterized. Recently, we reported that miR-196b is highly expressed in gastric cancers. Herein, we demonstrate that miR-196b expression was significantly repressed by ETS2 during gastric cancer oncogenesis. We demonstrate that knockdown of endogenous ETS2 expression increases miR-196b expression. A genomic region between -751 and -824 bp upstream of the miR-196b transcriptional start site was found to be critical for the repression activity. This putative regulatory promoter region contains three potential ETS2-binding motifs. Mutations within the ETS2 binding sites blocked the repression activity of ETS2. Furthermore, knockdown of ETS2 or overexpression of miR-196b significantly induced migration and invasion in gastric cancer cells. In addition, alterations in ETS2 and miR-196b expression in gastric cancer cell lines affected the expression of epithelial-mesenchymal transition-related genes. The levels of vimentin, matrix metalloproteinase (MMP)-2 and MMP9 were drastically induced, but levels of E-cadherin were decreased in shETS2- or miR-196b-transfected cells. Our data indicate that ETS2 plays a key role in controlling the expression of miR-196b, and miR-196b may mediate the tumor suppressor effects of ETS2. We demonstrated that miR-196b was transcriptionally regulated by ETS2 and there was an inverse expression profile between miR-196b and ETS2 in clinical samples. This finding could be beneficial for the development of effective cancer diagnostic and alternative therapeutic strategies.
PMID19066217	MicroRNA regulation of a cancer network: consequences of the feedback loops involving miR-17-92, E2F, and Myc. The transcription factors E2F and Myc participate in the control of cell proliferation and apoptosis, and can act as oncogenes or tumor suppressors depending on their levels of expression. Positive feedback loops in the regulation of these factors are predicted-and recently shown experimentally-to lead to bistability, which is a phenomenon characterized by the existence of low and high protein levels ("off" and "on" levels, respectively), with sharp transitions between levels being inducible by, for example, changes in growth factor concentrations. E2F and Myc are inhibited at the posttranscriptional step by members of a cluster of microRNAs (miRs) called miR-17-92. In return, E2F and Myc induce the transcription of miR-17-92, thus forming a negative feedback loop in the interaction network. The consequences of the coupling between the E2F/Myc positive feedback loops and the E2F/Myc/miR-17-92 negative feedback loop are analyzed using a mathematical model. The model predicts that miR-17-92 plays a critical role in regulating the position of the off-on switch in E2F/Myc protein levels, and in determining the on levels of these proteins. The model also predicts large-amplitude protein oscillations that coexist with the off steady state levels. Using the concept and model prediction of a "cancer zone," the oncogenic and tumor suppressor properties of miR-17-92 is demonstrated to parallel the same properties of E2F and Myc.
PMID18818396	The c-myb proto-oncogene and microRNA-15a comprise an active autoregulatory feedback loop in human hematopoietic cells. The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment, proliferation, and differentiation. Given its critical functions, c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)-15a. Using a luciferase reporter assay, we found that miR-15a directly binds the 3'-UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic, functionality of binding was shown. The mimic decreased c-Myb expression, and blocked the cells in the G(1) phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3'-UTR partially rescued the miR-15a induced cell-cycle block. Of interest, the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally, in studies using normal human CD34(+) cells, we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation, and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate, these findings suggest the presence of a c-Myb-miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis.
PMID21711587	Wnt/beta-catenin signaling activates microRNA-181 expression in hepatocellular carcinoma. Hepatocellular carcinoma (HCC) is a malignant cancer with an observable heterogeneity and microRNAs are functionally associated with the tumorigenesis of HCC. We recently identified that EpCAM (CD326)-positive cells isolated from alpha-fetoprotein (AFP)-positive HCC samples are hepatic cancer stem cells (HepCSCs). EpCAM+AFP+ HepCSCs have an activated Wnt/β-catenin signaling with a parallel increased expression of all four microRNA-181 family members. We hypothesized that Wnt/β-catenin signaling transcriptionally activates microRNA-181s in HCC. Using both western blot and quantitative reverse transcriptase-PCR analyses, we found that the expression of all four microRNA-181 family members was positively correlated with β-catenin expression in HCC cell lines. MicroRNA-181 expression could be directly induced upon an activation of Wnt/β-catenin signaling, which includes Wnt10B overexpression, inhibition of GSK3β signaling by LiCl, or forced expression of β-catenin/Tcf4. Moreover, microRNA-181 expression was inhibited upon an inactivation of Wnt/β-catenin signaling by an induction of adenomatosis polyposis coli (APC) expression or silencing β-catenin via RNA interference. In addition, seven putative β-catenin/Tcf4 binding sites were identified in the promoter region of the microRNA-181a-2 and microRNA-181b-2 transcripts. Consistently, we found that Tcf4 interacted with these regions in vivo using chromatin immunoprecipitation assay. Taken together, our results demonstrate that microRNA-181s are transcriptionally activated by the Wnt/beta-catenin signaling pathway in HCC.
PMID18829540	A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition. Epithelial to mesenchymal transition occurs during embryologic development to allow tissue remodeling and is proposed to be a key step in the metastasis of epithelial-derived tumors. The miR-200 family of microRNAs plays a major role in specifying the epithelial phenotype by preventing expression of the transcription repressors, ZEB1/deltaEF1 and SIP1/ZEB2. We show here that miR-200a, miR-200b, and the related miR-429 are all encoded on a 7.5-kb polycistronic primary miRNA (pri-miR) transcript. We show that the promoter for the pri-miR is located within a 300-bp segment located 4 kb upstream of miR-200b. This promoter region is sufficient to confer expression in epithelial cells and is repressed in mesenchymal cells by ZEB1 and SIP1 through their binding to a conserved pair of ZEB-type E-box elements located proximal to the transcription start site. These findings establish a double-negative feedback loop controlling ZEB1-SIP1 and miR-200 family expression that regulates cellular phenotype and has direct relevance to the role of these factors in tumor progression.
PMID19029138	Twist-1 regulates the miR-199a/214 cluster during development. MicroRNAs are known to regulate developmental processes but their mechanism of regulation remains largely uncharacterized. We show the transcription factor Twist-1 drives the expression of a 7.9-kb noncoding RNA transcript (from the Dynamin-3 gene intron) that encodes a miR-199a and miR-214 cluster. We also show that knocking down Twist-1 with shRNAs decreased miR-199a/214 levels and that Twist-1 bound an E-Box promoter motif to developmentally regulate the expression of these miRNAs. The expression of HIF-1 (known to mediate Twist-1 transcription), miR-199a and miR-214 was maximal at E12.5 and the miRNAs were expressed specifically in mouse cerebellum, midbrain, nasal process and fore- and hindlimb buds. This study shows the expression of the miR199a/214 cluster is controlled by Twist-1 via an E-Box promoter element and supports a role for these miRNAs as novel intermediates in the pathways controlling the development of specific neural cell populations.
PMID21403400	The Notch ligand Jagged2 promotes lung adenocarcinoma metastasis through a miR-200-dependent pathway in mice. Epithelial tumor cells transit to a mesenchymal state in response to extracellular cues, in a process known as epithelial-to-mesenchymal transition (EMT). The precise nature of these cues has not been fully defined, an important issue given that EMT is an early event in tumor metastasis. Here, we have found that a population of metastasis-prone mouse lung adenocarcinoma cells expresses Notch and Notch ligands and that the Notch ligand Jagged2 promotes metastasis. Mechanistically, Jagged2 was found to promote metastasis by increasing the expression of GATA-binding (Gata) factors, which suppressed expression of the microRNA-200 (miR-200) family of microRNAs that target the transcriptional repressors that drive EMT and thereby induced EMT. Reciprocally, miR-200 inhibited expression of Gata3, which reversed EMT and abrogated metastasis, suggesting that Gata3 and miR-200 are mutually inhibitory and have opposing effects on EMT and metastasis. Consistent with this, high levels of Gata3 expression correlated with EMT in primary tumors from 2 cohorts of lung adenocarcinoma patients. These findings reveal what we believe to be a novel Jagged2/miR-200-dependent pathway that mediates lung adenocarcinoma EMT and metastasis in mice and may have implications for the treatment of human epithelial tumors.
PMID19034270	p53-Repressed miRNAs are involved with E2F in a feed-forward loop promoting proliferation. Normal cell growth is governed by a complicated biological system, featuring multiple levels of control, often deregulated in cancers. The role of microRNAs (miRNAs) in the control of gene expression is now increasingly appreciated, yet their involvement in controlling cell proliferation is still not well understood. Here we investigated the mammalian cell proliferation control network consisting of transcriptional regulators, E2F and p53, their targets and a family of 15 miRNAs. Indicative of their significance, expression of these miRNAs is downregulated in senescent cells and in breast cancers harboring wild-type p53. These miRNAs are repressed by p53 in an E2F1-mediated manner. Furthermore, we show that these miRNAs silence antiproliferative genes, which themselves are E2F1 targets. Thus, miRNAs and transcriptional regulators appear to cooperate in the framework of a multi-gene transcriptional and post-transcriptional feed-forward loop. Finally, we show that, similarly to p53 inactivation, overexpression of representative miRNAs promotes proliferation and delays senescence, manifesting the detrimental phenotypic consequence of perturbations in this circuit. Taken together, these findings position miRNAs as novel key players in the mammalian cellular proliferation network.
PMID19197161	Repressing the repressor: a new mode of MYC action in lymphomagenesis. The MYC oncogene is often mutated or amplified in human tumors resulting in increased activity of the transcription factor. Recent evidence suggested that MYC not only regulates expression of protein-coding genes directly, but also controls expression of miRNAs thereby using a second mode to impact on gene expression programs. The importance of miRNAs in MYC-driven tumorigenesis has been enlightened by studying cell line and murine lymphoma models with conditional expression of MYC. The application of microarray technology revealed both MYC-induced and MYC-repressed miRNAs. A miRNA consistently repressed by MYC in multiple tumors was miR-26a indicating that this miRNA might have strong tumor suppressor function for MYC-induced lymphomas. Indeed, ectopic miR-26a expression in MYC-dependent cells resulted in attenuated proliferation and impaired cell cycle progression. When the effector pathway for miR-26a was elucidated, the Polycomb complex protein EZH2, a global regulator of gene expression, was identified as a direct target. The suppression of miR-26a mediated attenuation of EZH2 expression by MYC was shown to play a critical role in lymphomagenesis. Thus, MYC-induced oncogenesis does not only depend on direct targeting of protein-coding genes, but also on modulating them via deregulation of their targeting miRNAs, thereby significantly impacting lymphomagenesis.
PMID19285018	Characterization of function and regulation of miR-24-1 and miR-31. To date, numerous microRNAs (miRNAs) have been discovered. However, the function of these miRNAs is largely unknown. While our knowledge of miRNA post-transcriptional processing has greatly expanded in recent years, we have a limited understanding of the regulation and transcription of miRNA genes. In this study, we characterized two BMP-2 upregulated miRNAs, miR-24-1 and miR-31, in mesenchymal stem cells and showed their opposing function in controlling cellular proliferation, and adipogenesis. Furthermore, we are the first to identify and characterize mouse intronic miR-23b~27b~24-1 and intergenic miR-31 genes. Moreover, we found that pri-miR-23b, pri-miR-27b, and pri-miR-24-1 are transcribed independently and their expression profiles are unique when cells are treated with BMP-2, even though they are located closely together.
PMID22327366	Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs. The shear-responsive transcription factor Krüppel-like factor 2 (KLF2) is a critical regulator of endothelial gene expression patterns induced by atheroprotective flow. As microRNAs (miRNAs) post-transcriptionally control gene expression in many pathogenic and physiological processes, we investigated the regulation of miRNAs by KLF2 in endothelial cells. KLF2 binds to the promoter and induces a significant upregulation of the miR-143/145 cluster. Interestingly, miR-143/145 has been shown to control smooth muscle cell (SMC) phenotypes; therefore, we investigated the possibility of transport of these miRNAs between endothelial cells and SMCs. Indeed, extracellular vesicles secreted by KLF2-transduced or shear-stress-stimulated HUVECs are enriched in miR-143/145 and control target gene expression in co-cultured SMCs. Extracellular vesicles derived from KLF2-expressing endothelial cells also reduced atherosclerotic lesion formation in the aorta of ApoE(-/-) mice. Combined, our results show that atheroprotective stimuli induce communication between endothelial cells and SMCs through an miRNA- and extracellular-vesicle-mediated mechanism and that this may comprise a promising strategy to combat atherosclerosis.
PMID19088023	miR-192 Regulates dihydrofolate reductase and cellular proliferation through the p53-microRNA circuit. The purpose of this study is to investigate the molecular mechanism of miR-192 in colon cancer. Human colon cancer cell lines with different p53 status were used as our model system to study the effect of miR-192 on cell proliferation, cell cycle control, and mechanism of regulation. Our results show that one of the key miR-192 target genes is dihydrofolate reductase (DHFR). miR-192 affects cellular proliferation through the p53-miRNA circuit. Western immunoblot analyses indicated that the expression of DHFR was significantly decreased by miR-192. Further investigation revealed that such suppression was due to translational arrest rather than mRNA degradation. More profound inhibition of cellular proliferation was observed by ectopic expression of miR-192 in colon cancer cell lines containing wild-type p53 than cells containing mutant p53. Thus, the effect of miR-192 on cellular proliferation is mainly p53 dependent. Overexpression of miR-192 triggered both G1 and G2 arrest in HCT-116 (wt-p53) cells but not in HCT-116 (null-p53) cells. The cell cycle checkpoint control genes p53 and p21 were highly overexpressed in cells that overexpressed miR-192. Endogenous miR-192 expression was increased in HCT-116 (wt-p53) and RKO (wt-p53) cells treated with methotrexate, which caused an induction of p53 expression. Chromatin immunoprecipitation-quantitative reverse transcription-PCR analysis revealed that the p53 protein interacted with the miR-192 promoter sequence. These results indicate that miR-192 may be another miRNA candidate that is involved in the p53 tumor suppressor network with significant effect on cell cycle control and cell proliferation.
PMID22134354	miR-34 and SNAIL form a double-negative feedback loop to regulate epithelial-mesenchymal transitions. Recently, the inhibition of epithelial-mesenchymal-transition (EMT) by p53 has been described as a new mode of tumor suppression which presumably prevents metastasis. Here we report that activation of p53 down-regulates the EMT-inducing transcription factor SNAIL via induction of the miR-34a/b/c genes. Suppression of miR-34a/b/c caused up-regulation of SNAIL and cells displayed EMT markers and related features, as enhanced migration and invasion.  Ectopic miR-34a induced mesenchymal-epithelial-transition (MET) and down-regulation of SNAIL, which was mediated by a conserved miR-34a/b/c seed-matching sequence in the SNAIL 3'-UTR. miR-34a also down-regulated SLUG and ZEB1, as well as the stemness factors BMI1, CD44, CD133, OLFM4 and c-MYC. Conversely, the transcription factors SNAIL and ZEB1 bound to E-boxes in the miR-34a/b/c promoters, thereby repressing miR-34a and miR-34b/c expression. Since ectopic miR-34a prevented TGF-β-induced EMT, the repression of miR-34 genes by SNAIL and related factors is part of the EMT program. In conclusion, the frequent inactivation of p53 and/or miR-34a/b/c found in cancer may shift the equilibrium of these reciprocal regulations towards the mesenchymal state and thereby lock cells in a metastatic state.
PMID20080637	MYCN-regulated microRNAs repress estrogen receptor-alpha (ESR1) expression and neuronal differentiation in human neuroblastoma. MYCN, a proto-oncogene normally expressed in the migrating neural crest, is in its amplified state a key factor in the genesis of human neuroblastoma (NB). However, the mechanisms underlying MYCN-mediated NB progression are poorly understood. Here, we present a MYCN-induced miRNA signature in human NB involving the activation and transrepression of several miRNA genes from paralogous clusters. Several family members derived from the miR-17 approximately 92 cluster, including miR-18a and miR-19a, were among the up-regulated miRNAs. Expression analysis of these miRNAs in NB tumors confirmed increased levels in MYCN-amplified samples. Specifically, we show that miR-18a and miR-19a target and repress the expression of estrogen receptor-alpha (ESR1), a ligand-inducible transcription factor implicated in neuronal differentiation. Immunohistochemical staining demonstrated ESR1 expression in human fetal sympathetic ganglia, suggesting a role for ESR1 during sympathetic nervous system development. Concordantly, lentiviral restoration of ESR1 in NB cells resulted in growth arrest and neuronal differentiation. Moreover, lentiviral-mediated inhibition of miR-18a in NB cells led to severe growth retardation, outgrowth of varicosity-containing neurites, and induction of neuronal sympathetic differentiation markers. Bioinformatic analyses of microarray data from NB tumors revealed that high ESR1 expression correlates with increased event-free survival in NB patients and favorable disease outcome. Thus, MYCN amplification may disrupt estrogen signaling sensitivity in primitive sympathetic cells through deregulation of ESR1, thereby preventing the normal induction of neuroblast differentiation. Collectively, our findings demonstrate the molecular consequences of abnormal miRNA transcription in a MYCN-driven tumor and offer unique insights into the pathology underlying MYCN-amplified NB.
PMID22093444	Inhibition of the miR-143/145 cluster attenuated neutrophil differentiation of APL cells. MicroRNAs can influence hematopoietic cell lineage commitment and aberrant expression of hematopoietic miRNAs contributes to AML pathology. We found that miR-143 and miR-145 expression is significantly repressed in primary AML patient samples as compared to neutrophils of healthy donors. Further analysis revealed impaired neutrophil differentiation of APL cells upon inhibition of miR-145 expression. Lastly, we identified p73 as transcriptional regulator of miR-143/145 during neutrophil differentiation of APL cells. Our data suggest that low miR-145 levels in APL, possibly due to aberrant expression of p73 transcription factors, contribute to the differentiation block seen in this disease.
PMID19330006	A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination. MicroRNAs have been implicated as having important roles in stem cell biology. MicroRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain and may be involved in neural stem cell self-renewal and differentiation. We showed previously that the nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector that is not prone to miR-9 regulation rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses expression of the miR-9 pri-miRNA. By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.
PMID18508316	Upregulation of miR-23a approximately 27a approximately 24 decreases transforming growth factor-beta-induced tumor-suppressive activities in human hepatocellular carcinoma cells. Transforming growth factor-beta (TGF-beta) plays a dual and complex role in human cancer. In this report, we observe a specific set of MicroRNAs (miRNAs) changed in response to TGF-beta in human hepatocellular carcinoma (HCC) cells by miRNA microarray screening. A cluster of miRNA, miR-23a approximately 27a approximately 24, is induced in an early stage by TGF-beta in Huh-7 cells. Knockdown of Smad4, Smad2 or Smad3 expression by RNA interference can attenuate the response of miR-23a approximately 27a approximately 24 to TGF-beta addition, indicating that this induction is dependent on Smad pathway. We also explore that miR-23a approximately 27a approximately 24 can function as an antiapoptotic and proliferation-promoting factor in liver cancer cells. In addition, expression of this miRNA cluster is found to be remarkably upregulated in HCC tissues versus normal liver tissues. These findings suggest a novel, alternative mechanism through which TGF-beta could induce specific miRNA expression to escape from tumor-suppressive response in HCC cells.
PMID21888875	MicroRNA-135a contributes to the development of portal vein tumor thrombus by promoting metastasis in hepatocellular carcinoma. Portal vein tumor thrombus (PVTT) has previously been demonstrated to correlate with poor prognosis of hepatocellular carcinoma. Approximately 50-80% of HCC is accompanied by portal or hepatic vein invasion. The underlying mechanisms of PVTT development remain unclear. This study aimed to elucidate the role of miR-135a in PVTT tumorigenesis. In the present study, we investigated the expression of microRNAs and mRNAs in PVTT tissues using advanced microRNA and cDNA microarray techniques. MicroRNA (miR)-135a was noted to be highly over-expressed in PVTT and the cell line CSQT-2 and was selected for further study. We characterized the function of miR-135a in vitro and in vivo. We also analyzed the clinical relevance of miR-135a in relation to the prognosis and survival of HCC patients with PVTT. Our analyses found that the miRNA and mRNA expression profiles of PVTT were distinct from the parenchyma tumor. Overexpression of miR-135a favors invasive and metastatic behavior in vitro. Furthermore, in a CSQT-2 orthotopic transplantation nude mouse model, blockade of miR-135a significantly reduced PVTT incidence. We also found that miR-135a was transcribed by forkhead box M1 (FOXM1), and metastasis suppressor 1 (MTSS1) was identified as the direct and functional target of miR-135a. Additionally, the cohort analysis revealed the relevance of miR-135a with respect to the prognosis and survival of HCC patients with PVTT. Our data suggest an important role for miR-135a in promoting PVTT tumorigenesis and indicate the potential application of miR-135a in PVTT therapy.
PMID20375304	miR-146a is differentially expressed by myeloid dendritic cell subsets and desensitizes cells to TLR2-dependent activation. Langerhans cells (LCs) in epithelia and interstitial dendritic cells (intDCs) in adjacent connective tissues represent two closely related myeloid-derived DC subsets that exert specialized functions in the immune system and are of clinical relevance for cell therapy. Both subsets arise from monocyte-committed intermediates in response to tissue-associated microenvironmental signals; however, molecular mechanisms underlying myeloid DC subset specification and function remain poorly defined. Using microarray profiling, we identified microRNA (miRNA) miR-146a to be constitutively expressed at higher levels in human LCs compared with intDCs. Moreover, miR-146a levels were low in monocytes and nondetectable in neutrophil granulocytes. Interestingly, constitutive high miR-146a expression in LCs is induced by the transcription factor PU.1 in response to TGF-beta1, a key microenvironmental signal for epidermal LC differentiation. We identified miR-146a as a regulator of monocyte and DC activation but not myeloid/DC subset differentiation. Ectopic miR-146a in monocytes and intDCs interfered with TLR2 downstream signaling and cytokine production, without affecting phenotypic DC maturation. Inversely, silencing of miR-146a in LCs enhanced TLR2-dependent NF-kappaB signaling. We therefore conclude that high constitutive miR-146a levels are induced by microenvironmental signals in the epidermis and might render LCs less susceptible to inappropriate activation by commensal bacterial TLR2 triggers at body surfaces.
PMID22348059	Regulation of the let-7a-3 promoter by NF-κB. Changes in microRNA expression have been linked to a wide array of pathological states. However, little is known about the regulation of microRNA expression. The let-7 microRNA is a tumor suppressor that inhibits cellular proliferation and promotes differentiation, and is frequently lost in tumors. We investigated the transcriptional regulation of two let-7 family members, let-7a-3 and let-7b, which form a microRNA cluster and are located 864 bp apart on chromosome 22q13.31. Previous reports present conflicting data on the role of the NF-κB transcription factor in regulating let-7. We cloned three fragments upstream of the let-7a-3/let-7b miRNA genomic region into a plasmid containing a luciferase reporter gene. Ectopic expression of subunits of NF-κB (p50 or p65/RelA) significantly increased luciferase activity in HeLa, 293, 293T and 3T3 cells, indicating that the let-7a-3/let-7b promoter is highly responsive to NF-κB. Mutation of a putative NF-κB binding site at bp -833 reduced basal promoter activity and decreased promoter activity in the presence of p50 or p65 overexpression. Mutation of a second putative binding site, at bp -947 also decreased promoter activity basally and in response to p65 induction, indicating that both sites contribute to NF-κB responsiveness. While the levels of the endogenous primary let-7a and let-7b transcript were induced in response to NF-κB overexpression in 293T cells, the levels of fully processed, mature let-7a and let-7b miRNAs did not increase. Instead, levels of Lin-28B, a protein that blocks let-7 maturation, were induced by NF-κB. Increased Lin-28B levels could contribute to the lack of an increase in mature let-7a and let-7b. Our results suggest that the final biological outcome of NF-κB activation on let-7 expression may vary depending upon the cellular context. We discuss our results in the context of NF-κB activity in repressing self-renewal and promoting differentiation.
PMID21711453	Constitutive activation of the ETS-1-miR-222 circuitry in metastatic melanoma. MicroRNAs-221 and -222 are highly upregulated in several solid tumors, including melanomas. We demonstrate that the proto-oncogene ETS-1, involved in the pathogenesis of cancers of different origin, is a transcriptional regulator of miR-222 by direct binding to its promoter region. Differently from 293FT cells or early stage melanomas, where unphosphorylated ETS-1 represses miR-222 transcription, in metastatic melanoma the constitutively Thr-38 phosphorylated fraction of ETS-1 induces miR-222. Despite its stepwise decreased expression along with melanoma progression, the oncogenic activity of ETS-1 relies on its RAS/RAF/ERK-dependent phosphorylation status more than on its total amount. To close the loop, we demonstrate ETS-1 as a direct target of miR-222, but not miR-221, showing the novel option of their uncoupled functions. In addition, a spatial redistribution of ETS-1 protein from the nucleus to the cytoplasm is also evidenced in advanced melanoma cells. Finally, in vivo studies confirmed the contribution of miR-222 to the increased invasive potential obtained by ETS- silencing.
PMID19949084	Cutting edge: TNF-induced microRNAs regulate TNF-induced expression of E-selectin and intercellular adhesion molecule-1 on human endothelial cells: feedback control of inflammation. MicroRNAs (miRNAs) pair with target sequences in the 3' untranslated region of mRNAs to posttranscriptionally repress gene expression. In this study, we report that TNF-mediated induction of endothelial adhesion molecules can be regulated by miRNAs that are induced by TNF. Specifically, E-selectin and ICAM-1 are targets of TNF-induced miRNAs miR-31 and miR-17-3p, respectively. Specific antagonism of these TNF-induced miRNAs increased neutrophil adhesion to cultured endothelial cells. Conversely, transfections with mimics of these miRNAs decreased neutrophil adhesion to endothelial cells. These data suggest that miRNAs provide negative feedback control of inflammation.
PMID19148830	Activation of miR-17-92 by NK-like homeodomain proteins suppresses apoptosis via reduction of E2F1 in T-cell acute lymphoblastic leukemia. The NK-like family of homeobox genes includes TLX1, TLX3 and NKX2-5, which are ectopically activated in distinct subsets of T-cell acute lymphoblastic leukemia (T-ALL) cells. Here we analysed their effect on the miR-17-92 cluster overexpressed in several types of cancer, including T-ALL. The pri-miR-17-92 polycistron encodes micro-RNAs (miRNAs), which decrease E2F1 protein expression, regulating proliferation and/or apoptosis. Quantification of pri-miR-17-92 in T-ALL cell lines suggested an implication of the NK-like homeodomain proteins in transcriptional regulation. Lentiviral-mediated overexpression of NKX2-5 in the T-ALL cell line MOLT-4 consistently resulted in increased miR-17-92 pri-miRNA levels and decreased amounts of E2F1 protein. Induction of apoptosis by treating miR17-92 or E2F1 transduced T-ALL cells with etoposide led to reduced or enhanced cell viability, respectively. Furthermore, analysis of pri-miR-17-92 in T-ALL patients indicated elevated expression in those bearing TLX1/3 positive cells. These data support an activatory effect of NK-like homeodomain proteins on pri-miR-17-92 expression and concomitantly reduced E2F1 protein levels, thereby enhancing survival of leukemic T-cells.
PMID22389215	The miR-17/92 cluster is targeted by STAT5 but dispensable for mammary development. Genome wide analysis revealed the miR-17/92 cluster as a STAT5 target. This cluster encodes six microRNAs, which predictably target genes that play a role in mammary gland development. In this study, we have deleted the miR-17/92 cluster in mammary stem cells and evaluated in the mouse its function during mammary gland development. Loss of the miR-17/92 cluster did not affect mammary development from prepuberty to lactation. Our studies demonstrated that, while expression of the miR-17/92 cluster is under control of the key mammary transcription factor STAT5, its presence is not required for normal mammary development or lactation.
PMID21868360	miR-221/222 targeting of trichorhinophalangeal 1 (TRPS1) promotes epithelial-to-mesenchymal transition in breast cancer. Compared with the luminal subtype, the basal-like subtype of breast cancer has an aggressive clinical behavior, but the reasons for this difference between the two subtypes are poorly understood. We identified microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) as basal-like subtype-specific miRNAs that decrease expression of epithelial-specific genes and increase expression of mesenchymal-specific genes. In addition, expression of these miRNAs increased cell migration and invasion, which collectively are characteristics of the epithelial-to-mesenchymal transition (EMT). The basal-like transcription factor FOSL1 (also known as Fra-1) directly stimulated the transcription of miR-221/222, and the abundance of these miRNAs decreased with inhibition of MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), placing miR-221/222 downstream of the RAS pathway. The miR-221/222-mediated reduction in E-cadherin abundance depended on their targeting of the 3' untranslated region (3'UTR) of TRPS1 (trichorhinophalangeal syndrome type 1), which is a member of the GATA family of transcriptional repressors. TRPS1 inhibited EMT by directly repressing expression of ZEB2 (Zinc finger E-box-binding homeobox 2). Therefore, miR-221/222 may contribute to the aggressive clinical behavior of basal-like breast cancers.
PMID22354042	A novel miR-155/miR-143 cascade controls glycolysis by regulating hexokinase 2 in breast cancer cells. Cancer cells preferentially metabolize glucose through aerobic glycolysis. This phenomenon, known as the Warburg effect, is an anomalous characteristic of glucose metabolism in cancer cells. Chronic inflammation is a key promoting factor of tumourigenesis. It remains, however, largely unexplored whether and how pro-tumourigenic inflammation regulates glucose metabolism in cancer cells. Here, we show that pro-inflammatory cytokines promote glycolysis in breast cancer cells, and that the inflammation-induced miR-155 functions as an important mediator in this process. We further show that miR-155 acts to upregulate hexokinase 2 (hk2), through two distinct mechanisms. First, miR-155 promotes hk2 transcription by activation of signal transducer and activator of transcription 3 (STAT3), a transcriptional activator for hk2. Second, via targeting C/EBPβ (a transcriptional activator for mir-143), miR-155 represses mir-143, a negative regulator of hk2, thus resulting in upregulation of hk2 expression at the post-transcriptional level. The miR-155-mediated hk2 upregulation also appears to operate in other types of cancer cells examined. We suggest that the miR-155/miR-143/HK2 axis may represent a common mechanism linking inflammation to the altered metabolism in cancer cells.
PMID22370643	MiR-1 and miR-200 inhibit EMT via Slug-dependent and tumorigenesis via Slug-independent mechanisms. Epithelial-mesenchymal transition (EMT) is a developmental program of signaling pathways that determine commitment to epithelial and mesenchymal phenotypes. In the prostate, EMT processes have been implicated in benign prostatic hyperplasia and prostate cancer progression. In a model of Pten- and TP53-null prostate adenocarcinoma that progresses via transforming growth factor β-induced EMT, mesenchymal transformation is characterized by plasticity, leading to various mesenchymal lineages and the production of bone. Here we show that SLUG is a major regulator of mesenchymal differentiation. As microRNAs (miRs) are pleiotropic regulators of differentiation and tumorigenesis, we evaluated miR expression associated with tumorigenesis and EMT. Mir-1 and miR-200 were reduced with progression of prostate adenocarcinoma, and we identify Slug as one of the phylogenetically conserved targets of these miRs. We demonstrate that SLUG is a direct repressor of miR-1 and miR-200 transcription. Thus, SLUG and miR-1/miR-200 act in a self-reinforcing regulatory loop, leading to amplification of EMT. Depletion of Slug inhibited EMT during tumorigenesis, whereas forced expression of miR-1 or miR-200 inhibited both EMT and tumorigenesis in human and mouse model systems. Various miR targets were analyzed, and our findings suggest that miR-1 has roles in regulating EMT and mesenchymal differentiation through Slug and functions in tumor-suppressive programs by regulating additional targets.
PMID20008931	Genetic dissection of the miR-17~92 cluster of microRNAs in Myc-induced B-cell lymphomas. The miR-17 approximately 92 cluster is frequently amplified or overexpressed in human cancers and has emerged as the prototypical oncogenic polycistron microRNA (miRNA). miR-17 approximately 92 is a direct transcriptional target of c-Myc, and experiments in a mouse model of B-cell lymphomas have shown cooperation between these two oncogenes. However, both the molecular mechanism underlying this cooperation and the individual miRNAs that are responsible for it are unknown. By using a conditional knockout allele of miR-17 approximately 92, we show here that sustained expression of endogenous miR-17 approximately 92 is required to suppress apoptosis in Myc-driven B-cell lymphomas. Furthermore, we show that among the six miRNAs that are encoded by miR-17 approximately 92, miR-19a and miR-19b are absolutely required and largely sufficient to recapitulate the oncogenic properties of the entire cluster. Finally, by combining computational target prediction, gene expression profiling, and an in vitro screening strategy, we identify a subset of miR-19 targets that mediate its prosurvival activity.
PMID21455217	MiR-148a promotes apoptosis by targeting Bcl-2 in colorectal cancer. Apoptosis has a vital role in maintaining tissue homeostasis, and dysregulation of the apoptotic pathway is now widely recognized as a key step in tumourigenesis. Increasingly, evidence has demonstrated that microRNA (miRNA) can exert various biological functions in tumours by targeting oncogenes or tumour suppressors. Nevertheless, the role of miRNA in apoptosis remains unclear. Here we show that ectopical expression of miR-148a can induce apoptosis in colorectal cancer cells. In addition, MYB can inhibit miR-148a by directly acting on the transcription factor binding site in miR-148a gene and miR-148a can posttranscriptionally silence Bcl-2. Subsequently, the intrinsic apoptosis pathway is activated by releasing cytochrome c, cleaving caspase 9, caspase 3 and PARP, which eventually induce cancer-cell apoptosis. These findings are part of a hitherto undocumented apoptotic regulatory pathway in which a pleiotropic transcription factor controls the expression of a miRNA and the miRNA inhibits the target, leading to activation of an intrinsic mitochondrial pathway and tumour apoptosis.
PMID20364122	MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis. Within the circulatory system, blood flow regulates vascular remodelling, stimulates blood stem cell formation, and has a role in the pathology of vascular disease. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.
PMID18713946	MYC stimulates EZH2 expression by repression of its negative regulator miR-26a. The MYC oncogene, which is commonly mutated/amplified in tumors, represents an important regulator of cell growth because of its ability to induce both proliferation and apoptosis. Recent evidence links MYC to altered miRNA expression, thereby suggesting that MYC-regulated miRNAs might contribute to tumorigenesis. To further analyze the impact of MYC-regulated miRNAs, we investigated a murine lymphoma model harboring the MYC transgene in a Tet-off system to control its expression. Microarray-based miRNA expression profiling revealed both known and novel MYC targets. Among the miRNAs repressed by MYC, we identified the potential tumor suppressor miR-26a, which possessed the ability to attenuate proliferation in MYC-dependent cells. Interestingly, miR-26a was also found to be deregulated in primary human Burkitt lymphoma samples, thereby probably being of clinical relevance. Although today only few miRNA targets have been identified in human disease, we could show that ectopic expression of miR-26a influenced cell cycle progression by targeting the bona fide oncogene EZH2, a Polycomb protein and global regulator of gene expression yet unknown to be regulated by miRNAs. Thus, in addition to directly targeting protein-coding genes, MYC modulates genes important to oncogenesis via deregulation of miRNAs, thereby vitally contributing to MYC-induced lymphomagenesis.
PMID21903586	miR-125b, a target of CDX2, regulates cell differentiation through repression of the core binding factor in hematopoietic malignancies. MicroRNA-125b (miR-125b), a small noncoding RNA molecule, has been found to be deregulated and functions as an oncogene in many cancers including hematopoietic malignancies. However, the mechanisms accounting for miR-125b dysregulation remain to be elucidated. The present study aims to identify the factors that might contribute to up-regulation of miR-125b in human hematopoietic malignancies and its downstream targets for lineage-specific differentiation. We at first reported that CDX2, a homeobox transcription factor, binds to promoter regions of the miR-125b gene and activates transcriptional regulation of miR-125b in malignant myeloid cells. We further revealed that increasing levels of CDX2 in malignant myeloid cells activate miR-125b expression, which in turn inhibits core binding factor β (CBFβ) translation, thereby counteracting myeloid cell differentiation, at least for granulocytic lineage, and promoting leukemogenesis. Interestingly, we found that this novel pathway including CDX2, miR-125b, and CBFβ was mediated by undergoing all-trans-retinoic acid induction. Once differentiation ensues with all-trans-retinoic acid treatment, CDX2 activity decreases, leading to a reduction in miR-125b transcription and up-regulation of CBFβ in myeloid cells and in patients. The study provides a new mechanism that contributes to hematopoietic malignancies, which could involve deregulation of miR-125b and its up- and downstream factors. As altered expression of miRNAs has been reported in a wide range of malignancies, delineating the underlying molecular mechanisms of aberrant miRNA expression and characterizing the upstream and downstream factors will help to understand important steps in the pathogenesis of these afflictions.
PMID20146264	MicroRNA-dependent regulation of DNA methyltransferase-1 and tumor suppressor gene expression by interleukin-6 in human malignant cholangiocytes. Although the inflammation-associated cytokine interleukin-6 (IL-6) has been implicated in cholangiocarcinoma growth, the relationship between IL-6 and oncogenic changes is unknown. IL-6 can increase expression of DNA methyltransferase-1 (DNMT-1) and epigenetically regulate the expression of several genes, including microRNAs (miRNAs). DNMT-1 up-regulation occurs in hepatobiliary cancers and is associated with a poor prognosis. To understand the potential regulation of DNMT-1 by IL-6-dependent miRNAs, we examined the expression of a group of miRNAs which have sequence complementarity to the 3'-untranslated region of DNMT-1, namely miR-148a, miR-152, and miR-301. The expression of these miRNAs was decreased in cholangiocarcinoma cells. Moreover, the expression of all three miRNAs was decreased in IL-6-overexpressing malignant cholangiocytes in vitro and in tumor cell xenografts. There was a concomitant decrease in expression of the methylation-sensitive tumor suppressor genes Rassf1a and p16INK4a. Using luciferase reporter constructs, DNMT-1 was verified as a target for miR-148a and miR-152. Precursors to miR-148a and miR-152 decreased DNMT-1 protein expression, increased Rassf1a and p16INK4a expression, and reduced cell proliferation. These data indicate that IL-6 can regulate the activity of DNMT-1 and expression of methylation-dependent tumor suppressor genes by modulation of miR-148a and miR-152, and provide a link between this inflammation-associated cytokine and oncogenesis in cholangiocarcinoma.
PMID21872590	MiR-140 is co-expressed with Wwp2-C transcript and activated by Sox9 to target Sp1 in maintaining the chondrocyte proliferation. MiR-140 is a microRNA specially involved in chondrogenesis and osteoarthritis pathogenesis. However, its transcriptional regulation and target genes in cartilage development are not fully understood. Here we detected that miR-140 was uniquely expressed in chondrocyte and suppressed by Wnt/β-catenin signalling. The miR-140 primary transcript was an intron-retained RNA co-expressed with Wwp2-C isoform, which was directly induced by Sox9 through binding to the intron 10 of Wwp2 gene. Knockdown of miR-140 in limb bud micromass cultures resulted in arrest of chondrogenic proliferation. Sp1, the activator of the cell cycle regulator p15(INK4b), was identified as a target of miR-140 in maintaining the chondrocyte proliferation. Collectively, our findings expand our understanding of the transcriptional regulation and the chondrogenic role of miR-140 in chondrogenesis.
PMID20956382	MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3. The molecules that regulate the apoptosis cascade are also involved in differentiation and syncytial fusion in skeletal muscle. MyoD is a myogenic transcription factor that plays essential roles in muscle differentiation. We noticed that MyoD(-/-) myoblasts display remarkable resistance to apoptosis by down-regulation of miR-1 (microRNA-1) and miR-206 and by up-regulation of Pax3. This resulted in transcriptional activation of antiapoptotic factors Bcl-2 and Bcl-xL. Forced MyoD expression induces up-regulation of miR-1 and miR-206 and down-regulation of Pax3, Bcl-2, and Bcl-xL along with increased apoptosis in MyoD(-/-) myoblasts. In contrast, MyoD gene knockdown increases cell survival of wild-type myoblasts. The 3' untranslated region of Pax3 mRNA contains two conserved miR-1/miR-206-binding sites, which are required for targeting of these microRNAs (miRNAs). Therefore, these data suggest that MyoD not only regulates terminal differentiation but also apoptosis through miRNA-mediated down-regulation of Pax3. Finally, MyoD, miR-1, and miR-206 are all down-regulated in quiescent satellite cells, which may be required for maintenance of muscle stem cells.
PMID21436470	A microRNA-dependent circuit controlling p63/p73 homeostasis: p53 family cross-talk meets therapeutic opportunity. The p53 family transcription factors p53, p63 and p73 make diverse contributions in development and cancer. Mutation or deletion of p53 is observed in the majority of human cancers. In contrast, p63 and p73 are not lost in cancer but mediate distinct genetic roles in normal and tumor-specific contexts: p73 promotes genome stability and mediates chemosensitivity, while p63 largely lacks these p53-like functions and instead promotes proliferation and cell survival. We recently uncovered a mechanism which maintains p63/p73 homeostasis within the epithelium through direct transcriptional regulation of microRNAs (miRs). We discovered that several of the top p63-regulated miRs target p73 for inhibition, including miR-193a-5p, a direct p63/p73 transcriptional target which is repressed by p63 and activated by p73 both in vitro and in vivo. The resulting feed-forward circuit involving p63, miR-193a-5p and p73 controls p73 levels, cell viability and DNA damage susceptibility in certain cancers including squamous cell carcinoma. Here, we discuss the evolutionary implications of this regulatory circuit, which may point to a general mechanism of miR-mediated cross-talk within transcription factor gene families. Additionally, we suggest that inducible chemoresistance mediated by this miR-dependent mechanism might be an attractive target for therapeutic intervention.
PMID21372146	Gonadotropin-releasing hormone induces miR-132 and miR-212 to regulate cellular morphology and migration in immortalized LbetaT2 pituitary gonadotrope cells. GnRH is central to the regulation of reproductive function. It acts on pituitary gonadotropes to stimulate LH and FSH synthesis and secretion. We had previously presented evidence for translational control of LHβ synthesis; therefore we investigated whether micro-RNAs might play a role in GnRH regulation in LβT2 cells. We show here that GnRH strongly induces the AK006051 gene transcript that encodes two micro-RNAs, miR-132 and miR-212, within the first intron. We show furthermore that the AK006051 promoter region is highly GnRH responsive. We verify that the p250Rho GTPase activating protein (GAP) is a target of miR-132/212 and show that GnRH treatment leads to a decrease in mRNA and protein expression. This reduction is blocked by an anti-miR to miR-132/212 and mimicked by a pre-miR-132. GnRH inhibits p250RhoGAP expression through a miR-132/212 response element within the 3'-untranslated region. The loss of p250RhoGAP expression leads to activation of Rac and marked increases in both the number and length of neurite-like processes extending from the cell. Knockdown of p250RhoGAP by small interfering RNA induces the same morphological changes observed with GnRH treatment. In addition, loss of p250RhoGAP causes an increase in cellular motility. Our findings suggest a novel pathway regulating long-term changes in cellular motility and process formation via the GnRH induction of miR-132/212 with the subsequent down-regulation of p250RhoGAP.
PMID20212154	p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication. The DNA damage response activates several pathways that stall the cell cycle and allow DNA repair. These consist of the well-characterized ATR (Ataxia telangiectasia and Rad-3 related)/CHK1 and ATM (Ataxia telangiectasia mutated)/CHK2 pathways in addition to a newly identified ATM/ATR/p38MAPK/MK2 checkpoint. Crucial to maintaining the integrity of the genome is the S-phase checkpoint that functions to prevent DNA replication until damaged DNA is repaired. Inappropriate expression of the proto-oncogene c-Myc is known to cause DNA damage. One mechanism by which c-Myc induces DNA damage is through binding directly to components of the prereplicative complex thereby promoting DNA synthesis, resulting in replication-associated DNA damage and checkpoint activation due to inappropriate origin firing. Here we show that following etoposide-induced DNA damage translation of c-Myc is repressed by miR-34c via a highly conserved target-site within the 3(') UTR. While miR-34c is induced by p53 following DNA damage, we show that in cells lacking p53 this is achieved by an alternative pathway which involves p38 MAPK signalling to MK2. The data presented here suggest that a major physiological target of miR-34c is c-Myc. Inhibition of miR-34c activity prevents S-phase arrest in response to DNA damage leading to increased DNA synthesis, DNA damage, and checkpoint activation in addition to that induced by etoposide alone, which are all reversed by subsequent c-Myc depletion. These data demonstrate that miR-34c is a critical regulator of the c-Myc expression following DNA damage acting downstream of p38 MAPK/MK2 and suggest that miR-34c serves to remove c-Myc to prevent inappropriate replication which may otherwise lead to genomic instability.
PMID21856745	miR-375 is activated by ASH1 and inhibits YAP1 in a lineage-dependent manner in lung cancer. Lung cancers with neuroendocrine (NE) features are often very aggressive but the underlying molecular mechanisms remain elusive. The transcription factor ASH1/ASCL1 is a master regulator of pulmonary NE cell development that is involved in the pathogenesis of lung cancers with NE features (NE-lung cancers). Here we report the definition of the microRNA miR-375 as a key downstream effector of ASH1 function in NE-lung cancer cells. miR-375 was markedly induced by ASH1 in lung cancer cells where it was sufficient to induce NE differentiation. miR-375 upregulation was a prerequisite for ASH1-mediated induction of NE features. The transcriptional coactivator YAP1 was determined to be a direct target of miR-375. YAP1 showed a negative correlation with miR-375 in a panel of lung cancer cell lines and growth inhibitory activities in NE-lung cancer cells. Our results elucidate an ASH1 effector axis in NE-lung cancers that is functionally pivotal in controlling NE features and the alleviation from YAP1-mediated growth inhibition.
PMID22356768	Phospho-ΔNp63α-dependent regulation of autophagic signaling through transcription and micro-RNA modulation. Cisplatin was shown to induce the ataxia telangiectasia mutated (ATM)-dependent phosphorylation of tumor protein p63 isoform, (ΔNp63α), leading to a transcriptional regulation of specific genes implicated in the control of cell death of squamous cell carcinoma (SCC) cells. We previously observed that the cisplatin-induced phosphorylated (p)-ΔNp63α transcriptionally regulates the expression of specific microRNAs (miRNAs) in SCC cells. We found here that cisplatin exposure of SCC cells led to modulation of the members of the autophagic pathway, such as Atg1/Ulk1, Atg3, Atg4A, Atg5, Atg6/Becn1, Atg7, Atg9A and Atg10, by a direct p-ΔNp63α-dependent transcriptional regulation. We further found that specific miRNAs (miR-181a, miR-519a, miR-374a and miR-630), which are critical downstream targets of the p-ΔNp63α, modulated the protein levels of ATG5, ATG6/BECN1, ATG10, ATG12, ATG16L1 and UVRAG, adding another level of expression control for autophagic pathways in SCC cells upon cisplatin exposure. Our data support the notion that the cisplatin-induced p-ΔNp63α could regulate key pathways implicated in response of cancer cells to chemotherapeutics.
PMID21897363	Epigenetic silencing of the oncogenic miR-17-92 cluster during PU.1-directed macrophage differentiation. The oncogenic cluster miR-17-92 encodes seven related microRNAs that regulate cell proliferation, apoptosis and development. Expression of miR-17-92 cluster is decreased upon cell differentiation. Here, we report a novel mechanism of the regulation of miR-17-92 cluster. Using transgenic PU.1(-/-) myeloid progenitors we show that upon macrophage differentiation, the transcription factor PU.1 induces the secondary determinant Egr2 which, in turn, directly represses miR-17-92 expression by recruiting histone demethylase Jarid1b leading to histone H3 lysine K4 demethylation within the CpG island at the miR-17-92 promoter. Conversely, Egr2 itself is targeted by miR-17-92, indicating existence of mutual regulatory relationship between miR-17-92 and Egr2. Furthermore, restoring EGR2 levels in primary acute myeloid leukaemia blasts expressing elevated levels of miR-17-92 and low levels of PU.1 and EGR2 leads to downregulation of miR-17-92 and restored expression of its targets p21CIP1 and BIM. We propose that upon macrophage differentiation PU.1 represses the miR-17-92 cluster promoter by an Egr-2/Jarid1b-mediated H3K4 demethylation mechanism whose deregulation may contribute to leukaemic states.
PMID21454377	MiR-15 and miR-16 are direct transcriptional targets of E2F1 that limit E2F-induced proliferation by targeting cyclin E. MicroRNAs (miR) are small noncoding RNA molecules that have recently emerged as critical regulators of gene expression and are often deregulated in cancer. In particular, miRs encoded by the miR-15a, miR-16-1 cluster seem to act as tumor suppressors. Here, we evidence that the miR-15a, miR-16-1 cluster and related miR-15b, miR-16-2 cluster comprise miRs regulated by E2F1, a pivotal transcription factor that can induce both proliferation and cell death. E2F1 is a critical downstream target of the tumor suppressor retinoblastoma (RB). The RB pathway is often inactivated in human tumors resulting in deregulated E2F activity. We show that expression levels of the 4 mature miRs, miR-15a, miR-16-1 and miR-15b, miR-16-2, as well as their precursor pri-miRNAs, are elevated upon activation of ectopic E2F1. Moreover, activation of endogenous E2Fs upregulates expression of these miRs and endogenous E2F1 binds their respective promoters. Importantly, we corroborate that miR-15a/b inhibits expression of cyclin E, the latter a key direct transcriptional target of E2F pivotal for the G(1)/S transition, raising the possibility that E2F1, miR-15, and cyclin E constitute a feed-forward loop that modulates E2F activity and cell-cycle progression. In support of this, ectopic expression of miR-15 inhibits the G(1)/S transition, and, conversely, inhibition of miR-15 expression enhances E2F1-induced upregulation of cyclin E1 levels. Furthermore, inhibition of both miR-15 and miR-16 enhances E2F1-induced G(1)/S transition. In summary, our data identify the miR-15 and miR-16 families as novel transcriptional targets of E2F, which, in turn, modulates E2F activity.
PMID19307576	Posttranscriptional regulation of interleukin-10 expression by hsa-miR-106a. IL-10 is a key regulator of the immune system that critically determines health and disease. Its expression is finely tuned both at the transcriptional and posttranscriptional levels. Although the importance of posttranscriptional regulation of IL-10 has been previously shown, understanding the underlying mechanisms is still in its infancy. In this study, using a combination of bioinformatics and molecular approaches, we report that microRNA (hsa-miR-106a) regulates IL-10 expression. The hsa-miR-106a binding site in the 3' UTR of IL10 has been identified by site-directed mutagenesis studies. Also, the involvement of transcription factors, Sp1 and Egr1, in the regulation of hsa-miR-106a expression and concomitant decrease in the IL-10 expression, has also been demonstrated. In summary, our results showed that IL-10 expression may be regulated by miR-106a, which is in turn transcriptionally regulated by Egr1 and Sp1.
PMID20023696	Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 suppresses CXCR4 expression by upregulating miR-146a. Kaposi's sarcoma (KS)-associated herpesvirus (KSHV)-encoded viral FLICE inhibitory protein (vFLIP) K13 is a potent activator of the nuclear factor-kappaB (NF-kappaB) pathway. In this study, we show that infection with KHSV and ectopic expression of K13, but not its NF-kappaB-defective mutant, suppressed the expression of CXCR4. Suppression of CXCR4 by KSHV and K13 was associated with upregulated expression of miR-146a, a microRNA that is known to bind to the 3'-untranslated region of CXCR4 mRNA. Reporter studies identified two NF-kappaB sites in the promoter of miR-146a that were essential for its activation by K13. Accordingly, ectopic expression of K13, but not its NF-kappaB-defective mutant or other vFLIPs, strongly stimulated the miR-146a promoter activity, which could be blocked by specific genetic and pharmacological inhibitors of the NF-kappaB pathway. Finally, expression of CXCR4 was downregulated in clinical samples of KS and this was accompanied by an increased expression of miR-146a. Our results show that K13-induced NF-kappaB activity suppresses CXCR4 through upregulation of miR-146a. Downregulation of CXCR4 expression by K13 may contribute to KS development by promoting premature release of KSHV-infected endothelial progenitors into the circulation.
PMID18316553	hsa-miR-210 Is induced by hypoxia and is an independent prognostic factor in breast cancer. MicroRNA (miRNA) expression alterations have been described in cancer. Many cancers are characterized by areas of hypoxia, enhanced hypoxia-inducible factor (HIF) levels, and increased expression of hypoxically regulated genes, all of which correlate with patient outcome. We examined hypoxia-induced miRNA expression changes to identify markers of survival in breast cancer. We used microarrays to analyze miRNA expression changes induced by hypoxia in MCF7 breast cancer cell lines and validated results by quantitative-PCR (Q-PCR). Small interfering RNA against HIF-1alpha and HIF-2alpha, and RCC4 cells transfected with the von Hippel-Lindau (VHL) protein were used to investigate HIF dependency of miRNA expression. miRNA Q-PCR assays were done on 219 early breast cancer samples with long-term follow-up. Correlation of expression with clinical variables was done using Pearson and Spearman's rank tests, univariate, and Cox multivariate analysis. hsa-miR-210 induction was the most significant change under hypoxia by microarray analysis (3.4-fold, P < 0.001). hsa-miR-210 expression changes were validated by Q-PCR and detected in other cancer cell lines. Using small interfering RNAs and RCC4 cells transfected with VHL, we showed that the regulation by hypoxia of hsa-miR-210 was mediated by the HIF-1alpha/VHL transcriptional system but not HIF-2alpha. hsa-miR-210 expression levels in breast cancer samples correlated directly with a hypoxia score based on the expression of 99 genes. hsa-miR-210 expression levels showed an inverse correlation with disease-free and overall survival, significant in both univariate and multivariate analyses. We show that hsa-miR-210 overexpression is induced by hypoxia in a HIF-1alpha- and VHL-dependent fashion and its expression levels in breast cancer samples are an independent prognostic factor.
PMID22392644	Androgen pathway stimulates microRNA-216a transcription to suppress the tumor suppressor in lung cancer-1 gene in early hepatocarcinogenesis. Deregulation of microRNAs (miRNAs) is common in advanced human hepatocellular carcinoma (HCC); however, the ones involved in early carcinogenesis have not yet been investigated. By examining the expression of 22 HCC-related miRNAs between precancerous and cancerous liver tissues, we found miR-216a and miR-224 were significantly up-regulated, starting from the precancerous stage. Furthermore, the elevation of miR-216a was mainly identified in male patients. To study this gender difference, we demonstrated that pri-miR-216a is activated transcriptionally by the androgen pathway in a ligand-dependent manner and is further enhanced by the hepatitis B virus X protein. The transcription initiation site for pri-miR-216a was delineated, and one putative androgen-responsive element site was identified within its promoter region. Mutation of this site abolished the elevation of pri-miR-216a by the androgen pathway. One target of miR-216a was shown to be the tumor suppressor in lung cancer-1 gene (TSLC1) messenger RNA (mRNA) through the three target sites at its 3' untranslated region. Finally, the androgen receptor level increased in male liver tissues during hepatocarcinogenesis, starting from the precancerous stage, with a concomitant elevation of miR-216a but a decrease of TSLC1. The current study discovered the up-regulation of miRNA-216a by the androgen pathway and a subsequent suppression of TSLC1 as a new mechanism for the androgen pathway in early hepatocarcinogenesis.
PMID18850008	An autoregulatory loop mediated by miR-21 and PDCD4 controls the AP-1 activity in RAS transformation. The transcription factor AP-1 plays key roles in tumorigenesis, by regulating a variety of protein-coding genes, implicated in multiple hallmarks of cancer. Among non-coding genes, no AP-1 target has been described yet in tumorigenesis. MicroRNAs (miRNAs) are negative post-transcriptional regulators of protein-coding genes. miRNA expression signatures are highly relevant in cancer and several tumor-associated miRNAs (oncomirs) play critical roles in oncogenesis. Here, we show that the miRNA miR-21, which represents the most frequently upregulated oncomir in solid tumors, is induced by AP-1 in response to RAS. By analyzing validated miR-21 targets, we have found that the tumor suppressors PTEN and PDCD4 are downregulated by RAS in an AP-1- and miR-21-dependent fashion. We further show that, given the role of PDCD4 as negative regulator of AP-1, the miR-21-mediated downregulation of PDCD4 is essential for the maximal induction of AP-1 activity in response to RAS. Our data reveal a novel mechanism of positive autoregulation of the AP-1 complex in RAS transformation and disclose the function of oncomirs as critical targets and regulators of AP-1 in tumorigenesis.
PMID20023698	TGFbeta-mediated upregulation of hepatic miR-181b promotes hepatocarcinogenesis by targeting TIMP3. To identify microRNAs (miRNAs) that may have a causal role in hepatocarcinogenesis, we used an animal model in which C57BL/6 mice fed choline-deficient and amino acid defined (CDAA) diet develop preneoplastic lesions at 65 weeks and hepatocellular carcinomas after 84 weeks. miRNA expression profiling showed significant upregulation of miR-181b and miR-181d in the livers of mice as early as 32 weeks that persisted at preneoplastic stage. The expression of tissue inhibitor of metalloprotease 3 (TIMP3), a tumor suppressor and a validated miR-181 target, was markedly suppressed in the livers of mice fed CDAA diet. Upregulation of hepatic transforming growth factor (TGF)beta and its downstream mediators Smad 2, 3 and 4 and increase in phospho-Smad2 in the liver nuclear extract correlated with elevated miR-181b/d in mice fed CDAA diet. The levels of the precursor and mature miR-181b were augmented on exposure of hepatic cells to TGFbeta and were significantly reduced by small interference RNA-mediated depletion of Smad4, showing the involvement of TGFbeta signaling pathway in miR-181b expression. Ectopic expression and depletion of miR-181b showed that miR-181b enhanced matrix metallopeptidases (MMP)2 and MMP9 activity and promoted growth, clonogenic survival, migration and invasion of hepatocellular carcinoma (HCC) cells that could be reversed by modulating TIMP3 level. Further, depletion of miR-181b inhibited tumor growth of HCC cells in nude mice. miR-181b also enhanced resistance of HCC cells to the anticancer drug doxorubicin. On the basis of these results, we conclude that upregulation of miR-181b at early stages of feeding CDAA diet promotes hepatocarcinogenesis.
PMID18303114	A GATA-1-regulated microRNA locus essential for erythropoiesis. MicroRNAs (miRNAs) control tissue development, but their mechanism of regulation is not well understood. We used a gene complementation strategy combined with microarray screening to identify miRNAs involved in the formation of erythroid (red blood) cells. Two conserved miRNAs, miR 144 and miR 451, emerged as direct targets of the critical hematopoietic transcription factor GATA-1. In vivo, GATA-1 binds a distal upstream regulatory element to activate RNA polymerase II-mediated transcription of a single common precursor RNA (pri-miRNA) encoding both mature miRNAs. Zebrafish embryos depleted of miR 451 by using antisense morpholinos form erythroid precursors, but their development into mature circulating red blood cells is strongly and specifically impaired. These results reveal a miRNA locus that is required for erythropoiesis and uncover a new regulatory axis through which GATA-1 controls this process.
PMID18765229	MIR-451 and Imatinib mesylate inhibit tumor growth of Glioblastoma stem cells. We examined the microRNA profiles of Glioblastoma stem (CD133+) and non-stem (CD133-) cell populations and found up-regulation of several miRs in the CD133- cells, including miR-451, miR-486, and miR-425, some of which may be involved in regulation of brain differentiation. Transfection of GBM cells with the above miRs inhibited neurosphere formation and transfection with the mature miR-451 dispersed neurospheres, and inhibited GBM cell growth. Furthermore, transfection of miR-451 combined with Imatinib mesylate treatment had a cooperative effect in dispersal of GBM neurospheres. In addition, we identified a target site for SMAD in the promoter region of miR-451 and showed that SMAD3 and 4 activate such a promoter-luciferase construct. Transfection of SMAD in GBM cells inhibited their growth, suggesting that SMAD may drive GBM stem cells to differentiate to CD133- cells through up-regulation of miR-451 and reduces their tumorigenicity. Identification of additional miRs and target genes that regulate GBM stem cells may provide new potential drugs for therapy.
PMID21730352	Macrophage development from HSCs requires PU.1-coordinated microRNA expression. The differentiation of HSCs into myeloid lineages requires the transcription factor PU.1. Whereas PU.1-dependent induction of myeloid-specific target genes has been intensively studied, negative regulation of stem cell or alternate lineage programs remains incompletely characterized. To test for such negative regulatory events, we searched for PU.1-controlled microRNAs (miRs) by expression profiling using a PU.1-inducible myeloid progenitor cell line model. We provide evidence that PU.1 directly controls expression of at least 4 of these miRs (miR-146a, miR-342, miR-338, and miR-155) through temporally dynamic occupation of binding sites within regulatory chromatin regions adjacent to their genomic coding loci. Ectopic expression of the most robustly induced PU.1 target miR, miR-146a, directed the selective differentiation of HSCs into functional peritoneal macrophages in mouse transplantation assays. In agreement with this observation, disruption of Dicer expression or specific antagonization of miR-146a function inhibited the formation of macrophages during early zebrafish (Danio rerio) development. In the present study, we describe a PU.1-orchestrated miR program that mediates key functions of PU.1 during myeloid differentiation.
PMID21777817	FGF-2 regulates cell proliferation, migration, and angiogenesis through an NDY1/KDM2B-miR-101-EZH2 pathway. The histone H3K27 methyltransferase EZH2 plays an important role in oncogenesis, by mechanisms that are incompletely understood. Here, we show that the JmjC domain histone H3 demethylase NDY1 synergizes with EZH2 to silence the EZH2 inhibitor miR-101. NDY1 and EZH2 repress miR-101 by binding its promoter in concert, via a process triggered by upregulation of NDY1. Whereas EZH2 binding depends on NDY1, the latter binds independently of EZH2. However, both are required to repress transcription. NDY1 and EZH2 acting in concert upregulate EZH2 and stabilize the repression of miR-101 and its outcome. NDY1 is induced by FGF-2 via CREB phosphorylation and activation, downstream of DYRK1A, and mediates the FGF-2 and EZH2 effects on cell proliferation, migration, and angiogenesis. The FGF-2-NDY1/EZH2-miR-101-EZH2 axis described here was found to be active in bladder cancer. These data delineate an oncogenic pathway that functionally links FGF-2 with EZH2 via NDY1 and miR-101.
PMID21903590	MYC protein inhibits transcription of the microRNA cluster MC-let-7a-1~let-7d via noncanonical E-box. The human microRNA cluster MC-let-7a-1∼let-7d, with three members let-7a-1, let-7f-1, and let-7d, is an important cluster of the let-7 family. These microRNAs play critical roles in regulating development and carcinogenesis. Therefore, precise control of MC-let-7a-1∼let-7d level is critical for cellular functions. In this study, we first showed that the expression of these three members was significantly reduced in human hepatocellular carcinoma HepG2 cells as compared with the immortalized human liver L02 cells. We demonstrated that the MC-let-7a-1∼let-7d cluster was encoded by a single polycistronic transcript driven by a 10-kb upstream promoter, with two MYC-binding sites. Importantly, MYC inhibited MC-let-7a-1∼let-7d promoter activity via binding to the noncanonical E-box 3 downstream of the transcription start sites, whereas it enhanced promoter activity by binding to the canonical E-box 2 upstream of the transcription start sites. We found that although the binding affinity of MYC to E-box 2 was stronger than E-box 3, the binding quantum of MYC to E-box 3 was significantly higher in cancerous HepG2 cells as compared with the noncancerous L02 cells. In addition, forced expression of let-7 could reverse the MYC-mediated cell proliferation. These findings suggested that in L02 cells with a low level of MYC, MYC binds mainly to E-box 2 to enhance MC-let-7a-1∼let-7d expression. However, in HepG2 cells with an elevated MYC, the extra MYC could bind to E-box 3 to suppress the transcription of MC-let-7a-1∼let-7d and thus enable HepG2 cells to maintain a high level of MYC and a low level of let-7 microRNAs simultaneously.
PMID21151097	A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation. Multiple levels of control are in play to regulate pluripotency and differentiation in human embryonic stem cells (hESCs). At the transcriptional level, the core factors OCT4, NANOG and SOX2 form a positive autoregulatory loop that is pivotal for maintaining the undifferentiated state. At the post-transcriptional level, microRNAs (miRNAs) belonging to the miR-302 family are emerging as key players in the control of proliferation and cell fate determination during differentiation. Here, we show that the transcriptional factors OCT4 and NR2F2 (COUP-TFII) and the miRNA miR-302 are linked in a regulatory circuitry that critically regulate both pluripotency and differentiation in hESCs. In the undifferentiated state, both OCT4 and the OCT4-induced miR-302 directly repress NR2F2 at the transcriptional and post-transcriptional level, respectively. Conversely, NR2F2 directly inhibits OCT4 during differentiation, triggering a positive feedback loop for its own expression. In addition, we show that regulation of NR2F2 activity itself relies on alternative splicing and transcriptional start site choice to generate a full-length transcriptionally active isoform and shorter variants, which enhance the activity of the long isoform. During hESC differentiation, NR2F2 is first detected at the earliest steps of neural induction and thus is among the earliest human embryonic neural markers. Finally, our functional analysis points to a crucial role for NR2F2 in the activation of neural genes during early differentiation in humans. These findings introduce a new molecular player in the context of early embryonic stem cell state and cell fate determination in humans.
PMID21712382	Transforming growth factor-beta1 (TGF-beta1) utilizes distinct pathways for the transcriptional activation of microRNA 143/145 in human coronary artery smooth muscle cells. MicroRNA 143/145 (miR143/145) is restricted to adult smooth muscle cell (SMC) lineages and mediates, in part, the expression of several SMC contractile genes. Although the function of miR143/145 has begun to be elucidated, its transcriptional regulation in response to various signaling inputs is poorly understood. In an effort to define a miR signature for SMC differentiation, we screened human coronary artery SMCs for miRs modulated by TGF-β1, a known stimulus for SMC differentiation. Array analysis revealed a number of TGF-β1-induced miRs, including miR143/145. Validation studies showed that TGF-β1 stimulated miR143/145 expression in a dose- and time-dependent manner. We utilized several chemical inhibitors and found that SB203580, a specific inhibitor of p38MAPK, significantly decreased TGF-β1-induced miR143/145 expression. siRNA studies demonstrated that the effect of TGF-β1 on miR143/145 was dependent upon the myocardin and serum response factor transcriptional switch as well as SMAD4. TGF-β1 stimulated a 580-bp human miR143/145 enhancer, and mutagenesis studies revealed a critical role for both a known CArG box and an adjacent SMAD-binding element for full TGF-β1-dependent activation of the enhancer. Chromatin immunoprecipitation assays documented TGF-β1-mediated enrichment of SMAD3 and SMAD4 binding over the enhancer region containing the SMAD-binding element. Pre-miR145 strongly promoted SMC differentiation, whereas an anti-miR145 partially blocked TGF-β1-induced SMC differentiation. These results demonstrate a dual pathway for TGF-β1-induced transcription of miR143/145, thus revealing a novel mechanism underlying TGF-β1-induced human vascular SMC differentiation.
PMID20811575	Global Egr1-miRNAs binding analysis in PMA-induced K562 cells using ChIP-Seq. Although much is known about microRNAs' regulation in gene expression and their contributions in cell fate, to date, globally lineage-(cell-) specific identification of the binding events between a transcription factor and its targeting microRNA genes is still waiting for elucidation. In this paper, we performed a ChIP-Seq experiment to find the targeting microRNA genes of a transcription factor, Egr1, in human erythroleukemia cell line K562. We found Egr1 binding sites near the promoters of 124 distinct microRNA genes, accounting for about 42% of the miRNAs which have high-confidence predicted promoters (294). We also found EGR1 bind to another 63 pre-miRNAs. We chose 12 of the 187 microRNAs with Egr1 binding sites to perform ChIP-PCR assays and the positive binding signal from ChIP-PCR confirmed the ChIP-Seq results. Our experiments provide the first global binding profile between Egr1 and its targeting microRNA genes in PMA-treated K562 cells, which may facilitate the understanding of pathways controlling microRNA biology in this specific cell line.
PMID20883704	MicroRNA, SND1, and alterations in translational regulation in colon carcinogenesis. Post-transcriptional regulation of gene expression by microRNA (miRNA) has recently attracted major interest in relation to its involvement in cancer development. miRNA is a member of small non-coding RNA, consists of 22-24 nucleotides and regulates expression of target mRNA species in a post-transcriptional manner by being incorporated with RNA-induced silencing complex (RISC). Staphylococcal nuclease homology domain containing 1 (SND1), a component of RISC, is frequently up-regulated in human colon cancers and also chemically induced colon cancers in animals. We here showed that SDN1 is involved in miRNA-mediated gene suppression and overexpression of SND1 in colon cancer cells causes down-regulation of APC without altering APC mRNA levels. As for the miRNA expression profile in human colon cancer, miR-34a was among the list of down-regulated miRNA. Expression of miR-34a is tightly regulated by p53, and ectopic expression of miR-34a in colon cancer cells causes remarkable reduction of cell proliferation and induces senescence-like phenotypes. MiR-34a also participates in the positive feedback loop of the p53 tumor suppressor network. This circuitry mechanism for p53 activation is of interest in understanding the tumor suppressive function of miR-34a in colon carcinogenesis. miRNA should also be considered as novel anti-cancer agents in tumor suppressive therapeutic applications.
PMID21685392	The miR-143/145 cluster is a novel transcriptional target of Jagged-1/Notch signaling in vascular smooth muscle cells. Activation of Notch signaling by Jagged-1 (Jag-1) in vascular smooth muscle cells (VSMC) promotes a differentiated phenotype characterized by increased expression of contractile proteins. Recent studies show that microRNAs (miR)-143/145 regulates VSMC phenotype. The serum response factor (SRF)/myocardin complex binds to CArG sequences to activate miR-143/145 transcription, but no other regulators are known in VSMC. Using miR arrays, we found miR-143/145 induced following expression of a constitutively active Notch1 intracellular domain (N1ICD). We hypothesized that miR-143/145 is required for Jag-1/Notch-induced VSMC differentiation. Activation of Notch receptors by Jag-1 caused CBF1-dependent up-regulation of miR-143/145, increased differentiation, and decreased proliferation. Conversely, inhibiting basal Notch signaling decreased steady state levels of miR-143/145. Using SRF knockdown, we found that Jag-1/Notch induction of miR-143/145 is SRF independent, although full acquisition of contractile markers requires SRF. Using miR-143/145 promoter reporter constructs we show Jag-1/Notch increases promoter activity, and this is dependent on intact CBF1 consensus sites within the promoter. Chromatin immunoprecipitation (ChIP) assays revealed that N1ICD-containing complexes bind to CBF1 sites in the miR-143/145 promoter. We also identified N1ICD complex binding to CBF1 sites within the endogenous human miR-143/145 promoter. Using miR-143/145-interfering oligonucleotides, we demonstrate that Jag-1/Notch signaling requires induction of both miR-143 and miR-145 to promote the VSMC contractile phenotype. Thus, miR-143/145 is a novel transcriptional target of Jag-1/Notch signaling in VSMC. We propose miR-143/145 as activated independently by Jag-1/Notch and SRF in parallel pathways. Multiple pathways converging on miR-143/145 provides potential for fine-tuning or amplification of VSMC differentiation signals.
PMID20889924	C/EBPα regulated microRNA-34a targets E2F3 during granulopoiesis and is down-regulated in AML with CEBPA mutations. The transcription factor, CCAAT enhancer binding protein alpha (C/EBPα), is crucial for granulopoiesis and is deregulated by various mechanisms in acute myeloid leukemia (AML). Mutations in the CEBPA gene are reported in 10% of human patients with AML. Even though the C/EBPα mutants are known to display distinct biologic function during leukemogenesis, the molecular basis for this subtype of AML remains elusive. We have recently showed the significance of deregulation of C/EBPα-regulated microRNA (miR) in AML. In this study, we report that miR-34a is a novel target of C/EBPα in granulopoiesis. During granulopoiesis, miR-34a targets E2F3 and blocks myeloid cell proliferation. Analysis of AML samples with CEBPA mutations revealed a lower expression of miR-34a and elevated levels of E2F3 as well as E2F1, a transcriptional target of E2F3. Manipulation of miR-34a reprograms granulocytic differentiation of AML blast cells with CEBPA mutations. These results define miR-34a as a novel therapeutic target in AML with CEBPA mutations.
PMID22262409	In hepatocellular carcinoma miR-519d is up-regulated by p53 and DNA hypomethylation and targets CDKN1A/p21, PTEN, AKT3 and TIMP2. MiR-519d belongs to the chromosome 19 miRNA cluster (C19MC), the largest human miRNA cluster. One of its members, miR-519d, is over-expressed in hepatocellular carcinoma (HCC) and we characterized its contribution to hepatocarcinogenesis. In HCC cells, the over-expression of miR-519d promotes cell proliferation, invasion and impairs apoptosis following anticancer treatments. These functions are, at least in part, exerted through the direct targeting of CDKN1A/p21, PTEN, AKT3 and TIMP2. The mechanisms underlying miR-519d aberrant expression in HCC were assayed by genomic DNA amplification, methylation analysis and ChIP assay. The aberrant hypomethylation of C19MC and TP53 were respectively identified as an epigenetic change allowing the aberrant expression of miR-519d and one of the factors able to activate its transcription. In conclusion, we assessed the oncogenic role of miR-519d in HCC by characterizing its biological functions, including the modulation of response to anticancer treatments and by identifying CDKN1A/p21, PTEN, AKT3 and TIMP2 among its targets.
PMID19167416	The tumor-promoting activity of 2-acetylaminofluorene is associated with disruption of the p53 signaling pathway and the balance between apoptosis and cell proliferation. The aromatic amine 2-acetylaminofluore (2-AAF) is a powerful complete genotoxic rat liver carcinogen that induces tumors without any additional interventions. While the tumor-initiating genotoxic activity of 2-AAF is well established, its tumor-promotion activity is far less understood. It is believed that the tumor-promoting property of 2-AAF is associated with selective enhancement of cell replication and sustained suppression of apoptosis in initiated cells. In the present study, we investigated the underlying mechanisms of tumor-promoting events induced by 2-AAF-exposure. Male Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 12 and 24 weeks, and the expression pattern of genes associated with the p53-signaling pathway and microRNA genes was determined in the livers of control and 2-AAF-fed rats. The results indicate that the tumor-promoting property of 2-AAF during hepatocarcinogenesis is associated predominantly with the up-regulation of anti-apoptotic growth-related genes and down-regulation of expression of pro-apoptotic genes. This disrupts the balance between cell proliferation and apoptosis, which leads to consequential unrestricted cell proliferation, especially of initiated cells. Also, the long-term-administration of 2-AAF resulted in disruption of regulatory miR-34a-p53 feed-back loop that mediates apoptosis. This was evidenced by an increased expression of miR-34a in response to genotoxic effects of 2-AAF in the absence of p53 up-regulation, and loss of regulatory control of mir-34a on SIRT1 function. Additionally, the livers of 2-AAF-exposed rats were characterized by the substantial deregulation of expression of miR-18, miR-21, miR-182, and miR-200 family, microRNAs involved in control of apoptosis/cell proliferation and cell-cell contact pathways, two major pathways disrupted during the promotion stage of hepatocarcinogenesis.
PMID20061417	Nuclear factor-{kappa}B contributes to anaplastic thyroid carcinomas through up-regulation of miR-146a. Micro-RNAs (miRNAs) have been recently involved in the modulation of several biological activities including cancer. Many human tumors show deregulated expression of miRNAs targeting oncogenes and/or tumor suppressors, thus identifying miRNAs as new molecular targets for cancer therapy. Nuclear factor (NF)-kappaB is strongly activated in human anaplastic thyroid carcinomas (ATCs). Because the regulation of miRNA expression is under control of RNA polymerase II-dependent transcription factors, we stably inactivated NF-kappaB in the ATC-derived FRO cell line and analyzed its miRNA profile in comparison with the parental counterpart by using a miRNA chip microarray. The analysis revealed that a number of miRNAs were differentially expressed in the two cell lines. Among others, the miR-146a showed a strong down-regulation that was confirmed by quantitative real time RT-PCR. The expression of miR-146a was almost undetectable in mouse embryonic fibroblasts isolated from the RelA knockout mice and was restored after reexpression of RelA, thus indicating that miR-146a transcription was controlled by NF-kappaB. The inhibition of miR-146a expression in FRO cells decreased their oncogenic potential and increased the susceptibility to chemotherapeutic drug-induced apoptosis. No difference was found in the growth rate between untransfected and miR-146a-null FRO cells. Importantly, the miR-146a resulted in overexpression of human ATC specimens compared with the normal thyroid tissue. Our results show that NF-kappaB contributes to anaplastic thyroid cancer up-regulating the expression of miR-146a.
PMID20935646	The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes. After being activated by antigen, helper T lymphocytes switch from a resting state to clonal expansion. This switch requires inactivation of the transcription factor Foxo1, a suppressor of proliferation expressed in resting helper T lymphocytes. In the early antigen-dependent phase of expansion, Foxo1 is inactivated by antigen receptor-mediated post-translational modifications. Here we show that in the late phase of expansion, Foxo1 was no longer post-translationally regulated but was inhibited post-transcriptionally by the interleukin 2 (IL-2)-induced microRNA miR-182. Specific inhibition of miR-182 in helper T lymphocytes limited their population expansion in vitro and in vivo. Our results demonstrate a central role for miR-182 in the physiological regulation of IL-2-driven helper T cell-mediated immune responses and open new therapeutic possibilities.
PMID22086602	RNA helicase DDX5 regulates microRNA expression and contributes to cytoskeletal reorganization in basal breast cancer cells. RNA helicase DDX5 (also p68) is involved in all aspects of RNA metabolism and serves as a transcriptional coregulator, but its functional role in breast cancer remains elusive. Here, we report an integrative biology study of DDX5 in breast cancer, encompassing quantitative proteomics, global MicroRNA profiling, and detailed biochemical characterization of cell lines and human tissues. We showed that protein expression of DDX5 increased progressively from the luminal to basal breast cancer cell lines, and correlated positively with that of CD44 in the basal subtypes. Through immunohistochemistry analyses of tissue microarrays containing over 200 invasive human ductal carcinomas, we observed that DDX5 was up-regulated in the majority of malignant tissues, and its expression correlated strongly with those of Ki67 and EGFR in the triple-negative tumors. We demonstrated that DDX5 regulated a subset of MicroRNAs including miR-21 and miR-182 in basal breast cancer cells. Knockdown of DDX5 resulted in reorganization of actin cytoskeleton and reduction of cellular proliferation. The effects were accompanied by up-regulation of tumor suppressor PDCD4 (a known miR-21 target); as well as up-regulation of cofilin and profilin, two key proteins involved in actin polymerization and cytoskeleton maintenance, as a consequence of miR-182 down-regulation. Treatment with miR-182 inhibitors resulted in morphologic phenotypes resembling those induced by DDX5 knockdown. Using bioinformatics tools for pathway and network analyses, we confirmed that the network for regulation of actin cytoskeleton was predominantly enriched for the predicted downstream targets of miR-182. Our results reveal a new functional role of DDX5 in breast cancer via the DDX5→miR-182→actin cytoskeleton pathway, and suggest the potential clinical utility of DDX5 and its downstream MicroRNAs in the theranostics of breast cancer.
PMID17656095	p53-mediated activation of miRNA34 candidate tumor-suppressor genes. In response to varied cell stress signals, the p53 tumor-suppressor protein activates a multitude of genes encoding proteins with functions in cell-cycle control, DNA repair, senescence, and apoptosis. The role of p53 in transcription of other types of RNAs, such as microRNAs (miRNAs) is essentially unknown. Using gene-expression analyses, reporter gene assays, and chromatin-immunoprecipitation approaches, we present definitive evidence that the abundance of the three-member miRNA34 family is directly regulated by p53 in cell lines and tissues. Using array-based approaches and algorithm predictions, we define genes likely to be directly regulated by miRNA34, with cell-cycle regulatory genes being the most prominent class. In addition, we provide functional evidence, obtained via antisense oligonucleotide transfection and the use of mouse embryonic stem cells with loss of miRNA34a function, that the BCL2 protein is regulated directly by miRNA34. Finally, we demonstrate that the expression of two miRNA34s is dramatically reduced in 6 of 14 (43%) non-small cell lung cancers (NSCLCs) and that the restoration of miRNA34 expression inhibits growth of NSCLC cells. Taken together, the data suggest the miRNA34s might be key effectors of p53 tumor-suppressor function, and their inactivation might contribute to certain cancers.
PMID18460397	Differential expression of PTEN-targeting microRNAs miR-19a and miR-21 in Cowden syndrome. Germline mutations in the gene encoding phosphatase and tensin homolog deleted on chromosome ten (PTEN [MIM 601728]) are associated with a number of clinically distinct heritable cancer syndromes, including both Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRRS). Seemingly identical pathogenic PTEN mutations have been observed in patients with CS and BRRS, as well as in patients with incomplete features of CS, referred to as CS-like (CSL) patients. These observations indicate that additional, unidentified, genetic and epigenetic factors act as phenotypic modifiers in these disorders. These genetic factors could also contribute to disease in patients with CS, CSL, or BRRS without identifiable PTEN mutations. Two potential modifiers are miR-19a and miR-21, which are previously identified PTEN-targeting miRNAs. We investigated the role of these miRNAs by characterizing their relative expression levels in PTEN-mutation-positive and PTEN-mutation-negative patients with CS, CSL, or BRRS. Interestingly, we observed differential expression of miR-19a and miR-21 in our PTEN-mutation-positive patients. Both were found to be significantly overexpressed within this group (p < 0.01) and were inversely correlated with germline PTEN protein levels. Similarly, the relative expression of miR-19a and miR-21 was differentially expressed in a series of PTEN-mutation-negative patients with CS or CSL with variable clinical phenotypes and decreased full-length PTEN protein expression. Among PTEN-mutation-positive patients with CS, both miRNAs were significantly overexpressed (p = 0.006-0.013). Taken together, our study results suggest that differential expression of PTEN-targeting miR-19a and miR-21 modulates the PTEN protein levels and the CS and CSL phenotypes, irrespective of the patient's mutation status, and support their roles as genetic modifiers in CS and CSL.
PMID21464222	Repression of tumor suppressor miR-451 is essential for NOTCH1-induced oncogenesis in T-ALL. The NOTCH1 signaling pathway is a critical determinant of cell fate decisions and drives oncogenesis through mechanisms that are incompletely understood. Using an established mouse model of T cell acute lymphoblastic leukemia (T-ALL), here we report that induction of intracellular Notch1 (ICN1) leads to repression of miR-451 and miR-709. ICN1 decreases expression of these miRNAs by inducing degradation of the E2a tumor suppressor, which transcriptionally activates the genes encoding miR-451 and miR-709. Both miR-451 and miR-709 directly repress Myc expression. In addition, miR-709 directly represses expression of the Akt and Ras-GRF1 oncogenes. We also show that repression of miR-451 and miR-709 expression is required for initiation and maintenance of mouse T-ALL. miR-451 but not miR-709 is conserved in humans, and human T-ALLs with activating NOTCH1 mutations have decreased miR-451 and increased MYC levels compared with T-ALLs with wild-type NOTCH1. Thus, miR-451 and miR-709 function as potent suppressors of oncogenesis in NOTCH1-induced mouse T-ALL, and miR-451 influences MYC expression in human T-ALL bearing NOTCH1 mutations.
PMID22319554	A Novel YY1-miR-1 regulatory circuit in skeletal myogenesis revealed by genome-wide prediction of YY1-miRNA network. microRNAs (miRNAs) are non-coding RNAs that regulate gene expression post-transcriptionally, and mounting evidence supports the prevalence and functional significance of their interplay with transcription factors (TFs). Here we describe the identification of a regulatory circuit between muscle miRNAs (miR-1, miR-133 and miR-206) and Yin Yang 1 (YY1), an epigenetic repressor of skeletal myogenesis in mouse. Genome-wide identification of potential down-stream targets of YY1 by combining computational prediction with expression profiling data reveals a large number of putative miRNA targets of YY1 during skeletal myoblasts differentiation into myotubes with muscle miRs ranking on top of the list. The subsequent experimental results demonstrate that YY1 indeed represses muscle miRs expression in myoblasts and the repression is mediated through multiple enhancers and recruitment of Polycomb complex to several YY1 binding sites. YY1 regulating miR-1 is functionally important for both C2C12 myogenic differentiation and injury-induced muscle regeneration. Furthermore, we demonstrate that miR-1 in turn targets YY1, thus forming a negative feedback loop. Together, these results identify a novel regulatory circuit required for skeletal myogenesis and reinforce the idea that regulatory circuitries involving miRNAs and TFs are prevalent mechanisms.
PMID21045151	Double-negative feedback loop between reprogramming factor LIN28 and microRNA let-7 regulates aldehyde dehydrogenase 1-positive cancer stem cells. A relatively rare aldehyde dehydrogenase 1 (ALDH1)-positive "stem cell-like" subpopulation of tumor cells has the unique ability to initiate and perpetuate tumor growth; moreover, it is highly resistant to chemotherapy and significantly associated with poor clinical outcomes. The development of more effective therapies for cancer requires targeting of this cell population. Using cDNA microarray analysis, we identified that the expression of the Caenorhabditis elegans lin-28 homologue (LIN28) was positively correlated with the percentage of ALDH1+ tumor cells; this was further validated in an independent set of tissue arrays (n=197). Both loss-of-function and gain-of-function studies showed that LIN28 plays a critical role in the maintenance of ALDH1+ tumor cells. In addition, we found that there is a double-negative feedback loop between LIN28 and let-7 in tumor cells, and that let-7 negatively regulates ALDH1+ tumor cells. Finally, we report that a LIN28/let-7 loop modulates self-renewal and differentiation of mammary gland epithelial progenitor cells. Our data provide evidence that cancer stem cells may arise through a "reprogramming-like" mechanism. A rebalancing of the LIN28/let-7 regulatory loop could be a novel therapeutic strategy to target ALDH1+ cancer stem cells.
PMID22135361	Negative feedback regulation between microRNA let-7g and the oxLDL receptor LOX-1. Lectin-like oxidized LDL receptor-1 (LOX-1) is a surface scavenger receptor for oxidized low-density lipoprotein (oxLDL). Several transcription factors have been reported to regulate LOX-1 expression. MicroRNAs are small noncoding RNAs that control gene expression, but there have been no reports of LOX-1 expression being regulated by microRNAs. Because the microRNA let-7g has been predicted to bind to LOX-1 mRNA, we investigated whether let-7g can regulate LOX-1 expression. Our experiments first demonstrated that oxLDL can reduce let-7g expression. We later confirmed that there is a let-7g binding site on the 3'-untranslated region of LOX-1 mRNA. We showed that intracellular Ca(2+)-activated protein kinase C is involved in the oxLDL-LOX-1-let-7g pathway. Bioinformatics predicted that the let-7g promoter has a binding site for the transcriptional repressor OCT-1. We used a promoter assay and chromatin immunoprecipitation to confirm this binding. Consequently, knockdown of OCT-1 was found to increase let-7g expression. Transfection of let-7g inhibited oxLDL-induced LOX-1 and OCT-1 expression, cell proliferation and migration. Mice fed with a high-fat diet showed a decrease in let-7g and an increase in LOX-1 and OCT-1. A study on humans showed the serum levels of let-7g are lower in subjects with hypercholesterolemia compared with normal controls. Our findings identify a negative feedback regulation between let-7g and LOX-1, and indicate that let-7g could be a target to treat cardiovascular disease.
PMID21170085	Cyclin-dependent kinase 1 expression is inhibited by p16(INK4a) at the post-transcriptional level through the microRNA pathway. The p16(INK4a) protein regulates cell cycle progression mainly by inhibiting the activity of G1-phase cyclin-dependent kinases (CDKs) 4 and 6, the subsequent retinoblastoma protein (pRb) phosphorylation and E2F transcription factor release. The p16(INK4a) protein can also repress the activity of other transcription factors, such as c-myc, nuclear factor-kappaB and c-Jun/AP1. Here, we report that, in two p16(-/-), pRb(WT) and p53(WT) cell lines (MCF7 and U87), p16(INK4a) overexpression induces a dramatic decrease in CDK1 protein expression. In response to p16(INK4a), the decreased rate of CDK1 protein synthesis, its unchanged protein half-life, unreduced CDK1 mRNA steady-state levels and mRNA half-life allow us to hypothesize that p16(INK4a) could regulate CDK1 expression at the post-transcriptional level. This CDK1 downregulation is mediated by the 3'-untranslated region (3'UTR) of CDK1 mRNA as shown by translational inhibition in luciferase assays and is associated with a modified expression balance of microRNAs (miRNAs) that potentially regulate CDK1, analyzed by TaqMan Human microRNA Array. The p16(INK4a)-induced expression of two miRNAs (miR-410 and miR-650 chosen as an example) in MCF7 cells is confirmed by individual reverse transcription-qPCR. Furthermore, we show the interaction of miR-410 or miR-650 with CDK1-3'UTR by luciferase assays. Endogenous CDK1 expression decreases upon both miRNA overexpression and increases with their simultaneous inhibition. The induction of miR-410, but not miR-650 could be related to the pRb/E2F pathway. These results demonstrate the post-transcriptional inhibition of CDK1 by p16(INK4a). We suggest that p16(INK4a) may regulate gene expression by modifying the functional equilibrium of transcription factors and consequently the expression balance of miRNAs.
PMID20689156	Controlling SIRT1 expression by microRNAs in health and metabolic disease. SIRT1 is a NAD+-dependent deacetylase implicated in longevity and diverse physiological processes. SIRT1, as a key mediator of beneficial effects of caloric restriction, regulates lipid and glucose metabolism by deacetylating metabolic regulators, as well as histones, in response to nutritional deprivation. Here we discuss how SIRT1 levels are regulated by microRNAs (miRs) which are emerging as important metabolic regulators; the recently identified nuclear receptor FXR/SHP cascade pathway that controls the expression of miR-34a and its target SIRT1; and a FXR/SIRT1 positive feedback regulatory loop, which is deregulated in metabolic disease states. The FXR/miR-34a pathway and other miRs controlling SIRT1 may be useful therapeutic targets for age-related diseases, including metabolic disorders.
PMID20382746	Interferons modulate mitogen-induced protein synthesis in airway smooth muscle. Severe asthma is characterized by increased airway smooth muscle (ASM) mass due, in part, to ASM cell growth and contractile protein expression associated with increased protein synthesis. Little is known regarding the combined effects of mitogens and interferons on ASM cytosolic protein synthesis. We demonstrate that human ASM mitogens including PDGF, EGF, and thrombin stimulate protein synthesis. Surprisingly, pleiotropic cytokines IFN-beta and IFN-gamma, which inhibit ASM proliferation, also increased cytosolic protein content in ASM cells. Thus IFN-beta alone significantly increased protein synthesis by 1.62 +/- 0.09-fold that was further enhanced by EGF to 2.52 +/- 0.17-fold. IFN-gamma alone also stimulated protein synthesis by 1.91 +/- 0.15-fold; treatment of cells with PDGF, EGF, and thrombin in the presence of IFN-gamma stimulated protein synthesis by 2.24 +/- 0.3-, 1.25 +/- 0.17-, and 2.67 +/- 0.34-fold, respectively, compared with growth factors alone. The mammalian target of rapamycin (mTOR)/S6 kinase 1 (S6K1) inhibition with rapamycin inhibited IFN- and EGF-induced protein synthesis, suggesting that IFN-induced protein synthesis is modulated by mTOR/S6K1 activation. Furthermore, overexpression of tumor suppressor protein tuberous sclerosis complex 2 (TSC2), which is an upstream negative regulator of mTOR/S6K1 signaling, also inhibited mitogen-induced protein synthesis in ASM cells. IFN-beta and IFN-gamma stimulated miR143/145 microRNA expression and increased SM alpha-actin accumulation but had little effect on ASM cell size. In contrast, EGF increased ASM cell size but had little effect on miR143/145 expression. Our data demonstrate that both IFNs and mitogens stimulate protein synthesis but have differential effects on cell size and contractile protein expression and suggest that combined effects of IFNs and mitogens may contribute to ASM cell growth, contractile protein expression, and ASM remodeling in asthma.
PMID20797623	STAT3 activation of miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic switch linking inflammation to cancer. A transient inflammatory signal can initiate an epigenetic switch from nontransformed to cancer cells via a positive feedback loop involving NF-kappaB, Lin28, let-7, and IL-6. We identify differentially regulated microRNAs important for this switch and putative transcription factor binding sites in their promoters. STAT3, a transcription factor activated by IL-6, directly activates miR-21 and miR-181b-1. Remarkably, transient expression of either microRNA induces the epigenetic switch. MiR-21 and miR-181b-1, respectively, inhibit PTEN and CYLD tumor suppressors, leading to increased NF-kappaB activity required to maintain the transformed state. These STAT3-mediated regulatory circuits are required for the transformed state in diverse cell lines and tumor growth in xenografts, and their transcriptional signatures are observed in colon adenocarcinomas. Thus, STAT3 is not only a downstream target of IL-6 but, with miR-21, miR-181b-1, PTEN, and CYLD, is part of the positive feedback loop that underlies the epigenetic switch that links inflammation to cancer.
PMID20523723	miR-22 forms a regulatory loop in PTEN/AKT pathway and modulates signaling kinetics. The tumor suppressor PTEN (phosphatase and tensin homolog) is a lipid phosphatase that converts PIP3 into PIP2 and downregulates the kinase AKT and its proliferative and anti-apoptotic activities. The FoxO transcription factors are PTEN downstream effectors whose activity is negatively regulated by AKT-mediated phosphorylation. PTEN activity is frequently lost in many types of cancer, leading to increased cell survival and cell cycle progression. Here we characterize the widely expressed miR-22 and report that miR-22 is a novel regulatory molecule in the PTEN/AKT pathway. miR-22 downregulates PTEN levels acting directly through a specific site on PTEN 3'UTR. Interestingly, miR-22 itself is upregulated by AKT, suggesting that miR-22 forms a feed-forward circuit in this pathway. Time-resolved live imaging of AKT-dependent FoxO1 phosphorylation revealed that miR-22 accelerated AKT activity upon growth factor stimulation, and attenuated its down regulation by serum withdrawal. Our results suggest that miR-22 acts to fine-tune the dynamics of PTEN/AKT/FoxO1 pathway.
PMID20185821	A pathway involving farnesoid X receptor and small heterodimer partner positively regulates hepatic sirtuin 1 levels via microRNA-34a inhibition. Sirtuin 1 (SIRT1) is a NAD-dependent deacetylase that is critically involved in diverse cellular processes including metabolic disease, cancer, and possibly aging. Despite extensive studies on SIRT1 function, how SIRT1 levels are regulated remains relatively unknown. Here, we report that the nuclear bile acid receptor farnesoid X receptor (FXR) inhibits microRNA-34a (miR-34a) in the liver, which results in a positive regulation of SIRT1 levels. Activation of FXR by the synthetic agonist GW4064 decreases hepatic miR-34a levels in normal mice, and consistently, hepatic miR-34a levels are elevated in FXR-null mice. FXR induces expression of small heterodimer partner (SHP), an orphan nuclear receptor and transcriptional corepressor, which in turn results in repression of p53, a key activator of the miR-34a gene, by inhibiting p53 occupancy at the promoter. MiR-34a decreased SIRT1 levels by binding to the 3'-untranslated region of SIRT1 mRNA, and adenovirus-mediated overexpression of miR-34a substantially decreased SIRT1 protein levels in mouse liver. Remarkably, miR-34a levels were elevated, and SIRT1 protein levels were reduced in diet-induced obese mice, and FXR activation in these mice reversed the miR-34a and SIRT1 levels, indicating an intriguing link among FXR activation, decreased miR-34a, and subsequently, increased SIRT1 levels. Our study demonstrates an unexpected role of the FXR/SHP pathway in controlling SIRT1 levels via miR-34a inhibition and that elevated miR-34a levels in obese mice contribute to decreased SIRT1 levels. Manipulation of this regulatory network may be useful for treating diseases of aging, such as metabolic disease and cancer.
PMID18568019	A three-step pathway comprising PLZF/miR-146a/CXCR4 controls megakaryopoiesis. MicroRNAs (miRNAs or miRs) regulate diverse normal and abnormal cell functions. We have identified a regulatory pathway in normal megakaryopoiesis, involving the PLZF transcription factor, miR-146a and the SDF-1 receptor CXCR4. In leukaemic cell lines PLZF overexpression downmodulated miR-146a and upregulated CXCR4 protein, whereas PLZF knockdown induced the opposite effects. In vitro assays showed that PLZF interacts with and inhibits the miR-146a promoter, and that miR-146a targets CXCR4 mRNA, impeding its translation. In megakaryopoietic cultures of CD34(+) progenitors, PLZF was upregulated, whereas miR-146a expression decreased and CXCR4 protein increased. MiR-146a overexpression and PLZF or CXCR4 silencing impaired megakaryocytic (Mk) proliferation, differentiation and maturation, as well as Mk colony formation. Mir-146a knockdown induced the opposite effects. Rescue experiments indicated that the effects of PLZF and miR-146a are mediated by miR-146a and CXCR4, respectively. Our data indicate that megakaryopoiesis is controlled by a cascade pathway, in which PLZF suppresses miR-146a transcription and thereby activates CXCR4 translation.
PMID21642433	Heparin impairs angiogenesis through inhibition of microRNA-10b. Heparin, which has been used as an anticoagulant drug for decades, inhibits angiogenesis, whereas thrombin promotes tumor-associated angiogenesis. However, the mechanisms underlying the regulation of angiogenesis by heparin and thrombin are not well understood. Here, we show that microRNA-10b (miR-10b) is down-regulated by heparin and up-regulated by thrombin in human microvascular endothelial cells (HMEC-1). Overexpression of miR-10b induces HMEC-1 cell migration, tube formation, and angiogenesis, and down-regulates homeobox D10 (HoxD10) expression via direct binding of miR-10b to the putative 3' UTR of HoxD10. In addition, HMEC-1 cell migration and tube formation are induced by HoxD10 knockdown, whereas angiogenesis is arrested when HoxD10 expression is increased after anti-miR-10b or heparin treatments. Furthermore, expression of miR-10b and its transcription factor Twist are up-regulated by thrombin, whereas HoxD10 expression is impaired by thrombin. Using quartz crystal microbalance analysis, we show that heparin binds to thrombin, thereby inhibiting thrombin-induced expression of Twist and miR-10b. However, the expression of miR-10b is not attenuated by heparin any more after thrombin expression is silenced by its siRNA. Interestingly, we find that heparin attenuates miR-10b expression and induces HoxD10 expression in vivo to inhibit angiogenesis and impair the growth of MDA-MB-231 tumor xenografts. These results provide insight into the molecular mechanism by which heparin and thrombin regulate angiogenesis.
PMID17496199	Interleukin-6 dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21 through a highly conserved enhancer. Signal transducer and activator of transcription 3 (Stat3) is implicated in the pathogenesis of many malignancies and essential for IL-6-dependent survival and growth of multiple myeloma cells. Here, we demonstrate that the gene encoding oncogenic microRNA-21 (miR-21) is controlled by an upstream enhancer containing 2 Stat3 binding sites strictly conserved since the first observed evolutionary appearance of miR-21 and Stat3. MiR-21 induction by IL-6 was strictly Stat3 dependent. Ectopically raising miR-21 expression in myeloma cells in the absence of IL-6 significantly reduced their apoptosis levels. These data provide strong evidence that miR-21 induction contributes to the oncogenic potential of Stat3.
PMID21336307	p53 regulates epithelial-mesenchymal transition and stem cell properties through modulating miRNAs. The epithelial-mesenchymal transition (EMT) has recently been linked to stem cell phenotype. However, the molecular mechanism underlying EMT and regulation of stemness remains elusive. Here, using genomic approaches, we show that tumour suppressor p53 has a role in regulating both EMT and EMT-associated stem cell properties through transcriptional activation of the microRNA miR-200c. p53 transactivates miR-200c through direct binding to the miR-200c promoter. Loss of p53 in mammary epithelial cells leads to decreased expression of miR-200c and activates the EMT programme, accompanied by an increased mammary stem cell population. Re-expressing miR-200c suppresses genes that mediate EMT and stemness properties and thereby reverts the mesenchymal and stem-cell-like phenotype caused by loss of p53 to a differentiated epithelial cell phenotype. Furthermore, loss of p53 correlates with a decrease in the level of miR-200c, but an increase in the expression of EMT and stemness markers, and development of a high tumour grade in a cohort of breast tumours. This study elucidates a role for p53 in regulating EMT-MET (mesenchymal-epithelial transition) and stemness or differentiation plasticity, and reveals a potential therapeutic implication to suppress EMT-associated cancer stem cells through activation of the p53-miR-200c pathway.
PMID21296997	MYB transcriptionally regulates the miR-155 host gene in chronic lymphocytic leukemia. Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.
PMID22286762	Sulindac inhibits tumor cell invasion by suppressing NF-κB-mediated transcription of microRNAs. Non-steroidal anti-inflammatory drugs (NSAIDs) have been widely reported to display strong efficacy for cancer chemoprevention, although their mechanism of action is poorly understood. The most well-documented effects of NSAIDs include inhibition of tumor cell proliferation and induction of apoptosis, but their effect on tumor cell invasion has not been well studied. Here, we show that the NSAID, sulindac sulfide (SS) can potently inhibit the invasion of human MDA-MB-231 breast and HCT116 colon tumor cells in vitro at concentrations less than those required to inhibit tumor cell growth. To study the molecular basis for this activity, we investigated the involvement of microRNA (miRNA). A total of 132 miRNAs were found to be altered in response to SS treatment, including miR-10b, miR-17, miR-21 and miR-9, which have been previously implicated in tumor invasion and metastasis. We confirmed that these miRNA can stimulate tumor cell invasion and show that SS can attenuate their invasive effects by downregulating their expression. Employing luciferase and chromatin immunoprecipitation assays, NF-κB was found to bind the promoters of all four miRNAs to suppress their expression at the transcriptional level. We show that SS can inhibit the translocation of NF-κB to the nucleus by decreasing the phosphorylation of IKKβ and IκB. Analysis of the promoter sequences of the miRNAs suppressed by SS revealed that 81 of 115 sequences contained NF-κB-binding sites. These results show that SS can inhibit tumor cell invasion by suppressing NF-κB-mediated transcription of miRNAs.
PMID21081469	NF-κB targets miR-16 and miR-21 in gastric cancer: involvement of prostaglandin E receptors. Cigarette smoke is one of the risk factors for gastric cancer and nicotine has been reported to promote tumor growth. Deregulation of microRNA (miRNA) and cyclooxygenase-2 (COX-2) expressions are hallmarks of many cancers including gastric cancer. Here, we used an miRNA array platform covering a panel of 95 human miRNAs to examine the expression profile in nicotine-treated gastric cancer cells. We found that miR-16 and miR-21 were upregulated upon nicotine stimulation, transfection with anti-miR-16 or anti-miR-21 significantly abrogated cell proliferation. In contrast, ectopic miR-16 or miR-21 expression exhibited a similar stimulatory effect on cell proliferation as nicotine. Nicotine-mediated IkappaBα degradation and nuclear factor-kappa B (NF-κB) translocation dose-dependently. Knockdown of NF-κB by short interfering RNA (siRNA) or specific inhibitor (Bay-11-7085) markedly suppressed nicotine-induced cell proliferation and upregulation of miR-16 and miR-21. Interestingly, NF-κB-binding sites were located in both miR-16 and miR-21 gene transcriptional elements and we showed that nicotine enhanced the binding of NF-κB to the promoters of miR-16 and miR-21. Furthermore, activation of COX-2/prostaglandin E₂ (PGE₂) signaling in response to nicotine was mediated by the action of prostaglandin E receptors (EP2 and EP4). EP2 or EP4 siRNA or antagonists impaired the nicotine-mediated NF-κB activity, upregulation of miR-16 and miR-21 and cell proliferation. Taken together, these results suggest that miR-16 and miR-21 are directly regulated by the transcription factor NF-κB and yet nicotine-promoted cell proliferation is mediated via EP2/4 receptors. Perhaps this study may shed light on the development of anticancer drugs to improve the chemosensitivity in smokers.
PMID19211792	Lin-28B transactivation is necessary for Myc-mediated let-7 repression and proliferation. Direct control of microRNA (miRNA) expression by oncogenic and tumor suppressor networks results in frequent dysregulation of miRNAs in cancer cells and contributes to tumorigenesis. We previously demonstrated that activation of the c-Myc oncogenic transcription factor (Myc) broadly influences miRNA expression and in particular leads to widespread miRNA down-regulation. miRNA transcripts repressed by Myc include several with potent tumor suppressor activity such as miR-15a/16-1, miR-34a, and let-7 family members. In this study, we have investigated mechanisms downstream of Myc that contribute to miRNA repression. Consistent with transcriptional down-regulation, Myc activity results in the decreased abundance of multiple miRNA primary transcripts. Surprisingly, however, primary transcripts encoding several let-7 miRNAs are not reduced in the high Myc state, suggesting a posttranscriptional mechanism of repression. The Lin-28 and Lin-28B RNA binding proteins were recently demonstrated to negatively regulate let-7 biogenesis. We now show that Myc induces Lin-28B expression in multiple human and mouse tumor models. Chromatin immunoprecipitation and reporter assays reveal direct association of Myc with the Lin-28B promoter resulting in transcriptional transactivation. Moreover, we document that activation of Lin-28B is necessary and sufficient for Myc-mediated let-7 repression. Accordingly, Lin-28B loss-of-function significantly impairs Myc-dependent cellular proliferation. These findings highlight an important role for Lin-28B in Myc-driven cellular phenotypes and uncover an orchestration of transcriptional and posttranscriptional mechanisms in Myc-mediated reprogramming of miRNA expression.
PMID20732742	MicroRNA-10b induced by Epstein-Barr virus-encoded latent membrane protein-1 promotes the metastasis of human nasopharyngeal carcinoma cells. MicroRNA-10b (miR-10b) has been reported to facilitate the metastasis of breast cancer. However, little is known about the role of miR-10b in the metastasis of nasopharyngeal carcinoma (NPC). Here, we show that high levels of miR-10b expression in Epstein-Barr virus (EBV)-positive latent membrane protein-1 (LMP1)-expressing NPC cells, and its expression is down-regulated by silencing LMP1 or Twist. Induction of miR-10b over-expression in LMP1-silent C666-1 cells promoted significant wound healing and transmembrane invasiveness in vitro. More importantly, miR-10b over-expression promoted the metastasis of NPC and accelerated the death of tumor-bearing nude mice. These findings strongly suggest that miR-10b positively regulates the metastasis of NPC.
PMID22286765	SIX1 promotes epithelial-mesenchymal transition in colorectal cancer through ZEB1 activation. Epithelial-mesenchymal transition (EMT) has a major role in cancer progression, as well as normal organ development and human pathology such as organ fibrosis and wound healing. Here, we performed a gene expression array specialized in EMT of colorectal cancer (CRC). From a comprehensive gene expression analysis using epithelial- and mesenchymal-like CRC cell lines, and following the ontology (GO) analysis, SIX1 gene was identified to be an EMT-related gene in CRC. Using SW480 cells stably transfected with a SIX1 expression construct and their control counterparts, we demonstrated that SIX1 overexpression represses CDH1 expression and promotes EMT in CRC. SIX1-induced CDH1 repression and EMT in CRC cells were correlated at least in part with posttranscriptional ZEB1 activation and miR-200-family transcriptional repression. In primary tumors of CRC, in accord with the functional findings, aberrant expression of SIX1 in cancer cells was observed at the disruption of the basement membrane and at the tumor invasive front, where tumor cells underwent EMT in vivo. Taken together, SIX1 overexpression is suggested to occur in carcinogenesis, and contribute to repression of CDH1 expression and promotion of EMT partly through repression of miR-200-family expression and activation of ZEB1 in CRC.
PMID22064487	Developmental regulation of microRNA expression in Schwann cells. Schwann cell differentiation and subsequent myelination of the peripheral nervous system require the action of several transcription factors, including Sox10, which is vital at multiple stages of development. The transition from immature to myelinating Schwann cell is also regulated posttranscriptionally and depends upon Dicer-mediated processing of microRNAs (miRNAs). Although specific miRNA targets have begun to be identified, the mechanisms establishing the dynamic regulation of miRNA expression have not been elucidated. We performed expression profiling studies and identified 225 miRNAs differentially expressed during peripheral myelination. A subset of 9 miRNAs is positively regulated by Sox10, including miR-338 which has been implicated in oligodendrocyte maturation. In vivo chromatin immunoprecipitation (ChIP) of sciatic nerve cells revealed a Sox10 binding site upstream of an alternate promoter within the Aatk gene, which hosts miR-338. Sox10 occupied this site in spinal cord ChIP experiments, suggesting a similar regulatory mechanism in oligodendrocytes. Cancer profiling studies have identified clusters of miRNAs that regulate proliferation, termed "oncomirs." In Schwann cells, the expression of many of these proproliferative miRNAs was reduced in the absence of Sox10. Finally, Schwann cells with reduced Sox10 and oncomir expression have an increase in the CDK inhibitor p21 and a concomitant reduction in cell proliferation.
PMID20130193	BMPR1a and BMPR1b signaling exert opposing effects on gliosis after spinal cord injury. Astrogliosis following spinal cord injury (SCI) involves an early hypertrophic response that is beneficial and a subsequent formation of a dense scar. We investigated the role of bone morphogenetic protein (BMP) signaling in gliosis after SCI and find that BMPR1a and BMPR1b signaling exerts opposing effects on hypertrophy. Conditional ablation of BMPR1a from glial fibrillary acidic protein (GFAP)-expressing cells leads to defective astrocytic hypertrophy, increased infiltration by inflammatory cells, and reduced axon density. BMPR1b-null mice conversely develop "hyperactive" reactive astrocytes and consequently have smaller lesion volumes. The effects of ablation of either receptor are reversed in the double knock-out animals. These findings indicate that BMPR1a and BMPR1b exert directly opposing effects on the initial reactive astrocytic hypertrophy. Also, BMPR1b knock-out mice have an attenuated glial scar in the chronic stages following injury, suggesting that it has a greater role in glial scar progression. To elucidate the differing roles of the two receptors in astrocytes, we examined the effects of ablation of either receptor in serum-derived astrocytes in vitro. We find that the two receptors exert opposing effects on the posttranscriptional regulation of astrocytic microRNA-21. Further, overexpression of microRNA-21 in wild-type serum-derived astrocytes causes a dramatic reduction in cell size accompanied by reduction in GFAP levels. Hence, regulation of microRNA-21 by BMP signaling provides a novel mechanism for regulation of astrocytic size. Targeting specific BMPR subunits for therapeutic purposes may thus provide an approach for manipulating gliosis and enhancing functional outcomes after SCI.
PMID20173740	miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. MicroRNAs (miRNAs) are increasingly implicated in regulating the malignant progression of cancer. Here we show that miR-9, which is upregulated in breast cancer cells, directly targets CDH1, the E-cadherin-encoding messenger RNA, leading to increased cell motility and invasiveness. miR-9-mediated E-cadherin downregulation results in the activation of beta-catenin signalling, which contributes to upregulated expression of the gene encoding vascular endothelial growth factor (VEGF); this leads, in turn, to increased tumour angiogenesis. Overexpression of miR-9 in otherwise non-metastatic breast tumour cells enables these cells to form pulmonary micrometastases in mice. Conversely, inhibiting miR-9 by using a 'miRNA sponge' in highly malignant cells inhibits metastasis formation. Expression of miR-9 is activated by MYC and MYCN, both of which directly bind to the mir-9-3 locus. Significantly, in human cancers, miR-9 levels correlate with MYCN amplification, tumour grade and metastatic status. These findings uncover a regulatory and signalling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis-suppressing protein E-cadherin.
PMID20404092	Multiple E2F-induced microRNAs prevent replicative stress in response to mitogenic signaling. Transcription of microRNAs (miRNAs) is thought to be regulated similarly to that of protein-coding genes. However, how miRNAs are regulated during the cell division cycle is not well understood. We have analyzed the transcription profiles of miRNAs in response to mitogenic stimulation in primary fibroblasts. About 33% of the miRNAs expressed in these cells are induced upon exit from quiescence. Many of these miRNAs are specifically induced by E2F1 or E2F3 during the G(1)/S transition and are repressed in E2F1/3-knockout cells. At least four miRNA clusters, let-7a-d, let-7i, mir-15b-16-2, and mir-106b-25, are direct targets of E2F1 and E2F3 during G(1)/S and are repressed in E2F1/3-null cells. Interestingly, these miRNAs do not contribute to E2F-dependent entry into S phase but rather inhibit the G(1)/S transition by targeting multiple cell cycle regulators and E2F targets. In fact, E2F1 expression results in a significant increase in S-phase entry and DNA damage in the absence of these microRNAs. Thus, E2F-induced miRNAs contribute to limiting the cellular responses to E2F activation, thus preventing replicative stress. Given the known function of E2F of inducing other oncogenic miRNAs, control of miRNAs by E2F is likely to play multiple roles in cell proliferation and in proliferative diseases such as cancer.
PMID17996649	Epigenetic silencing of the myelopoiesis regulator microRNA-223 by the AML1/ETO oncoprotein. Hematopoietic transcription factors are involved in chromosomal translocations, which generate fusion proteins contributing to leukemia pathogenesis. Analysis of patient's primary leukemia blasts revealed that those carrying the t(8;21) generating AML1/ETO, the most common acute myeloid leukemia-associated fusion protein, display low levels of a microRNA-223 (miR-223), a regulator of myelopoiesis. Here, we show that miR-223 is a direct transcriptional target of AML1/ETO. By recruiting chromatin remodeling enzymes at an AML1-binding site on the pre-miR-223 gene, AML1/ETO induces heterochromatic silencing of miR-223. Ectopic miR-223 expression, RNAi against AML1/ETO, or demethylating treatment enhances miR-223 levels and restores cell differentiation. Here, we identify an additional action for a leukemia fusion protein linking the epigenetic silencing of a microRNA locus to the differentiation block of leukemia.
PMID19289835	Induction and regulatory function of miR-9 in human monocytes and neutrophils exposed to proinflammatory signals. Inflammation involves a coordinated, sequential, and self limiting sequence of events controlled by positive and negative regulatory mechanisms. Recent studies have shown that microRNAs (miRNAs), an evolutionarily conserved class of endogenous 22-nucleotide noncoding RNAs, contribute to the regulation of inflammation by repressing gene expression at the posttranscriptional level. In this study, we characterize the profile of miRNAs induced by LPS in human polymorphonuclear neutrophils (PMN) and monocytes. In particular, we identify miR-9 as the only miRNA (among 365 analyzed) up-regulated in both cell types after TLR4 activation. miR-9 is also induced by TLR2 and TLR7/8 agonists and by the proinflammatory cytokines TNF-alpha and IL-1beta, but not by IFNgamma. Among the 3 different genes encoding miR-9 precursors in humans, we show that LPS selectively induces the transcription of miR-9-1 located in the CROC4 locus, in a MyD88- and NF-kappaB-dependent manner. In PMN and monocytes, LPS regulates NFKB1 at both the transcriptional and posttranscriptional levels, and a conserved miR-9 seed sustained a miR-9-dependent inhibition of the NFKB1 transcript. Overall, these data suggest that TLR4-activated NF-kappaB rapidly increases the expression of miR-9 that operates a feedback control of the NF-kappaB-dependent responses by fine tuning the expression of a key member of the NF-kappaB family.
PMID20154725	Hypoxia and RAS-signaling pathways converge on, and cooperatively downregulate, the RECK tumor-suppressor protein through microRNAs. Cancer cells show characteristic gene expression profiles. Recent studies support the potential importance of microRNA (miRNA) expression signatures as biomarkers and therapeutic targets. The membrane-anchored protease regulator RECK is downregulated in many cancers, and forced expression of RECK in tumor cells results in decreased malignancy in animal models. RECK is also essential for mammalian development. In this study, we found that RECK is a target of at least three groups of miRNAs (miR-15b/16, miR-21 and miR-372/373); that RECK mutants lacking the target sites for these miRNA show augmented tumor/metastasis-suppressor activities; and that miR-372/373 are upregulated in response to hypoxia through HIF1alpha and TWIST1, whereas miR-21 is upregulated by RAS/ERK signaling. These data indicate that the hypoxia- and RAS-signaling pathways converge on RECK through miRNAs, cooperatively downregulating this tumor suppressor and thereby promoting malignant cell behavior.
PMID20019669	Molecular basis for antagonism between PDGF and the TGFbeta family of signalling pathways by control of miR-24 expression. Modulation of the vascular smooth-muscle-cell (vSMC) phenotype from a quiescent 'contractile' phenotype to a proliferative 'synthetic' phenotype has been implicated in vascular injury repair, as well as pathogenesis of vascular proliferative diseases. Both bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta)-signalling pathways promote a contractile phenotype, while the platelet-derived growth factor-BB (PDGF-BB)-signalling pathway promotes a switch to the synthetic phenotype. Here we show that PDGF-BB induces microRNA-24 (miR-24), which in turn leads to downregulation of Tribbles-like protein-3 (Trb3). Repression of Trb3 coincides with reduced expression of Smad proteins and decrease in BMP and TGFbeta signalling, promoting a synthetic phenotype in vSMCs. Inhibition of miR-24 by antisense oligonuclotides abrogates the downregulation of Trb3 as well as pro-synthetic activity of the PDGF-signalling pathway. Thus, this study provides a molecular basis for the antagonism between the PDGF and TGFbeta pathways, and its effect on the control of the vSMC phenotype.
PMID19114653	A regulatory interplay between miR-27a and Runx1 during megakaryopoiesis. The transcription factor Runx1 is a key regulator of definitive hematopoiesis in the embryo and the adult. Lineage-specific expression of Runx1 involves transcription and post-transcription control through usage of alternative promoters and diverse 3'UTR isoforms, respectively. We identified and mapped microRNA (miR) binding sites on Runx1 3'UTR and show that miR-27a, miR-9, miR-18a, miR-30c, and miR-199a* bind and post-transcriptionally attenuate expression of Runx1. miR-27a impacts on both the shortest (0.15 kb) and longest (3.8 kb) 3'UTRs and, along with additional miRs, might contribute to translation attenuation of Runx1 mRNA in the myeloid cell line 416B. Whereas levels of Runx1 mRNA in 416B and the B cell line 70Z were similar, the protein levels were not. Large amounts of Runx1 protein were found in 70Z cells, whereas only minute amounts of Runx1 protein were made in 416B cells and overexpression of Runx1 in 416B induced terminal differentiation associated with megakaryocytic markers. Induction of megakaryocytic differentiation in K562 cells by 12-o-tetradecanoylphorbol-13-acetate markedly increased miR-27a expression, concomitantly with binding of Runx1 to miR-27a regulatory region. The data indicate that miR-27a plays a regulatory role in megakaryocytic differentiation by attenuating Runx1 expression, and that, during megakaryopoiesis, Runx1 and miR-27a are engaged in a feedback loop involving positive regulation of miR-27a expression by Runx1.
PMID21518763	miR-365, a novel negative regulator of interleukin-6 gene expression, is cooperatively regulated by Sp1 and NF-kappaB. Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a central role in host defense. IL-6 expression can be regulated at both a transcriptional and a post-transcriptional level. We used a combination of bioinformatics and experimental techniques to demonstrate that the miR-365 is a direct negative regulator of IL-6. Overexpression of miR-365 mimics decreased activity of a luciferase reporter containing the IL-6 3'-UTR and led to repression of IL-6 protein. In contrast, ectopic expression of a miR-365 inhibitor elevated IL-6 expression. The negative regulation of miR-365 was strictly dependent on a microRNA binding element in the 3'-UTR of IL-6 mRNA. Deletion mutant analysis of the miR-365 promoter showed that two transcription factors, Sp1 and NF-κB, are essential for the transcriptional regulation of miR-365. We also demonstrate that the MAPK/ERK pathway contributes to the regulation of miR-365. Furthermore, miR-365 exhibited a greater negative regulatory effect on IL-6 than hsa-let-7a, a previously identified microRNA negatively regulating IL-6. Taken together, our results show that miR-365 is a novel negative regulator of IL-6.
PMID20473948	Coordinated epigenetic repression of the miR-200 family and miR-205 in invasive bladder cancer. MicroRNAs (miRNA) are small noncoding RNAs commonly deregulated in cancer. The miR-200 family (miR-200a, -200b, -200c, -141 and -429) and miR-205 are frequently silenced in advanced cancer and have been implicated in epithelial to mesenchymal transition (EMT) and tumor invasion by targeting the transcriptional repressors of E-cadherin, ZEB1 and ZEB2. ZEB1 is also known to repress miR-200c-141 transcription in a negative feedback loop, but otherwise little is known about the transcriptional regulation of the miR-200 family and miR-205. Recently, miR-200 silencing was also reported in cancer stem cells, implying that miR-200 deregulation is a key event in multiple levels of tumor biology. However, what prevents miR-200 expression remains largely unanswered. Here we report concerted transcriptional regulation of the miR-200 and miR-205 loci in bladder tumors and bladder cell lines. Using a combination of miRNA expression arrays, qPCR assays and mass spectrometry DNA methylation analyses, we show that the miR-200 and miR-205 loci are specifically silenced and gain promoter hypermethylation and repressive chromatin marks in muscle invasive bladder tumors and undifferentiated bladder cell lines. Moreover, we report that miR-200c expression is significantly correlated with early stage T1 bladder tumor progression, and propose miR-200 and miR-205 silencing and DNA hypermethylation as possible prognostic markers in bladder cancer. In addition, we observe that the mesoderm transcription factor TWIST1 and miR-200 expression are inversely correlated in bladder tumor samples and cell lines. TWIST1 associates directly with the miR-200 and miR-205 promoters, and may act as a repressor of miR-200 and miR-205 expression.
PMID20621051	Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expression of defined embryonic factors. However, little is known of the molecular mechanisms underlying the reprogramming process. Here we explore somatic cell reprogramming by exploiting a secondary mouse embryonic fibroblast model that forms iPSCs with high efficiency upon inducible expression of Oct4, Klf4, c-Myc, and Sox2. Temporal analysis of gene expression revealed that reprogramming is a multistep process that is characterized by initiation, maturation, and stabilization phases. Functional analysis by systematic RNAi screening further uncovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. We show that this is linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs that are key regulators of MET. These studies thus define a multistep mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. PAPERCLIP:
PMID20935637	Ascl1a regulates Müller glia dedifferentiation and retinal regeneration through a Lin-28-dependent, let-7 microRNA signalling pathway. Unlike mammals, teleost fish mount a robust regenerative response to retinal injury that culminates in restoration of visual function. This regenerative response relies on dedifferentiation of Müller glia into a cycling population of progenitor cells. However, the mechanism underlying this dedifferentiation is unknown. Here, we report that genes encoding pluripotency factors are induced following retinal injury. Interestingly, the proneural transcription factor, Ascl1a, and the pluripotency factor, Lin-28, are induced in Müller glia within 6 h following retinal injury and are necessary for Müller glia dedifferentiation. We demonstrate that Ascl1a is necessary for lin-28 expression and that Lin-28 suppresses let-7 microRNA (miRNA) expression. Furthermore, we demonstrate that let-7 represses expression of regeneration-associated genes such as, ascl1a, hspd1, lin-28, oct4, pax6b and c-myc. hspd1, oct4 and c-myc(a) exhibit basal expression in the uninjured retina and let-7 may inhibit this expression to prevent premature Müller glia dedifferentiation. The opposing actions of Lin-28 and let-7 miRNAs on Müller glia differentiation and dedifferentiation are similar to that of embryonic stem cells and suggest novel targets for stimulating Müller glia dedifferentiation and retinal regeneration in mammals.
PMID20156358	A novel microRNA and transcription factor mediated regulatory network in schizophrenia. Schizophrenia is a complex brain disorder with molecular mechanisms that have yet to be elucidated. Previous studies have suggested that changes in gene expression may play an important role in the etiology of schizophrenia, and that microRNAs (miRNAs) and transcription factors (TFs) are primary regulators of this gene expression. So far, several miRNA-TF mediated regulatory modules have been verified. We hypothesized that miRNAs and TFs might play combinatory regulatory roles for schizophrenia genes and, thus, explored miRNA-TF regulatory networks in schizophrenia. We identified 32 feed-forward loops (FFLs) among our compiled schizophrenia-related miRNAs, TFs and genes. Our evaluation revealed that these observed FFLs were significantly enriched in schizophrenia genes. By converging the FFLs and mutual feedback loops, we constructed a novel miRNA-TF regulatory network for schizophrenia. Our analysis revealed EGR3 and hsa-miR-195 were core regulators in this regulatory network. We next proposed a model highlighting EGR3 and miRNAs involved in signaling pathways and regulatory networks in the nervous system. Finally, we suggested several single nucleotide polymorphisms (SNPs) located on miRNAs, their target sites, and TFBSs, which may have an effect in schizophrenia gene regulation. This study provides many insights on the regulatory mechanisms of genes involved in schizophrenia. It represents the first investigation of a miRNA-TF regulatory network for a complex disease, as demonstrated in schizophrenia.
PMID21743493	FOXP3 and FOXP3-regulated microRNAs suppress SATB1 in breast cancer cells. The transcription factor FOXP3 has been identified as a tumour suppressor in the breast and prostate epithelia, but little is known about its specific mechanism of action. We have identified a feed-forward regulatory loop in which FOXP3 suppresses the expression of the oncogene SATB1. In particular, we demonstrate that SATB1 is not only a direct target of FOXP3 repression, but that FOXP3 also induces two miRs, miR-7 and miR-155, which specifically target the 3'-UTR of SATB1 to further regulate its expression. We conclude that FOXP3-regulated miRs form part of the mechanism by which FOXP3 prevents the transformation of the healthy breast epithelium to a cancerous phenotype. Approaches aimed at restoring FOXP3 function and the miRs it regulates could help provide new approaches to target breast cancer.
PMID20173743	Myc-modulated miR-9 makes more metastases. The microRNA miR-9 is induced by Myc in breast cancer cells where it targets the major epithelial adherens junction protein, E-cadherin. This primes the cancer cells for epithelial-mesenchymal transition (EMT) and also stimulates angiogenesis in tumours.
PMID20008792	STAT5 requires the N-domain for suppression of miR15/16, induction of bcl-2, and survival signaling in myeloproliferative disease. Phosphorylated signal transducer and activator of transcription 5 (STAT5) is a biomarker and potential molecular target for hematologic malignancies. We have shown previously that lethal myeloproliferative disease (MPD) in mice mediated by persistently activated STAT5 (STAT5a(S711F)) requires the N-domain, but the mechanism was not defined. We now demonstrate by retrovirally complementing STAT5ab(null/null) primary mast cells that relative to wild-type STAT5a, STAT5a lacking the N-domain (STAT5aDeltaN) ineffectively protected against cytokine withdrawal-induced cell death. Both STAT5a and STAT5aDeltaN bound to a site in the bcl-2 gene and both bound near the microRNA 15b/16 cluster. However, only STAT5a could effectively induce bcl-2 mRNA and reciprocally suppress miR15b/16 leading to maintained bcl-2 protein levels. After retroviral complementation of STAT5ab(null/null) fetal liver cells and transplantation, persistently active STAT5a(S711F) lacking the N-domain (STAT5aDeltaN(S711F)) was insufficient to protect c-Kit(+)Lin(-)Sca-1(+) (KLS) cells from apoptosis and unable to induce bcl-2 expression, whereas STAT5a(S711F) caused robust KLS cell expansion, induction of bcl-2, and lethal MPD. Severe attenuation of MPD by STAT5aDeltaN(S711F) was reversed by H2k/bcl-2 transgenic expression. Overall, these studies define N-domain-dependent survival signaling as an Achilles heel of persistent STAT5 activation and highlight the potential therapeutic importance of targeting STAT5 N-domain-mediated regulation of bcl-2 family members.
PMID22074923	MicroRNA 34c gene down-regulation via DNA methylation promotes self-renewal and epithelial-mesenchymal transition in breast tumor-initiating cells. Tumor-initiating cells (T-ICs), a subpopulation of cancer cells with stem cell-like properties, are related to tumor relapse and metastasis. Our previous studies identified a distinct profile of microRNA (miRNA) expression in breast T-ICs (BT-ICs), and the dysregulated miRNAs contribute to the self-renewal and tumorigenesis of these cells. However, the underlying mechanisms for miRNA dysregulation in BT-ICs remain obscure. In the present study, we demonstrated that the expression and function of miR-34c were reduced in the BT-ICs of MCF-7 and SK-3rd cells, a breast cancer cell line enriched for BT-ICs. Ectopic expression of miR-34c reduced the self-renewal of BT-ICs, inhibited epithelial-mesenchymal transition, and suppressed migration of the tumor cells via silencing target gene Notch4. Furthermore, we identified a single hypermethylated CpG site in the promoter region of miR-34c gene that contributed to transcriptional repression of miR-34c in BT-ICs by reducing DNA binding activities of Sp1. Therefore, miR-34c reduction in BT-ICs induced by a single hypermethylated CpG site in the promoter region promotes self-renewal and epithelial-mesenchymal transition of BT-ICs.
PMID21956205	Genome-wide identification of TCF7L2/TCF4 target miRNAs reveals a role for miR-21 in Wnt-driven epithelial cancer. Transcription factor 7 like 2 (TCF7L2, also known as TCF4) is a Wnt signaling pathway transcription factor involved in regulation of numerous Wnt targeted genes. Recently, thousands of high-confidence TCF4 binding sites were reported in LS174T colon carcinoma cells, however, potential TCF4 target miRNAs remain largely unknown. Here, we utilized a bioinformatics approach to discover 26 miRNA transcription start sites (TSSs) within close proximity to TCF4 chromatin occupancy sites, and validated these sites as bona fide TCF4 targets in LS174T colon carcinoma cells, MCF-7 breast cancer cells and U87 glioma cells by ChIP-PCR. We then selected miR-21 to demonstrate for the first time direct TCF4 transcriptional activation of a miRNA via binding to the promoter region. Tissue array analysis supported this finding, revealing a positive correlation between activation of the β-catenin pathway and in situ expression of miR-21. Finally, based upon the well known but poorly understood preventive effect of aspirin on colorectal cancer incidence and mortality, we report downregulation of miR-21 upon administration of aspirin. In sum, our findings identify direct transcriptional regulation of miR-21 by TCF4 and suggest a role for miR-21 in cancer cell proliferation and invasion upon activation of β-catenin/TCF4 signaling.
PMID18353861	Transforming growth factor-beta-regulated miR-24 promotes skeletal muscle differentiation. MicroRNAs (miRNAs) have recently been proposed as a versatile class of molecules involved in regulation of a variety of biological processes. However, the role of miRNAs in TGF-beta-regulated biological processes is poorly addressed. In this study, we found that miR-24 was upregulated during myoblast differentiation and could be inhibited by TGF-beta1. Using both a reporter assay and Northern blot analysis, we showed that TGF-beta1 repressed miR-24 transcription which was dependent on the presence of Smad3 and a Smads binding site in the promoter region of miR-24. TGF-beta1 was unable to inhibit miR-24 expression in Smad3-deficient myoblasts, which exhibited accelerated myogenesis. Knockdown of miR-24 led to reduced expression of myogenic differentiation markers in C2C12 cells, while ectopic expression of miR-24 enhanced differentiation, and partially rescued inhibited myogenesis by TGF-beta1. This is the first study demonstrating a critical role for miRNAs in modulating TGF-beta-dependent inhibition of myogenesis, and provides a novel mechanism of the genetic regulation of TGF-beta signaling during skeletal muscle differentiation.
PMID22020340	Smad3 regulates E-cadherin via miRNA-200 pathway. To identify potential microRNA (miRNA) links between Smad3, a mediator of TGF-β (transforming growth factor-β) signaling, and E-cadherin, we characterized the miRNA profiles of two gastric cancer cell lines: SNU484-LPCX, which does not express Smad3, and SNU484-Smad3, in which Smad3 is overexpressed. We found that among differentially expressed miRNAs, miR-200 family members are overexpressed in SNU484-Smad3 cells. Subsequent studies, including analysis of the effects of silencing Smad3 in SNU484-Smad3 cells and a luciferase reporter assay, revealed that Smad3 directly binds to a Smad-binding element located in the promoter region of miR-200b/a, where it functions as a transcriptional activator. TGF-β did not affect the regulatory role of Smad3 in transcription of miR-200 and expression of epithelial-mesenchymal transition markers. We conclude that Smad3 regulates, at the transcriptional level, miR-200 family members, which themselves regulate ZEB1 and ZEB2, known transcriptional repressors of E-cadherin, at the posttranscriptional level in a TGF-β-independent manner. This represents a novel link between Smad3 and posttranscriptional regulation by miRNAs in epithelial-mesenchymal transition in gastric cancer cells.
PMID22178270	Transcriptional regulation of miR-224 upregulated in human HCCs by NFκB inflammatory pathways. miR-224 is up-regulated in human HCCs as compared to both paired peri-tumoral cirrhotic tissues and cirrhotic livers without HCC. Here, we have cloned the miR-224 regulatory region and characterized its transcriptional regulation by the NFκB-dependent inflammatory pathways. Mature miRNA expression was evaluated by a 2 step stem-loop real-time RT-PCR. The recruitment of polymerase II and transcription factors on the pre-miR-224 promoter has been assessed by ChIPSeq and ChIP. We found miR-224 levels strongly up-regulated in both peri-tumoral cirrhotic livers and HCC samples as compared to normal livers. In silico analysis of the putative miR-224 promoter revealed multiple NFκB sites. We showed that LTα and TNFα activate transcription from the miR-224 promoter and of endogenous miR-224 expression in HCC cell lines, whereas the expression of miR-224 target API5 was reduced. Exogenously expressed p65/RelA activates the miR-224 promoter and a dominant negative form of IκBα (IκBSR) represses it. ChIP analysis showed that p65/NFκB is recruited on the miR-224 promoter and that its binding sharply increases after exposure to LPS, TNFα, and LTα. Altogether these findings link the inflammatory signals to NFκB-mediated activation of miR-224 expression. An antago-miR specific for miR-224 blocked LPS and LTα stimulated HCC cells migration and invasion. Conversely, the IKK inhibitor BMS-345541 blocks pre-miR-224-induced cellular migration and invasion. Our results identify p65/NFκB as a direct transcriptional regulator of miR-224 expression and link miR-224 up-regulation with the activation of the LPS, LTα, and TNFα inflammatory pathways and cell migration/invasion in HCC.
PMID20708014	Synergistic effects of the GATA-4-mediated miR-144/451 cluster in protection against simulated ischemia/reperfusion-induced cardiomyocyte death. Among the identified microRNAs (miRs) thus far, ~50% of mammalian miRs are clustered in the genome and transcribed as polycistronic primary transcripts. However, whether clustered miRs mediate non-redundant and cooperative functions remains poorly understood. In this study, we first identified activation of the promoter of miR-144/451 by GATA-4, a critical transcription factor in the heart. Next, we observed that ectopic expression of miR-144 and -451 individually augmented cardiomyocyte survival, which was further improved by overexpression of miR-144/451, compared to control cells in response to simulated ischemia/reperfusion. In contrast, knockdown of endogenous miR-144 and -451 revealed opposite effects. Using luciferase reporter assay and western blot analysis, we also validated that both miR-144 and miR-451 target CUG triplet repeat-binding protein 2 (CUGBP2), a ubiquitously expressed RNA-binding protein, known to interact with COX-2 3'UTR and inhibit its translation. Accordingly, protein levels of CUGBP2 were greatly reduced and COX-2 activity was markedly increased in miR-144-, miR-451-, and miR-144/451-overexpressing cardiomyocytes, compared to GFP cells. Furthermore, inhibition of COX-2 activity by either NS-398 or DUP-697 partially offset protective effects of the miR-144/451 cluster. Together, these data indicate that both partners of the miR-144/451 cluster confer protection against simulated I/R-induced cardiomyocyte death via targeting CUGBP2-COX-2 pathway, at least in part. Thus, both miR-144 and miR-451 may represent new therapeutic agents for the treatment of ischemic heart disease.
PMID21365266	Characterization and functional analysis of the human microRNA let-7a2 promoter in lung cancer A549 cell lines. Recent studies have revealed that microRNAs have a strong association with cancer in humans. The miRNA let-7 is highly expressed in normal lung tissue, but frequently expressed at reduced levels in lung cancers. Let-7a2 is a member of the let-7 family. So far, little is known about the transcriptional regulation of let-7a2. Our study is focused on the characterization and functional analysis of the promoter of the human miRNA let-7a2 in A549 cell lines. Firstly, 5' rapid amplification of cDNA ends (5' RACE) was carried out and a 2.8 kb fragment in the upstream of let-7a2 gene was then cloned into pGL3-basic vector. Sequence analysis with the MatInspector database revealed that there were putative binding sites for some important transcriptional factors in the promoter region of let-7a2, such as p53, c-Myc, Ras, CEBPα, RORA, RXR, TCF, and GR. Additionally, a series of transfection and luciferase reporter assays were carried out to test let-7a2 promoter activity. RT-PCR and transfection of let-7a target sequence-reporter plasmid were performed to detect transcription levels of the let-7a2 gene in A549 cells treated with 9-cis-RA, all-trans-RA, lithium chloride or dexamethasone. Our results showed that the recombinant pGL3-p7a2 could acts as a promoter. The promoter activity of the 2.8 kb fragment could be downregulated by transfection with CEBPα or treatment with lithium chloride and enhanced by 9-cis-RA or all-trans-RA treatment. Furthermore, the results of RT-PCR analysis and transfection of let-7a target sequence-reporter plasmid showed that 9-cis-RA and all-trans-RA both upregulated let-7a2 expression, while lithium chloride downregulated its expression. Our results suggest that 9-cis-RA, all-trans-RA,lithium chloride and CEBPα might play important regulatory roles in let-7a2 gene expression in A549 cells.
PMID18362916	REST maintains self-renewal and pluripotency of embryonic stem cells. The neuronal repressor REST (RE1-silencing transcription factor; also called NRSF) is expressed at high levels in mouse embryonic stem (ES) cells, but its role in these cells is unclear. Here we show that REST maintains self-renewal and pluripotency in mouse ES cells through suppression of the microRNA miR-21. We found that, as with known self-renewal markers, the level of REST expression is much higher in self-renewing mouse ES cells than in differentiating mouse ES (embryoid body, EB) cells. Heterozygous deletion of Rest (Rest+/-) and its short-interfering-RNA-mediated knockdown in mouse ES cells cause a loss of self-renewal-even when these cells are grown under self-renewal conditions-and lead to the expression of markers specific for multiple lineages. Conversely, exogenously added REST maintains self-renewal in mouse EB cells. Furthermore, Rest+/- mouse ES cells cultured under self-renewal conditions express substantially reduced levels of several self-renewal regulators, including Oct4 (also called Pou5f1), Nanog, Sox2 and c-Myc, and exogenously added REST in mouse EB cells maintains the self-renewal phenotypes and expression of these self-renewal regulators. We also show that in mouse ES cells, REST is bound to the gene chromatin of a set of miRNAs that potentially target self-renewal genes. Whereas mouse ES cells and mouse EB cells containing exogenously added REST express lower levels of these miRNAs, EB cells, Rest+/- ES cells and ES cells treated with short interfering RNA targeting Rest express higher levels of these miRNAs. At least one of these REST-regulated miRNAs, miR-21, specifically suppresses the self-renewal of mouse ES cells, corresponding to the decreased expression of Oct4, Nanog, Sox2 and c-Myc. Thus, REST is a newly discovered element of the interconnected regulatory network that maintains the self-renewal and pluripotency of mouse ES cells.
PMID20972335	Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis. Adaptive changes to oxygen availability are critical for cell survival and tissue homeostasis. Prolonged oxygen deprivation due to reduced blood flow to cardiac or peripheral tissues can lead to myocardial infarction and peripheral vascular disease, respectively. Mammalian cells respond to hypoxia by modulating oxygen-sensing transducers that stabilize the transcription factor hypoxia-inducible factor 1α (HIF-1α), which transactivates genes governing angiogenesis and metabolic pathways. Oxygen-dependent changes in HIF-1α levels are regulated by proline hydroxylation and proteasomal degradation. Here we provide evidence for what we believe is a novel mechanism regulating HIF-1α levels in isolated human ECs during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in ECs. miR-424 targeted cullin 2 (CUL2), a scaffolding protein critical to the assembly of the ubiquitin ligase system, thereby stabilizing HIF-α isoforms. Hypoxia-induced miR-424 was regulated by PU.1-dependent transactivation. PU.1 levels were increased in hypoxic endothelium by RUNX-1 and C/EBPα. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by a specific morpholino. The rodent homolog of human miR-424, mu-miR-322, was significantly upregulated in parallel with HIF-1α in experimental models of ischemia. These results suggest that miR-322/424 plays an important physiological role in post-ischemic vascular remodeling and angiogenesis.
PMID19745602	LIN28B confers radio-resistance through the posttranscriptional control of KRAS. To screen the differentially expressed microRNAs related to radio-resistance, we compared the microRNA profiles of lung cancer cells with different responses to ionizing radiation (IR). Of 328 microRNAs in microarray, 27 microRNAs were differentially expressed in NCI-H460 (H460) and NCI-H1299 (H1299) cells. Among them, let-7g was down-regulated in radio-resistant H1299 cells, and the level of let-7g was higher in radio-sensitive cells like Caski, H460, and ME180 in qRT-PCR analysis than in radio-resistant cells like A549, H1299, DLD1, and HeLa. Over-expression of let-7g in H1299 cells could suppress the translation of KRAS, and increase the sensitivity to IR. When we knockdown the expression of LIN28B, an upstream regulator of let-7g, the level of mature let-7g was increased in H1299 cells and the sensitivity to IR was also enhanced in LIN28B knockdown cells. From these data, we suggest that LIN28B plays an important role in radiation responses of lung cancer cells through inhibiting let-7g processing and increasing translation of KRAS.
PMID19202062	p53 represses c-Myc through induction of the tumor suppressor miR-145. The tumor suppressor p53 negatively regulates a number of genes, including the proto-oncogene c-Myc, in addition to activating many other genes. One mechanism of the p53-mediated c-Myc repression may involve transcriptional regulation. However, it is not clear whether microRNAs (miRNAs) play a role in the p53-mediated posttranscriptional regulation of c-Myc. In this study, we show that a putative tumor suppressor, miR-145, is expressed through the phosphoinositide-3 kinase (PI-3K)/Akt and p53 pathways. Importantly, p53 transcriptionally induces the expression of miR-145 by interacting with a potential p53 response element (p53RE) in the miR-145 promoter. We further show that c-Myc is a direct target for miR-145. Although miR-145 silences the expression of c-Myc, anti-miR-145 enhances its expression. This specific silencing of c-Myc by miR-145 accounts at least in part for the miR-145-mediated inhibition of tumor cell growth both in vitro and in vivo. Finally, the blockade of miR-145 by anti-miR-145 is able to reverse the p53-mediated c-Myc repression. Together, these results define the role of miR-145 in the posttranscriptional regulation of c-Myc by p53 and suggest that, as a new member of the p53 regulatory network, miR-145 provides a direct link between p53 and c-Myc in this gene regulatory network.
PMID21482675	LIN28B polymorphisms influence susceptibility to epithelial ovarian cancer. Defective microRNA (miRNA) biogenesis contributes to the development and progression of epithelial ovarian cancer (EOC). In this study, we examined the hypothesis that single nucleotide polymorphisms (SNP) in miRNA biogenesis genes may influence EOC risk. In an initial investigation, 318 SNPs in 18 genes were evaluated among 1,815 EOC cases and 1,900 controls, followed up by a replicative joint meta-analysis of data from an additional 2,172 cases and 3,052 controls. Of 23 SNPs from 9 genes associated with risk (empirical P < 0.05) in the initial investigation, the meta-analysis replicated 6 SNPs from the DROSHA, FMR1, LIN28, and LIN28B genes, including rs12194974 (G>A), an SNP in a putative transcription factor binding site in the LIN28B promoter region (summary OR = 0.90, 95% CI: 0.82-0.98; P = 0.015) which has been recently implicated in age of menarche and other phenotypes. Consistent with reports that LIN28B overexpression in EOC contributes to tumorigenesis by repressing tumor suppressor let-7 expression, we provide data from luciferase reporter assays and quantitative RT-PCR to suggest that the inverse association among rs12194974 A allele carriers may be because of reduced LIN28B expression. Our findings suggest that variants in LIN28B and possibly other miRNA biogenesis genes may influence EOC susceptibility.
PMID20170545	Teratogen-induced alterations in microRNA-34, microRNA-125b and microRNA-155 expression: correlation with embryonic p53 genotype and limb phenotype. In a large number of studies, members of the microRNA (miRNA)-34 family such as miRNA-34a, miRNA-34b, miRNA-34c, as well as miRNA-125b and miRNA-155, have been shown to be regulators of apoptosis. The ability of these miRNAs to perform this function is mainly attributed to their ability to interact with the p53 tumor suppressor, which is a powerful regulator of the teratologic susceptibility of embryos. We chose to explore whether miRNA-34a/b/c, miRNA-125b and miRNA-155 may play a role in teratogenesis by using p53+/- pregnant mice treated with cyclophosphamide (CP) as a model. We evaluated how CP-induced alterations in the expression of these miRNAs in the embryonic limbs correlate with embryonic p53 genotype and CP-induced limb phenotypes. The limbs of p53 positive embryos were more sensitive to CP-induced teratogenic insult than the limbs of p53 negative embryos. The hindlimbs were more severely affected than the forelimbs. Robust miRNA-34a expression was observed in the fore- and hindlimbs of p53+/+ embryos exposed to 12.5 mg/kg CP. The dose of 20 mg/kg CP induced almost a two-fold increase in the level of miRNA-34a expression as compared to that exhibited by p53+/+ embryos exposed to a lower dose. Increased miRNA-34b and miRNA-34c expression was also observed. Of note, this dose activated miRNA-34a and miRNA-34c in the forelimbs of p53-/- embryos. When embryos were exposed to 40 mg/kg CP, the expression pattern of the miRNA-34a/b/c was identical to that registered in the limbs of embryos exposed to 20 mg/kg CP. However, this dose suppressed miRNA-125b and miRNA-155 expression in the fore- and hindlimbs of p53+/+ embryos. This study demonstrates that teratogen-induced limb dysmorphogenesis may be associated with alterations in miRNA-34, miRNA-125b and miRNA-155 expression. It also suggests for the first time that p53-independent mechanisms exist contributing to teratogen-induced activation of miRNA-34a and miRNA-34c. At the same time, teratogen-induced suppression of miRNA-125b and miRNA-155 expression may be p53 dependent. The analysis of correlations between the expression pattern of the tested miRNAs and CP induced limb phenotypes implies that miRNAs regulating apoptosis may differ from each other with respect to their functional role in teratogenesis: some miRNAs act to protect embryos, whereas other miRNAs boost a teratogen-induced process of maldevelopment to induce embryonic death.
PMID22093834	miR-125b suppresses the proliferation and migration of osteosarcoma cells through down-regulation of STAT3. There is accumulating evidence that microRNAs are involved in multiple processes in development and tumor progression. Abnormally expressed miR-125b was found to play a fundamental role in several types of cancer; however, whether miR-125b participates in regulating the initiation and progress of osteosarcoma still remains unclear. Here we demonstrate that miR-125b is frequently down-regulated in osteosarcoma samples and human osteosarcoma cell lines. The ectopic restoration of miR-125b expression in human osteosarcoma cells suppresses proliferation and migration in vitro and inhibits tumor formation in vivo. We further identified signal transducer and activator of transcription 3 (STAT3) as the direct and functional downstream target of miR-125b. Interestingly, we discovered that the expression of miR-125b is regulated by STAT3 at the level of transcription. STAT3 binds to the promoter region of miR-125b in vitro and serves as a transactivator. Taken together, our findings point to an important role in the molecular etiology of osteosarcoma and suggest that miR-125b is a potential target in the treatment of osteosarcoma.
PMID20546595	High-throughput sequencing identifies STAT3 as the DNA-associated factor for p53-NF-kappaB-complex-dependent gene expression in human heart failure. Genome-wide maps of DNA regulatory elements and their interaction with transcription factors may form a framework for understanding regulatory circuits and gene expression control in human disease, but how these networks, comprising transcription factors and DNA-binding proteins, form complexes, interact with DNA and modulate gene expression remains largely unknown. Using microRNA-21 (mir-21), which is an example of genes that are regulated in heart failure, we performed chromatin immunoprecipitation (ChIP) assays to determine the occupancy of transcription factors at this genetic locus. Tissue ChIP was further performed using human hearts and genome-wide occupancies of these transcription factors were analyzed by high-throughput sequencing. We show that the transcription factor p53 piggy-backs onto NF-kappaB/RELA and utilizes the kappaB-motif at a cis-regulatory region to control mir-21 expression. p53 behaves as a co-factor in this complex because despite a mutation in its DNA binding domain, mutant p53 was still capable of binding RELA and the cis-element, and inducing mir-21 expression. In dilated human hearts where mir-21 upregulation was previously demonstrated, the p53-RELA complex was also associated with this cis-element. Using high-throughput sequencing, we analyzed genome-wide binding sites for the p53-RELA complex in diseased and control human hearts and found a significant overrepresentation of the STAT3 motif. We further determined that STAT3 was necessary for the p53-RELA complex to associate with this cis-element and for mir-21 expression. Our results uncover a mechanism by which transcription factors cooperate in a multi-molecular complex at a cis-regulatory element to control gene expression.
PMID18384814	miR-21 Gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism. miR-21 has been reported to be highly expressed in various cancers and to be inducible in a human promyelocytic cell line, HL-60, after phorbol 12-myristate 13-acetate (PMA) treatment. To examine molecular mechanisms involved in miR-21 expression, we analyzed the structure of the miR-21 gene by determining its promoter and primary transcripts. We show that activation protein 1 (AP-1) activates the miR-21 transcription in conjugation with the SWI/SNF complex, after PMA stimulation, through the conserved AP-1 and PU.1 binding sites in the promoter identified here. The previous findings of enhanced miR-21 expression in several cancers may therefore reflect the elevated AP-1 activity in these carcinomas. A single precursor RNA containing miR-21 was transcribed just downstream from the TATA box in this promoter, which is located in an intron of a coding gene, TMEM49. More important, expression of this overlapping gene is completely PMA-independent and all its transcripts are polyadenylated before reaching the miR-21 hairpin embedding region, indicating that miRNAs could have their own promoter even if overlapped with other genes. By available algorithms that predict miRNA target using a conservation of sequence complementary to the miRNA seed sequence, we next predicted and confirmed that the NFIB mRNA is a target of miR-21. NFIB protein usually binds the miR-21 promoter in HL-60 cells as a negative regulator and is swept off from the miR-21 promoter during PMA-induced macrophage differentiation of HL-60. The translational repression of NFIB mRNA by miR-21 accelerates clearance of NFIB in parallel with the simultaneous miR-21-independent transcriptional repression of NFIB after PMA stimulation. Since exogenous miR-21 expression moderately induced endogenous miR-21, an evolutionarily conserved double-negative feedback regulation would be operating as a mechanism to sustain miR-21 expression.
PMID21670079	Inhibition of miR-193a expression by Max and RXRα activates K-Ras and PLAU to mediate distinct aspects of cellular transformation. MicroRNA profiling in isogenic models of cellular transformation involving either breast epithelial cells or fibroblasts reveals that expression of miR-193a is lower in transformed cells than in nontransformed cells. The transcription factors Max and RXRα bind directly to the miR-193a promoter and inhibit miR-193a expression during transformation. miR-193a inhibits cellular transformation by directly targeting the 3' untranslated regions of PLAU and K-Ras. Interestingly, miR-193a controls anchorage-independent growth in soft agar through K-Ras, whereas it affects invasive growth through PLAU. miR-193a overexpression inhibits the tumorigenicity of developmentally diverse but not all cancer cell types, and it inhibits tumor growth in colon- and breast-derived xenografts. Finally, expression of miR-193a is inversely correlated with PLAU and K-Ras in human colon adenocarcinomas. Thus, a pathway in which Max and RXRα inhibit miR-193a expression, thereby activating the PLAU and K-Ras oncogenes is important for distinct aspects of cellular transformation, as well as tumor growth and colon (and perhaps other types of) cancer.
PMID21088493	MicroRNA-449a levels increase by several orders of magnitude during mucociliary differentiation of airway epithelia. MicroRNAs of the miR-34/449 family mediate cell cycle arrest and tumor suppression.  Here we show that the expression of microRNA miR-449a, unlike its paralogue miR-34a, is highly tissue specific and largely restricted to pulmonary and testicular tissue. MiR-449a levels in the murine lung are particularly high shortly before and after birth, coinciding with terminal differentiation of lung epithelia. Strikingly, miR-449a is upregulated by more than 1000-fold when epithelial cells from human airways are lifted from a liquid environment to air, allowing them to undergo mucociliary differentiation. The induction of miR-449a occurs in parallel to its host gene CDC20B and the transcription factor FoxJ1. Exposure to tobacco smoke induces a moderate further increase in the levels of miR-449a, and also miR-34a, in differentiated airway epithelia. We propose that miR-449a can serve as an exquisitely sensitive and specific biomarker for the differentiation of bronchial epithelia. Moreover, miR-449a may actively promote mucociliary differentiation through its ability to block cell cycle progression, and it may conribute to a first line of defence against genotoxic stress by its proapoptotic functions.
PMID20053927	microRNA-141 is involved in a nasopharyngeal carcinoma-related genes network. microRNAs (miRNAs) are small non-coding RNAs and have been implicated in the pathology of various diseases, including cancer. Here we report that the miRNA profiles have been changed after knockdown of one of the most important oncogene c-MYC or re-expression of a candidate tumor suppressor gene SPLUNC1 in nasopharyngeal carcinoma (NPC) cells. Both c-MYC knockdown and SPLUNC1 re-expression can down-regulate microRNA-141 (miR-141). miR-141 is up-regulated in NPC specimens in comparison with normal nasopharyngeal epithelium. Inhibition of miR-141 could affect cell cycle, apoptosis, cell growth, migration and invasion in NPC cells. We found that BRD3, UBAP1 and PTEN are potential targets of miR-141, which had been confirmed following luciferase reporter assays and western blotting. BRD3 and UBAP1 are both involved in NPC carcinogenesis as confirmed through our previous studies and PTEN is a crucial tumor suppressor in many tumor types. BRD3 is involved in the regulation of the Rb/E2F pathway. Inhibition of miR-141 could affect some important molecules in the Rb/E2F, JNK2 and AKT pathways. It is well known that carcinogenesis of NPC is involved in the networks of genetic and epigenetic alteration events. We propose that miR-141- and tumor-related genes c-MYC, SPLUNC1, BRD3, UBAP1 and PTEN may constitute a gene-miRNA network to contribute to NPC development.
PMID20371612	Foxo3a regulates apoptosis by negatively targeting miR-21. MicroRNAs are a class of small non-coding RNAs and participate in the regulation of apoptotic program. Although miR-21 is able to inhibit apoptosis, its expression regulation and downstream targets remain to be fully elucidated. Here we report that the transcriptional factor Foxo3a initiates apoptosis by transcriptionally repressing miR-21 expression. Our results showed that doxorubicin could simultaneously induce the translocation of Foxo3a to the cell nuclei and a reduction in miR-21 expression. Knockdown of Foxo3a resulted in an elevation in miR-21 levels, whereas enforced expression of Foxo3a led to a decrease in miR-21 expression. In exploring the molecular mechanism by which Foxo3a regulates miR-21, we observed that Foxo3a bound to the promoter region of miR-21 and suppressed its promoter activity. These results indicate that Foxo3a can transcriptionally repress miR-21 expression. In searching for the downstream targets of miR-21 in apoptosis, we found that miR-21 suppressed the translation of Fas ligand (FasL), a pro-apoptotic factor. Furthermore, Foxo3a was able to up-regulate FasL expression through down-regulating miR-21. Our data suggest that Foxo3a negatively regulates miR-21 in initiating apoptosis.
PMID17911593	Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock. We report here that miR-155 and miR-125b play a role in innate immune response. LPS stimulation of mouse Raw 264.7 macrophages resulted in the up-regulation of miR-155 and down-regulation of miR-125b levels. The same changes also occurred when C57BL/6 mice were i.p. injected with LPS. Furthermore, the levels of miR-155 and miR-125b in Raw 264.7 cells displayed oscillatory changes in response to TNF-alpha. These changes were impaired by pretreating the cells with the proteasome inhibitor MG-132, suggesting that these two microRNAs (miRNAs) may be at least transiently under the direct control of NF-kappaB transcriptional activity. We show that miR-155 most probably directly targets transcript coding for several proteins involved in LPS signaling such as the Fas-associated death domain protein (FADD), IkappaB kinase epsilon (IKKepsilon), and the receptor (TNFR superfamily)-interacting serine-threonine kinase 1 (Ripk1) while enhancing TNF-alpha translation. In contrast, miR-125b targets the 3'-untranslated region of TNF-alpha transcripts; therefore, its down-regulation in response to LPS may be required for proper TNF-alpha production. Finally, Emu-miR-155 transgenic mice produced higher levels of TNF-alpha when exposed to LPS and were hypersensitive to LPS/d-galactosamine-induced septic shock. Altogether, our data suggest that the LPS/TNF-alpha-dependent regulation of miR-155 and miR-125b may be implicated in the response to endotoxin shock, thus offering new targets for drug design.
PMID20080624	ATM is down-regulated by N-Myc-regulated microRNA-421. Ataxia-telangiectasia mutated (ATM) is a high molecular weight protein serine/threonine kinase that plays a central role in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of DNA double-strand breaks. Little is known about the regulatory mechanisms for ATM expression itself. MicroRNAs are naturally existing regulators that modulate gene expression in a sequence-specific manner. Here, we show that a human microRNA, miR-421, suppresses ATM expression by targeting the 3'-untranslated region (3'UTR) of ATM transcripts. Ectopic expression of miR-421 resulted in S-phase cell cycle checkpoint changes and an increased sensitivity to ionizing radiation, creating a cellular phenotype similar to that of cells derived from ataxia-telangiectasia (A-T) patients. Blocking the interaction between miR-421 and ATM 3'UTR with an antisense morpholino oligonucleotide rescued the defective phenotype caused by miR-421 overexpression, indicating that ATM mediates the effect of miR-421 on cell cycle checkpoint and radiosensitivity. Overexpression of the N-Myc transcription factor, an oncogene frequently amplified in neuroblastoma, induced miR-421 expression, which, in turn, down-regulated ATM expression, establishing a linear signaling pathway that may contribute to N-Myc-induced tumorigenesis in neuroblastoma. Taken together, our findings implicate a previously undescribed regulatory mechanism for ATM expression and ATM-dependent DNA damage response and provide several potential targets for treating neuroblastoma and perhaps A-T.
PMID21715655	IL-2R common gamma-chain is epigenetically silenced by nucleophosphin-anaplastic lymphoma kinase (NPM-ALK) and acts as a tumor suppressor by targeting NPM-ALK. Anaplastic lymphoma kinase (ALK), physiologically expressed only by certain neural cells, becomes highly oncogenic, when aberrantly expressed in nonneural tissues as a fusion protein with nucleophosphin (NPM) and other partners. The reason why NPM-ALK succeeds in transforming specifically CD4(+) T lymphocytes remains unknown. The IL-2R common γ-chain (IL-2Rγ) is shared by receptors for several cytokines that play key roles in the maturation and growth of normal CD4(+) T lymphocytes and other immune cells. We show that IL-2Rγ expression is inhibited in T-cell lymphoma cells expressing NPM-ALK kinase as a result of DNA methylation of the IL-2Rγ gene promoter. IL-2Rγ promoter methylation is induced in malignant T cells by NPM-ALK. NPM-ALK acts through STAT3, a transcription factor that binds to the IL-2Rγ gene promoter and enhances binding of DNA methyltransferases (DNMTs) to the promoter. In addition, STAT3 suppresses expression of miR-21, which selectively inhibits DNMT1 mRNA expression. Reconstitution of IL-2Rγ expression leads to loss of the NPM-ALK protein and, consequently, apoptotic cell death of the lymphoma cells. These results demonstrate that the oncogenic tyrosine kinase NPM-ALK induces epigenetic silencing of the IL-2Rγ gene and that IL-2Rγ acts as a tumor suppressor by reciprocally inhibiting expression of NPM-ALK.
PMID19706389	The estrogen receptor-alpha-induced microRNA signature regulates itself and its transcriptional response. Following estrogenic activation, the estrogen receptor-alpha (ERalpha) directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) modulated by ERalpha have the potential to fine tune these regulatory systems and also provide an alternate mechanism that could impact on estrogen-dependent developmental and pathological systems. Through a microarray approach, we identify the subset of microRNAs (miRNAs) modulated by ERalpha, which include upregulation of miRNAs derived from the processing of the paralogous primary transcripts (pri-) mir-17-92 and mir-106a-363. Characterization of the mir-17-92 locus confirms that the ERalpha target protein c-MYC binds its promoter in an estrogen-dependent manner. We observe that levels of pri-mir-17-92 increase earlier than the mature miRNAs derived from it, implicating precursor cleavage modulation after transcription. Pri-mir-17-92 is immediately cleaved by DROSHA to pre-miR-18a, indicating that its regulation occurs during the formation of the mature molecule from the precursor. The clinical implications of this novel regulatory system were confirmed by demonstrating that pre-miR-18a was significantly upregulated in ERalpha-positive compared to ERalpha-negative breast cancers. Mechanistically, miRNAs derived from these paralogous pri-miRNAs (miR-18a, miR-19b, and miR-20b) target and downregulate ERalpha, while a subset of pri-miRNA-derived miRNAs inhibit protein translation of the ERalpha transcriptional p160 coactivator, AIB1. Therefore, different subsets of miRNAs identified act as part of a negative autoregulatory feedback loop. We propose that ERalpha, c-MYC, and miRNA transcriptional programs invoke a sophisticated network of interactions able to provide the wide range of coordinated cellular responses to estrogen.
PMID19117988	A feed-forward loop involving protein kinase Calpha and microRNAs regulates tumor cell cycle. Protein kinase Calpha (PKCalpha) has been implicated in cancer, but the mechanism is largely unknown. Here, we show that PKCalpha promotes head and neck squamous cell carcinoma (SCCHN) by a feed-forward network leading to cell cycle deregulation. PKCalpha inhibitors decrease proliferation in SCCHN cell lines and xenografted tumors. PKCalpha inhibition or depletion in tumor cells decreases DNA synthesis by suppressing extracellular signal-regulated kinase phosphorylation and cyclin E synthesis. Additionally, PKCalpha down-regulates miR-15a, a microRNA that directly inhibits protein synthesis of cyclin E, as well as other cell cycle regulators. Furthermore, both PKCalpha and cyclin E protein expression are increased in primary tumors, and PKCalpha inversely correlates with miR-15a expression in primary tumors. Finally, PKCalpha is associated with poor prognosis in SCCHN. These results identify PKCalpha as a key regulator of SCCHN tumor cell growth by a mechanism involving activation of mitogen-activated protein kinase, an initiator of the cell cycle, and suppression of miR-15a, an inhibitor of DNA synthesis. Although the specific components may be different, this type of feed-forward loop network, consisting of a stimulus that activates a positive signal and removes a negative brake, is likely to be a general one that enables induction of DNA synthesis by a variety of growth or oncogenic stimuli.
PMID22249264	MicroRNA-29b is involved in the Src-ID1 signaling pathway and is dysregulated in human lung adenocarcinoma. The c-Src kinase regulates cancer cell invasion through inhibitor of DNA binding/differentiation 1 (ID1). Src and ID1 are frequently overexpressed in human lung adenocarcinoma. The current study aimed at identifying microRNAs (miRNAs) involved in the Src-ID1 signaling in lung cancer. Incubation of lung cancer cells with the Src inhibitor saracatinib led to the upregulation of several miRNAs including miR-29b, which was the most highly upregulated miRNA with predicted binding to the ID1 3'-untranslated region (UTR). Luciferase reporter assays confirmed direct binding of miR-29b to the ID1 3'-UTR. Expression of miR-29b suppressed ID1 levels and significantly reduced migration and invasion. Expression of antisense-miR-29b (anti-miR-29b), on the other hand, enhanced ID1 mRNA and protein levels, and significantly increased lung cancer cell migration and invasion, a hallmark of the Src-ID1 pathway. The ectopic expression of ID1 in miR-29b-overexpressing cells was able to rescue the migratory potential of these cells. Both, anti-miR-29b and ID1 overexpression diminished the effects of the Src inhibitors saracatinib and dasatinib on migration and invasion. Saracatinib and dasatinib decreased c-Myc transcriptional repression on miR-29b and led to increased ID1 protein levels, whereas forced expression of c-Myc repressed miR-29b and induced ID1. In agreement, we showed direct recruitment of c-Myc to the miR-29b promoter. miR-29b was significantly downregulated in primary lung adenocarcinoma samples compared with matched alveolar lung tissue, and miR-29b expression was a significant prognostic factor for patient outcome. These results suggest that miR-29b is involved in the Src-ID1 signaling pathway, is dysregulated in lung adenocarcinoma and is a potential predictive marker for Src kinase inhibitors.
PMID20842122	EVI1 controls proliferation in acute myeloid leukaemia through modulation of miR-1-2. The EVI1(ecotropic virus integration site 1) gene codes for a zinc-finger transcription factor, whose transcriptional activation leads to a particularly aggressive form of acute myeloid leukaemia (AML). Although, EVI1 interactions with key proteins in hematopoiesis have been previously described, the precise role of this transcription factor in promoting leukaemic transformation is not completely understood. Recent works have identified specific microRNA (miRNA) signatures in different AML subgroups. However, there is no analysis of miRNAs profiles associated with EVI1 overexpression in humans. We performed QT-RT-PCR to assess the expression of 250 miRNAs in cell lines with or without EVI1 overexpression and in patient samples. We used ChIP assays to evaluated the possible binding of EVI1 binding to the putative miRNA promoter. Proliferation of the different cell lines transfected with the anti- or pre-miRs was quantified by MTT. Our data showed that EVI1 expression was significantly correlated with the expression of miR-1-2 and miR-133-a-1 in established cell lines and in patient samples. ChIP assays confirmed that EVI1 binds directly to the promoter of these two miRNAs. However, only miR-1-2 was involved in abnormal proliferation in EVI1 expressing cell lines. Our data showed that EVI1 controls proliferation in AML through modulation of miR-1-2. This study contributes to further understand the transcriptional networks involving transcription factors and miRNAs in AML.
PMID18725401	Embryonic stem cell-specific miR302-367 cluster: human gene structure and functional characterization of its core promoter. MicroRNAs (miRNAs) play a central role in the regulation of multiple biological processes including the maintenance of stem cell self-renewal and pluripotency. Recently, the miRNA cluster miR302-367 was shown to be differentially expressed in embryonic stem cells (ESCs). Unfortunately, very little is known about the genomic structure of miRNA-encoding genes and their transcriptional units. Here, we have characterized the structure of the gene coding for the human miR302-367 cluster. We identify the transcriptional start and functional core promoter region which specifically drives the expression of this miRNA cluster. The promoter activity depends on the ontogeny and hierarchical cellular stage. It is functional during embryonic development, but it is turned off later in development. From a hierarchical standpoint, its activity decays upon differentiation of ESCs, suggesting that its activity is restricted to the ESC compartment and that the ESC-specific expression of the miR302-367 cluster is fully conferred by its core promoter transcriptional activity. Furthermore, algorithmic prediction of transcription factor binding sites and knockdown studies suggest that ESC-associated transcription factors, including Nanog, Oct3/4, Sox2, and Rex1 may be upstream regulators of miR302-367 promoter. This study represents the first identification, characterization, and functional validation of a human miRNA promoter in stem cells. This study opens up new avenues to further investigate the upstream transcriptional regulation of the miR302-367 cluster and to dissect how these miRNAs integrate in the complex molecular network conferring stem cell properties to ESCs.
PMID18048365	B-cell receptor activation induces BIC/miR-155 expression through a conserved AP-1 element. microRNA-155 is an oncogenic microRNA that has been shown to be critical for B-cell maturation and immunoglobulin production in response to antigen. In line with its function in B-cell activation, miR-155, and its primary transcript, B-cell integration cluster (BIC), is induced by B-cell receptor (BCR) cross-linking. Using pharmacological inhibitors in the human B-cell line, Ramos, we show that activation of BIC and miR-155 expression by BCR signaling occurs through the extracellular signaling-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways but not the p38 pathway. BCR activation results in the induction of c-Fos, FosB, and JunB, and expression of these are suppressed by ERK and JNK inhibitors. Reporter analysis established a key role for a conserved AP-1 site approximately 40 bp upstream from the site of initiation but not an upstream NF-kappaB site or a putative c-Ets located at the site of initiation. Lastly, chromatin immunoprecipitation analysis demonstrated the recruitment of FosB and JunB to the miR-155 promoter following BCR activation. These results identify key determinants of BCR-mediated signaling that lead to the induction of BIC/miR-155.
PMID22359598	ER stress negatively modulates the expression of the miR-199a/214 cluster to regulates tumor survival and progression in human hepatocellular cancer. Recent studies have emphasized causative links between microRNAs (miRNAs) deregulation and tumor development. In hepatocellular carcinoma (HCC), more and more miRNAs were identified as diagnostic and prognostic cancer biomarkers, as well as additional therapeutic tools. This study aimed to investigate the functional significance and regulatory mechanism of the miR-199a2/214 cluster in HCC progression. In this study, we showed that miR-214, as well as miR-199a-3p and miR-199a-5p levels were significantly reduced in the majority of examined 23 HCC tissues and HepG2 and SMMC-7721 cell lines, compared with their nontumor counterparts. To further explore the role of miR-214 in hepatocarcinogenesis, we disclosed that the ER stress-induced pro-survival factor XBP-1 is a target of miR-214 by using western blot assay and luciferase reporter assay. Re-expression of miR-214 in HCC cell lines (HepG2 and SMMC-7721) inhibited proliferation and induced apoptosis. Furthermore, ectopic expression of miR-214 dramatically suppressed the ability of HCC cells to form colonies in vitro and to develop tumors in a subcutaneous xenotransplantation model of the BALB/c athymic nude mice. Moreover, reintroduction of XBP-1s attenuated miR-214-mediated suppression of HCC cells proliferation, colony and tumor formation. To further understand the mechanism of the miR-199a/214 cluster down-expression in HCC, we found that thapsigargin (TG) and tunicamycin (TM) or hypoxia-induced unfolded protein response (UPR) suppresses the expression of the miR-199a/214 cluster in HCC cells. By promoter analysis of the miR-199a2/214 gene, we conjectured NFκB as a potential negative regulator. We further found that UPR and LPS-induced NFκB activation suppressed miR-199a2/214 transcription, and this suppression was reversed by NFκB inhibition in HCC cells. Our study suggest that modulation of miR-214 levels may provide a new therapeutic approach for cancer treatment and revealed that UPR may offer a new explanation for why the miR-199a/214 cluster were down-regulated in the progression in HCC.
PMID20029046	Cell-cycle regulator E2F1 and microRNA-223 comprise an autoregulatory negative feedback loop in acute myeloid leukemia. Transcription factor CCAAT enhancer binding protein alpha (C/EBPalpha) is essential for granulopoiesis and its function is deregulated in leukemia. Inhibition of E2F1, the master regulator of cell-cycle progression, by C/EBPalpha is pivotal for granulopoiesis. Recent studies show microRNA-223 (miR-223), a transcriptional target of C/EBPalpha, as a critical player during granulopoiesis. In this report, we demonstrate that during granulopoiesis microRNA-223 targets E2F1. E2F1 protein was up-regulated in miR-223 null mice. We show that miR-223 blocks cell-cycle progression in myeloid cells. miR-223 is down-regulated in different subtypes of acute myeloid leukemia (AML). We further show that E2F1 binds to the miR-223 promoter in AML blast cells and inhibits miR-223 transcription, suggesting that E2F1 is a transcriptional repressor of the miR-223 gene in AML. Our study supports a molecular network involving miR-223, C/EBPalpha, and E2F1 as major components of the granulocyte differentiation program, which is deregulated in AML.
PMID21768389	MicroRNA-21 regulates the self-renewal of mouse spermatogonial stem cells. MicroRNAs (miRs) play a key role in the control of gene expression in a wide array of tissue systems, where their functions include the regulation of self-renewal, cellular differentiation, proliferation, and apoptosis. However, the functional importance of individual miRs in controlling spermatogonial stem cell (SSC) homeostasis has not been investigated. Using high-throughput sequencing, we profiled the expression of miRs in the Thy1(+) testis cell population, which is highly enriched for SSCs, and the Thy1(-) cell population, composed primarily of testis somatic cells. In addition, we profiled the global expression of miRs in cultured germ cells, also enriched for SSCs. Our results demonstrate that miR-21, along with miR-34c, -182, -183, and -146a, are preferentially expressed in the Thy1(+) SSC-enriched population, compared with Thy1(-) somatic cells. Importantly, we demonstrate that transient inhibition of miR-21 in SSC-enriched germ cell cultures increased the number of germ cells undergoing apoptosis and significantly reduced the number of donor-derived colonies of spermatogenesis formed from transplanted treated cells in recipient mouse testes, indicating that miR-21 is important in maintaining the SSC population. Moreover, we show that in SSC-enriched germ cell cultures, miR-21 is regulated by the transcription factor ETV5, known to be critical for SSC self-renewal.
PMID20308559	P53-induced microRNA-107 inhibits HIF-1 and tumor angiogenesis. The pathway involving the tumor suppressor gene TP53 can regulate tumor angiogenesis by unclear mechanisms. Here we show that p53 regulates hypoxic signaling through the transcriptional regulation of microRNA-107 (miR-107). We found that miR-107 is a microRNA expressed by human colon cancer specimens and regulated by p53. miR-107 decreases hypoxia signaling by suppressing expression of hypoxia inducible factor-1beta (HIF-1beta). Knockdown of endogenous miR-107 enhances HIF-1beta expression and hypoxic signaling in human colon cancer cells. Conversely, overexpression of miR-107 inhibits HIF-1beta expression and hypoxic signaling. Furthermore, overexpression of miR-107 in tumor cells suppresses tumor angiogenesis, tumor growth, and tumor VEGF expression in mice. Finally, in human colon cancer specimens, expression of miR-107 is inversely associated with expression of HIF-1beta. Taken together these data suggest that miR-107 can mediate p53 regulation of hypoxic signaling and tumor angiogenesis.
PMID19273269	A network of miRNAs expressed in the ovary are regulated by FSH. The process of folliculogenesis requires a tightly regulated series of gene expression that are a pre-requisite to the development of ovarian follicle. Among these genes, follicle-stimulating hormone (FSH) is notable for its dual role in development of follicles as well as proliferation and differentiation of granulosa cells. The post-transcriptional expression of these genes is under the control of microRNAs (miRNAs), a class of small, endogenous RNAs that negatively impact gene expression. This study was carried out to determine the role of several miRNAs including mir-143, let-7a, mir-125b, let-7b, let-7c, mir-21 in follicular development in the mouse. The expression of these RNAs was very low in primordial follicles but these became readily detectable in the granulosa cells of primary, secondary and antral follicles. We show that this expression of some miRNAs (mir-143, let-7a, mir-15b) is under negative control of FSH. Together, these findings suggest that FSH regulates folliculogenesis by a novel pathway of miRNAs.
PMID17943719	MYCN regulates oncogenic MicroRNAs in neuroblastoma. MYCN amplification is a common feature of aggressive tumour biology in neuroblastoma. The MYCN transcription factor has been demonstrated to induce or repress expression of numerous genes. MicroRNAs (miRNA) are a recently discovered class of short RNAs that repress translation and promote mRNA degradation by sequence-specific interaction with mRNA. Here, we sought to analyse the role of MYCN in regulation of miRNA expression. Using a miRNA microarray containing 384 different miRNAs and a set of 160 miRNA real-time PCR assays to validate the microarray results, 7 miRNAs were identified that are induced by MYCN in vitro and are upregulated in primary neuroblastomas with MYCN amplification. Three of the seven miRNAs belong to the miR-106a and miR-17 clusters, which have previously been shown to be regulated by c-Myc. The miR-17-92 polycistron also acts as an oncogene in haematopoietic progenitor cells. We show here that miR-221 is also induced by MYCN in neuroblastoma. Previous studies have reported miR-221 to be overexpressed in several other cancer entities, but its regulation has never before been associated with Myc. We present evidence of miRNA dysregulation in neuroblastoma. Additionally, we report miRNA induction to be a new mechanism of gene expression downregulation by MYCN.
PMID20878079	Suppression of p21 by c-Myc through members of miR-17 family at the post-transcriptional level. The miR-17 family, composed of miR-17-5p, miR-20a/b, miR-106a/b and miR-93, has been demonstrated to take part in critical pathways that regulate cellular life and death decisions during normal development and in malignancy. Previous studies have shown that the expression of miR-17 family members has close relationship with the expression of c-Myc. Another study has reported p21 is a direct target of miR-17 family and their silencing of p21 contributes to tumor cell proliferation in part. Since c-Myc is a potent transcriptional repressor of p21, these findings suggest that c-Myc may negatively modulate p21 expression through an additional pathway except transcriptional suppression. In the study presented here, we compared p21 mRNA in the nucleus and cytoplasm of c-Myc stable transfectants with its control, which indicated further repression of p21 by c-Myc at the post-transcriptional level. Stem-loop and conventional real-time PCR showed elevated expression of some members in miR-17 family and their primary transcripts when c-Myc was stably overexpressed. To further investigate the relationship of c-Myc, miR-17 family and p21, we antagonized the expression of each miR-17 family member by transfection of their antisense oligonucleotides respectively in transfectants constitutively overexpressing c-Myc or not, and found that the restoration of p21 expression by treatment above was much stronger in the presence of c-Myc. These results suggest c-Myc could further repress p21 expression at the post-transcriptional level through some, but not all, members of miR-17 family.
PMID21837748	The histone deacetylase 4/SP1/microrna-200a regulatory network contributes to aberrant histone acetylation in hepatocellular carcinoma. As an important epigenetic mechanism, histone acetylation modulates the transcription of many genes and plays important roles in hepatocellular carcinoma (HCC). Aberrations in histone acetylation have been observed in HCC, but the factors that contribute to the aberrations have not been fully elucidated. MicroRNAs (miRNAs), which are noncoding RNAs that regulate gene expression, are involved in important epigenetic mechanisms. In this study, we determined that miR-200a and the level of histone H3 acetylation at its promoter were reduced in human HCC tissues in comparison with adjacent noncancerous hepatic tissues. Furthermore, our results suggested that the histone deacetylase 4 (HDAC4) inhibited the expression of miR-200a and its promoter activity and reduced the histone H3 acetylation level at the mir-200a promoter through a Sp1-dependent pathway. Interestingly, we observed that the miR-200a directly targeted the 3'-untranslated region of the HDAC4 messenger RNA and repressed expression of HDAC4. Therefore, miR-200a ultimately induced its own transcription and increased the histone H3 acetylation level at its own promoter. Through targeting HDAC4, miR-200a also induced the up-regulation of total acetyl-histone H3 levels and increased the histone H3 acetylation level at the p21(WAF/Cip1) promoter. Finally, we determined that miR-200a inhibited the proliferation and migration of HCC cells in vivo and in vitro. Our findings suggest that the HDAC4/Sp1/miR-200a regulatory network induces the down-regulation of miR-200a and the up-regulation of HDAC4 in HCC. As a result, down-regulation of miR-200a enhances the proliferation and migration of HCC cells and induces aberrant histone acetylation in HCC. These findings highlight a potential therapeutic approach in targeting the HDAC4/Sp1/miR-200a regulatory network for the treatment of HCC.
PMID19190326	Breast cancer metastasis suppressor 1 up-regulates miR-146, which suppresses breast cancer metastasis. Breast cancer metastasis suppressor 1 (BRMS1) is a predominantly nuclear protein that differentially regulates expression of multiple genes, leading to suppression of metastasis without blocking orthotopic tumor growth in multiple human and murine cancer cells of diverse origins. We hypothesized that miR-146 may be involved in the ability of BRMS1 to supress metastasis because miR-146 expression is altered by BRMS1 and because BRMS1 and miR-146 are both associated with decreased signaling through the nuclear factor-kappaB pathway. BRMS1 significantly up-regulates miR-146a by 6- to 60-fold in metastatic MDA-MB-231 and MDA-MB-435 cells, respectively, and miR-146b by 40-fold in MDA-MB-435 as measured by real-time quantitative reverse transcription-PCR. Transduction of miR-146a or miR-146b into MDA-MB-231 down-regulated expression of epidermal growth factor receptor, inhibited invasion and migration in vitro, and suppressed experimental lung metastasis by 69% and 84%, respectively (mean +/- SE: empty vector = 39 +/- 6, miR-146a = 12 +/- 1, miR-146b = 6 +/- 1). These results further support the recent notion that modulating the levels of miR-146a or miR-146b could have a therapeutic potential to suppress breast cancer metastasis.
PMID22002058	The unfolded protein response (UPR)-activated transcription factor X-box-binding protein 1 (XBP1) induces microRNA-346 expression that targets the human antigen peptide transporter 1 (TAP1) mRNA and governs immune regulatory genes. To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3'-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress.
PMID22264233	Transforming growth factor β1 represses proximal tubular cell microRNA-192 expression through decreased hepatocyte nuclear factor DNA binding. miR (microRNA)-192 plays key roles in renal pathological and physiological responses, by repressing targets including Zeb1, Zeb2 and Wnk1. In the present study, we have assessed the regulation of miR-192 expression. We found that TGF-β1 (transforming growth factor β1) down-regulates miR-192 and miR-194, co-transcribed in the shared precursor pri-miR (primary miR transcript)-192/194. Luciferase reporter analysis showed constitutive promoter activity within nucleotides +21 to -223. We identified HNF (hepatocyte nuclear factor) and p53 binding sites within this region that were required for constitutive promoter activity, which was decreased by TGF-β1 through an Alk5-dependent mechanism. TGF-β1 treatment decreased HNF binding to the miR-194-2/192 promoter, whereas knockdown of HNF-1 inhibited mature miR-192 and miR-194 expression. miR-192, miR-194 and HNF expression were restricted to a defined subset of human tissues including kidney, small intestine, colon and liver. Our results from the present study identify co-ordinated regulation of miR-192 and miR-194, with binding of HNF and p53 transcription factors necessary for activation of transcription, and TGF-β1-mediated repression through decreased HNF binding to its cognate promoter element.
PMID21730150	The microRNA-21-PDCD4 axis prevents type 1 diabetes by blocking pancreatic beta cell death. Death of pancreatic β cells is a pathological hallmark of type 1 diabetes (T1D). However, the molecular mechanisms of β cell death and its regulation are poorly understood. Here we describe a unique regulatory pathway of β cell death that comprises microRNA-21, its target tumor suppressor PDCD4, and its upstream transcriptional activator nuclear factor-κB (NF-κB). In pancreatic β cells, c-Rel and p65 of the NF-κB family activated the mir21 gene promoter and increased miR-21 RNA levels; miR-21 in turn decreased the level of PDCD4, which is able to induce cell death through the Bax family of apoptotic proteins. Consequently, PDCD4 deficiency in pancreatic β cells renders them resistant to death, and PDCD4 deficiency in NOD or C57BL/6 mice conferred resistance to spontaneous diabetes and diabetes induced by autoimmune T cells or the β cell toxin streptozotocin (STZ). Thus, the NF-κB-microRNA-21-PDCD4 axis plays a crucial role in T1D and represents a unique therapeutic target for treating the disease.
PMID18698484	Hedgehog signaling, epithelial-to-mesenchymal transition and miRNA (review). SHH, IHH, and DHH are lipid-modified secreted proteins binding to Patched receptors, and CDON, BOC or GAS1 co-receptors. In the absence of Hedgehog signaling, GLI1 is transcriptionally repressed, GLI2 is phosphorylated by GSK3 and CK1 for the FBXW11 (betaTRCP2)-mediated degradation, and GLI3 is processed to a cleaved repressor. In the presence of Hedgehog signaling, Smoothened is relieved from Patched-mediated suppression due to the Hedgehog-dependent internalization of Patched, which leads to MAP3K10 (MST) activation and SUFU inactivation for the stabilization and nuclear accumulation of GLI family members. GLI activators then upregulate CCND1, CCND2 for cell cycle acceleration, FOXA2, FOXC2, FOXE1, FOXF1, FOXL1, FOXP3, POU3F1, RUNX2, SOX13, TBX2 for cell fate determination, JAG2, INHBC, and INHBE for stem cell signaling regulation. Hedgehog signals also upregulate SFRP1 in mesenchymal cells for WNT signaling regulation. Epithelial-to-mesenchymal transition (EMT) during embryogenesis, adult tissue homeostasis and carcinogenesis is characterized by class switch from E-cadherin to N-cadherin. SNAI1 (Snail), SNAI2 (Slug), SNAI3, ZEB1, ZEB2 (SIP1), KLF8, TWIST1, and TWIST2 are EMT regulators repressing CDH1 gene encoding E-cadherin. Hedgehog signals induce JAG2 upregulation for Notch-CSL-mediated SNAI1 upregulation, and also induce TGFbeta1 secretion for ZEB1 and ZEB2 upregulation via TGFbeta receptor and NF-kappaB. TGFbeta-mediated downregulation of miR-141, miR-200a, miR-200b, miR-200c, miR-205, and miR-429 results in upregulation of ZEB1 and ZEB2 proteins. Hedgehog signaling activation indirectly leads to EMT through FGF, Notch, TGFbeta signaling cascades, and miRNA regulatory networks. miRNAs targeted to stem cell signaling components or EMT regulators are potent drug targets; however, off-target effects should be strictly controlled before clinical application of synthetic miRNA. Peptide mimetic and RNA aptamer could also be utilized as Hedgehog signaling inhibitors or EMT suppressors.
PMID20978187	Epigenetically deregulated microRNA-375 is involved in a positive feedback loop with estrogen receptor alpha in breast cancer cells. Estrogen receptor α (ERα) upregulation causes abnormal cell proliferation in about two thirds of breast cancers, yet understanding of the underlying mechanisms remains incomplete. Here, we show that high expression of the microRNA miR-375 in ERα-positive breast cell lines is a key driver of their proliferation. miR-375 overexpression was caused by loss of epigenetic marks including H3K9me2 and local DNA hypomethylation, dissociation of the transcriptional repressor CTCF from the miR-375 promoter, and interactions of ERα with regulatory regions of miR-375. Inhibiting miR-375 in ERα-positive MCF-7 cells resulted in reduced ERα activation and cell proliferation. A combination of expression profiling from tumor samples and miRNA target prediction identified RASD1 as a potential miR-375 target. Mechanistic investigations revealed that miR-375 regulates RASD1 by targeting the 3' untranslated region in RASD1 mRNA. Additionally, we found that RASD1 negatively regulates ERα expression. Our findings define a forward feedback pathway in control of ERα expression, highlighting new strategies to treat ERα-positive invasive breast tumors.
PMID20385090	A splicing-independent function of SF2/ASF in microRNA processing. Both splicing factors and microRNAs are important regulatory molecules that play key roles in posttranscriptional gene regulation. By miRNA deep sequencing, we identified 40 miRNAs that are differentially expressed upon ectopic overexpression of the splicing factor SF2/ASF. Here we show that SF2/ASF and one of its upregulated microRNAs (miR-7) can form a negative feedback loop: SF2/ASF promotes miR-7 maturation, and mature miR-7 in turn targets the 3'UTR of SF2/ASF to repress its translation. Enhanced microRNA expression is mediated by direct interaction between SF2/ASF and the primary miR-7 transcript to facilitate Drosha cleavage and is independent of SF2/ASF's function in splicing. Other miRNAs, including miR-221 and miR-222, may also be regulated by SF2/ASF through a similar mechanism. These results underscore a function of SF2/ASF in pri-miRNA processing and highlight the potential coordination between splicing control and miRNA-mediated gene repression in gene regulatory networks.
PMID19234151	Cutting edge: the Foxp3 target miR-155 contributes to the development of regulatory T cells. Foxp3 is a transcription factor that is essential for the normal development of regulatory T cells (Tregs). In the absence of microRNAs (miRNAs), Foxp3(+) Tregs develop but fail to maintain immune homeostasis, leading to a scurfy-like disease. Global analysis of the network of genes regulated by Foxp3 has identified the miRNA miR-155, which is highly expressed in Tregs, as a direct target of Foxp3. In this study we report that miR-155-deficient mice have reduced numbers of Tregs, both in the thymus and periphery, due to impaired development. However, we found no evidence for defective suppressor activity of miR-155-deficient Tregs, either in vitro or in vivo. Our results indicate that miR-155 contributes to Treg development, but that additional miRNAs control Treg function.
PMID20628397	The tumour-suppressive miR-29a/b1 cluster is regulated by CEBPA and blocked in human AML. CCAAT/enhancer-binding protein-alpha (CEBPA) is crucial for normal granulopoiesis and is frequently disrupted in acute myeloid leukaemia (AML). Increasing evidence suggests that CEBPA exerts its effects, in parts, by regulating specific microRNAs (miRNAs), as previously shown for miR-223. The aim of this study was to investigate the genome-wide pattern of miRNAs regulated by CEBPA in myeloid cells. In Kasumi-1 cells, conditionally expressing CEBPA, we assessed the expression of 470 human miRNAs by microarray analysis. We further investigated the microarray results by qRT-PCR, luciferase reporter assays, and chromatin immunoprecipitation assays. In all, 18 miRNAs were more than two-fold suppressed or induced after CEBPA restoration. Among these 18 miRNAs, we focused on CEBPA-mediated regulation of the tumour-suppressive miR-29b. We observed that miR-29b is suppressed in AML patients with impaired CEBPA function or loss of chromosome 7q. We found that CEBPA selectively regulates miR-29b expression on its miR-29a/b1 locus on chromosome 7q32.3, whereas miR-29b2/c on chromosome 1q32.2 is not affected. This study reports the activation of the tumour-suppressive miR-29b by the haematopoietic key transcription factor CEBPA. Our data provide a rationale for miR-29b suppression in AML patients with loss of chromosome 7q or CEBPA deficiency.
PMID22042699	miR-135b mediates NPM-ALK-driven oncogenicity and renders IL-17-producing immunophenotype to anaplastic large cell lymphoma. Many transformed lymphoma cells show immune-phenotypes resembling the corresponding normal lymphocytes; thus, they provide a guide for proper diagnosis and present promising routes to improve their pathophysiologic understanding and to identify novel therapeutic targets. However, the underlying molecular mechanism(s) of these aberrant immune-phenotypes is largely unknown. Here, we report that microRNA-135b (miR-135b) mediates nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-driven oncogenicity and empowers IL-17-producing immunophenotype in anaplastic large cell lymphoma (ALCL). NPM-ALK oncogene strongly promoted the expression of miR-135b and its host gene LEMD1 through activation of signal transducer and activator of transcription (STAT) 3. In turn, elevated miR-135b targeted FOXO1 in ALCL cells. miR-135b introduction also decreased chemosensitivity in Jurkat cells, suggesting its contribution to oncogenic activities of NPM-ALK. Interestingly, miR-135b suppressed T-helper (Th) 2 master regulators STAT6 and GATA3, and miR-135b blockade attenuated IL-17 production and paracrine inflammatory response by ALCL cells, indicating that miR-135b-mediated Th2 suppression may lead to the skewing to ALCL immunophenotype overlapping with Th17 cells. Furthermore, antisense-based miR-135b inhibition reduced tumor angiogenesis and growth in vivo, demonstrating significance of this "Th17 mimic" pathway as a therapeutic target. These results collectively illuminated unique contribution of oncogenic kinase-linked microRNA to tumorigenesis through modulation of tumor immune-phenotype and microenvironment.
PMID21555452	A hypoxia-induced positive feedback loop promotes hypoxia-inducible factor 1alpha stability through miR-210 suppression of glycerol-3-phosphate dehydrogenase 1-like. Oxygen-dependent regulation of the transcription factor HIF-1α relies on a family of prolyl hydroxylases (PHDs) that hydroxylate hypoxia-inducible factor 1α (HIF-1α) protein at two prolines during normal oxygen conditions, resulting in degradation by the proteasome. During low-oxygen conditions, these prolines are no longer hydroxylated and HIF-1α degradation is blocked. Hypoxia-induced miRNA-210 (miR-210) is a direct transcriptional target of HIF-1α, but its complete role and targets during hypoxia are not well understood. Here, we identify the enzyme glycerol-3-phosphate dehydrogenase 1-like (GPD1L) as a novel regulator of HIF-1α stability and a direct target of miR-210. Expression of miR-210 results in stabilization of HIF-1α due to decreased levels of GPD1L resulting in an increase in HIF-1α target genes. Altering GPD1L levels by overexpression or knockdown results in a decrease or increase in HIF-1α stability, respectively. GPD1L-mediated decreases in HIF-1α stability can be reversed by pharmacological inhibition of the proteasome or PHD activity. When rescued from degradation by proteasome inhibition, elevated amounts of GPD1L cause hyperhydroxylation of HIF-1α, suggesting increases in PHD activity. Importantly, expression of GPD1L attenuates the hypoxic response, preventing complete HIF-1α induction. We propose a model in which hypoxia-induced miR-210 represses GPD1L, contributing to suppression of PHD activity, and increases of HIF-1α protein levels.
PMID21941025	Myc enforces overexpression of EZH2 in early prostatic neoplasia via transcriptional and post-transcriptional mechanisms. EZH2 is part of the PRC2 polycomb repressive complex that is overexpressed in multiple cancer types and has been implicated in prostate cancer initiation and progression. Here, we identify EZH2 as a target of the MYC oncogene in prostate cancer and show that MYC coordinately regulates EZH2 through transcriptional and post-transcriptional means. Although prior studies in prostate cancer have revealed a number of possible mechanisms of EZH2 upregulation, these changes cannot account for the overexpression EZH2 in many primary prostate cancers, nor in most cases of high grade PIN. We report that upregulation of Myc in the mouse prostate results in overexpression of EZH2 mRNA and protein which coincides with reductions in miR-26a and miR-26b, known regulators of EZH2 in some non-prostate cell types, albeit not in others. Further, in human prostate cancer cells, Myc negatively regulates miR-26a and miR-26b via direct binding to their parental Pol II gene promoters, and forced overexpression of miR-26a and miR-26b in prostate cancer cells results in decreased EZH2 levels and suppressed proliferation. In human clinical samples, miR-26a and miR-26b are downregulated in most primary prostate cancers. As a separate mechanism of EZH2 mRNA upregulation, we find that Myc binds directly to and activates the transcription of the EZH2 promoter. These results link two major pathways in prostate cancer by providing two additional and complementary Myc-regulated mechanisms by which EZH2 upregulation occurs and is enforced during prostatic carcinogenesis. Further, the results implicate EZH2-driven mechanisms by which Myc may stimulate prostate tumor initiation and disease progression.
PMID19308091	BMP-6 inhibits microRNA-21 expression in breast cancer through repressing deltaEF1 and AP-1. MicroRNAs (miRNAs), which are small noncoding RNA molecules, play important roles in the post-transcriptional regulation process. The microRNA-21 gene (miR-21) has been reported to be highly expressed in various solid tumors, including breast cancer. Bone morphogenetic protein-6 (BMP-6) has been identified as an inhibitor of breast cancer epithelial-mesenchymal transition (EMT) through rescuing E-cadherin expression. We initiated experiments to identify the relationships between miR-21 and BMP-6 in breast cancer progression. Real-time PCR analysis showed that miR-21 expression was very high in MDA-MB-231 cells that expressed little BMP-6. A reverse correlation between BMP-6 and miR-21 was also determined in breast cancer tissue samples. Moreover, BMP-6 inhibited miR-21 transcription in MDA-MB-231 cells. In order to investigate how BMP-6 inhibited the miR-21 promoter (miPPR-21), we constructed a series of miPPR-21 reporters. Luciferase assay results indicated that BMP-6 inhibited miPPR-21 activity through the E2-box and AP-1-binding sites. We also demonstrated that both deltaEF1 and TPA induced miR-21 expression. Using site-directed mutation and CHIP assay, we found that deltaEF1 induced miPPR-21 activity by binding to the E2-box on miPPR-21. Moreover, TPA triggered miPPR-21 activity through the AP-1 binding sites. BMP-6 treatment significantly reduced the binding of these factors to miPPR-21 by decreasing the expression of deltaEF1 and c-Fos/c-Jun. We also demonstrated that BMP-6-induced downregulation of miR-21 modified the activity of PDCD4 3'UTR and inhibited MDA-MB-231 cell invasion. deltaEF1 overexpression and TPA induction blocked this inhibitory effect of BMP-6. In conclusion, BMP-6-induced inhibition of miR-21 suggests that BMP-6 may function as an anti-metastasis factor by a mechanism involving transcriptional repression of miR-21 in breast cancer.
PMID19150961	Stemness is only a state of the cell. How the programming and reprogramming of stem/progenitor cells regulate normal cell development and cancer is still not well known. One of the tools that we have chosen to use to investigate stem cell regulation is the transcriptional repressor element 1-silencing transcription factor (REST). REST contains a DNA-binding domain and two repressor domains. Once bound to its target genes, REST can interact with several cellular corepressors to regulate epigenetic modifications. REST is expressed in most nonneural cells, including neural stem/progenitor cells (NSCs), but it is absent in most neuronal cells. REST was originally found to be a major transcriptional repressor of neural differentiation. Previously, we found that activation of REST target genes in NSCs was sufficient to cause neuronal differentiation. Furthermore, the activation of REST target genes in myoblasts was sufficient to override the muscle differentiation pathway and produce a physiologically active neuronal phenotype. Although REST is normally not expressed in most neural cells, we previously found that approximately 50% of human medulloblastomas, a malignant pediatric brain tumor, express REST and that abnormal expression of REST in NSCs causes medulloblastoma-like cerebellar tumors by blocking neuronal differentiation. Interestingly, REST is also expressed at high levels in mouse embryonic stem (mES) cells, but its role in these cells is not understood. Recently, we found that REST maintains self-renewal and pluripotency in mES cells through suppression of microRNA-21 (miRNA21). Thus, REST is a newly discovered element of the interconnected regulatory network that maintains the self-renewal and pluripotency of mES cells. Taken together, the results of several different studies indicate that stem/progenitor cells are more flexible than previously believed and that a simple alteration of transcriptional regulators in these cells can affect both normal cell development and cancer.
PMID19264808	Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells. Select changes in microRNA (miRNA) expression correlate with estrogen receptor alpha (ER alpha) expression in breast tumors. miR-21 is higher in ER alpha positive than negative tumors, but no one has examined how estradiol (E(2)) regulates miR-21 in breast cancer cells. Here we report that E(2) inhibits miR-21 expression in MCF-7 human breast cancer cells. The E(2)-induced reduction in miR-21 was inhibited by 4-hydroxytamoxifen (4-OHT), ICI 182 780 (Faslodex), and siRNA ER alpha indicating that the suppression is ER alpha-mediated. ER alpha and ER beta agonists PPT and DPN inhibited and 4-OHT increased miR-21 expression. E(2) increased luciferase activity from reporters containing the miR-21 recognition elements from the 3'-UTRs of miR-21 target genes, corroborating that E(2) represses miR-21 expression resulting in a loss of target gene suppression. The E(2)-mediated decrease in miR-21 correlated with increased protein expression of endogenous miR-21-targets Pdcd4, PTEN and Bcl-2. siRNA knockdown of ER alpha blocked the E(2)-induced increase in Pdcd4, PTEN and Bcl-2. Transfection of MCF-7 cells with antisense (AS) to miR-21 mimicked the E(2)-induced increase in Pdcd4, PTEN and Bcl-2. These results are the first to demonstrate that E(2) represses the expression of an oncogenic miRNA, miR-21, by activating estrogen receptor in MCF-7 cells.
PMID21515911	MicroRNA-155 is involved in the remodelling of human-trophoblast-derived HTR-8/SVneo cells induced by lipopolysaccharides. A low dose injection of lipopolysaccharides (LPS) may induce pre-eclampsia-like symptoms in rats, and microRNA-155 (miR-155) is elevated in the placentas of patients with pre-eclampsia. Our goal was to investigate the association of miR-155 with pre-eclampsia and the pathways involved using human-trophoblast-derived cell line (HTR-8/SVneo) stimulated with LPS. We measured miR-155 in HTR-8/SVneo cells treated with LPS (25-800 ng/ml) using real-time PCR. Western blotting was used to study transcription factor activated protein 1 (AP-1) (JunB and FosB subunits) and nuclear factor (NF)-κB p65 in the HTR-8/SVneo cells and placentas from patients with pre-eclampsia. DNA precipitation assays and luciferase reporter analysis were used to evaluate the regulation of miR-155 by AP-1 and NF-κB. Cell migration was determined by scratch assay. Syncytialization of HTR-8/SVneo cells was analysed following transfection with miR-155. miR-155 was increased together with AP-1 and NF-κB in HTR-8/SVneo cells incubated with low dose of LPS (≤100 ng/ml; P < 0.05 versus baseline). Both JunB/FosB and p65 were increased in placenta from women with severe pre-eclampsia versus a normal pregnancy, with elevated expression of miR-155 (P < 0.05). For specific DNA-binding sites upstream of BIC/miR-155 gene promoter, the AP-1 site was more important than the NF-κB site for increasing miR-155 in HTR-8/SVneo cells. The cells with enforced expression of miR-155 showed a reduced ability to migrate (P < 0.05) and an increased number of syncytiotrophoblast-like multinuclear cells (P < 0.05). LPS may induce remodelling of the human-trophoblast-derived HTR-8/SVneo cells by increasing miR-155, acting in part through the AP-1 and NF-κB pathways.
PMID19088079	Induction of microRNA-221 by platelet-derived growth factor signaling is critical for modulation of vascular smooth muscle phenotype. The platelet-derived growth factor (PDGF) signaling pathway is a critical regulator of animal development and homeostasis. Activation of the PDGF pathway leads to neointimal proliferative responses to artery injury; it promotes a switch of vascular smooth muscle cells (vSMC) to a less contractile phenotype by inhibiting the SMC-specific gene expression and increasing the rate of proliferation and migration. The molecular mechanism for these pleiotropic effects of PDGFs has not been fully described. Here, we identify the microRNA-221 (miR-221), a small noncoding RNA, as a modulator of the phenotypic change of vSMCs in response to PDGF signaling. We demonstrate that miR-221 is transcriptionally induced upon PDGF treatment in primary vSMCs, leading to down-regulation of the targets c-Kit and p27Kip1. Down-regulation of p27Kip1 by miR-221 is critical for PDGF-mediated induction of cell proliferation. Additionally, decreased c-Kit causes inhibition of SMC-specific contractile gene transcription by reducing the expression of Myocardin (Myocd), a potent SMC-specific nuclear coactivator. Our study demonstrates that PDGF signaling, by modulating the expression of miR-221, regulates two critical determinants of the vSMC phenotype; they are SMC gene expression and cell proliferation.
PMID19169275	Regulation of microRNA expression by HMGA1 proteins. The High Mobility Group proteins HMGA1 are nuclear architectural factors that play a critical role in a wide range of biological processes. Since recent studies have identified the microRNAs (miRNAs) as important regulators of gene expression, modulating critical cellular functions such as proliferation, apoptosis and differentiation, the aim of our work was to identify the miRNAs that are physiologically regulated by HMGA1 proteins. To this purpose, we have analysed the miRNA expression profile of mouse embryonic fibroblasts (MEFs) carrying two, one or no Hmga1 functional alleles using a microarray (miRNA microarray). By this approach, we found a miRNA expression profile that differentiates Hmga1-null MEFs from the wild-type ones. In particular, a significant decrease in miR-196a-2, miR-101b, miR-331 and miR-29a was detected in homozygous Hmga1-knockout MEFs in comparison with wild-type cells. Consistently, these miRNAs are downregulated in most of the analysed tissues of Hmga1-null mice in comparison with the wild-type mice. ChIP assay shows that HMGA1 is able to bind regions upstream of these miRNAs. Moreover, we identified the HMGA2 gene product as a putative target of miR-196a-2, suggesting that HMGA1 proteins are able to downregulate the expression of the other member of the HMGA family through the regulation of the miR-196a-2 expression. Finally, ATXN1 and STC1 gene products have been identified as targets of miR-101b. Therefore, it is reasonable to hypothesize that HMGA1 proteins are involved in several functions by regulating miRNA expression.
PMID22231442	Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer. Estrogen effects on mammary epithelial and breast cancer (BC) cells are mediated by the nuclear receptors ERα and ERβ, transcription factors that display functional antagonism with each other, with ERβ acting as oncosuppressor and interfering with the effects of ERα on cell proliferation, tumor promotion and progression. Indeed, hormone-responsive, ERα+ BC cells often lack ERβ, which when present associates with a less aggressive clinical phenotype of the disease. Recent evidences point to a significant role of microRNAs (miRNAs) in BC, where specific miRNA expression profiles associate with distinct clinical and biological phenotypes of the lesion. Considering the possibility that ERβ might influence BC cell behavior via miRNAs, we compared miRNome expression in ERβ+ vs ERβ- hormone-responsive BC cells and found a widespread effect of this ER subtype on the expression pattern of these non-coding RNAs. More importantly, the expression pattern of 67 miRNAs, including 10 regulated by ERβ in BC cells, clearly distinguishes ERβ+, node-negative, from ERβ-, metastatic, mammary tumors. Molecular dissection of miRNA biogenesis revealed multiple mechanisms for direct regulation of this process by ERβ+ in BC cell nuclei. In particular, ERβ downregulates miR-30a by binding to two specific sites proximal to the gene and thereby inhibiting pri-miR synthesis. On the other hand, the receptor promotes miR-23b, -27b and 24-1 accumulation in the cell by binding in close proximity of the corresponding gene cluster and preventing in situ the inhibitory effects of ERα on pri-miR maturation by the p68/DDX5-Drosha microprocessor complex. These results indicate that cell autonomous regulation of miRNA expression is part of the mechanism of action of ERβ in BC cells and could contribute to establishment or maintenance of a less aggressive tumor phenotype mediated by this nuclear receptor.
PMID22388088	Tyrosine phosphatase SHP2 promotes breast cancer progression and maintains tumor-initiating cells via activation of key transcription factors and a positive feedback signaling loop. New cancer therapies are likely to arise from an in-depth understanding of the signaling networks influencing tumor initiation, progression and metastasis. We show a fundamental role for Src-homology 2 domain-containing phosphatase 2 (SHP2) in these processes in human epidermal growth factor receptor 2 (HER2)-positive and triple-negative breast cancers. Knockdown of SHP2 eradicated breast tumor-initiating cells in xenograft models, and SHP2 depletion also prevented invasion in three-dimensional cultures and in a transductal invasion assay in vivo. Notably, SHP2 knockdown in established breast tumors blocked their growth and reduced metastasis. Mechanistically, SHP2 activated stemness-associated transcription factors, including v-myc myelocytomatosis viral oncogene homolog (c-Myc) and zinc finger E-box binding homeobox 1 (ZEB1), which resulted in the repression of let-7 microRNA and the expression of a set of 'SHP2 signature' genes. We found these genes to be simultaneously activated in a large subset of human primary breast tumors that are associated with invasive behavior and poor prognosis. These results provide new insights into the signaling cascades influencing tumor-initiating cells as well as a rationale for targeting SHP2 in breast cancer.
PMID20308562	Hypoxia inducible microRNA 210 attenuates keratinocyte proliferation and impairs closure in a murine model of ischemic wounds. Ischemia complicates wound closure. Here, we are unique in presenting a murine ischemic wound model that is based on bipedicle flap approach. Using this model of ischemic wounds we have sought to elucidate how microRNAs may be implicated in limiting wound re-epithelialization under hypoxia, a major component of ischemia. Ischemia, evaluated by laser Doppler as well as hyperspectral imaging, limited blood flow and lowered tissue oxygen saturation. EPR oximetry demonstrated that the ischemic wound tissue had pO(2) <10 mm Hg. Ischemic wounds suffered from compromised macrophage recruitment and delayed wound epithelialization. Specifically, epithelial proliferation, as determined by Ki67 staining, was compromised. In vivo imaging showed massive hypoxia inducible factor-1alpha (HIF-1alpha) stabilization in ischemic wounds, where HIF-1alpha induced miR-210 expression that, in turn, silenced its target E2F3, which was markedly down-regulated in the wound-edge tissue of ischemic wounds. E2F3 was recognized as a key facilitator of cell proliferation. In keratinocytes, knock-down of E2F3 limited cell proliferation. Forced stabilization of HIF-1alpha using Ad-VP16- HIF-1alpha under normoxic conditions up-regulated miR-210 expression, down-regulated E2F3, and limited cell proliferation. Studies using cellular delivery of miR-210 antagomir and mimic demonstrated a key role of miR-210 in limiting keratinocyte proliferation. In summary, these results are unique in presenting evidence demonstrating that the hypoxia component of ischemia may limit wound re-epithelialization by stabilizing HIF-1alpha, which induces miR-210 expression, resulting in the down-regulation of the cell-cycle regulatory protein E2F3.
PMID21857159	MicroRNA-449 in cell fate determination. The microRNAs 449a, b, and c (miR-449) are potent inducers of cell death, cell cycle arrest, and/or cell differentiation. They belong to the same family as the p53-responsive microRNAs miR-34. Instead of p53, however, the cell cycle regulatory transcription factor E2F1 induces miR-449. All members of this microRNA family are capable of mediating cell cycle arrest and apoptosis and might thereby contribute to tumor suppression. Underlying mechanisms include the downregulation of histone acetyl transferases and consecutive activation of p53, but also the targeting of cyclin dependent kinases and their association partners. Thus, miR-34 and miR-449 provide an asymmetric feedback loop to balance E2F and p53 activities. More recently, it was discovered that miR-449 displays strong tissue specificity, with high levels in lung and testes. Two model systems (Xenopus embryos and cultured human cells) revealed that miR-449 is essential for the development of ciliated epithelia, and this appears to depend on miR-449-mediated modulation of the Notch signaling pathway. Here we summarize our current knowledge on cell fate determination by miR-449, and we propose future directions to explore the function of miR-449 in cell regulation and organismal development. MiR-449 helps to ensure proper cell function but also to avoid cancer, marking a close link between cell differentiation and tumor suppression.
PMID21738483	A functional variant in microRNA-146a promoter modulates its expression and confers disease risk for systemic lupus erythematosus. Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a strong genetic predisposition, characterized by an upregulated type I interferon pathway. MicroRNAs are important regulators of immune homeostasis, and aberrant microRNA expression has been demonstrated in patients with autoimmune diseases. We recently identified miR-146a as a negative regulator of the interferon pathway and linked the abnormal activation of this pathway to the underexpression of miR-146a in SLE patients. To explore why the expression of miR-146a is reduced in SLE patients, we conducted short parallel sequencing of potentially regulatory regions of miR-146a and identified a novel genetic variant (rs57095329) in the promoter region exhibiting evidence for association with SLE that was replicated independently in 7,182 Asians (P(meta) = 2.74×10(-8), odds ratio = 1.29 [1.18-1.40]). The risk-associated G allele was linked to reduced expression of miR-146a in the peripheral blood leukocytes of the controls. Combined functional assays showed that the risk-associated G allele reduced the protein-binding affinity and activity of the promoter compared with those of the promoter containing the protective A allele. Transcription factor Ets-1, encoded by the lupus-susceptibility gene ETS1, identified in recent genome-wide association studies, binds near this variant. The manipulation of Ets-1 levels strongly affected miR-146a promoter activity in vitro; and the knockdown of Ets-1, mimicking its reduced expression in SLE, directly impaired the induction of miR-146a. We also observed additive effects of the risk alleles of miR-146a and ETS1. Our data identified and confirmed an association between a functional promoter variant of miR-146a and SLE. This risk allele had decreased binding to transcription factor Ets-1, contributing to reduced levels of miR-146a in SLE patients.
PMID22226905	OxLDL causes both epigenetic modification and signaling regulation on the microRNA-29b gene: novel mechanisms for cardiovascular diseases. MicroRNA-29b has been reported to epigenetically regulate proatherogenic genes in response to oxLDL. Since transcription factors and epigenetic regulations are important mechanisms to regulate gene expression, we investigated whether these mechanisms are involved in oxLDL-induced microRNA-29b upregulation. First, we confirmed that microRNA-29b expression was increased in the aorta of mice fed with a high-fat diet, which was consistent with our previous in vitro findings. Next, we found that oxLDL only activated the microRNA-29b-1/microRNA-29a cluster gene on chromosome 7 but not the other distinct microRNA-29b gene located on chromosome 1. Using the promoter reporter assay and chromatin immunoprecipitation, activator protein-1 (AP-1) was shown to bind to the microRNA-29b-1 promoter. We further identified the signaling pathway of LOX-1/Ca(2+)/ROS/ERK/c-Fos was involved in oxLDL-mediated microRNA-29b overexpression after treating with the MAPTAM (Ca(2+) chelator), NAC (ROS scavenger), U0126 (ERK inhibitor) and c-Fos (one of the AP-1 proteins) shRNA, respectively. To investigate epigenetic regulations, we found that microRNA-29b promoter contained no CpG islands for DNA methylation. Therefore we investigated whether histone modifications influence microRNA-29b promoter activity. We showed that down-regulation of HDAC1 and the modifications on histone 3 lysine 4 (H3K4) and H3K9 significantly affected microRNA-29b expression. Furthermore, knockdown of c-Fos expression attenuated the effect of oxLDL-induced histone modifications on the microRNA-29b gene expression. Taken together, our data suggest that both transcription factor activation and histone modifications are important regulatory mechanisms of oxLDL-induced atherogenic process. This article is part of a Special Issue entitled OxLDL causes both epigenetic modification and signaling regulation on the microRNA-29b gene: Novel mechanisms for cardiovascular diseases.
PMID21673316	TRPS1 targeting by miR-221/222 promotes the epithelial-to-mesenchymal transition in breast cancer. The basal-like subtype of breast cancer has an aggressive clinical behavior compared to that of the luminal subtype. We identified the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) as basal-like subtype-specific miRNAs and showed that expression of miR-221/222 decreased expression of epithelial-specific genes and increased expression of mesenchymal-specific genes, and increased cell migration and invasion in a manner characteristic of the epithelial-to-mesenchymal transition (EMT). The transcription factor FOSL1 (also known as Fra-1), which is found in basal-like breast cancers but not in the luminal subtype, stimulated the transcription of miR-221/222, and the abundance of these miRNAs decreased with inhibition of the epidermal growth factor receptor (EGFR) or MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), placing miR-221/222 downstream of the RAS pathway. Furthermore, miR-221/222-mediated reduction in E-cadherin abundance depended on their targeting the 3' untranslated region of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1), which inhibited EMT by decreasing ZEB2 (zinc finger E-box-binding homeobox2) expression. We conclude that by promoting EMT, miR-221/222 may contribute to the more aggressive clinical behavior of basal-like breast cancers.
PMID17823410	MicroRNA-34b and MicroRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth. MicroRNAs (miRNA) are a recently discovered class of noncoding RNAs that negatively regulate gene expression. Recent evidence indicates that miRNAs may play an important role in cancer. However, the mechanism of their deregulation in neoplastic transformation has only begun to be understood. To elucidate the role of tumor suppressor p53 in regulation of miRNAs, we have analyzed changes in miRNA microarray expression profile immediately after conditional inactivation of p53 in primary mouse ovarian surface epithelium cells. Among the most significantly affected miRNAs were miR-34b and miR-34c, which were down-regulated 12-fold according to quantitative reverse transcription-PCR analysis. Computational promoter analysis of the mir-34b/mir-34c locus identified the presence of evolutionarily conserved p53 binding sites approximately 3 kb upstream of the miRNA coding sequence. Consistent with evolutionary conservation, mir-34b/mir-34c were also down-regulated in p53-null human ovarian carcinoma cells. Furthermore, as expected from p53 binding to the mir-34b/c promoter, doxorubicin treatment of wild-type, but not p53-deficient, cells resulted in an increase of mir-34b/mir-34c expression. Importantly, miR-34b and miR-34c cooperate in suppressing proliferation and soft-agar colony formation of neoplastic epithelial ovarian cells, in agreement with the partially overlapping spectrum of their predicted targets. Taken together, these results show the existence of a novel mechanism by which p53 suppresses such critical components of neoplastic growth as cell proliferation and adhesion-independent colony formation.
PMID19617899	Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. Growing evidence indicates that microRNAs have a significant role in tumor development and may constitute robust biomarkers for cancer diagnosis and prognosis. In this study, we evaluated the clinical and functional relevance of microRNA-122 (miR-122) expression in human hepatocellular carcinoma (HCC). We report that miR-122 is specifically repressed in a subset of primary tumors that are characterized by poor prognosis. We further show that the loss of miR-122 expression in tumor cells segregates with specific gene expression profiles linked to cancer progression, namely the suppression of hepatic phenotype and the acquisition of invasive properties. We identify liver-enriched transcription factors as central regulatory molecules in the gene networks associated with loss of miR-122, and provide evidence suggesting that miR-122 is under the transcriptional control of HNF1A, HNF3A and HNF3B. We further show that loss of miR-122 results in an increase of cell migration and invasion and that restoration of miR-122 reverses this phenotype. In conclusion, miR-122 is a marker of hepatocyte-specific differentiation and an important determinant in the control of cell migration and invasion. From a clinical point of view, our study emphasizes miR-122 as a diagnostic and prognostic marker for HCC progression.
PMID20018373	Complexity of miR-223 regulation by CEBPA in human AML. microRNA-223 (miR-223) can trigger normal granulopoiesis. miR-223 expression is regulated by two distinct CEBPA (CCAAT/enhancer binding protein-alpha) sites. Here, we report that miR-223 is largely suppressed in cells from acute myeloid leukemia (AML) patients. By sequencing, we found that miR-223 suppression in AML is not caused by DNA sequence alterations, nor is it mediated by promoter hypermethylation. The analysis of the individual contribution of both CEBPA sites to miR-223 regulation identified the site upstream of the miR-223 primary transcript as the predominant regulatory element. Our results suggest that miR-223 suppression in AML is caused by impaired miR-223 upstream factors.
PMID19269368	The TRIM-NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural progenitors. In the mouse neocortex, neural progenitor cells generate both differentiating neurons and daughter cells that maintain progenitor fate. Here, we show that the TRIM-NHL protein TRIM32 regulates protein degradation and microRNA activity to control the balance between those two daughter cell types. In both horizontally and vertically dividing progenitors, TRIM32 becomes polarized in mitosis and is concentrated in one of the two daughter cells. TRIM32 overexpression induces neuronal differentiation while inhibition of TRIM32 causes both daughter cells to retain progenitor cell fate. TRIM32 ubiquitinates and degrades the transcription factor c-Myc but also binds Argonaute-1 and thereby increases the activity of specific microRNAs. We show that Let-7 is one of the TRIM32 targets and is required and sufficient for neuronal differentiation. TRIM32 is the mouse ortholog of Drosophila Brat and Mei-P26 and might be part of a protein family that regulates the balance between differentiation and proliferation in stem cell lineages.
PMID18922890	MicroRNA-7, a homeobox D10 target, inhibits p21-activated kinase 1 and regulates its functions. MicroRNAs are noncoding RNAs that inhibit the expression of their targets in a sequence-specific manner and play crucial roles during oncogenesis. Here we show that microRNA-7 (miR-7) inhibits p21-activated kinase 1 (Pak1) expression, a widely up-regulated signaling kinase in multiple human cancers, by targeting the 3'-untranslated region (UTR) of Pak1 mRNA. We noticed an inverse correlation between the levels of endogenous miR-7 and Pak1 expression in human cancer cells. We discovered that endogenous miR-7 expression is positively regulated by a homeodomain transcription factor, HoxD10, the loss of which leads to an increased invasiveness. HoxD10 directly interacts with the miR-7 chromatin. Accordingly, the levels of Pak1 protein are progressively up-regulated whereas those of miR-7 and its upstream activator HoxD10 are progressively down-regulated in a cellular model of breast cancer progression from low to highly invasive phenotypes. Furthermore, HoxD10 expression in highly invasive breast cancer cells resulted in an increased miR-7 expression but reduced Pak1 3'-UTR-luciferase activity and reduced Pak1 protein. Finally, we show that miR-7 introduction inhibits the motility, invasiveness, anchorage-independent growth, and tumorigenic potential of highly invasive breast cancer cells. Collectively, these findings establish for the first time that Pak1 is a target of miR-7 and that HoxD10 plays a regulatory role in modifying the expression of miR-7 and, consequently, the functions of the miR-7-Pak1 pathway in human cancer cells.
PMID20833636	p53 activates the PANK1/miRNA-107 gene leading to downregulation of CDK6 and p130 cell cycle proteins. The tumor suppressor p53 is a central regulator of cell-cycle arrest and apoptosis by acting as a transcription factor to regulate numerous genes. We identified all human p53-regulated mRNAs by microarray analyses and searched for protein-coding genes which contain intronic miRNAs. Among others, this analysis yielded the panthothenate kinase 1 (PANK1) gene and its intronic miRNA-107. We showed that miRNA-107 and PANK1 are coregulated by p53 in different cell systems. The PANK1 protein, which catalyzes the rate-limiting step of coenzyme A biosynthesis, is also upregulated by p53. We observed that p53 directly activates PANK1 and miRNA-107 transcription through a binding site in the PANK1 promoter. Furthermore, p53 is recruited to the PANK1 promoter after DNA damage. In order to get more insight into miRNA-107 function we investigated its potential target genes. Cell-cycle regulators are significantly enriched among predicted miRNA-107 targets. We found miRNA-107-dependent regulation of two important regulators of G(1)/S progression, CDK6 and the RB-related 2 gene RBL2 (p130). CDK6 and p130 proteins are downregulated upon miRNA-107 expression. Our results uncover a novel miRNA-dependent signaling pathway which leads to downregulation of cell cycle proteins in the absence of transcriptional repression.
PMID20399246	MIR-23A microRNA cluster inhibits B-cell development. The transcription factor PU.1 (encoded by Sfpi1) promotes myeloid differentiation, but it is unclear what downstream genes are involved. Micro RNAs (miRNAs) are a class of small RNAs that regulate many cellular pathways, including proliferation, survival, and differentiation. The objective of this study was to identify miRNAs downstream of PU.1 that regulate hematopoietic development. miRNAs that change expression in a PU.1-inducible cell line were identified with microarrays. The promoter for an miRNA cluster upregulated by PU.1 induction was analyzed for PU.1 binding by electrophoretic mobility shift and chromatin immunoprecipitation assays. Retroviral transduction of hematopoietic progenitors was performed to evaluate the effect of miRNA expression on hematopoietic development in vitro and in vivo. We identified an miRNA cluster whose pri-transcript is regulated by PU.1. The pri-miRNA encodes three mature miRNAs: miR-23a, miR-27a, and miR-24-2. Each miRNA is more abundant in myeloid cells compared to lymphoid cells. When hematopoietic progenitors expressing the 23a cluster miRNAs were cultured in B-cell-promoting conditions, we observed a dramatic decrease in B lymphopoiesis and an increase in myelopoiesis compared to control cultures. In vivo, hematopoietic progenitors expressing the miR-23a cluster generate reduced numbers of B cells compared to control cells. The miR-23a cluster is a downstream target of PU.1 involved in antagonizing lymphoid cell fate acquisition. Although miRNAs have been identified downstream of PU.1 in mediating development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in regulating development of myeloid vs lymphoid cells.