PMID23015123	Ion channels/transporters as epigenetic regulators? -a microRNA perspective. MicroRNA (miRNA) alterations in response to changes in an extracellular microenvironment have been observed and considered as one of the major mechanisms for epigenetic modifications of the cell. While enormous efforts have been made in the understanding of the role of miRNAs in regulating cellular responses to the microenvironment, the mechanistic insight into how extracellular signals can be transduced into miRNA alterations in cells is still lacking. Interestingly, recent studies have shown that ion channels/transporters, which are known to conduct or transport ions across the cell membrane, also exhibit changes in levels of expression and activities in response to changes of extracellular microenvironment. More importantly, alterations in expression and function of ion channels/transporters have been shown to result in changes in miRNAs that are known to change in response to alteration of the microenvironment. In this review, we aim to summarize the recent data demonstrating the ability of ion channels/transporters to transduce extracellular signals into miRNA changes and propose a potential link between cells and their microenvironment through ion channels/transporters. At the same time, we hope to provide new insights into epigenetic regulatory mechanisms underlying a number of physiological and pathological processes, including embryo development and cancer metastasis.
PMID23436935	Regulation of cystic fibrosis transmembrane conductance regulator by microRNA-145, -223, and -494 is altered in ΔF508 cystic fibrosis airway epithelium. Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is altered in individuals with the ΔF508 CFTR mutation. We previously reported differential expression of microRNA (miRNA) in CF airway epithelium; however, the role of miRNA in regulation of CFTR expression here remains unexplored. In this study, we investigated the role of upregulated miRNAs in CFTR regulation in vivo in bronchial brushings from individuals homozygous or heterozygous for ΔF508 CFTR, validated our observations in vitro, and assessed the impact of defective chloride ion conductance, genotype, and colonization status on miRNA expression. miRNA target prediction was performed in silico, and expression of miRNA and target genes were measured by quantitative real-time PCR and/or Western blotting. Overexpression and inhibition studies were performed with pre-miRs or antimiRs, respectively, and a luciferase reporter gene was used to elucidate direct miRNA-target interactions. miR-145, miR-223, and miR-494 were upregulated in CF versus non-CF bronchial brushings and cell lines; in ΔF508 CFTR homozygotes versus heterozygotes; in subjects positive for P. aeruginosa; and in cells treated with a CFTR inhibitor or IL-1β. Reciprocal downregulation or upregulation of CFTR gene and/or protein expression was observed after miRNA manipulation and direct miRNA-target relationships demonstrated via a reporter system containing a wild type or mutated full-length CFTR 3' untranslated region. Increased expression of miR-145, miR-223, and miR-494 in vivo in bronchial epithelium of individuals carrying the ΔF508 CFTR mutation correlates with decreased CFTR expression. Defective CFTR function, Pseudomonas colonization, and inflammation may affect miRNA expression and contribute to the regulation of ΔF508 CFTR.
PMID24909068	Emerging role of cystic fibrosis transmembrane conductance regulator as an epigenetic regulator: linking environmental cues to microRNAs. Although microRNAs (miRNAs) have been recognized as one of the important epigenetic mechanisms that regulate gene expression in response to changes in the environment, the links between environmental cues and changes in miRNAs remain largely unknown. Localized to the cell membrane and recognized as an anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR) has recently been shown to mediate important signalling pathways leading to activation of miRNAs. This brief review summarizes the related findings and discusses the emerging role of CFTR as an epigenetic regulator, possibly involved in a wide spectrum of physiological and pathological processes.
PMID21936826	SLPI and inflammatory lung disease in females. During the course of certain inflammatory lung diseases, SLPI (secretory leucoprotease inhibitor) plays a number of important roles. As a serine antiprotease it functions to protect the airways from proteolytic damage due to neutrophil and other immune cell-derived serine proteases. With respect to infection it has known antimicrobial and anti-viral properties that are likely to contribute to host defence. Another of its properties is the ability to control inflammation within the lung where it can interfere with the transcriptional induction of pro-inflammatory gene expression induced by NF-κB (nuclear factor κB). Thus, factors that regulate the expression of SLPI in the airways can impact on disease severity and outcome. Gender represents once such idiosyncratic factor. In females with CF (cystic fibrosis), it is now thought that circulating oestrogen contributes, in part, to the observed gender gap whereby females have worse disease and poorer prognosis than males. Conversely, in asthma, sufferers who are females have more frequent exacerbations at times of low-circulating oestrogen. In the present paper, we discuss how SLPI participates in these events and speculate on whether regulatory mechanisms such as post-transcriptional modulation by miRNAs (microRNAs) are important in the control of SLPI expression in inflammatory lung disease.
PMID22664907	CFTR mediates bicarbonate-dependent activation of miR-125b in preimplantation embryo development. Although HCO(3)(-) is known to be required for early embryo development, its exact role remains elusive. Here we report that HCO(3)(-) acts as an environmental cue in regulating miR-125b expression through CFTR-mediated influx during preimplantation embryo development. The results show that the effect of HCO(3)(-) on preimplantation embryo development can be suppressed by interfering the function of a HCO(3)(-)-conducting channel, CFTR, by a specific inhibitor or gene knockout. Removal of extracellular HCO(3)(-) or inhibition of CFTR reduces miR-125b expression in 2 cell-stage mouse embryos. Knockdown of miR-125b mimics the effect of HCO(3)(-) removal and CFTR inhibition, while injection of miR-125b precursor reverses it. Downregulation of miR-125b upregulates p53 cascade in both human and mouse embryos. The activation of miR-125b is shown to be mediated by sAC/PKA-dependent nuclear shuttling of NF-κB. These results have revealed a critical role of CFTR in signal transduction linking the environmental HCO(3)(-) to activation of miR-125b during preimplantation embryo development and indicated the importance of ion channels in regulation of miRNAs.
PMID24940638	miR-31 dysregulation in cystic fibrosis airways contributes to increased pulmonary cathepsin S production. Cathepsin S (CTSS) activity is increased in bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis (CF). This activity contributes to lung inflammation via degradation of antimicrobial proteins, such as lactoferrin and members of the β-defensin family. In this study, we investigated the hypothesis that airway epithelial cells are a source of CTSS, and mechanisms underlying CTSS expression in the CF lung. Protease activity was determined using fluorogenic activity assays. Protein and mRNA expression were analyzed by ELISA, Western blotting, and reverse-transcriptase polymerase chain reaction. In contrast to neutrophil elastase, CTSS activity was detectable in 100% of CF BAL fluid samples from patients without Pseudomonas aeruginosa infection. In this study, we identified epithelial cells as a source of pulmonary CTSS activity with the demonstration that CF airway epithelial cells express and secrete significantly more CTSS than non-CF control cells in the absence of proinflammatory stimulation. Furthermore, levels of the transcription factor IRF-1 correlated with increased levels of its target gene CTSS. We discovered that miR-31, which is decreased in the CF airways, regulates IRF-1 in CF epithelial cells. Treating CF bronchial epithelial cells with a miR-31 mimic decreased IRF-1 protein levels with concomitant knockdown of CTSS expression and secretion. The miR-31/IRF-1/CTSS pathway may play a functional role in the pathogenesis of CF lung disease and may open up new avenues for exploration in the search for an effective therapeutic target.
PMID22797075	CFTR suppresses tumor progression through miR-193b targeting urokinase plasminogen activator (uPA) in prostate cancer. Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is expressed in the epithelial cells of a wide range of organs/tissues from which most cancers are derived. Although accumulating reports have indicated the association of cancer incidence with genetic variations in CFTR gene, the exact role of CFTR in cancer development and the possible underlying mechanism have not been elucidated. Here, we report that CFTR expression is significantly decreased in both prostate cancer cell lines and human prostate cancer tissue samples. Overexpression of CFTR in prostate cancer cell lines suppresses tumor progression (cell growth, adhesion and migration), whereas knockdown of CFTR leads to enhanced malignancies both in vitro and in vivo. In addition, we demonstrate that CFTR knockdown-enhanced cell proliferation, cell invasion and migration are significantly reversed by antibodies against either urokinase plasminogen activator (uPA) or uPA receptor (uPAR), which are known to be involved in various malignant traits of cancer development. More interestingly, overexpression of CFTR suppresses uPA by upregulating the recently described tumor suppressor microRNA-193b (miR-193b), and overexpression of pre-miR-193b significantly reverses CFTR knockdown-enhanced malignant phenotype and abrogates elevated uPA activity in prostate cancer cell line. Finally, we show that CFTR gene transfer results in significant tumor repression in prostate cancer xenografts in vivo. Taken together, the present study has demonstrated a previously undefined tumor-suppressing role of CFTR and its involvement in regulation of miR-193b in prostate cancer development.
PMID26869937	Targeting MicroRNA Function in Respiratory Diseases: Mini-Review. MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate expression of the majority of genes by inhibiting protein translation. Growing literature has identified functional roles for miRNAs across a broad range of biological processes. As such, miRNAs are recognized as potential disease biomarkers and novel targets for therapies. While several miRNA-targeted therapies are currently in clinical trials (e.g., for the treatment of hepatitis C virus infection and cancer), no therapies have targeted miRNAs in respiratory diseases in the clinic. In this mini-review, we review the current knowledge on miRNA expression and function in respiratory diseases, intervention strategies to target miRNA function, and considerations specific to respiratory diseases. Altered miRNA expression profiles have been reported in a number of respiratory diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, and idiopathic pulmonary fibrosis. These include alterations in isolated lung tissue, as well as sputum, bronchoalveolar lavage fluids and peripheral blood or serum. The observed alterations in easily accessible body fluids (e.g., serum) have been proposed as new biomarkers that may inform disease diagnosis and patient management. In a subset of studies, miRNA-targeted interventions also improved disease outcomes, indicating functional roles for altered miRNA expression in disease pathogenesis. In fact, direct administration of miRNA-targeting molecules to the lung has yielded promising results in a number of animal models. The ability to directly administer compounds to the lung holds considerable promise and may limit potential off-target effects and side effects caused by the systemic administration required to treat other diseases.
PMID26166255	MicroRNA expression profiles differentiate chronic pain condition subtypes. Chronic pain is a significant health care problem, ineffectively treated because of its unclear etiology and heterogeneous clinical presentation. Emerging evidence demonstrates that microRNAs (miRNAs) regulate the expression of pain-relevant genes, yet little is known about their role in chronic pain. Here, we evaluate the relationship among pain, psychological characteristics, plasma cytokines, and whole blood miRNAs in 22 healthy controls (HCs); 33 subjects with chronic pelvic pain (vestibulodynia, VBD); and 23 subjects with VBD and irritable bowel syndrome (VBD + IBS). VBD subjects were similar to HCs in self-reported pain, psychological profiles, and remote bodily pain. VBD + IBS subjects reported decreased health and function; and an increase in headaches, somatization, and remote bodily pain. Furthermore, VBD subjects exhibited a balance in proinflammatory and anti-inflammatory cytokines, whereas VBD + IBS subjects failed to exhibit a compensatory increase in anti-inflammatory cytokines. VBD subjects differed from controls in expression of 10 miRNAs of predicted importance for pain and estrogen signaling. VBD + IBS subjects differed from controls in expression of 11 miRNAs of predicted importance for pain, cell physiology, and insulin signaling. miRNA expression was correlated with pain-relevant phenotypes and cytokine levels. These results suggest that miRNAs represent a valuable tool for differentiating VBD subtypes (localized pain with apparent peripheral neurosensory disruption vs widespread pain with a central sensory contribution) that may require different treatment approaches.
PMID25186262	Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis. The CFTR gene displays a tightly regulated tissue-specific and temporal expression. Mutations in this gene cause cystic fibrosis (CF). In this study we wanted to identify trans-regulatory elements responsible for CFTR differential expression in fetal and adult lung, and to determine the importance of inhibitory motifs in the CFTR-3'UTR with the aim of developing new tools for the correction of disease-causing mutations within CFTR. We show that lung development-specific transcription factors (FOXA, C/EBP) and microRNAs (miR-101, miR-145, miR-384) regulate the switch from strong fetal to very low CFTR expression after birth. By using miRNome profiling and gene reporter assays, we found that miR-101 and miR-145 are specifically upregulated in adult lung and that miR-101 directly acts on its cognate site in the CFTR-3'UTR in combination with an overlapping AU-rich element. We then designed miRNA-binding blocker oligonucleotides (MBBOs) to prevent binding of several miRNAs to the CFTR-3'UTR and tested them in primary human nasal epithelial cells from healthy individuals and CF patients carrying the p.Phe508del CFTR mutation. These MBBOs rescued CFTR channel activity by increasing CFTR mRNA and protein levels. Our data offer new understanding of the control of the CFTR gene regulation and new putative correctors for cystic fibrosis.
PMID24829907	Exploitation of a very small peptide nucleic acid as a new inhibitor of miR-509-3p involved in the regulation of cystic fibrosis disease-gene expression. Computational techniques, and in particular molecular dynamics (MD) simulations, have been successfully used as a complementary technique to predict and analyse the structural behaviour of nucleic acids, including peptide nucleic acid- (PNA-) RNA hybrids. This study shows that a 7-base long PNA complementary to the seed region of miR-509-3p, one of the miRNAs involved in the posttranscriptional regulation of the CFTR disease-gene of Cystic Fibrosis, and bearing suitable functionalization at its N- and C-ends aimed at improving its resistance to nucleases and cellular uptake, is able to revert the expression of the luciferase gene containing the 3'UTR of the gene in A549 human lung cancer cells, in agreement with the MD results that pointed at the formation of a stable RNA/PNA heteroduplex notwithstanding the short sequence of the latter. The here reported results widen the interest towards the use of small PNAs as effective anti-miRNA agents.
PMID26160865	miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production. Interleukin (IL)-8 levels are higher than normal in cystic fibrosis (CF) airways, causing neutrophil infiltration and non-resolving inflammation. Overexpression of microRNAs that target IL-8 expression in airway epithelial cells may represent a therapeutic strategy for cystic fibrosis. IL-8 protein and mRNA were measured in cystic fibrosis and non-cystic fibrosis bronchoalveolar lavage fluid and bronchial brushings (n=20 per group). miRNAs decreased in the cystic fibrosis lung and predicted to target IL-8 mRNA were quantified in βENaC-transgenic, cystic fibrosis transmembrane conductance regulator (Cftr)-/- and wild-type mice, primary cystic fibrosis and non-cystic fibrosis bronchial epithelial cells and a range of cystic fibrosis versus non-cystic fibrosis airway epithelial cell lines or cells stimulated with lipopolysaccharide, Pseudomonas-conditioned medium or cystic fibrosis bronchoalveolar lavage fluid. The effect of miRNA overexpression on IL-8 protein production was measured. miR-17 regulates IL-8 and its expression was decreased in adult cystic fibrosis bronchial brushings, βENaC-transgenic mice and bronchial epithelial cells chronically stimulated with Pseudomonas-conditioned medium. Overexpression of miR-17 inhibited basal and agonist-induced IL-8 protein production in F508del-CFTR homozygous CFTE29o(-) tracheal, CFBE41o(-) and/or IB3 bronchial epithelial cells. These results implicate defective CFTR, inflammation, neutrophilia and mucus overproduction in regulation of miR-17. Modulating miR-17 expression in cystic fibrosis bronchial epithelial cells may be a novel anti-inflammatory strategy for cystic fibrosis and other chronic inflammatory airway diseases.
PMID25687559	New insights about miRNAs in cystic fibrosis. The molecular basis of cystic fibrosis (CF) is a mutation-related defect in the epithelial-cell chloride channel called CF transmembrane conductance regulator (CFTR). This defect alters chloride ion transport and impairs water transport across the cell membrane. Marked clinical heterogeneity occurs even among patients carrying the same mutation in the CFTR gene. Recent studies suggest that such heterogeneity could be related to epigenetic factors and/or miRNAs, which are small noncoding RNAs that modulate the expression of various proteins via post-transcriptional inhibition of gene expression. In the respiratory system, it has been shown that the dysregulation of miRNAs could participate in and lead to pathogenicity in several diseases. In CF airways, recent studies have proposed that miRNAs may modulate disease progression by affecting the production of either CFTR or various proteins that are dysregulated in the CF lung. Herein, we provide an overview of studies showing how miRNAs may modulate CF pathology and the efforts to develop miRNA-based treatments and/or to consider miRNAs as biomarkers. The identification of miRNAs involved in CF disease progression opens up new avenues toward treatments targeting selected clinical components of CF, independently from the CFTR mutation.
PMID23916755	Downregulation of CFTR promotes epithelial-to-mesenchymal transition and is associated with poor prognosis of breast cancer. The epithelial-to-mesenchymal transition (EMT), a process involving the breakdown of cell-cell junctions and loss of epithelial polarity, is closely related to cancer development and metastatic progression. While the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) and HCO3(-) conducting anion channel expressed in a wide variety of epithelial cells, has been implicated in the regulation of epithelial polarity, the exact role of CFTR in the pathogenesis of cancer and its possible involvement in EMT process have not been elucidated. Here we report that interfering with CFTR function either by its specific inhibitor or lentiviral miRNA-mediated knockdown mimics TGF-β1-induced EMT and enhances cell migration and invasion in MCF-7. Ectopic overexpression of CFTR in a highly metastatic MDA-231 breast cancer cell line downregulates EMT markers and suppresses cell invasion and migration in vitro, as well as metastasis in vivo. The EMT-suppressing effect of CFTR is found to be associated with its ability to inhibit NFκB targeting urokinase-type plasminogen activator (uPA), known to be involved in the regulation of EMT. More importantly, CFTR expression is found significantly downregulated in primary human breast cancer samples, and is closely associated with poor prognosis in different cohorts of breast cancer patients. Taken together, the present study has demonstrated a previously undefined role of CFTR as an EMT suppressor and its potential as a prognostic indicator in breast cancer.
PMID23517647	Reflections on microRNAs in chronic pulmonary disease: looking into the miR-ror and crystal ball. Chronic respiratory diseases are a significant health problem requiring novel approaches to both complement existing therapies and provide breakthrough medicines. Recent clinical advances in understanding the behavior of inhaled oligonucleotides provide the impetus for application of this technology to microRNA therapeutics. MicroRNAs are evolutionarily conserved small regulatory RNA molecules involved in tuning gene networks controlling biological and pathological processes. Deletion or overexpression of microRNAs results in phenotypic changes in animal models of disease such as cancer, fibrosis, diabetes, and inflammation. Inhibition of microRNAs in preclinical models of asthma, cystic fibrosis, and idiopathic pulmonary fibrosis has shown therapeutic promise. In animals, inhibitors of microRNAs directly delivered to the airway at doses suitable for nebulizers or hand-held inhalers up-regulate expression of cohorts of genes containing complementary "seed" sequences for specific and directed microRNA binding within their mRNA untranslated regions. These observations suggest the opportunity to exploit intervention in microRNA biology to create new therapies for chronic pulmonary disorders.
PMID26654095	High-throughput transcriptomics reveals common and strain-specific responses of human macrophages to infection with Mycobacterium abscessus Smooth and Rough variants. Mycobacterium abscessus (MAB) is an emerging pathogen causing pulmonary infections in those with inflammatory lung disorders, such as Cystic Fibrosis (CF), and is associated with the highest fatality rate among rapidly growing mycobacteria (RGM). Phenotypically, MAB manifests as either a Smooth (MAB-S) or a Rough (MAB-R) morphotype, which differ in their levels of cell wall glycopeptidolipids (GPLs) and in their pathogenicity in vivo. As one of the primary immune cells encountered by MAB, we sought to examine the early transcriptional events within macrophages, following infection with both MAB-S or MAB-R. We sampled the transcriptomes (mRNA and miRNA) of THP-1-derived macrophages infected with both MAB-R and MAB-S at 1, 4 and 24 h post-infection (hpi) using RNA-seq. A core set of 606 genes showed consistent expression profiles in response to both morphotypes, corresponding to the early transcriptional response to MAB. The core response is type I Interferon (IFN)-driven, involving the NF-κB and MAPK signaling pathways with concomitant pro-inflammatory cytokine production, and network analysis identified STAT1, EGR1, and SRC as key hub and bottleneck genes. MAB-S elicited a more robust transcriptional response at both the mRNA and miRNA levels, which was reflected in higher cytokine levels in culture supernatants. The transcriptional profiles of macrophages infected with both morphotypes were highly correlated, however, and a direct comparison identified few genes to distinguish them. Most of the induced miRNAs have previously been associated with mycobacterial infection and overall miRNA expression patterns were similarly highly correlated between the morphotypes. The report here details the first whole transcriptome analysis of the early macrophage response to MAB infection. The overall picture at the early stages of macrophage infection is similar to that of other mycobacteria, reflected in a core type I IFN and pro-inflammatory cytokine response. Large-scale transcriptional differences in the host response to the different MAB morphotypes are not evident in the early stages of infection, however the subset of genes with distinct expression profiles suggest potentially interesting differences in internal trafficking of MAB within macrophages.
PMID23646886	Post-transcriptional regulation of cystic fibrosis transmembrane conductance regulator expression and function by microRNAs. MicroRNAs (miRNAs) are increasingly recognized as important posttranscriptional regulators of gene expression, and changes in their actions can contribute to disease states. Little is understood regarding miRNA functions in the airway epithelium under normal or diseased conditions. We profiled miRNA expression in well-differentiated primary cultures of human cystic fibrosis (CF) and non-CF airway epithelia, and discovered that miR-509-3p and miR-494 concentrations were increased in CF epithelia. Human non-CF airway epithelia, transfected with the mimics of miR-509-3p or miR-494, showed decreased cystic fibrosis transmembrane conductance regulator (CFTR) expression, whereas their respective anti-miRs exerted the opposite effect. Interestingly, the two miRNAs acted cooperatively in regulating CFTR expression. Upon infecting non-CF airway epithelial cells with Staphylococcus aureus, or upon stimulating them with the proinflammatory cytokines TNF-α or IL-1β, we observed an increased expression of both miRNAs and a concurrent decrease in CFTR expression and function, suggesting that inflammatory mediators may regulate these miRNAs. Transfecting epithelia with anti-miRs for miR-509-3p and miR-494, or inhibiting NF-κB signaling before stimulating cells with TNFα or IL-1β, suppressed these responses, suggesting that the expression of both miRNAs was responsive to NF-κB signaling. Thus, miR-509-3p and miR-494 are dynamic regulators of CFTR abundance and function in normal, non-CF airway epithelia.
PMID26319446	MicroRNAs in lung diseases: Recent findings and their pathophysiological implications. Lung diseases are one of the leading causes of mortality and morbidity worldwide and effective therapies are imperfect. Nonetheless, recently some novel strategies have been developed to treat and curtail their debilitating impact. Some of the treatments include the role of MicroRNAs (miRNAs) in stemming the spread of lung morbidities. Micro RNAs are small non-coding RNAs which are known as important players in the posttranscriptional regulation of gene expression in mammalian cells by regulating translation. MiRNAs are involved in basic regulatory mechanisms of cells including influencing inflammation. MiRNA dysregulation, resulting in aberrant expression of a gene, is suggested to play a key role in susceptibility of diseases. MiRNAs are involved in the pathogenesis of lung diseases such as cystic fibrosis, lung cancer, asthma, chronic obstructive pulmonary disease, and Idiopathic pulmonary fibrosis. A better understanding of the involvement of miRNAs in pathogenesis of these diseases could result in the development of new therapeutic and diagnostic tools. In this review, we provide an overview of the current understanding of miRNA biogenesis and role as well as recent insights into role of some miRNAs in different pulmonary diseases.
PMID25665524	Pharmacological modulation of the AKT/microRNA-199a-5p/CAV1 pathway ameliorates cystic fibrosis lung hyper-inflammation. In cystic fibrosis (CF) patients, hyper-inflammation is a key factor in lung destruction and disease morbidity. We have previously demonstrated that macrophages drive the lung hyper-inflammatory response to LPS in CF mice, because of reduced levels of the scaffold protein CAV1 with subsequent uncontrolled TLR4 signalling. Here we show that reduced CAV1 and, consequently, increased TLR4 signalling, in human and murine CF macrophages and murine CF lungs, is caused by high microRNA-199a-5p levels, which are PI3K/AKT-dependent. Downregulation of microRNA-199a-5p or increased AKT signalling restores CAV1 expression and reduces hyper-inflammation in CF macrophages. Importantly, the FDA-approved drug celecoxib re-establishes the AKT/miR-199a-5p/CAV1 axis in CF macrophages, and ameliorates lung hyper-inflammation in Cftr-deficient mice. Thus, we identify the AKT/miR-199a-5p/CAV1 pathway as a regulator of innate immunity, which is dysfunctional in CF macrophages contributing to lung hyper-inflammation. In addition, we found that this pathway can be targeted by celecoxib.
PMID23878802	MicroRNAs: new insights into chronic childhood diseases. Chronic diseases are the major cause of morbidity and mortality worldwide and have shown increasing incidence rates among children in the last decades. Chronic illnesses in the pediatric population, even if well managed, affect social, psychological, and physical development and often limit education and active participation and increase the risk for health complications. The significant pediatric morbidity and mortality rates caused by chronic illnesses call for serious efforts toward better understanding of the pathogenesis of these disorders. Recent studies have shown the involvement of microRNAs (miRNAs) in various aspects of major pediatric chronic non-neoplastic diseases. This review focuses on the role of miRNAs in four major pediatric chronic diseases including bronchial asthma, diabetes mellitus, epilepsy and cystic fibrosis. We intend to emphasize the importance of miRNA-based research in combating these major disorders, as we believe this approach will result in novel therapies to aid securing normal development and to prevent disabilities in the pediatric population.
PMID23524258	Regulation of miR-155 biogenesis in cystic fibrosis lung epithelial cells: antagonistic role of two mRNA-destabilizing proteins, KSRP and TTP. Cystic fibrosis (CF) is characterized by a massive pro-inflammatory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyper-expression of IL-8 protein through elevated expression of miR-155. We therefore investigated what factors promote the enhanced aberrant expression of miR-155 in CF. Here we report for the first time, the role of mRNA-destabilizing inflammatory RNA-binding proteins, KSRP and TTP, in the regulation of miR-155 biogenesis in CF lung epithelial cells. We find that KSRP and TTP have an antagonistic role in miR-155 biogenesis. While KSRP promotes enhanced processing of miR-155 precursors to mature miR-155, over-expression of TTP in the CF lung epithelial cells suppresses expression of miR-155. We find that TTP induces the expression of miR-1, which appears to be a regulator of miR-155 biogenesis in CF lung epithelial cells. These data provide novel insights into the mechanisms that induce hyper-inflammatory phenotype of CF, and are potential candidates for anti-inflammatory therapeutics for CF.
PMID23289731	Differential regulation of inflammation by inflammatory mediators in cystic fibrosis lung epithelial cells. Cystic fibrosis (CF) is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which cause a massively proinflammatory phenotype in the CF airway. The chemical basis of the inflammation is hyperproduction of interleukin-8 (IL-8) by CF airway epithelial cells, based on both an intrinsic mutation-dependent mechanism and by infection. In infection-free, cultured CF lung epithelial cells, high levels of the microRNA (miR), miR-155, is responsible for hyperexpression of IL-8. However, whether infection-induced IL-8 expression in CF cells is also mediated by miR-155 is not known. We have hypothesized that miR-155 might be a general mediator of enhanced IL-8 expression in CF cells, either in response to other cytokine/chemokine mediators of inflammation, or after exposure to infectious agents. Here we find that a reduction in miR-155 accompanies suppression of IL-8 by either the anti-inflammatory cytokine IL-10 or by inhibition of ambient IL-1β with a neutralizing antibody. However, attempts to elevate IL-8 levels with either intact bacteria [viz. a mucoid strain of Pseudomonas aeruginosa (PA)], or lipopolysaccharide were unable to elevate miR-155 above its intrinsically high level in the absence of these agents. Instead, in response to PA infection, the CF cells modestly suppress the expression of miR-155, and express a novel set of miRs, including miR-215. We find that ex vivo CF lung epithelial cells also express high levels of both miR-155 and miR-215. The predicted module of infection-induced mRNA targets focuses on activation of the NFκB-signaling pathway, and on the proapoptotic p53-signaling pathway. We interpret these data to suggest that that CF lung epithelial cells respond to PA or bacterial cell products with a novel miR program that may carry with it serious challenges to survival.
PMID26055330	MicroRNA 224 Regulates Ion Transporter Expression in Ameloblasts To Coordinate Enamel Mineralization. Enamel mineralization is accompanied by the release of protons into the extracellular matrix, which is buffered to regulate the pH value in the local microenvironment. The present study aimed to investigate the role of microRNA 224 (miR-224) as a regulator of SLC4A4 and CFTR, encoding the key buffering ion transporters, in modulating enamel mineralization. miR-224 was significantly downregulated as ameloblasts differentiated, in parallel with upregulation of SLC4A4 and CFTR. Overexpression of miR-224 downregulated SLC4A4 and CFTR expression in cultured human epithelial cells. A microRNA luciferase assay confirmed the specific binding of miR-224 to the 3' untranslated regions (UTRs) of SLC4A4 and CFTR mRNAs, thereby inhibiting protein translation. miR-224 agomir injection in mouse neonatal incisors resulted in normal enamel length and thickness, but with disturbed organization of the prism structure and deficient crystal growth. Moreover, the enamel Ca/P ratio and microhardness were markedly reduced after miR-224 agomir administration. These results demonstrate that miR-224 plays a pivotal role in fine tuning enamel mineralization by modulating SLC4A4 and CFTR to maintain pH homeostasis and support enamel mineralization.
PMID26133785	miR-16 rescues F508del-CFTR function in native cystic fibrosis epithelial cells. Cystic fibrosis (CF) is due to mutations in the CFTR gene, which prevents correct folding, trafficking and function of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. The dysfunctional effect of CFTR mutations, principally the F508del-CFTR mutant, is further manifested by hypersecretion of the pro-inflammatory chemokine interleukin-8 into the airway lumen, which further contributes to morbidity and mortality. We have hypothesized that microRNA (miR)-based therapeutics could rescue the dysfunctional consequences of mutant CFTR. Here we report that a miR-16 mimic can effectively rescue F508del-CFTR protein function in airway cell lines and primary cultures, of differentiated human bronchial epithelia from F508del homozygotes, which express mutant CFTR endogenously. We also identify two other miRs, miR-1 and miR-302a, which are also active. Although miR-16 is expressed at basal comparable levels in CF and control cells, miR-1 and miR-302a are undetectable. When miR mimics are expressed in CF lung or pancreatic cells, the expression of the F508del-CFTR protein is significantly increased. Importantly, miR-16 promotes functional rescue of the cyclic AMP-activated apical F508del-CFTR chloride channel in primary lung epithelial cells from CF patients. We interpret these findings to suggest that these miRs may constitute novel targets for CF therapy.
PMID27043239	Toll-Like Receptors in Cystic Fibrosis: Impact of Dysfunctional microRNA on Innate Immune Responses in the Cystic Fibrosis Lung. Toll-like receptors (TLRs) are a class of pattern recognition receptors that are particularly expressed in the sentinel and epithelial cells in the body, including the lung. They are central players in the innate immune system in response to microbial infection, and are the triggers of a complex pathway network that both promotes the inflammatory response and influences the adaptive immune response. These pathways are transiently and finely tuned by cellular factors, including a cell's microRNA response program. MicroRNAs are small, non-coding RNAs that specifically regulate gene expression. In this article, we review the disease-specific microRNA regulatory network of cystic fibrosis, a debilitating and ultimately fatal disease and, specifically, its effect on TLR signalling.
PMID24970162	MicroRNA Expression in Cystic Fibrosis Airway Epithelium. MicroRNAs (miRs) have emerged as major regulators of the protein content of a cell. In the most part, miRs negatively regulate target mRNA expression, with sets of miRs predicted to regulate certain signaling pathways. The miR expression profile of endobronchial brushings is altered in people with cystic fibrosis (CF) compared to those without CF. How this impacts on CF has important implications for our growing understanding of the pathophysiology of CF lung disease and the development of new therapeutics to treat its pulmonary manifestations. Herein we discuss the potential consequences of altered miR expression in CF airway epithelium particularly with respect to cystic fibrosis transmembrane conductance regulator (CFTR) expression, innate immunity and toll-like receptor signalling and explore how best to exploit these changes for therapeutic benefit.
PMID25625579	Circulating microRNAs as noninvasive diagnostic biomarkers of liver disease in children with cystic fibrosis. Cystic fibrosis liver disease (CFLD), resulting from progressive hepatobiliary fibrosis, causes significant morbidity and mortality in up to 20% of children with cystic fibrosis (CF). Both pathogenesis and early detection of CFLD are elusive. Current diagnostic procedures to detect early CFLD and stage fibrosis severity are inadequate. Recent studies highlight a role for microRNAs (miRNAs) in the pathogenesis of many diseases and have suggested that serum miRNAs could be used as diagnostic biomarkers. We profiled circulating serum miRNA levels in patients with CFLD (n = 52), patients with CF without liver disease (CFnoLD, n = 30), and non-CF pediatric controls (n = 20). Extracted RNA was subjected to polymerase chain reaction (PCR) array of 84 miRNAs detectable in human serum. Seven candidate miRNAs identified were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), normalizing data to geNorm-determined stable reference genes, miR-19b and miR-93. miR-122 was significantly elevated in patients with CFLD versus patients with CFnoLD and controls (P < 0.0001). miR-25 (P = 0.0011) and miR-21 (P = 0.0133) were elevated in patients with CFnoLD versus patients with CFLD and controls. CFLD was discriminated by both miR-122 (area under the curve [AUC] 0.71, P = 0.002) and miR-25 (AUC 0.65, P = 0.026). Logistic regression combining 3 miRNAs (-122, -25, -21) was greatly predictive of detecting CFLD (AUC 0.78, P < 0.0001). A combination of 6 miRNAs (-122, -21, -25, -210, -148a, -19a) distinguished F0 from F3-F4 fibrosis (AUC 0.73, P = 0.04), and miR-210 combined with miR-22 distinguished F0 fibrosis from any fibrosis, that is, F1-F4 (AUC 0.72, P = 0.02). These data provide the first evidence of changes to circulating miRNA levels in CF, suggesting that serum-based miRNA analysis may complement and extend current CFLD screening strategies with potential to predict early hepatic fibrosis.
PMID23226399	MiR-101 and miR-144 regulate the expression of the CFTR chloride channel in the lung. The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that plays a critical role in the lung by maintaining fluid homeostasis. Absence or malfunction of CFTR leads to Cystic Fibrosis, a disease characterized by chronic infection and inflammation. We recently reported that air pollutants such as cigarette smoke and cadmium negatively regulate the expression of CFTR by affecting several steps in the biogenesis of CFTR protein. MicroRNAs (miRNAs) have recently received a great deal of attention as both biomarkers and therapeutics due to their ability to regulate multiple genes. Here, we show that cigarette smoke and cadmium up-regulate the expression of two miRNAs (miR-101 and miR-144) that are predicted to target CFTR in human bronchial epithelial cells. When premature miR-101 and miR-144 were transfected in human airway epithelial cells, they directly targeted the CFTR 3'UTR and suppressed the expression of the CFTR protein. Since miR-101 was highly up-regulated by cigarette smoke in vitro, we investigated whether such increase also occurred in vivo. Mice exposed to cigarette smoke for 4 weeks demonstrated an up-regulation of miR-101 and suppression of CFTR protein in their lungs. Finally, we show that miR-101 is highly expressed in lung samples from patients with severe chronic obstructive pulmonary disease (COPD) when compared to control patients. Taken together, these results suggest that chronic cigarette smoking up-regulates miR-101 and that this miRNA could contribute to suppression of CFTR in the lungs of COPD patients.
PMID21873408	Low oxygen induces the type III secretion system in Pseudomonas aeruginosa via modulation of the small RNAs rsmZ and rsmY. A steep oxygen gradient within the mucus of the cystic fibrosis (CF) lung combined with the biofilm mode of bacterial growth forces respiratory pathogens to adapt to varying oxygen availability. This study presents the novel finding that the Pseudomonas aeruginosa response to limiting oxygen stress includes induction of its type III secretion system (T3SS), which subsequently contributes towards host cell cytotoxicity. In P. aeruginosa, the global anaerobic response regulator Anr perceives low oxygen and subsequently triggers gene expression of a range of target genes, including the response regulator narL. Here we demonstrate that microaerobic induction of the T3SS is dependent on Anr, and that this is mediated through direct NarL transcriptional repression of the sRNAs rsmY and rsmZ, allowing free RsmA protein to positively regulate the T3SS. This study reveals a novel interplay between the Anr-NarL and RsmAYZ regulatory circuits, and introduces RsmA as an important regulator during P. aeruginosa adaptation to a low-oxygen environment.
PMID23632450	Elevated levels of miR-145 correlate with SMAD3 down-regulation in cystic fibrosis patients. MicroRNAs (miRNAs) have recently emerged as important gene regulators in Cystic Fibrosis (CF), a common monogenic disease characterized by severe infection and inflammation, especially in the airway compartments. In the current study, we show that both miR-145 and miR-494 are significantly up-regulated in nasal epithelial tissues from CF patients compared with healthy controls (p<0.001 and p<0.01, respectively) by Quantitative Real-Time PCR. Only miR-494 levels showed a trend of correlation with reduced CFTR mRNA expression and positive sweat test values, supporting the negative regulatory role of this miRNA on CFTR synthesis. Using computational prediction algorithms and luciferase reporter assays, SMAD family member 3 (SMAD3), a key element of the TGF-β1 inflammatory pathway, was identified as a target of miR-145. Indeed, miR-145 synthetic mimics suppressed by approximately 40% the expression of a reporter construct containing the SMAD3 3'-UTR. Moreover, we observed an inverse correlation between SMAD3 mRNA expression and miR-145 in CF nasal tissues (r=-0.68, p=0.0018, Pearson's correlation). Taken together, these results confirm the pivotal role of miRNAs in the CF physio-pathogenesis and suggest that miRNA deregulation play a role in the airway disease severity by modulating CFTR levels as well as the expression of important molecules involved in the inflammatory response. miR-494 and miR-145 may, therefore, be potential biomarker and therapeutic target to specific CF clinical manifestations.
PMID23590310	Correction of nonsense BMPR2 and SMAD9 mutations by ataluren in pulmonary arterial hypertension. Heritable pulmonary arterial hypertension (HPAH) is a serious lung vascular disease caused by heterozygous mutations in the bone morphogenetic protein (BMP) pathway genes, BMPR2 and SMAD9. One noncanonical function of BMP signaling regulates biogenesis of a subset of microRNAs. We have previously shown that this function is abrogated in patients with HPAH, making it a highly sensitive readout of BMP pathway integrity. Ataluren (PTC124) is an investigational drug that permits ribosomal readthrough of premature stop codons, resulting in a full-length protein. It exhibits oral bioavailability and limited toxicity in human trials. Here, we tested ataluren in lung- or blood-derived cells from patients with HPAH with nonsense mutations in BMPR2 (n = 6) or SMAD9 (n = 1). Ataluren significantly increased BMP-mediated microRNA processing in six of the seven cases. Moreover, rescue was achieved even for mutations exhibiting significant nonsense-mediated mRNA decay. Response to ataluren was dose dependent, and complete correction was achieved at therapeutic doses currently used in clinical trials for cystic fibrosis. BMP receptor (BMPR)-II protein levels were normalized and ligand-dependent phosphorylation of downstream target Smads was increased. Furthermore, the usually hyperproliferative phenotype of pulmonary artery endothelial and smooth muscle cells was reversed by ataluren. These results indicate that ataluren can effectively suppress a high proportion of BMPR2 and SMAD9 nonsense mutations and correct BMP signaling in vitro. Approximately 29% of all HPAH mutations are nonsense point mutations. In light of this, we propose ataluren as a potential new personalized therapy for this significant subgroup of patients with PAH.
PMID25847505	Respiratory syncytial virus infection of airway cells: Role of microRNAs. MicroRNAs (miRNAs) are small single-stranded RNA molecules involved in the regulation of gene expression at the post-transcriptional level. In the airways, miRNAs are implicated in the modulation of antiviral defense, through modulation of both innate and adaptive immune response in inflammatory and immune effector cells but also in parenchymal cells. The first target of respiratory viruses are airway epithelial cells. Following infection, an altered expression of distinct miRNAs occurs in airway cells aimed at inhibiting viral replication and preserving the airway epithelial barrier, while at the same time viruses induce or repress the expression of other miRNAs that favor viral replication. Understanding the changes in miRNA expression profile, identification of miRNAs target genes and their contribution to the pathogenesis of the disease may help the intricate mechanisms of virus-host interaction. Further understanding of these molecular mechanisms could lead to development of new antiviral treatments in common, high impact, respiratory disorders for which specific treatments are not available. Respiratory syncytial virus (RSV) airway infection is a common example of virus modifying miRNAs expression to favor immune evasion, and constitutes the salient feature of this review.
PMID23555973	Gene mutation in microRNA target sites of CFTR gene: a novel pathogenetic mechanism in cystic fibrosis? Cystic fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians. It depends on alterations of a chloride channel expressed by most epithelial cells and encoded by CFTR gene. Also using scanning techniques to analyze the whole coding regions of CFTR gene, mutations are not identified in up to 10% of CF alleles, and such figure increases in CFTR-related disorders (CFTR-RD). Other gene regions may be the site of causing-disease mutations. We searched for genetic variants in the 1500 bp of CFTR 3' untranslated region, typical target of microRNA (miRNA) posttranscriptional gene regulation, in either CF patients with the F508del homozygous genotype and different clinical expression (n = 20), CF (n = 32) and CFTR-RD (n = 43) patients with one or none mutation after CFTR scanning and in controls (n = 50). We identified three SNPs, one of which, the c.*1043A>C, was located in a region predicted to bind miR-433 and miR-509-3p. Such mutation was peculiar of a CFTR-RD patient that had Congenital Bilateral Absence of Vas Deferens (CBAVD), diffuse bronchiectasis, a borderline sweat chloride test and the heterozygous severe F508del mutation on the other allele. The expression analysis demonstrated that the c.*1043A>C increases the affinity for miR-509-3p and slightly decreases that for the miR-433. Both miRNAs cause in vitro a reduced expression of CFTR protein. Thus, the c.*1043A>C may act as a mild CFTR mutation enhancing the affinity for inhibitory miRNAs as a novel pathogenetic mechanism in CF.
PMID24143095	Targeting miRNA-based medicines to cystic fibrosis airway epithelial cells using nanotechnology. Cystic fibrosis (CF) is an inherited disorder characterized by chronic airway inflammation. microRNAs (miRNAs) are endogenous small RNAs which act on messenger (m) RNA at a post transcriptional level, and there is a growing understanding that altered expression of miRNA is involved in the CF phenotype. Modulation of miRNA by replacement using miRNA mimics (premiRs) presents a new therapeutic paradigm for CF, but effective and safe methods of delivery to the CF epithelium are limiting clinical translation. Herein, polymeric nanoparticles are investigated for delivery of miRNA mimics into CF airway epithelial cells, using miR-126 as a proof-of-concept premiR cargo to determine efficiency. Two polymers, polyethyleneimine (PEI) and chitosan, were used to prepare miRNA nanomedicines, characterized for their size, surface (zeta) potential, and RNA complexation efficiency, and screened for delivery and cytotoxicity in CFBE41o- (human F508del cystic fibrosis transmembrane conductance regulator bronchial epithelial) cells using a novel high content analysis method. RNA extraction was carried out 24 hours post transfection, and miR-126 and TOM1 (target of Myb1) expression (a validated miR-126 target) was assessed. Manufacture was optimized to produce small nanoparticles that effectively complexed miRNA. Using high content analysis, PEI-based nanoparticles were more effective than chitosan-based nanoparticles in facilitating uptake of miRNA into CFBE41o- cells and this was confirmed in miR-126 assays. PEI-premiR-126 nanoparticles at low nitrogen/phosphate (N/P) ratios resulted in significant knockdown of TOM1 in CFBE41o- cells, with the most significant reduction of 66% in TOM1 expression elicited at an N/P ratio of 1:1 while chitosan-based miR-126 nanomedicines failed to facilitate statistically significant knockdown of TOM1 and both nanoparticles appeared relatively nontoxic. miRNA nanomedicine uptake can be qualitatively and quantitatively assessed rapidly by high content analysis and is highly polymer-dependent but, interestingly, there is not a direct correlation between the levels of miRNA uptake and the downstream gene knockdown. Polymeric nanoparticles can deliver premiRs effectively to CFBEs in order to modulate gene expression but must be tailored specifically for miRNA delivery.
PMID26542291	miRNA-221 is elevated in cystic fibrosis airway epithelial cells and regulates expression of ATF6. MicroRNA (miRNA) and messenger RNA (mRNA) expression differs in cystic fibrosis (CF) versus non-CF bronchial epithelium. Here, the role of miRNA in basal regulation of the transcription factor ATF6 was investigated in bronchial epithelial cells in vitro and in vivo. Using in silico analysis, miRNAs predicted to target the 3'untranslated region (3'UTR) of the human ATF6 mRNA were identified. Three of these miRNAs, miR-145, miR-221 and miR-494, were upregulated in F508del-CFTR homozygous CFBE41o- versus non-CF 16HBE14o- bronchial epithelial cells and also in F508del-CFTR homozygous or heterozygous CF (n = 8) versus non-CF (n = 9) bronchial brushings. ATF6 was experimentally validated as a molecular target of these miRNAs through the use of a luciferase reporter vector containing the full-length 3'UTR of ATF6. Expression of ATF6 was observed to be decreased in CF both in vivo and in vitro. miR-221 was also predicted to regulate murine ATF6, and its expression was significantly increased in native airway tissues of 6-week-old βENaC-overexpressing transgenic mice with CF-like lung disease versus wild-type littermates. These results implicate miR-145, miR-221 and miR-494 in the regulation of ATF6 in CF bronchial epithelium, with miR-221 demonstrating structural and functional conservation between humans and mice. The altered miRNA expression evident in CF bronchial epithelial cells can affect expression of transcriptional regulators such as ATF6.
PMID25010962	Emerging role of MicroRNAs and long noncoding RNAs in respiratory disease. The advent of techniques such as microarrays and high-throughput sequencing has revolutionized our ability to examine messenger RNA (mRNA) expression within the respiratory system. Importantly, these approaches have also uncovered the widespread expression of "noncoding RNAs," including microRNAs and long noncoding RNAs, which impact biologic responses through the regulation of mRNA transcription and/or translation. To date, most studies of the role of noncoding RNAs have focused on microRNAs, which regulate mRNA translation via the RNA interference pathway. These studies have shown changes in microRNA expression in cells and tissues derived from patients with asthma, pulmonary fibrosis, cystic fibrosis, COPD, and non-small cell lung cancer. Although the evidence is currently limited, we review the work that has been carried out in cell and animal models that has identified the function and mechanism of action of a small number of these microRNAs in disease etiology. In addition to microRNAs, we assess the emerging evidence that long noncoding RNAs regulate respiratory phenotype. Because these investigations into long noncoding RNAs were performed almost exclusively in non-small cell lung cancer, future work will need to extend these into other respiratory diseases and to analyze how microRNAs and long noncoding RNAs interact to regulate mRNA expression. From a clinical perspective, the targeting of noncoding RNAs as a novel therapeutic approach will require a deeper understanding of their function and mechanism of action. However, in the short term, changes in miRNA and long noncoding RNA expression are likely to be of use as biomarkers for disease stratification and/or assessment of drug action.
PMID27541364	Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages. Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly-regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.
PMID27307950	Expression Profiling of LPS Responsive miRNA in Primary Human Macrophages. microRNAs (miRNAs) have emerged as important regulators of the innate and adaptive immune response. The purpose of the present study was to interrogate miRNA profiles of primary human macrophages challenged with bacterial lipopolysaccharide (LPS) with focus on expression kinetics. We employed Nanostring platform to precisely characterize the changes in miRNA expression following different doses and durations of LPS exposure. Differentially expressed miRNAs were identified in response to LPS challenge with convergent and divergent expression profiles. Pathway analysis of LPS-responsive miRNAs revealed regulation of biological processes linked to key cell signaling (including PIK3-Akt, MAP kinase, ErbB) and pathogen response pathways. Our data provide a comprehensive miRNA profiling of human primary macrophages treated with LPS. These results show that bacterial Toll like receptor (TLR) ligands can temporally modulate macrophage miRNA expression.
PMID22853952	A microRNA network regulates expression and biosynthesis of wild-type and DeltaF508 mutant cystic fibrosis transmembrane conductance regulator. Production of functional proteins requires multiple steps, including gene transcription and posttranslational processing. MicroRNAs (miRNAs) can regulate individual stages of these processes. Despite the importance of the cystic fibrosis transmembrane conductance regulator (CFTR) channel for epithelial anion transport, how its expression is regulated remains uncertain. We discovered that miRNA-138 regulates CFTR expression through its interactions with the transcriptional regulatory protein SIN3A. Treating airway epithelia with an miR-138 mimic increased CFTR mRNA and also enhanced CFTR abundance and transepithelial Cl(-) permeability independent of elevated mRNA levels. An miR-138 anti-miR had the opposite effects. Importantly, miR-138 altered the expression of many genes encoding proteins that associate with CFTR and may influence its biosynthesis. The most common CFTR mutation, ΔF508, causes protein misfolding, protein degradation, and cystic fibrosis. Remarkably, manipulating the miR-138 regulatory network also improved biosynthesis of CFTR-ΔF508 and restored Cl(-) transport to cystic fibrosis airway epithelia. This miRNA-regulated network directs gene expression from the chromosome to the cell membrane, indicating that an individual miRNA can control a cellular process more broadly than recognized previously. This discovery also provides therapeutic avenues for restoring CFTR function to cells affected by the most common cystic fibrosis mutation.
PMID26542979	miR-1343 attenuates pathways of fibrosis by targeting the TGF-β receptors. Irreversible respiratory obstruction resulting from progressive airway damage, inflammation and fibrosis is a feature of several chronic respiratory diseases, including cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). The cytokine transforming growth factor β (TGF-β) has a pivotal role in promoting lung fibrosis and is implicated in respiratory disease severity. In the present study, we show that a previously uncharacterized miRNA, miR-1343, reduces the expression of both TGF-β receptor 1 and 2 by directly targeting their 3'-UTRs. After TGF-β exposure, elevated intracellular miR-1343 significantly decreases levels of activated TGF-β effector molecules, pSMAD2 (phosphorylated SMAD2) and pSMAD3 (phosphorylated SMAD3), when compared with a non-targeting control miRNA. As a result, the abundance of fibrotic markers is reduced, cell migration into a scratch wound impaired and epithelial-to-mesenchymal transition (EMT) repressed. Mature miR-1343 is readily detected in human neutrophils and HL-60 cells and is activated in response to stress in A549 lung epithelial cells. miR-1343 may have direct therapeutic applications in fibrotic lung disease.
PMID24015331	Role of microRNAs in lung development and pulmonary diseases. MicroRNAs (miRNAs) are a class of small noncoding RNA which exert post-transcriptional gene regulation activity by targeting messenger RNAs. miRNAs have been found to be involved in various fundamental biological processes and deregulation of miRNAs is known to result in pathological conditions. In this review, we provide an overview of recent discoveries on the role played by this class of molecules in lung development and in pulmonary diseases, such as asthma, cystic fibrosis, chronic obstructive pulmonary disease, and pulmonary artery hypertension. Considering the relevant role of these miRNAs under physiological and pathological conditions, they represent new clinical targets as well as diagnostic and prognostic tools. Therefore, this review pays special attention to recent advances and possible future directions for the use of miRNAs for clinical applications.
PMID22028919	Synergistic post-transcriptional regulation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) by miR-101 and miR-494 specific binding. microRNAs (miRNAs) are a class of regulatory small non-coding molecules that control gene expression at post-transcriptional level. Deregulation of miRNA functions affects a variety of biological processes also involved in the etiology of several human mendelian and complex diseases. Recently, aberrant miRNA expression has been observed in Cystic Fibrosis (CF), an autosomal-recessive genetic disorder caused by mutations in the CFTR gene, in which a genotype-phenotype correlation is not always found. In order to determine miRNA role in CFTR post-transcriptional regulation, we searched for miR-responsive elements in the CFTR 3'-UTR. In silico analysis, performed using different computational on-line programs, identified some putative miRNAs. Both miR-101 and miR-494 synthetic mimics significantly inhibited the expression of a reporter construct containing the 3'-UTR of CFTR in luciferase assays. Interestingly, miR-101/miR-494 combination was able to markedly suppress CFTR activity by approximately 80% (p<0.001). This is one of the first in vitro studies implicating microRNAs as negative regulators of the CFTR gene expression. miRNA aberrant expression and function might explain the wide phenotypic variability observed among CF patients.
PMID25484921	Deciphering miRNA transcription factor feed-forward loops to identify drug repurposing candidates for cystic fibrosis. Cystic fibrosis (CF) is a fatal genetic disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that primarily affects the lungs and the digestive system, and the current drug treatment is mainly able to alleviate symptoms. To improve disease management for CF, we considered the repurposing of approved drugs and hypothesized that specific microRNA (miRNA) transcription factors (TF) gene networks can be used to generate feed-forward loops (FFLs), thus providing treatment opportunities on the basis of disease specific FFLs. Comprehensive database searches revealed significantly enriched TFs and miRNAs in CF and CFTR gene networks. The target genes were validated using ChIPBase and by employing a consensus approach of diverse algorithms to predict miRNA gene targets. STRING analysis confirmed protein-protein interactions (PPIs) among network partners and motif searches defined composite FFLs. Using information extracted from SM2miR and Pharmaco-miR, an in silico drug repurposing pipeline was established based on the regulation of miRNA/TFs in CF/CFTR networks. In human airway epithelium, a total of 15 composite FFLs were constructed based on CFTR specific miRNA/TF gene networks. Importantly, nine of them were confirmed in patient samples and CF epithelial cells lines, and STRING PPI analysis provided evidence that the targets interacted with each other. Functional analysis revealed that ubiquitin-mediated proteolysis and protein processing in the endoplasmic reticulum dominate the composite FFLs, whose major functions are folding, sorting, and degradation. Given that the mutated CFTR gene disrupts the function of the chloride channel, the constructed FFLs address mechanistic aspects of the disease and, among 48 repurposing drug candidates, 26 were confirmed with literature reports and/or existing clinical trials relevant to the treatment of CF patients. The construction of FFLs identified promising drug repurposing candidates for CF and the developed strategy may be applied to other diseases as well.
PMID25800681	The hsa-miR-125a/hsa-let-7e/hsa-miR-99b cluster is potentially implicated in Cystic Fibrosis pathogenesis. Cystic Fibrosis (CF) is an autosomal recessive disorder implicating the Cystic Fibrosis Transmembrane Regulator (CFTR). Even though CF is mainly considered an inherited monogenic disease, numerous findings over the last few years argue for a more complicated multifactorial disease involving modifier genes. The 19q13.2-19q13.4 region is suspected to contain genetic modifiers that correlate to the severity of CF. Here we studied a cohort of p.F508del patients for potential SNPs in the hsa-miR-99b/hsa-let-7e/hsa-miR-125a cluster, which is found within the 19q13.2-19q13.4 region. Three polymorphisms were identified in the hsa-miR-99b/hsa-let-7e/hsa-miR-125a cluster. Using a cell based model, we analysed whether expression of DeltaF508-CFTR influences the expression of mature hsa-miR-99b, hsa-let-7e, and hsa-miR-125a. We found that hsa-miR-99b and hsa-miR-125a were significantly increased in DeltaF508-CFTR expressing cells. The three miRNAs appear to be derived from the same precursor but differ in their expression levels suggesting differential maturation of these miRNAs in CF. In silico analysis revealed that two out of the three polymorphisms we identified in a CF p.F508del patients cohort could modulate miRNA maturation and therefore impact on hsa-miR-99b/hsa-let-7e/hsa-miR-125a expression levels. Ingenuity Pathway Analysis indicated that hsa-miR-99b and hsa-miR-125a could be associated with the phenotypes manifested by p.F508del patients. Here we provide novel elements in the mechanism of hsa-miR-99b and hsa-miR-125a biogenesis, and for the role of CFTR and DeltaF508-CFTR on the expression of this miRNA cluster. These findings augment existing data implicating miRNAs as putative CF modifiers.
PMID24433094	Expression of microRNA-93 and Interleukin-8 during Pseudomonas aeruginosa-mediated induction of proinflammatory responses. In this study we analyzed the microRNA profile of cystic fibrosis (CF) bronchial epithelial IB3-1 cells infected with Pseudomonas aeruginosa by microarray and quantitative RT-PCR, demonstrating that microRNA 93 (miR-93), which is highly expressed in basal conditions, decreases during infection in parallel with increased expression of the IL-8 gene. The down-regulation of miR-93 after P. aeruginosa infection was confirmed in other bronchial cell lines derived from subjects with and without CF, namely CuFi-1 and NuLi-1 cells. Sequence analysis shows that the 3'-UTR region of IL-8 mRNA is a potential target of miR-93 and that the consensus sequence is highly conserved throughout molecular evolution. The possible involvement of miR-93 in IL-8 gene regulation was validated using three luciferase vectors, including one carrying the complete 3'-UTR region of the IL-8 mRNA and one carrying the same region with a mutated miR-93 site. Up-modulation of IL-8 after P. aeruginosa infection was counteracted in IB3-1, CuFi-1, and NuLi-1 cells by pre-miR-93 transfection. In addition, IL-8 was up-regulated in uninfected cells treated with antagomiR-93. Our results support the concept of a possible link between microRNA expression and IL-8 induction in bronchial epithelial cells infected with P. aeruginosa. Specifically, the data presented here indicate that, in addition to NF-κB-dependent up-regulation of IL-8 gene transcription, IL-8 protein expression is posttranscriptionally regulated by interactions of the IL-8 mRNA with the inhibitory miR-93.
PMID25378544	Non-coding RNAs and respiratory disease. Recent scientific developments have radically changed the way we look at the vast 'non-coding' part of our genome. It is now clear that this genomic 'dark matter' is transcribed into myriads of RNA species that act behind the scenes to veto, or boost, the production of proteins in our cells. As a consequence, non-coding RNAs (ncRNAs) represent an additional layer of regulation for fundamental biological processes such as organ development, tissue repair and immunity. It also follows that disturbances in ncRNA networks (among which microRNAs and long ncRNAs are the best studied) can give rise to a whole range of pathological conditions. Increasing preclinical and translational evidence places ncRNAs as key players in a wide spectrum of diseases affecting the lung. In this concise review, we will provide essential concepts of ncRNA science, with special emphasis on discoveries relevant to the pulmonary physician.
PMID21745567	Fibrosis and progression of autosomal dominant polycystic kidney disease (ADPKD). The age on onset of decline in renal function and end-stage renal disease (ESRD) in autosomal polycystic kidney disease (ADPKD) is highly variable and there are currently no prognostic tools to identify patients who will progress rapidly to ESRD. In ADPKD, expansion of cysts and loss of renal function are associated with progressive fibrosis. Similar to the correlation between tubulointerstitial fibrosis and progression of chronic kidney disease (CKD), in ADPKD, fibrosis has been identified as the most significant manifestation associated with an increased rate of progression to ESRD. Fibrosis in CKD has been studied extensively. In contrast, little is known about the mechanisms underlying progressive scarring in ADPKD although some commonality may be anticipated. Current data suggest that fibrosis associated with ADPKD shares at least some of the "classical" features of fibrosis in CKD (increased interstitial collagens, changes in matrix metalloproteinases (MMPs), over-expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), over-expression of plasminogen activator inhibitor-1 (PAI-1) and increased transforming growth factor beta (TGFβ) but that there are also some unique and stage-specific features. Epithelial changes appear to precede and to drive interstitial changes leading to the proposal that development of fibrosis in ADPKD is biphasic with alterations in cystic epithelia precipitating changes in interstitial fibroblasts and that reciprocal interactions between these cell types drives progressive accumulation of extracellular matrix (ECM). Since fibrosis is a major component of ADPKD it follows that preventing or slowing fibrosis should retard disease progression with obvious therapeutic benefits. The development of effective anti-fibrotic strategies in ADPKD is dependent on understanding the precise mechanisms underlying initiation and progression of fibrosis in ADPKD and the role of the intrinsic genetic defect in these processes. This article is part of a Special Issue entitled: Polycystic Kidney Disease.
PMID26185362	MicroRNA Dysregulation in Cystic Fibrosis. The cystic fibrosis lung is a complex milieu comprising multiple factors that coordinate its physiology. MicroRNAs are regulatory factors involved in most biological processes and it is becoming increasingly clear that they play a key role in the development and manifestations of CF lung disease. These small noncoding RNAs act posttranscriptionally to inhibit protein production. Their involvement in the pathogenesis of CF lung disease stems from the fact that their expression is altered in vivo in the CF lung due to intrinsic and extrinsic factors; to date defective chloride ion conductance, endoplasmic reticulum stress, inflammation, and infection have been implicated in altering endogenous miRNA expression in this setting. Here, the current state-of-the-art and biological consequences of altered microRNA expression in cystic fibrosis are reviewed.
PMID26813518	Genetic evaluation of male infertility. Men with severe oligospermia (<5 million sperm/mL ejaculate fluid) or azoospermia should receive genetic testing to clarify etiology of male infertility prior to treatment. Categorization by obstructive azoospermia (OA) or non-obstructive azoospermia (NOA) is critical since genetic testing differs for the former with normal testicular function, testicular volume (~20 mL), and follicle-stimulating hormone (FSH) (1-8 IU/mL) when compared to the latter with small, soft testes and increased FSH. History and physician examination along with laboratory testing (following appropriate genetic counseling) is critical to accurate selection of genetic testing appropriate for azoospermia due to primary testicular failure as compared with congenital hypogonadotropic hypogonadism (HH). Genetic testing options include cystic fibrosis transmembrane conductance regulator (CFTR) testing for men with congenital absence of the vas, while karyotype, Y chromosome microdeletions (YCMD), and other specific genetic tests may be warranted depending on the clinical context of severe oligospermia or NOA. The results of genetic testing guide management options. The most recent techniques for genetic analysis, including sperm microRNA (miRNA) and epigenetics, are forming the foundation for future genetic diagnosis and therapeutic targets in male infertility.
PMID22397568	Therapeutic modulation of miRNA for the treatment of proinflammatory lung diseases. miRNAs are short, nonprotein coding RNAs that regulate target gene expression principally by causing translational repression and/or mRNA degradation. miRNAs are involved in most mammalian biological processes and have pivotal roles in controlling the expression of factors involved in basal and stimulus-induced signaling pathways. Considering their central role in the regulation of gene expression, miRNAs represent therapeutic drug targets. Here we describe how miRNAs are involved in the regulation of aspects of innate immunity and inflammation, what happens when this goes awry, such as in the chronic inflammatory lung diseases cystic fibrosis and asthma, and discuss the current state-of-the-art miRNA-targeted therapeutics.
PMID21903582	Elevated CO(2) levels cause mitochondrial dysfunction and impair cell proliferation. Elevated CO(2) concentrations (hypercapnia) occur in patients with severe lung diseases. Here, we provide evidence that high CO(2) levels decrease O(2) consumption and ATP production and impair cell proliferation independently of acidosis and hypoxia in fibroblasts (N12) and alveolar epithelial cells (A549). Cells exposed to elevated CO(2) died in galactose medium as well as when glucose-6-phosphate isomerase was knocked down, suggesting mitochondrial dysfunction. High CO(2) levels led to increased levels of microRNA-183 (miR-183), which in turn decreased expression of IDH2 (isocitrate dehydrogenase 2). The high CO(2)-induced decrease in cell proliferation was rescued by α-ketoglutarate and overexpression of IDH2, whereas proliferation decreased in normocapnic cells transfected with siRNA for IDH2. Also, overexpression of miR-183 decreased IDH2 (mRNA and protein) as well as cell proliferation under normocapnic conditions, whereas inhibition of miR-183 rescued the normal proliferation phenotype in cells exposed to elevated levels of CO(2). Accordingly, we provide evidence that high CO(2) induces miR-183, which down-regulates IDH2, thus impairing mitochondrial function and cell proliferation. These results are of relevance to patients with hypercapnia such as those with chronic obstructive pulmonary disease, asthma, cystic fibrosis, bronchopulmonary dysplasia, and muscular dystrophies.