microRNA and Cancer

Abstract

MicroRNAs (miRNAs), a class of small, regulatory, non-coding RNA molecules, display aberrant expression patterns and functional abnormalities in human diseases including cancers. This review summarizes the abnormally expressed miRNAs in various types of human cancers, possible mechanisms underlying such abnormalities, and miRNA-modulated molecular pathways critical for cancer development. Practical implications of miRNAs as biomarkers, novel drug targets and therapeutic tools for diagnosis, prognosis, and treatments of human cancers are also discussed.

INTRODUCTION

MicroRNAs (miRNAs) are small, endogenous non-coding RNA molecules that contribute to modulating the expression level of specific proteins based on sequence complementarities with their target mRNA molecules . Most of miRNA species identified thus far are encoded in portions of the genome that had been previously thought to be non-coding regions. Since first discovered in 1993, miRNAs have attracted wide attention due to their unique functional significance and modes of action, providing a new dimension of, and the latest addendum to, the central dogma of molecular biology. Because one miRNA can target multiple mRNA targets, it is estimated that more than one third of human genes are regulated by miRNAs, and the number of genes found to be under the modulation of miRNAs has increased sharply. The role of miRNAs as key regulatory molecules that control a wide variety of fundamental cellular processes, such as proliferation, death, differentiation, motility, invasiveness, etc., is increasingly recognized in almost all fields of biological and biomedical fields.

Understanding the significance of miRNAs in the pathogenesis of human diseases represents an important dimension in miRNA research as it may lead to the development of miRNA-based novel therapeutic strategies or diagnostic/prognostic biomarkers. Among diseases that most seriously threaten human lives, cancer, which has been found by recent studies to be associated with deregulation or genetic changes of miRNAs as previously reviewed by others (1,2), apparently represents an outstanding public health problem that causes 7.6 million deaths annually as estimated by WHO. This review attempts to briefly outline our current knowledge on the abnormalities of miRNAs found to be associated with cancer pathogenesis and possible mechanisms underlying the roles of miRNAs in cancer development and progression and to provide a perspective insight in using miRNAs as cancer biomarkers and therapeutic targets or tools.

miRNAs AND THEIR BIOLOGICAL FUNCTIONS

miRNAs, a class of 21- to 24-nucleotides (nt), non-coding, regulatory RNA molecules, was first discovered in developing nematode (3). Up to date, thousands of miRNAs have been identified in a wide variety of species. Over 10,883 miRNA sequences found in animals, plants, and viruses have been published in the miRBase database, including 721 entries from human and 579 from mouse (http://microrna.sanger.ac.uk; Release 14: September 2009). With the number of identified miRNA increasing rapidly, rules of annotation have been suggested to designate individual miRNAs, such as hsa-miR-199, with “hsa” standing for the homo sapiens, “miR” for microRNA, and the number “199” indicating the order of being discovered.

miRNA coding sequences can be found in introns or exons of a protein-coding gene or in the intergenic regions. It is quite common that several miRNA genes are clustered along the genome, sharing the same promoter, whereas they can also be present individually. miRNA genes are transcribed into a large, non-coding messenger RNA strand known as primary miRNA transcript (pri-miRNA), with coding capacity for one or more mature miRNAs. Pri-miRNA is subsequently processed into smaller, stem-looped, hairpin-like miRNA precursors (pre-miRNAs) of ∼70 nt in length by RNase III-type enzyme Drosha that forms a microprocessor complex with the double-stranded RNA-binding protein DGCR8. Afterwards, pre-miRNAs are exported from the nucleus across the nuclear membrane into the cytoplasm through an Exportin-5/Ran complex. Similar to the processing of an siRNA, a pre-miRNA is then cleaved by a highly conserved RNase III-type enzyme Dicer to generate a 19- to 23-nt RNA duplex and then incorporated into a RISC-like ribonucleoprotein complex (RNP), also known as microRNA-induced silencing complex (miRISC) or miRNP. Only one strand of the miRNA, known as the guide strand, is integrated into miRISC, while the other strand, miRNA*, also known as the anti-guide or passenger strand, is degraded by the RISC. Although the biosynthesis of most miRNAs is through the Drosha pathway, recent findings suggest the existence of an alternative Drosha-independent pathway. For example, in Drosophila or Caenorhabditis elegance, pre-miRNAs in intronic sequences are produced without Drosha-mediated cleavage. miRNAs that are generated through this pathway are mirtrons that may still function similarly to a regular miRNA [for a review, see (4)].

The miRNA strand guides the RISC to its target mRNA, subsequently cleaving or silencing the target mRNA. While it was thought that miRNA represses the expression of their targets by inducing deadenylation and destabilization of mRNA, recent evidence suggests that the repressive function of miRNA is mediated through preventing translation from the target mRNA (5). Although the precise mechanism of such inhibitory effect on translation remains to be validated, it is believed that miRNA interferes with protein factors involved in the elongation process during translation (6). It has been proposed that the degree of complementarity between miRNA and its target mRNA is a crucial factor on which the mode of miRNA function is determined, with the miRNAs imperfectly complementary to the binding sites in the 3′ untranslated regions (UTR) of their target mRNA repressing the protein expression through translational inhibition, whereas those miRNAs perfectly or nearly perfectly complementary to mRNA targets inducing mRNA degradation through the RNA-mediated interference pathway [reviewed in (4)]. On the other hand, a cytosolic site called “P-body” has been proposed as dynamic foci for mRNA sequestration leading to translational repression and mRNA turnover. Several studies have demonstrated that Ago1 and Ago2 are localized to mammalian P-bodies, indicating a functional link between P-bodies and a miRNA-induced repression (7).

Interestingly, recent studies have suggested that a number of miRNAs are able to activate the expression of certain target genes in a sequence-specific manner instead of silencing them. For instance, miR-373 induces expression of E-cadherin and cold-shock domain-containing protein C2 (CSDC2) genes with complementary sequences in their promoters (8). This novel phenomenon, although largely remaining uncharacterized, is termed “RNA activation” (RNAa). While thus far the exact mechanisms of RNAa remain to be elucidated, the process may require the Ago2 protein and could be associated with histone changes linked to gene activation (9).

It is estimated that over 30% of protein-coding genes in human genome are regulated by miRNAs, suggesting that most of individual miRNAs target multiple protein-coding genes (10). Therefore, it is convincible that miRNAs play important roles in a wide variety of biological processes. Indeed, accumulated evidence has demonstrated modulation effects of miRNA on development, cell proliferation, differentiation, apoptosis, adhesion, migration and invasion, as well as other biological processes. Thus, expression of this important class of molecules is usually correlated with an array of pathological conditions, among which cancer may represent one of the most relevant diseases related to aberrant expression and/or functions of miRNAs.

ABERRANT EXPRESSION OF miRNAs IN CANCER

The significance of miRNA in human cancers began to be revealed in 2002 when Croce and colleagues identified that a small genomic region in chromosome 13q14 that is commonly deleted in chronic lymphocytic leukemia (CLL) contained miR-15a and miR-16-1 genes, suggesting a link of these miRNAs to CLL (11). Following this observation, more and more miRNAs have been found to be aberrantly expressed in various types of cancer cell lines and clinical tumor specimens. In addition to the identified abnormal levels of specific miRNAs in certain types of human cancers, biological evidence that suggests an important role of miRNAs in cancer development and progression was also experimentally demonstrated in animal models (12). To date, a spectrum of cancer-associated miRNAs has been identified. While some miRNAs function as tumor suppressors and are downregulated in cancer cells, other miRNAs act as oncogenes, inducing or promoting cancer development or progression. In this context, a number of miRNAs could play an oncogenic role in one setting but suppress tumor formation in a different scenario (13).

Abnormal expression profiles of miRNAs have been found in both clinical tumor specimens and cancer cell lines when compared with chosen normal controls by microarray, Northern blotting, or real-time RT-PCR analyses. In some types of cancers, global deregulation of miRNAs has been found, indicating that miRNAs have a general potential to target genes involved in cell proliferation, apoptosis, differentiation, invasiveness, and motility that are critical for development or progression of human cancers (14). Examples of the miRNAs that are aberrantly expressed in tissue-specific cancers are summarized in Table I.

Table I.

miRNAs Aberrantly Expressed in Cancers

Cancer type	Upregulated	Downregulated	Reference
Breast cancer	miR-10b, miR-21, miR-22, miR-27a, miR-155, miR-210, miR-221, miR-222, miR-328, miR-373, miR-520c	let-7, miR-7, miR-9-1, miR-17/miR-20, miR-31, miR-125a, miR-125b, miR-146, miR-200 family, miR-205, miR-206, miR-335	(15–31)
CLL	miR-21,miR-155	miR-15, miR-16, miR-29b, miR-29c, miR-34a, miR-143, miR-145, miR-181b, miR-223	(11,32–36)
Lung cancer	miR-17-92 cluster, miR-21, miR-106a, miR-155	miR-1, let-7 family, miR-7, miR-15a/miR-16, miR-29 family	(12,22,37–40)
Lymphoma	miR-17-92 cluster, miR-155	miR-143, miR-145	(36,41,42)
Prostate cancer	miR-221, miR-222	miR-15a-miR-16-1 cluster, miR-101, miR-127, miR-449a	(43–47)
Glioblastoma	miR-21, miR-221, miR-222	miR-7	(22,48,49)
Hepatocellular carcinoma	miR-17-92 cluster, miR-21, miR-143, miR-224	miR-1, miR-101, miR-122a	(50–55)
Colorectal cancer	miR-17-92 cluster, miR-21	miR-34a, miR-34b/c, miR-127, miR-143, miR-145, miR-342	(46,56–60)
Gastric cancer	miR-21, miR-27a	miR-143, miR-145	(61–63)
Ovarian cancer	miR-214	miR-34b/c, miR-200 family	(64–66)
Melanoma	miR-221, miR-222	let-7a, miR-34a	(67–69)
Head and neck squamous cell carcinoma	miR-21	let-7d, miR-138, miR-205	(70–72)

CLL chronic lymphocytic leukemia, CNV copy number variation, CSDC2 cold-shock domain-containing protein C2, EBV Epstein–Barr virus, EMT epithelial-to-mesenchymal transformation, EZH2 enhancer of zeste homolog 2, HBV hepatitis B virus, LNA locked nucleic acid, miRISC microRNA-induced silencing complex, miRNA microRNA, miRNP microRNA-induced silencing complex, NPC nasopharyngeal carcinoma, NSCLC non-small-cell lung carcinoma, nt nucleotides, p53RE p53 response element, pRb retinoblastoma protein, pre-miRNA miRNA precursor, pri-miRNA primary miRNA transcript, PTC papillary thyroid carcinoma, RNAa RNA activation, RNP RISC-like ribonucleoprotein complex, SNP single nucleotide polymorphisms, TARBP2 TAR RNA-binding protein 2, UTR untranslated region

miRNA Modulation of Tumor Suppressor and Oncogenic Pathways

A number of oncogenic pathways have been found to be under the modulation of miRNAs. As aforementioned, because an individual miRNA usually targets multiple genes that are involved in various cellular signaling pathways, aberrant expression or malfunction of a miRNA could exhibit powerful capability in oncogenesis. The retinoblastoma (pRb) pathway represents one of regulatory networks frequently altered in cancer cells. pRb is a tumor suppressor that associates with and inhibits transcription factors of the E2F family, causing cell cycle arrest. On the other hand, pRb is inactivated through protein phosphorylation by cell cycle-dependent kinases such as Cdk2 and Cyclin E, which are also negatively regulated by cell cycle inhibitors of members of INK4 or Cip/Kip families such as p16, p11, and p27. Accumulating evidence suggests that miRNAs modulate the pRb pathway at almost all levels. 3′-UTR of the pRb gene can interact with and be suppressed by miR-106a, which is overexpressed in various types of human cancers, and such an inhibitory effect of miR-106a is associated with downregulation of the pRb protein (37). Moreover, E2F is downregulated by the miR-17-92 cluster (73), whereas miR-34a that could be induced by p53 suppresses the expression of cyclin D1 and CDK6 (74). Similarly, miR-192 and miR-215 that also are induced by p53 can enhance the accumulation of p21 and induce cell cycle arrest in colon cancer cells (75). Additionally, CDK6 is suppressed by miR-124 or miR-137 that are often inactivated by epigenetic changes in tumor cells (76). Furthermore, oncogenic cyclin D1 is a target of miR-17/20 that is frequently downregulated in breast cancers (77). Taken together, miRNAs have complicated the molecular networks that control the cell cycle and proliferation by adding another dimension of regulation, and yet from the evolutionary point of view, such a multi-dimension and multilayer regulatory mechanism provides more powerful and precise maintenance over the stability and efficacy of these critical pathways.

Oncogene Myc plays crucial roles in modulating cell proliferation through its proliferation-promoting function via E2F, Cdk2, and cdc25 (78) and induces cell apoptosis through upregulation of an apoptosis inducer, Bim (79). On one hand, Myc is regulated by miRNAs, including miR-145 and miR-34 (80,81); on the other hand, Myc induces transcription of miRNAs such as the miR-106b-25 cluster and the miR-17-92 cluster (73). Interestingly, some of the miRNAs in the above clusters inhibit the expression of Bim and E2F (73,82). Thus, the biological impact by miRNA-associated Myc modulation is complicated and can be bidirectional. Whether the net outcome of such modulatory mechanisms is oncogenic or tumor-suppressing is context-dependent in different microenvironments.

It was found that miR-29a, miR-29b, and miR-29c could directly suppress p85, the regulatory subunit of PI3K, and CDC42, a Rho family GTPase, both of which negatively regulate p53. Thus, the miR-29 family members could induce p53-dependent cell apoptosis (83). On the other hand, the miR-34 family members are direct targets for p53 (66). Low expression of miR-34a in CLL is associated with p53 inactivation, and p53-induced cell apoptosis could be dramatically attenuated by inactivation of miR-34a in cells exposed to genotoxic stress, whereas mildly increased by overexpressing miR-34a (84). Furthermore, miR-34a could also downregulate SIRT1, leading to an increase in acetylation of p53 and expression of p21 and PUMA that are transcription targets of p53 (85). miR-145 is another p53-induced miRNA and has a potential p53 response element (p53RE) in its promoter region. Interestingly, miR-145 also directly downregulates c-myc, thus linking the p53 pathway to the c-myc pathway (80).

Another interesting aspect of cancer-associated miRNAs is their roles in virus-induced human cancers. Numerous types of human cancer-associated viruses have been found to express miRNAs. Epstein–Barr virus (EBV), a herpesvirus associated with Burkitt’s lymphoma and nasopharyngeal carcinoma, expresses multiple miRNA species. One of EBV-coded miRNAs, miR-BART5, downregulates PUMA, contributing to increased host cell survival and establishment of latent infection of the virus (86). Using computation-assisted approaches, a candidate pre-miRNAs was identified in HBV. By searching the 3′ UTR sequences of human genome, a large number of cellular transcripts were predicted as potential targets of viral miRNAs, and these predicted miRNAs, if validated, could play important roles in virus infection (87).

miRNAs IN TUMOR ANGIOGENESIS AND METASTASIS

It is widely accepted that growth and dissemination of tumors require the development of neovasculature, a key process known as angiogenesis. Possible roles of miRNAs in angiogenic response of tumors have been recently demonstrated. miR-126 that is abundantly expressed in endothelial cells plays an important role in vascular development in mice and zebrafish. miR-126 enhances the pro-angiogenic effects of VEGF and FGF by directly targeting Spred-1, a negative regulator of the VEGF and FGF signaling pathways (88). Other miRNAs such as miR-130a and miR-296 are also involved in tumor angiogenesis through modulation of the expression of pro-angiogenic receptors and antiangiogenic factors such as HGS, GAX, and HOXA5, respectively (89,90).

A vast majority of cancer deaths are caused by metastasis. When metastasis occurs, the polarized immotile epithelial cells transform into motile mesenchymal cells, a phenomenon known as epithelial-to-mesenchymal transformation (EMT). Accumulating evidence shows that TGF-β and ZEB take important parts in the EMT process, characterized by high-level expression of transcription factors that bind to the E-Box of promoter regions in genes of E-cadherin and other polarity proteins, and repress their gene expression. In this context, miR-200 represses both TGF-β and ZEB, contributing to the maintenance of the epithelial phenotype of cancer cells (91). Loss of expression of members of the miR-200 family in several types of cancers such as ovarian cancers and breast cancers enhances the tumor metastasis by downregulating E-cadherin and promoting EMT during cancer progression (16,64). Additionally, correlations of other miRNAs with cancer metastasis have also been reported. For example, miR-10b and miR-373 were found to promote breast cancer invasion and metastasis, respectively (30,31). Regulation of cell survival and metastasis by miRNAs could also be mediated through the repression of histone methyltransferase. Enhancer of zeste homolog 2 (EZH2) is a mammalian histone methyltransferase that involves the epigenetic silencing of genes and cancer cell survival and metastasis. EZH2 is overexpressed in aggressive solid tumors such as prostate cancer, and miR-101 inhibits the expression of EZH2 in cancer cell lines (47). Thus, in addition to specifically targeting one or more signaling pathways, aberrantly expressed miRNAs also modulate the overall epigenetic status of the genome, leading to a global change in the expression profiles that consequently effect on initiation, progression, and metastasis of human cancers (47).

Taken together, it is evident that miRNAs regulate the expression of proteins involved in a wide variety of signaling pathways critical for cancer development and progression. Some of the miRNA-targeted proteins connect to different cellular signaling networks. Fully understanding the role of miRNAs in modulating the expression and functions of these pathway connector proteins will significantly advance our knowledge of the biology of human cancers. Possible roles of miRNA in development and progression of cancer are illustrated in Fig. 1.

Fig. 1.

Functions of miRNA in cancer development and progression. Possible interactions among oncogenic and tumor suppressor miRNAs and classical genes and consequences in cancer development are illustrated. Green color, downregulated or attenuated. Red color, upregulated or enhanced. ➀ Translation inhibition/mRNA degradation; ➁ Transcriptional inhibition (direct or indirect); ➂ Transcriptional activation (direct or indirect); ➃ Transcriptional inhibition/activity inhibition

Causes of Aberrant miRNA Expression in Cancers

SNP and Mutations in pri-miRNA Sequence

Single nucleotide polymorphism (SNP) and mutations in pri-miRNAs sequences could significantly alter the secondary structure of pre-miRNA, which subsequently affects the binding affinity between miRNA and mRNA. For example, the rs11614913 SNP in miR-196a-2 gene is associated with the survival probability of individuals suffering from non-small-cell lung carcinoma (NSCLC). In a genotype–phenotype correlation analysis of 23 human lung cancer specimens, rs11614913 CC was found to be associated with a statistically significant increase in the expression of mature miR-196a. The survival of patients with lung cancers was significantly decreased in individuals who carried homozygous CC at rs11614913, suggesting that the rs11614913 SNP might be a useful prognostic biomarker for NSCLC (92).

A G/C polymorphism (rs2910164) within the pre-miR-146a coding sequence is frequently found in papillary thyroid carcinoma (PTC). When compared with that from the G allele, the production of pre- and mature miR-146a from the C allele is reduced by 1.9- and 1.8-fold, respectively, possibly due to impeded binding of a nuclear factor to C allele-derived pre-miR-146a, which leads to reduced production of a mature miRNA. Reduced miR-146a production results in lowered efficiency in inhibiting target genes involved in the Toll-like receptor-mediated cytokine signaling and PTC1, a gene frequently rearranged with the RET proto-oncogene in PTC, increasing the risk of acquiring PTC (93). Heterozygosity for polymorphisms within a pre-miR sequence can also cause epistatic gene regulation due to the production of another mature miRNA derived from the passenger strand. Jazdzewski K et al. reported that miR-146a GC heterozygotes are different from both the GG and CC homozygotes in their abilities to produce three mature miRNAs, namely, one from the leading strand (miR-146a) and two from the passenger strand (miR-146a*G and miR-146a*C), each having a distinct target set. Analysis of paired tumor/normal samples showed a 0.5- to 2.6-fold upregulation of polymorphic miR-146a* in seven of eight PTC tumors tested when compared with the unaffected regions of the thyroid. Microarray data showed differential profile of transcriptomes distinct between patients of GC and GG genotypes. A majority of the altered genes are apoptosis regulators, suggesting a potential cause for the changed risk of acquiring thyroid cancer (94).

Recently, germline or somatic mutations of miRNA genes have been identified in clinical specimens of CLL and several types of solid tumors. Sequencing analysis of 75 CLL patients revealed mutations in 5 of 42 miRNA genes examined. Two of 75 CLL patients were found to carry mutations in primary miR-16-1 and miR-15a sequences with C to T mutations. This mutation was associated with lower expression of the mature miR-16-1 and miR-15a (95). In solid tumors, 15 cancer-associated miRNAs were analyzed in 91 cancer-derived cell lines, leading to the identification of one variation in a miRNA precursor and 15 variations in primary miRNAs (96).

Copy Number Ateration

Alteration in copy number of miRNA coding cassettes gives rise to copy number variation (CNV) in cells. CNV usually involves gains or losses of large segments of DNA sequence consisting of thousands to millions of nucleotides, leading to significant changes of expression level of miRNAs (for collections of CNVs, see http://www.sanger.ac.uk/humgen/cnv).

Calin et al. (97) mapped the genomic locations of 186 miRNAs and compared them with previously reported non-random genetic alterations. The study found that miRNA genes were frequently located at fragile sites of the genome, as well as in minimal regions of loss of heterozygosity, minimal regions of amplification (minimal amplicons), or common breakpoint regions. Overall, 52.5% of miRNA genes were in cancer-associated genomic regions or in fragile sites, and numerous miRNAs located in the deleted regions exhibited low levels of expression in cancer samples, suggesting important roles of CNV-induced alteration of miRNA expression in cancers (97). Additionally, the overall involvement of miRNA genes in CNV regions can be revealed by a genome-wide analysis (98).

Loss of chromosomal segments on chromosome 13 at cytoband 13q14, a most frequent genetic alteration in CLL, includes genotypic subtypes among which the type Ia deletions are relatively uniform in length (50.2–50.5 Mb) and commonly involve the miR15a/miR16 gene cluster. Deletion of miR-16-1 and miR-15a at 13q14 was found in over 15% of CLL patients. Due to the suppressive effect of both miRNAs on Bcl-2 expression, their deletion and consequent loss of expression might contribute to the development of CLL (99).

Abnormal Transcription

Recent expression profiling of miRNAs in human cancers showed an overall downregulation of miRNAs. Molecular mechanisms that account for such inhibition include failure of miRNA posttranscriptional regulation, transcriptional silencing associated with epigenetic changes, transcriptional repression by dysfunction of transcription factors, and defects in the miRNA biogenesis that involves enzymes and cofactors important for miRNA processing. For instance, in ovarian cancers, the level of total mature miRNAs was reduced and closely associated with a lowered level of Dicer protein. At the transcriptional level, global sequence analysis revealed that over 46% of 326 miRNA putative promoters contained potential p53 binding sites, and downregulation of wild-type p53 via specific siRNAs abolished the effect of wt-p53 in regulating miRNAs in HCT-116 colon cancer cells (100).

Protein TARBP2 (TAR RNA-binding protein 2), an integral component of a DICER1-containing complex, was found to have frame shift mutations in sporadic and hereditary carcinomas with microsatellite instability. This might result in a decrease in TRBP protein expression and a defect in the processing of miRNAs because reintroduction of TRBP in TRBP-deficient cells restored an efficient production of mature miRNAs and inhibited tumor growth (101).

Global upregulation of miRNA also has been found in certain types of human cancers. For example, microarray analysis of metastatic prostate adenocarcinomas in comparison with normal prostate tissues showed an upregulation of major components of the machinery for miRNA processing including Dicer. Importantly, increased expression of these proteins is associated with a global increase in miRNA expression and progression of clinical stage of prostate adenocarcinoma (102).

miRNAs AS BIOMARKERS FOR CANCER DIAGNOSIS

Distinguishable abnormalities in miRNA genes and expression patterns are being identified continuously in almost all types of cancer, thus providing a strong rationale for the application of miRNAs as diagnostic/prognostic biomarkers. Expression profiles of miRNAs have been found significantly altered in numerous types of human cancers when compared with their corresponding normal tissues, among different subtypes within the same type of cancers, or among individual patients suffering from a same type of cancer but having different prognoses. For example, miR-21 is expressed at high levels in glioblastoma as opposed to a low basal level in normal brain tissues, establishing miR-21 as a potential diagnostic marker for glioblastoma (48). In lung cancers, expression profiles of miRNAs correlate with the survival of patients with lung adenocarcinomas including those with stage I disease. High levels of miR-155 and low levels of let-7a-2 expression are associated with poorer patient survival (103). In breast cancers, overexpression of miR-21 correlates with certain pathophysiological features of the disease such as advanced tumor stage, lymph node metastasis, and poor survival of the patients (104). A study using microarray on 435 mature human miRNA oligonucleotide probes identifies miR-21 as a potential prognostic marker for diagnosis of breast cancers (104). Moreover, expression of miR-210 in breast cancer specimens reveals an inverse correlation with disease-free and overall survival, suggesting that miR-210 could be another independent prognostic factor for breast cancers (25). Additionally, miRNA signature might also be useful to distinguish different subtypes of human cancers. Comparative analysis on the expression patterns of miRNAs in 122 adenocarcinoma versus squamous lung cancer samples identified miR-205 as a highly specific marker for squamous cell lung carcinoma, with a sensitivity of 96% and a specificity of 90% (105).

It is noteworthy that miRNAs can be often found in the circulation and in microvesicles (also known as exosome when produced by tumor tissues) in the peripheral blood. This observation supports a proposition of using cancer-associated miRNAs as biomarkers for early cancer diagnosis (106). By evaluating specific exosomal miRNAs in 27 patients with lung adenocarcinoma, Rabinowits et al. (107) found that the mean concentrations of exosomes and miRNAs in exosomes varied significantly between cancer patients and normal controls, as opposed to an insignificance between miRNAs in exosomes and tumor tissues, indicating that the circulating exosomal miRNA might also be a useful diagnostic marker.

miRNAs as Novel Drug Targets for Cancer Treatment

Distinct functions of miRNAs in tumor initiation, progression, and metastasis in human cancers strongly suggest miRNAs as novel drug targets or therapeutic tools to develop novel strategies for the treatment of human cancers. Plausible approaches could be through either downregulating “oncogenic” miRNAs or upregulating “tumor suppressor” miRNAs. Two approaches have been tested for inhibition of the levels of miRNAs, namely, short oligonucleotides complementary to miRNAs (antagomir or antimir) (108) and miRNA sponges, which refer to synthetic mRNAs containing multiple binding sites for a specific miRNA and thereby competitively sequestering the endogenous miRNA (109). To achieve efficient binding with a miRNA, antagomirs and antimirs can be chemically modified as 2-O-methyl oligoribonucleotides and locked nucleic acid (LNA), respectively (108). In contrast, elevating the level of endogenous miRNAs could be achieved by the delivery of synthetic miRNAs or DNA constructs that code for specific miRNAs.

Similar to other gene therapy approaches, efficient, safe, and tissue-specific delivery of synthetic miRNA, antagomir, or antimir has been tested in laboratories with certain degree of success. Delivery strategies include uses of plasmids, viral vectors or transposons, as well as cationic liposomes coupled with monoclonal antibodies to direct membrane-permeable reagents-conjugated miRNA to specific organs such as the lung and the liver. PC3 prostate cells express miR-221/222 at high levels. Treatment of mice bearing established subcutaneous PC3 tumor xenografts with anti-miR-221/222 antagomirs significantly suppressed tumor growth with a long-term effect of tumor reduction, suggesting the clinical applicability of antagomir (110). LNA-antimir oligonucleotides have also been tested in vitro as well as in vivo. Treatments with LNA-antimirs against miR-16, miR-21, and the miR-17-92 cluster were tested in cancer cell lines derived from glioblastoma, colon cancers, breast cancers, and lung cancers (111). Administration of LNA-antimir to mice effectively blocks liver-specific miR-122 expression in vivo (112). Additionally, miR-127 is constitutively expressed in normal cells but epigenetically silenced in cancer cells. Treatment of cancer cells with chromatin-modifying drugs such as 5-Aza-CdR and PBA that inhibits DNA methylation significantly elevated the expression of miR-127 and inhibited cell proliferation, implicating an application of epigenetic approaches for cancer treatments (46).

SUMMARY

Investigation on the roles of miRNA in cancer represents a developing and promising research field in the war against cancer. As new knowledge and technologies continuously emerge, novel and effective anti-cancer strategies are becoming increasingly possible. Clinical management of human cancers will greatly benefit from the development of miRNA-based diagnostic and therapeutic approaches, and the pharmaceutical industry is also welcoming a new challenging opportunity in this exciting process. On the other hand, while the discovery of miRNA and the identification of its roles in cancer pathogenesis provide enormous promises in improving the outcome of cancer management, the research community as well as the industry face challenges in developing and delivering applicable and practically effective miRNA-based anti-cancer technologies. Like all other sequence specificity-based strategies, miRNA targeting can be accompanied by unwanted non-specific actions, and the resultant off-target effects could be even more daunting for miRNAs because miRNAs are characteristic of using imperfect complementarity to interact with their target sequences, displaying an intrinsic feature of the so-called multi-specificity. Furthermore, while the “multi-specific” feature enables a single miRNA to target more than one mRNA species, an individual mRNA can be synergistically regulated by multiple miRNA species. These features have obviously increased the complexity in developing effective miRNA-based anti-cancer approaches with minimal adverse effects. In addition, the development of new vehicle systems with low pharmacological toxicities is also urgently needed for the delivery of miRNAs or anti-miRNA inhibitors in vivo, particularly for systemic delivery. In this context, novel chemistry for miRNA modification, improved viral or non-viral vectors, and nanotechnology-based transportation tools are expected to find stages in the course of implementing safe and effective strategies for the detection and delivery of miRNAs.