miR-191: an emerging player in disease biology

1MicroRNAs are a class of small non-coding RNAs that function as post-transcriptional gene regulators. for the first time suggested the association of solid tumors with dysregulation of specific microRNAs. Since then miRNA-cancer link has extended beyond reports of abnormal miRNA levels in various cancers to miRNAs acting themselves as oncomiRs or tumor-suppressors. However, functional significance of majority of dysregulated miRNAs has not been elucidated. We have focused here on one such highly conserved miRNA, miR-191 that was found to be abnormally expressed in more than twenty different cancers and shown to be a major player in some of these. Further, altered expression of miR-191 has also been associated with various other diseases such as type 2 diabetes, neurodegenerative diseases, and more recently with innate immunity. This review is timely to highlight the crucial role miR-191 plays in a multitude of diseases including cancer and also in normal development and differentiation processes. We start off with a small introduction to miR-191/425 cluster and its regulation followed by summarizing studies on functional dissection of miR-191 in various cancers. Overall, current understanding of miR-191 biology suggests it to be a promising novel target for cancer prognosis and therapy.

2miR-191 is expressed as part of the miR-191/425 cluster which is highly conserved in several metazoan species (miR-425 in 26 species, miR-191 in 30 species) suggesting it to be an important player in higher eukaryotes. First discovered in mouse, its expression was later confirmed in humans in a leukemia cell line (HL-60) and subsequently in 20 different human tissues. This cluster is located in the first intron of DALRD3 gene on human chromosome 3 (3p21.31) and codes for four mature miRNAs – miR-191-5p, miR-191-3p, miR-425-5p, and miR-425-3p. It forms a sense antisense transcript pair with DALRD3, NDUFA3, and WDR6 mRNAs; however, the functional impact of this association has not been experimentally evaluated. miR-191 tends to be coexpressed with the DALRD3 and NDUFAF3 transcripts. miR-191 resides 381 nucleotides upstream of miR-425 and notably shows much higher expression or cloning frequency as compared to miR-425 in all the tissues studied so far. This is in compliance with recent reports showing that huge variations in expression levels of miRNA members may exist within the same cluster. Indeed, Croce and Kulshreshthas group have shown that both miR-191 and miR-425 promote cell proliferation in breast cancer exhibiting overlapping functions, but a previous study shows that miR-191 and not miR-425 promotes erythroid enucleation. Therefore, with very limited data, we assume that miR-191 and miR-425 may exhibit specific or overlapping functions depending on the tissue and conditions.

3The first report that miR-191 is developmentally regulated came from the observation that significantly high levels of miR-191 were present in (postmortem) human prefrontal cortex specimens of individuals older than 41 years than those under 15 years. It was suggested that miR-191 mediated downregulation of brain-derived neurotrophic factor (BDNF; predicted miR-191 target) may contribute to the cortical development. miR-191 is also one of the major miRNA expressed in rat neurons and its expression increases ~2–5 fold with time in rat cortical cultures suggestive of its role in neuronal development.

4Developmental association of miR-191 was further extended to senescence since its overexpression was shown to promote replicative senescence and inhibit proliferation in primary human keratinocytes. Role of miR-191 in regulation of circadian rhythm was also suggested as it was shown to exhibit a circadian pattern of expression in mouse liver and showed inverse correlation to Bma-1 (circadian transcription factor) levels.

5The connection of miR-191 and differentiation was also explored recently. miR-191 was found to be significantly downregulated during terminal erythroid differentiation (CFU-E to the Ter119+ stage) while its overexpression inhibited erythroid enucleation and chromatin condensation suggesting that miR-191 plays a crucial role in erythropoiesis. Levels of miR-191 were also found to be differentially expressed in monocytes vs. monocytes derived dendritic cells. Induced miR-191 levels were observed in osteoblast like cells following treatment with Bio-Oss (an organic bovine bone). As Bio-Oss plays an important role in bone regeneration thus miR-191 may be an important mediator for the same. Therefore, all these findings suggest that miR-191 plays an important role in cellular differentiation and development.

6miR-191 and cancer abnormalities have been reported in more than twenty different malignancies making the miR-191 a ubiquitously notorious miRNA like miR-21 or miR-155. Overexpression of miR-191 in 16 different cancer types [breast (female), colon, lung, liver, prostate, pancreas, stomach, ovarian cancer, pituitary adenoma, esophageal squamous carcinoma, oral squamous carcinoma, osteosarcoma, B-ALL, bladder, anaplastic large cell lymphoma, and acute myeloid leukemia (AML)] classifies it largely as an oncogenic miRNA. However, in six other cancer types (severe medulloblastomas, retinoblastoma, thyroid follicular tumor, male breast cancer, CALL, and melanoma) its levels are known to be downregulated. The nature of miR-191 deregulation, its functions and targets reported so far in various cancers are shown in Table 1.

7Since miR-191 is one of the highly expressed and stable miRNA in human serum or saliva, it shows potential of being used as non-invasive biomarker in human subjects. A new method for the disease characterization based upon blood circulating miRNAs was proposed recently. It involved both miR-191 and miR-425 as part of a unique combination of 12 miRNAs that could serve as potential biomarkers to distinguish disease/cancer from healthy controls in 10 out of 13 diseases (p = 0.046; ). recently filed a patent involving a group of 7 miRNAs including miR-191 that serve as markers for prediction, diagnosis, and progression of pancreatic cancer. Similarly, miR-191 (along with miR-182 and miR-199a) based methods for diagnosis and prognosis of AML have been recently patented. Higher expression of miR-191 was found to be significantly associated with poor overall survival (OS) and event free survival in adult AML patients. Also, in colorectal cancer patients, higher miR-191 expression has been shown to be associated with clinical stage, metastasis and tumor invasion. In contrast, reduced miR-191 levels have been reported to be associated with bad prognosis or short OS or disease relapse in melanoma, pediatric AML or male smoker squamous cell carcinoma (SCC) patients.

8Apart from miR-191 alone as a prognostic marker, the miR-146b/miR-191 ratio has also been reported as a negative prognostic indicator in lung SCC patients (0.29+0.25; ). Likewise, have shown that combined expression profile of two miRNAs (miR-125b/miR-191 and miR-21/miR-191) is more specific in discriminating between breast cancer and non-tumor tissue. Interestingly, presence of genetic variants in miR-191 precursor has been linked to genetic predisposition to familial ovarian cancer and could therefore serve as a diagnostic marker. Additionally, epigenetic regulation (hypomethylation) of miR-191 locus has been associated with poor prognosis in hepatocellular carcinoma (HCC; ). Hence, quantification of miR-191/its precursor variants/methylation patterns of its promoter using tissue/blood samples may emerge as a novel method for cancer diagnosis and prognosis.

9While there are ample reports on abnormal miR-191 levels in various cancers, the knowledge about its functional impact is quite limited. Studies in breast, colon, gastric, and hepatic cancer cell lines suggest miR-191 to be an oncomiR. miR-191 was shown to be involved in regulation of cell proliferation, apoptosis, and epithelial mesenchymal transition in HCC. In colon cancer, anti-miR-191 was shown to attenuate the invasiveness, suppress proliferation and induce apoptosis and in gastric cancer, miR-191 overexpression was shown to enhance cell proliferation and reduce apoptosis. However, functional analysis of miR-191 levels in pancreatic ductal carcinoma cells showed that although it is responsible for maintaining the transformed stage but cell proliferation is not affected. Recently, showed that miR-191 is responsible for promotion of various hallmarks of cancer (proliferation, metastasis, stress resistance) in ER+ breast cancer (MCF7) cells, while demonstrated that miR-191/425 cluster is responsible for reduced proliferation, tumorigenesis, and metastasis in aggressive ER- breast cancer cells (MDA-MB-231;; ). This dual effect of miR-191 could be due to the fact that miR-191 is an ER regulated miRNA that functions as a critical mediator of estrogen mediated cell proliferation. miR-191 has been shown to regulate cell cycle progression in breast cancer by promoting G1/S and G2/M transitions as the cells overexpressing miR-191 entered S-phase much faster with drastic reduction in G0/G1 phase.

10In contrast, miR-191 has been projected as a tumor-suppressor microRNA that leads to reduced growth and migration in thyroid follicular carcinoma. Notably, though miR-191 was shown to be differentially expressed (significantly upregulated in both squamous and non-squamous NSCLC, p < 1e-07) in lung cancer, it was shown to not alter cell cycle, proliferation or chemosensitivity of lung cancer cell lines.

11Till date only two groups have performed mouse xenograft assays to test miR-191 activity in vivo evaluating its therapeutic potential and the results are certainly encouraging. generated an orthotopic human liver tumor xenografts in nude mice and administered 2-O-metoxyethyl (MOE) anti-miR-191 through repeated intraperitoneal injections. A statistically significant reduction in tumor mass was observed without toxicity by the end of 40 days. Similarly, a subcutaneous transplantation of the anti-miR-191 transfected ER-alpha positive MCF7 or ZR-75-1 breast cancer cells in nude mice resulted in a 50% reduction in tumor mass. The effect of miR-191 on metastasis was also studied in aggressive breast cancer. Injection of miR-191 overexpressing ER negative breast cancer cells (MDA-MB-231) in NOD-SCID mice led to a significant reduction in a number of micrometastasis to lungs of mice as compared to the control tumor cells. Thus, regulated expression of miR-191 may serve as potential therapeutic treatment of the disease.

12Despite well documented miR-191 and cancer links, very little is known about the factors that lead to its deregulation in various tumor cells. In breast cancer, two independent groups demonstrated that estrogen and tumor microenvironment play a major role in regulation of miR-191. The promoter of miR-191 bears estrogen response elements which show dynamic binding of ER-alpha and ER-beta transcription factors in response to estrogen treatment. The levels of both miR-191 and miR-425 have been reported to be estrogen inducible. Tumor microenvironment (hypoxia and nutrient deprivation) has also been reported to strongly induce miR-191 in breast cancer. A report by suggests that the loss of p53 may be the reason behind higher expression (median 1.4-fold, p 0.0264) of miR-191 in colorectal tumor. Another study shows that the gain in expression of MDM4, a miR-191 target gene, in retinoblastoma leads to inactivation of p53 activity. Interestingly, the hypomethylation of CpG islands in miR-191 promoter has been linked to higher expression of miR-191 in HCC. While in melanoma, both, the genomic loss of the miR-191 locus and downregulation of miR-191 by a transcription factor, SNAIL have been shown as the reasons for low miR-191 levels. To conclude, transcription factors, tumor microenvironment and epigenetic mechanisms play major role in regulation of miR-191 in various cancers.

13Several recent reports suggest that miR-191 levels are also responsive to chemical drugs and radiation treatments. have shown miR-191 induction in response to X-ray treatment in human lymphoblast cells (TK6 cell line). However, miR-191 was shown to be rather downregulated in two colon cancer cell lines HCT116 and HCT-8 when treated with chemotherapeutic drugs (5-Fluorouracil and Oxaliplatin; ). Thus, the effects on miR-191 levels may vary depending on the stress or tissue/cell type. Work in our lab shows that miR-191 overexpressing breast cancer cells show increased survival in response to chemotherapeutic drug or hypoxia treatment. Altogether these reports suggest prospects of miR-191 being a target for studying chemotherapeutic interference.

14Interestingly, carcinogens too have been reported to mediate miR-191 regulation. showed that AOM (carcinogen) treated rats show downregulation of miR-191. Conversely, feeding rats with fish oil rich in polyunsaturated fatty acids (n = 3 PUFA; a chemoprotective agent) prevented AOM mediated downregulation of miR-191 and tumor formation. Environmental carcinogens also play a major role in regulation of miR-191, for example, cigarette smoke and TCDD (a dioxin family carcinogen) was shown to regulate miR-191 levels in rat lungs and hepatic cancer, respectively. Thus, these data provide evidence for hormonal, environmental and dietary regulation of miR-191.

15Association of miR-191 is not only limited to various cancers but its irregular expression and functional abnormalities have been reported in a variety of other diseases as well (Table 1). The functional impact of miR-191 is still unknown in these diseases but it does show potential as a diagnostic or prognostic marker for some of them. Recently reported that miR-191 may be a diagnostic biomarker candidate that could help to distinguish between the Crohn’s disease (CD) and ulcerative colitis (UC; ). Further, as the two disease types differ in their associated T- and B- cells, miR-191 may be involved in regulation of innate and adaptive immunity as well. Similarly, miR-191 was shown to be a potential biomarker for early detection and severity of pulmonary hypertension. Remarkably, miR-191-5p was shown to be one of the best biomarker candidates to predict the Alzheimer’s disease with >95% accuracy. miR-191 was also found to be significantly induced (3.79-fold, p < 0.0001) in nephrotic syndrome (NS) children making it a potential diagnostic marker for pediatric NS. Interestingly, NS patients under remission have significantly reduced level of miR-191 suggestive of its association with disease progression and therapy. Overall, miR-191 seems to be closely associated with the pathogenesis of diverse diseases and may also be involved in innate immune responses. A compiled list of diseases having an association with miR-191 is given in Table 1.

16A number of miR-191 targets have been validated and characterized functionally. Its targets range from chromatin remodelers to transcription factors to cell cycle regulators. A detailed analysis of the validated potential targets of miR-191 is given below:

17Special AT-rich sequence-binding protein-1 is a global chromatin remodeler and transcription factor that is known to be overexpressed in several cancers and also associated with aggressive phenotype in colorectal and breast cancer. miR-191 mediated downregulation of SATB1 has been linked to gain/loss of epithelial/mesenchymal markers in aggressive breast cancer and enhanced cell proliferation and migration in hormone dependent breast cancer. The miR-191/SATB1 functional link has been proposed to be helpful for prognosis and therapeutics of breast cancer. Additionally, suppression of SATB1 was demonstrated to be linked to cell senescence, as its suppression by miR-191 led to enhanced senescence in keratinocytes.

18RIO kinase 3 and max-interacting protein 1 are reported to be negatively regulated by miR-191 and play essential role in chromatin condensation and enucleation during mouse erythroid differentiation.

19Early growth response protein 1 is a zinc finger transcription factor mainly involved in cell growth, development and stem cell homeostasis. It was demonstrated to be an estrogen responsive miR-191 target, having regulatory role in cell proliferation in ER-alpha positive breast cancer cells. Additionally, it was reported to be downregulated by miR-191 in scrapie infection, a prion disease.

20Sex determining region Y-box 4 is a transcriptional factor found to be upregulated in various cancers. It is involved in regulation of development, differentiation, proliferation, apoptosis, and more recently epithelial-mesenchymal-transition. It was shown to be a novel target of miR-191 in HCC. Thus, downregulation of SOX4 by miR-191 may affect chromatin architecture and crucial cellular processes.

21Mdm4 p53 binding protein homolog is a negative regulator of transcription factor, p53 and is found to be overexpressed in 17% of all cancers. miR-191 is reported to regulate cancer progression and chemosensitivity in ovarian cancer by modulating the level of MDM4 through binding with an illegitimate site (rs4245739) within the 3′ UTR. Recently, the same SNP (rs4245739) was investigated and found to be a risk factor for esophageal SCC and breast cancer. The MDM4 rs4245739 SNP alone or in combination with the p53 variant Arg72Pro has been shown to be responsible for attenuating breast cancer risk in Chinese population.

22Cyclin-dependent kinase 6 is a protein kinase mainly associated with cell cycle progression and differentiation. It has been reported as a bona fide target of miR-191 in aggressive and hormone receptor positive breast cancer and thyroid and follicular carcinoma. Additionally, miR-191 mediated downregulation of CDK6 has been reported to trigger senescence in keratinocytes.

23Cyclin D2 is a cell cycle regulatory protein involved in estrogen mediated cell proliferation. It was recently established as a target of miR-191 and its downregulation led to suppression of PI3/AKT pathway thereby leading to reduced cell proliferation in aggressive breast cancer.

24N-deacetylase/N-sulfotransferase1 is a key enzyme involved in the biosynthesis of heparan sulfate chains and is responsible for maintenance of physiological functions. It regulates binding of FGF to mammary epithelial cells leading to branching morphogenesis. It was shown to be negatively regulated by miR-191 and its downregulation has suppressive effect on cell proliferation in gastric carcinoma.

25Brain-derived neurotrophic factor is a neurotrophin essential for differentiation, survival, and synaptic plasticity of CNS. Recently our group has shown it to be a target of miR-191 in ER positive breast cancer.

26Tissue inhibitor of metalloprotease 3 is a proapoptotic protein whose expression is negatively correlated with cell growth and invasiveness. It is a direct miR-191 target in colorectal carcinoma and its targeted downregulation is associated with enhanced invasiveness of the disease.

27An overview of miR-191 regulatory components along with its functional impact on direct and indirect targets is given in Figure 1.

28Our review brings to limelight an important player in cancer biology that has been shown to have profound effects on various hallmarks of cancer. Due to its ubiquitous role in various biological processes, the interest in miRNA-191 is likely to dramatically increase in coming years, especially in cancer. Though various studies document miR-191 association with cancer progression, but its link to cancer initiation has not yet been elucidated. However, since carcinogens have been shown to regulate the levels of miR-191, it may be involved in tumor initiation process as well.

29Considering the widespread association of miR-191 to several cancer types and recent studies documenting that its inhibition leads to reversal of cancer phenotype, it is tempting to speculate that miR-191 may qualify as a candidate for “oncogene addition.” However, the validation of this concept warrants careful and extensive experimentation using genetically engineered mouse models of miR-191. These models may also throw light on the role of miR-191 in various stages of tumor development. Nevertheless, based on miR-191-lung cancer studies, miR-191 may not necessarily functionally contribute to aggression of all cancer/diseases where its levels are abnormally expressed.

30Interestingly, several types of abnormalities have been linked to miR-191 in cancer. These include but may not be limited to aberrant mature miR-191 levels, hypomethylated miR-191 promoter, mutations in miR-191 target site or loss of genomic locus. However, there are very few reports depicting the basis behind its altered expression in disease states compared to that of normal. Till date transcription factors, tumor microenvironment and epigenetic regulation have been identified as the major players responsible for aberrant expression of miR-191 in various disease states. A strong estrogen and hypoxic regulation of miR-191 have been shown to regulate key pathways in breast cancer. In light of recent reports of interdependence of estrogen and hypoxia pathways, it would be interesting to know if miR-191 functions to connect the two pathways. Further, considering miR-191 to be stress inducible and its reported effects on apoptosis, it would be interesting to see if p53 is directly involved in its regulation.

31Owing to highly consistent and stable expression of miR-191 and the ability to detect it in human saliva or serum, miR-191 emerges as a strong candidate for non-invasive disease diagnostics. A strong association between miR-191 levels and disease prognosis/survival laid basis for two recent patents featuring miR-191 based diagnosis and prognosis in AML and pancreatic cancer. It is likely that miR-191 may emerge as an independent or a co-prognostic marker in other cancers as well. Use of disease specific mouse models, conditional transgenic as well as knockouts are required to elucidate its functional role in vivo and verify its therapeutic potential, both of which remain largely unexplored till now. More novel approaches such as combination of Argonaute protein immunoprecipitation, deep sequencing, or proteomic profiling approaches are required to identify its potential bona fide targets responsible for its functional impact. The fact that miR-191 may directly enhance target transcript levels is striking and needs further elucidation of the mechanism. In particular a combination of phenotypic, transcriptomic, proteomic, and metabolomic changes on manipulation of miR-191 levels in cell line or mouse models will provide a complete understanding of miR-191 functions. To conclude, achieving ways to regulate the level of this particular microRNA in patients (in a safe and consistent manner) might help to fight the underlying disease.

32The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.