1. EPIGENETIC REGULATION OF MICRORNA EXPRESSION IN PROSTATE CANCER Seodhna M. Lynch 1, Karla M. O’Neill 1, Michael M. McKenna 2, Colum P. Walsh 1, Declan J. McKenna 1 1 Transcriptional Regulation and Epigenetics Group, Biomedical Sciences Research Institute University of Ulster, Coleraine, Northern Ireland 2 Western Health & Social Care Trust, Altnagelvin Area Hospital, Derry, Northern Ireland ABSTRACT MicroRNAs are small, non-coding RNA species (19-25nts) which have been found to play a fundamental role in many molecular pathways including embryogenesis, cell differentiation, proliferation and apoptosis. Altered miRNA expression has been correlated with a wide range of disease states and pathologies, including cancer. Recently, there is an increasing body of evidence suggesting that epigenetic regulation of miRNAs via DNA methylation may be an important mechanism in contributing to their deregulation in cancer. We hypothesise that miRNAs with tumour suppressor function may be silenced by epigenetic modifications in prostate cancer (PCa). In this study we have carried out a screen for miRNAs showing altered expression in PCa cell lines. This identified three miRNAs (miR-205, miR-200c, miR-138) that are typically down-regulated in PCa cells and which may be controlled by methylation. We also show that the expression of these miRNAs is generally decreased in prostate tumour biopsy samples compared to matched normal tissue. We demonstrate that these miRNAs are up-regulated by the chemotherapy drug decitabine (5-aza- 2’deoxycytidine), which alters DNA methylation levels, and also by reduction in DNA methyltransferase 1 (DNMT1) levels in PCa cells. Methylation levels in the loci of each candidate miRNA were examined in PCa cells and biopsy tissue. We propose that the relative expression of these miRNAs is related to their respective methylation status. In addition, the functionality of the miRNAs in PCa cells has been investigated by examining their effect on cell proliferation, and by measuring expression levels of known and novel target genes. Our findings provide further evidence that profiling of miRNA expression and methylation status has great potential as a basis for a novel biomarker in the diagnosis and prognosis of PCa. BACKGROUND MicroRNAs are small, non-coding RNA species (19-25nts) which have been found to play a fundamental role in many molecular pathways including embryogenesis, cell differentiation, proliferation and apoptosis (1). Altered miRNA expression has been correlated with a wide range of disease states and pathologies, including cancer (2). MicroRNAs have been identified to be useful in PCa in terms of diagnosis, prognosis, signature profiling and promising potential as biomarkers for disease in tissues and serum (3,4). Recently, there is an increasing body of evidence suggesting that epigenetic regulation of miRNAs via DNA methylation may be an important mechanism in contributing to their deregulation in cancer (5,6). In our lab we have screened a panel of miRNAs in prostate cancer (PCa) cells to identify those which display altered differential expression. We hypothesise that the altered expression of certain miRNAs may be due to epigenetic modifications and we have selected three lead candidates, miR- 205, miR-200c and miR-138 to test this hypothesis. There is previous evidence that miR-205 is epigenetically regulated in PCa (5), but no study to date has similarly investigated epigenetic regulation of miR-200c and miR-138 in PCa. RESULTS Knockdown of DNA Methyltransferase 1 (DNMT1) in PCa cells (G & H) resulted in increased expression of miR-200c and miR-138 (I) (For each graph n=3,,** P<0.01 *** P<0.001). Fig.2 microRNA profiling in PCa Needle Core Biopsies Paired diseased and normal tissue from a panel of 15 FFPE embedded PCa needle core biopsies were analysed for miRNA expression by qRT-PCR. (A) miR-205 (B) miR-200c and (C) miR-138 all show decreased expression in 11/15 (73.3%) tumour biopsies compared to matched normal tissue. (Normalisation was to U6snRNA for each graph) DISCUSSION & FUTURE WORK This study investigated whether expression of the miRNAs miR- 205, miR-200c and miR-138 in PCa cells was related to epigenetic regulation. We are interested in miR-138, miR-200c and miR-205 because their expression appears to be generally down-regulated in PCa cell lines and prostate biopsies (Fig 1 & 2). miR-205 is known to be significantly reduced in prostate cancer (5) and our results corroborate those findings. miR-200c has been reported to be both elevated and reduced in prostate cancer (7, 8). Our results would suggest this differential expression is dependent on the stage of cancer, which is an important consideration in establishing its potential as a biomarker for early- or late-stage PCa. miR-138 has not been extensively studied in Pca to date. Here, for the first time, we show its expression is significantly lost in PCa cells relative to control cells, suggesting a possible tumour suppressor function. We hypothesised that expression of these miRNAs might be epigenetically regulated. We proceeded to validate that demethylation, by either DNA methyltransferase knockdown or treatment with de-methylation agent decitabine, impacts upon transcription expression of miR-138, miR-200c and miR-205 (Fig 3). We therefore designed assays to specifically measure methylation in the promoters and loci of these miRNAs (Figs 4-6). The preliminary data we present here shows that we can successfully analyse methylation in the selected regions by COBRA and pyrosequencing. We show that there is a general correlation between methylation status and expression in these cells, in that increased methylation silences the miRNA expression. Our observations are backed up by previous studies which have also suggested miR-205 and miR-200c may be epigenetically silenced in cancer (5, 9), although only miR-205 has been studied in this regard in PCa (5). miR-138 has not been shown to be epigenetically regulated, so this is the first report to suggest this novel mechanism. We also present preliminary data indicating that miR-200c may itself contribute to epigenetic regulation by targeting DNA methyltransferase 3A (Fig. 7). Luciferase assays will confirm this interaction and similar experiments are investigating novel targets of miR-138 and miR-205 to establish functionality in these cells. On-going and future experiments will attempt to further clarify the regulation of these miRNAs and their functional role in PCa. In addition, a clinical collaboration, using a panel of archived prostatectomy samples, will assess the biomarker potential of both methylation status and expression profiling of miR-138, miR-200c and miR-205 in prostate cancer. REFERENCES 1.FABBRI and CALIN, 2010. Advances in Genetics, 70, 87-99. 2.COPPOLA et al. 2010. Endocrine-related cancer, 17(1), 1-17. 3.SITA-LUMSDEN et al, 2013. British journal of cancer, 108,1925-1930. 4.VAN NESTE et al, 2012. The Prostate, 72, 1248-1261. 5.HULF et al, 2013. Oncogene, 32, 2891-2899. 6.PAONE et al, 2011. Frontiers in genetics, 2, 62. 7.PUHR et al, 2012. Am J Pathol. 181, 2188-201. 8.TANG et al, 2011.Methods Mol Biol.732, 69-88. 9.TANG et al, 2013.Clin Cancer Res, 19, 5602-12. Fig. 4 Methylation analysis of miR-200c Promoter (A) miR200c Primer Design Schematic shows the promoter region of miR200c which spans 406bp from -324 to +82. 21 CpG sites are analysed and represented by black filled circles, with individual CpG sites numbered. (B) CpG Site Methylation Analysis. Primers were designed to allow the same CpG sites to be analysed by three different methods; Combined Bisulfite Restriction Analysis (COBRA), Bisulfite Sequencing and pyrosequencing. (C) Schematic of CpG sites assessed by COBRA. The sizes of expected products resulting from complete digestion of a fully methylated product are indicated. (D) COBRA Methylation Analysis. Untreated lanes (UNT) show partial methylation in RWPE1 cells, no methylation in LNCaP cells and hypermethylation in PC3 cells. 5’-Aza treatment (AZA) appears to have little effect on methylation in this region. (Gel is representative of 3 experiments. u = unmethylated, m = methylated. Digest control is miniprep plasmid, + = BstU1 added, - = no BstU1 added) (E) Pyrosequencing Methylation Analysis gives quantitative confirmation of COBRA analysis. Analysed region is ~55% methylated in RWPE1 cells, ~6% methylated in LNCaP cells and ~93% methylated in PC3 cells. As with COBRA, 5’-Aza treatment has minimal effect on methylation in LNCap or PC3 cells (n = 3) (A) miR-205 Primer Design Schematic shows the promoter region of miR205 which spans 364bp. 10 CpG sites are analysed and represented by black filled circles, with individual CpG sites numbered. (B) CpG Site Methylation Analysis. (C) Schematic of CpG sites assessed by COBRA. The sizes of expected products resulting from complete digestion of a fully methylated product are indicated. (D) COBRA Methylation Analysis. Untreated lanes (UNT) reveal approximately 50% methylation of this region in RWPE1 cells and hypermethylation in LNCaP and PC3 cells. 5’-Aza treatment has minimal effect on methylation in this region. (Gel is representative of n= 3. u = unmethylated, m = methylated). Pyrosequencing will confirm and quantify these results Fig. 6 Methylation analysis of miR-205 promoter Fig. 5 Methylation analysis of putative miR-138 regulatory CpG island (A) miR138 Primer Design Schematic shows a CpG island (CGI) upstream of miR-138 as identified by UCSC genome browser. Within the CGI, the region analysed for methylation status spans 212bp from +156 to +367. 19 CpG sites are analysed and represented by black filled circles, with individual CpG sites numbered. (B) CpG Site Methylation Analysis for each assay. (C) Schematic of CpG sites assessed by COBRA. The sizes of expected products resulting from complete digestion of a fully methylated product are indicated. COBRA Methylation Analysis in (D) RWPE1, (E) LNCaP and (F) PC3. The CGI upstream of miR-138 loci appears to be partially methylated in untreated RWPE1 cells, less methylated in LNCaP cells. However, in PC3 cells the CGI appears hypermethylated. Pyrosequencing will confirm and quantify these results. Fig.1 microRNA profiling in PCa Cell lines (A) miR-205, (B) miR-200c and (C) miR-138 expression was profiled in normal RWPE1 prostate epithelial cells, androgen dependent LNCaP and 22RV1 PCa cells, and androgen-independent PC3 PCa cells by quantitative real-time PCR (qRT-PCR). Both miR-205 and miR-138 show significantly reduced expression in PCa cells relative to normal cells. miR-200c is significantly elevated in LNCaP and 22RV1, before it is significantly reduced in expression in PC3 cells (Normalisation was to U6snRNA for each graph, n=3,*** p<0.001). *** A BC ABC LNCaP and PC3 cells were treated with for 72hours with the demethylating agent decitabine (5-aza-2 deoxycytidine) (1µM). Effect upon methylation was confirmed by increased expression of the germline gene SYCP3 in 5’-Aza treated cells (AZA) relative to untreated cells (UNT) ( A & B, D & E). miR-138 and miR-205 were both up-regulated following 5’-Aza treatment in both cells lines (C & F). miR-200c was not increased by 5’-Aza treatment in LNCaP cells (C), but showed a small, but significant increase in PC3 cells (F) (n=3 LNCaP experiments, n=2 PC3 experiments, *p<0.05, **p<0.01) Fig.3 Demethylation induced by decitabine treatment or DNMT1 knockdown increases expression of miR-205, miR-200c & miR-138 GH I Fig. 7 DNMT3A is a potential target of miR-200c (A) Venn diagram displaying numbers of miRNAs computationally predicted to target DNMT3A by TargetScan (green), PicTar (red) and miRDB (blue). miR-200c is predicted by all three algorithms. (B) qRT-PCR shows that over-expression of miR-200c in PC3 cells results in significant down-regulation of DNMT3A. Known targets of miR- 200c, SOX2 and ZEB1, are included as controls. (n = 3, p<0.001) *** ** (Joshi et al., 2011) (Wiemer at al., 2007) AB RWPE1LNCaPPC3 D