Abstract 90: A comprehensive analysis to screen for epigenetically silenced miRNA genes in colorectal cancer

Abstract

Abstract Epigenetic alteration plays an important role in inactivating microRNA (miRNA) genes in cancer. In the present study, we performed comprehensive analysis of miRNA expression, histone modification and DNA methylation in order to determine the transcription start sites (TSSs) of miRNA genes as well as to identify epigenetically silenced miRNA genes in colorectal cancer (CRC). We used a CRC cell line HCT116 treated with our without 5-aza-2’-deoxycytidine (DAC) plus 4-phenylbutyric acid (PBA). We also used a HCT116 cells in which DNMT1 and DNMT3B are genetically disrupted (DNMTs KO). For miRNA expression profiling, we used Human miRNA Microarray v1 (Agilent Technologies) and TaqMan MicroRNA Array v2.0 (Applied Biosystems). We carried out chromatin immunoprecipitation (ChIP) using antibodies against trimethyl-histone H3K4 (H3K4me3), trimethyl-histone H3K27 (H3K27me3) or dimethyl-histone H3K79 (H3K79me2). We performed ChIP-on-chip analysis using Human Promoter ChIP-on-chip microarray (Agilent Technologies) and ChIP-seq analysis using SOLiD3plus (Applied Biosystems). DNA methylation was analyzed using methylated-specific PCR (MSP), bisulfite-pyrosequencing and methylated-CpG island amplification microarray (MCAM). miRNA microarray analysis revealed that 187 miRNAs were downregulated in HCT116 cells compared to normal colon, and more than half of the downregulated miRNAs were upregulated by demethylating treatment. By combining miRNA expression and H3K4me3 analysis, we identified presumed TSSs of 173 primary miRNA genes which encode 266 pre-miRNAs. Of the 266 pre-genes, TSSs of 114 genes are located in the proximal upstream (< 5 kb) of pre-miRNA-coding regions, and TSSs of 213 genes are associated with CpG islands (CGIs). Comparison between HCT116 and DNMTs KO cells suggested that 39 primary genes which encode 87 pre-miRNAs are epigenetically silenced in HCT116. Of the 38 genes, TSSs of 24 genes are associated with CGIs, and we observed hypermethylation in all of them. We conclude that the combined analysis of expression, histone modification and DNA methylation is a useful strategy to identify epigenetically silenced miRNA genes in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 90. doi:10.1158/1538-7445.AM2011-90