miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma

MicroRNAs (miRNAs) are a class of small non-coding RNAs that, in general, negatively regulate gene expression. They have been identified in various tumor types, showing that different sets of miRNAs are usually deregulated in different cancers. Some miRNA genes harboring CpG islands undergo methylation-mediated silencing, a characteristic of many tumor suppressor genes. To identify such miRNAs in hepatocellular carcinoma (HCC), we first examined the methylation status of 43 loci containing CpG islands around 39 mature miRNA genes in a panel of HCC cell lines and non-cancerous liver tissues as controls. Among 11 miRNA genes frequently methylated in HCC cell lines but not in non-cancerous liver tissues, three miRNA genes, i.e. miR-124, miR-203 and miR-375, were selected as silenced miRNAs through CpG-island methylation by comparing methylation and expression status and evaluating restored expression after treatment with 5-aza-2′-deoxycytidine. In primary tumors of HCC with paired non-tumorous liver tissues, only miR-124 and miR-203 showed frequent tumor-specific methylation, and their expression status was inversely correlated with methylation status. Ectopic expression of miR-124 or miR-203 in HCC cells lacking their expression inhibited cell growth, with direct downregulation of possible targets, cyclin-dependent kinase 6 (CDK6), vimentin (VIM), SET and MYND domain containing 3 (SMYD3) and IQ motif containing GTPase activating protein 1 (IQGAP1) or ATPbinding cassette, subfamily E, member 1 (ABCE1), respectively. Our results suggest that miR-124 and miR-203 are novel tumorsuppressive miRNAs for HCC epigenetically silenced and activating multiple targets during hepatocarcinogenesis.