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

Mayuko Furuta,Ken-Ich Kozaki,Johji Inazawa,Issei Imoto,Shigeki Arii,Shinji Tanaka

doi:10.1093/carcin/bgp250

Abstract

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 ATP-binding cassette, subfamily E, member 1 (ABCE1), respectively. Our results suggest that miR-124 and miR-203 are novel tumor-suppressive miRNAs for HCC epigenetically silenced and activating multiple targets during hepatocarcinogenesis.

Highlights

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide [1]
In the study presented here, we focused on miRNA genes containing CpG islands upstream or around them and screened miRNAs with tumor-specific methylation and silencing in cell lines and primary samples of hepatocellular carcinoma (HCC) to identify epigenetically inactivated miRNAs leading to HCC
In the course of a program to screen a panel of 19 HCC cell lines for tumor-suppressive miRNAs silenced by tumor-specific aberrant DNA methylation, we first selected miR-124, miR-203 and miR-375 as possible candidate genes because of (i) consistency in the inverse correlation between DNA methylation status around these miRNAs and their expression patterns in a panel of HCC cell lines; (ii) their higher expression and lower methylation pattern in non-tumorous liver tissues, including inflammatory or cirrhotic livers and (iii) restoration of their expression on treatment with 5-aza-2#-deoxycytidine in cells lacking the expression

Summary

Introduction

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide [1]. It shows a wide geographical variation with high incidence areas in Africa and Asia. Among various cancer-related gene-silencing mechanisms in the epigenetic pathways, DNA hypermethylation of CpG sites within CpG islands is known to lead to the inactivation of many TSGs [10] and several tumor-suppressive miRNAs [11]. In the study presented here, we focused on miRNA genes containing CpG islands upstream or around them and screened miRNAs with tumor-specific methylation and silencing in cell lines and primary samples of HCC to identify epigenetically inactivated miRNAs leading to HCC. Through this approach, two possible HCC-associated tumor-suppressive miRNAs, miR-124 and miR-203, and novel candidates for their putative targets were identified

Materials and methods

Results

Discussion