Identification of aberrant DNA methylation profiles in non-tumor liver tissues of patients with non-B, non-C hepatocellular carcinoma.

Abstract

249 Background: Percentage of patients with non-B non-C (NBNC) hepatocellular carcinoma (HCC) is rapidly increasing in Japan. We have previously shown that miRNA expression profiles in non-tumor liver tissues of patients with NBNC-HCC are different from those of patients with HBV-HCC and HCV-HCC. Moreover, we have recently reported that specific miRNA expression profiles in non-tumor liver tissues can predict a risk of multicentric recurrence after hepatectomy for HCC (Hepatol Res, 2013). In this study, we performed the genome-wide analysis of aberrant DNA methylation in non-tumor liver tissues of patients with NBNC-HCC. Methods: We divided the 23 patients, who underwent hepatectomy, into 3 groups; NBNC (HBcAb-, n=7), NBNC (HBcAb+, n=8), and normal control (NC, n=8). We analyzed DNA methylation status in fresh-frozen non-tumor liver tissues using Infinium Human Methylation 450 Bead Chip (illumina). Gene-expression levels of several identified genes were analyzed by quantitative RT-PCR to validate the correlation between DNA methylation status and gene-expressions. Results: We identified 87 differentially methylated genes in HBcAb- liver tissues, and 603 genes in HBcAb+ liver tissues in comparison to NC liver tissues (Beta value>0.2, P<0.05). Thirty CpG sites were commonly hyper- or hypo-methylated in both HBcAb- and HBcAb+ livers. High percentages of aberrant DNA methylation were observed in gene body. One hypermethylated gene (Lrig1, an intestinal stem cell marker that functions as a tumor suppressor) was confirmed to be down-regulated in its gene-expression level by quantitative RT-PCR. One hypomethylated gene (LEF1, a molecule enhancing hepatocarcinogenesis though Wnt/beta-catenin signaling) was also confirmed to be up-regulated. Ingenuity Pathway Analysisrevealed the association of Wnt/beta-catenin signaling with aberrant DNA methylation status. Conclusions: Genome-wide analysis of aberrant DNA methylation in non-tumor liver tissues may provide not only molecular mechanisms of epigenetic modulation during hepatocarcinogenesis but also early molecular diagnosis in patients with NBNC-HCC.