Study of promoter hypomethylation profiles of RAS oncogenes in hepatocellular carcinoma derived from hepatitis C virus genotype 3a in Pakistani population.

Abstract

Epigenetic modifications such as DNA methylation contribute to progression of hepatitis C virus (HCV) infection to life-threatening hepatocellular carcinoma (HCC) by promoting the silencing of tumor suppressor genes through DNA hypermethylation and by causing genomic instability through global hypomethylation. However few studies have addressed the promoter region hypomethylation status of the oncogenes involved in HCV derived HCC. In this study, we analyzed the promoter region methylation pattern of RAS oncogenes (HRAS, KRAS, and NRAS) using methylation-specific PCR for 50 chronic HCV patients infected with genotype 3a (27 HCC patients and 23 control non-HCC patients). Methylation-specific polymerase chain reaction analysis revealed that the NRAS oncogene promoter (P = .0025) was significantly hypomethylated in HCC patients compared to the non-HCC patients suggesting its contribution to the progression of HCV towards HCC. To identify the agent for alteration in the RAS oncogene expression, 7 HCV genes were expressed in the Huh-7 cell line followed by measurement of the NRAS expression level in Huh-7 by a quantitative real-time polymerase chain reaction. An increase in the messenger RNA level of the NRAS gene was detected when Huh-7 were transfected with Core, NS5a, and NS2 genes. Our findings suggest the involvement of NRAS oncogene in the pathogenesis of HCV3a derived HCC in Pakistani population and also identifies the HCV genes responsible for its enhanced expression. Our study raises the hypothesis that a single HCV gene may increase the chances of malignancy. Therefore, our study may have identified a useful epigenetic biomarker of HCC progression in HCV patients and may help to develop novel diagnostic tools.