Hepatitis C virus core protein modulates pRb2/p130 expression in human hepatocellular carcinoma cell lines through promoter methylation.

Anna Maria Mileo,Marco G Paggi,Anna Ruggieri,Stefano Mattarocci,Rodolfo Sacco,Paola Matarrese,Simona Anticoli,Claudia Abbruzzese,Stefania Catone

doi:10.1186/s13046-015-0255-1

Abstract

BackgroundHepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial. The nucleocapsid (core) protein of HCV has been shown to be directly implicated in cellular transformation and immortalization, enhancing the effect of oncogenes and decreasing the one of tumor suppressor genes, as RB1 and its protein product pRB. With the aim of identifying novel molecular mechanisms of hepatocyte transformation by HCV, we examined the effect of HCV core protein on the expression of the whole Retinoblastoma (RB) family of tumor and growth suppressor factors, i.e. pRb, p107 and pRb2/p130.MethodsWe used a model system consisting of the HuH-7, HCV-free, human hepatocellular carcinoma cell line and of the HuH-7-CORE cells derived from the former and constitutively expressing the HCV core protein. We determined pRb, p107 and pRb2/p130 protein and mRNA amount of the respective genes RB1, RBL1 and RBL2, RBL2 promoter activity and methylation as well as DNA methyltransferase 1 (DNMT1) and 3b (DNMT3b) expression level. The effect of pRb2/p130 over-expression on the HCV core-expressing HuH-7-CORE cells was also evaluated.ResultsWe found that the HCV core protein expression down-regulated pRb2/p130 protein and mRNA levels in HuH-7-CORE cells by inducing promoter hyper-methylation with the concomitant up-regulation of DNMT1 and DNMT3b expression. When pRb2/p130 expression was artificially re-established in HuH-7-CORE cells, cell cycle analysis outlined an accumulation in the G0/G1 phase, as expected.ConclusionsHCV core appears indeed able to significantly down-regulate the expression and the function of two out of three RB family tumor and growth suppressor factors, i.e. pRb and pRb2/p130. The functional consequences at the level of cell cycle regulation, and possibly of more complex cell homeostatic processes, may represent a plausible molecular mechanism involved in liver transformation by HCV.

Highlights

Hepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial
HCV core expression and cellular localization in a human HCC cell line To investigate the interplay between the HCV core protein and the cellular factors involved in cell growth and replication, we engineered the HCV-negative HuH-7 human HCC cell line in order to constitutively express high levels of the HCV core protein from genotype 1b, by transfection with the plasmid pcCAG39neo (Fig. 1, panel a)
In order to examine the effect of the HCV core protein on the expression of all the RB family proteins, we determined by Western blot pRb, p107 and pRb2/p130 protein expression in both HuH-7 and HuH-7CORE (HCV core-expressing) cells (Fig. 2)

Summary

Introduction

Hepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial. Hepatitis C Virus (HCV), a member of the Flaviviridae family, has a single-stranded, positive-sense RNA genome of approximately 9.6 kb in length, which encodes a large polyprotein precursor of about 3,000 amino acids. This precursor is subsequently cleaved, by a combination of host and viral proteases, into at least ten proteins, four structural proteins [core, envelope 1 (E1), envelope 2 (E2) and p7] and six non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) [1, 2]. Activation of cellular oncogenes, inactivation of tumor suppressor genes, and dysregulation of multiple signal transduction pathways have been reported as possible pro-oncogenic mechanisms of HCV associated tumor [6]. Several studies have reported the presence of HCV core mutant proteins in HCV-infected patients who developed HCC, the functional relevance of these mutations on malignant transformation is still not clear [18]

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