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Hepatitis C virus proteins modulate microRNA expression and chemosensitivity in malignant hepatocytes.

ABSTRACT: Hepatocellular cancer (HCC) is highly resistant to chemotherapy and is associated with poor prognosis. Chronic hepatitis C virus (HCV) infection is a major cause of HCC. However, the effect of viral proteins in mediating chemosensitivity in tumor cells is unknown. We postulated that HCV viral proteins could modulate therapeutic responses by altering host cell microRNA (miRNA) expression.HepG2 malignant hepatocytes were stably transfected with full-length HCV genome (Hep-394) or an empty vector (Hep-SWX). MiRNA profiling was done by using a custom microarray, and the expression of selected miRNAs was validated by real-time PCR. Protein expression was assessed by Western blotting, whereas caspase activation was assessed by a luminometric assay.The IC(50) to sorafenib was lower in Hep-394 compared with Hep-SWX control cells. Alterations in miRNA expression occurred with 10 miRNAs downregulated >2-fold and 23 miRNAs upregulated >2-fold in Hep-394 cells compared with controls. Of these, miR-193b was overexpressed by 5-fold in Hep-394 cells. miR-193b was predicted to target Mcl-1, an antiapoptotic protein that can modulate the response to sorafenib. The expression of Mcl-1 was decreased, and basal caspase-3/7 activity and poly ADP ribose polymerase cleavage were increased in Hep-394 cells compared with controls. Moreover, transfection with precursors to miR-193b decreased both Mcl-1 expression and the IC(50) to sorafenib.Cellular expression of full-length HCV increases sensitivity to sorafenib by the miRNA-dependent modulation of Mcl-1 and apoptosis. Modulation of miRNA responses may be a useful strategy to enhance response to chemotherapy in HCC.

SUBMITTER: Braconi C

PROVIDER: S-EPMC2818698 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Hepatitis C virus proteins modulate microRNA expression and chemosensitivity in malignant hepatocytes.

Braconi Chiara C Valeri Nicola N Gasparini Pierluigi P Huang Nianyuan N Taccioli Cristian C Nuovo Gerard G Suzuki Tetsuro T Croce Carlo Maria CM Patel Tushar T

Clinical cancer research : an official journal of the American Association for Cancer Research 20100126 3

<h4>Purpose</h4>Hepatocellular cancer (HCC) is highly resistant to chemotherapy and is associated with poor prognosis. Chronic hepatitis C virus (HCV) infection is a major cause of HCC. However, the effect of viral proteins in mediating chemosensitivity in tumor cells is unknown. We postulated that HCV viral proteins could modulate therapeutic responses by altering host cell microRNA (miRNA) expression.<h4>Experimental design</h4>HepG2 malignant hepatocytes were stably transfected with full-leng ...[more]

PMID: 20103677

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Project description:Hepatitis C virus (HCV) directly induces oxidative stress and liver injury. Bach1, a basic leucine zipper mammalian transcriptional repressor, negatively regulates heme oxygenase 1 (HMOX1), a key cytoprotective enzyme that has antioxidant and anti-inflammatory activities. microRNAs (miRNAs) are small noncoding RNAs ( approximately 22 nt) that are important regulators of gene expression. Whether and how miRNAs regulate Bach1 or HCV are largely unknown. The aims of this study were to determine whether miR-196 regulates Bach1, HMOX1, and/or HCV gene expression. HCV replicon cell lines (Con1 and 9-13) of the Con1 isolate and J6/JFH1-based HCV cell culture system were used in this study. The effects of miR-196 mimic on Bach1, HMOX1, and HCV RNA, and protein levels were measured by way of quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. The Dual Glo Luciferase Assay System was used to determine reporter activities. miR-196 mimic significantly down-regulated Bach1 and up-regulated HMOX1 gene expression and inhibited HCV expression. Dual luciferase reporter assays demonstrated that transfection of miR-196 mimic resulted in a significant decrease in Bach1 3'-untranslated region (UTR)-dependent luciferase activity but not in mutant Bach1 3'-UTR-dependent luciferase activity. Moreover, there was no detectable effect of mutant miR-196 on Bach1 3'-UTR-dependent luciferase activity.miR-196 directly acts on the 3'-UTR of Bach1 messenger RNA and translationally represses the expression of this protein, and up-regulates HMOX1. miR-196 also inhibits HCV expression in HCV replicon cell lines (genotype 1b) and in J6/JFH1 (genotype 2a) HCV cell culture system. Thus, miR-196 plays a role in both HMOX1/Bach1 expression and the regulation of HCV expression in human hepatocytes. Overexpression of miR-196 holds promise as a potential novel strategy to prevent or ameliorate hepatitis C infection, and to protect against liver injury in chronic HCV infection.

Project description:BACKGROUND: Virus-host interactions result in altered gene expression profiles in host cell nuclei and enable virus particle production, thus obligatorily involving changes in their epigenomes. Neither such epigenome changes nor their response to antiviral treatment have been extensively studied to date, although viral infections are known to contribute to the long-term development of severe secondary diseases, for example, hepatocellular carcinoma. This may be causally linked to virus-induced persistent plastic chromatin deformations. RESULTS: We studied whether impaired hepatitis B virus (HBV) replication can lead to the restitution of epigenome signatures hypothesizing that hepatocytes alternatively could adopt a 'memory' state of the infection; that is, the chromatin could persist in a HBV-induced configuration potentially inheritable between dividing hepatocytes. We therefore determined epigenomic signatures and gene expression changes altered by HBV and the effects of suppressed HBV replication in nontransformed hepatocytes of newborn mice. Further we investigated differential histone acetyltransferase and histone deacetylase activities in HBV-negative and HBVpositive hepatocytes, as well as the effects of HBV suppression on gene expression and the chromatin landscape. We show that the expression of several genes and the chromatin landscape become altered upon HBV infection, including global hypoacetylation of H2A.Z and H3K9. Reporter assays monitoring the activities of histone acetyltransferases or histone deacetylases, respectively, suggest that hypoacetylation most probably depends on elevated sirtuin deacetylase activity, but not on class I/II histone deacetylases. Using Micrococcus nuclease to study the chromatin accessibility in met murine-D3 and hepatitis B virus met murine hepatocytes, we demonstrate that the observed differences in H2A.Z/H3K9 acetylation lead to global chromatin structure changes. At all selected sites examined by chromatin immunoprecipitation and quantitative real-time PCR, these effects can be partly restituted via the nucleoside analog reverse transcriptase inhibitor 3TC or using anti-HBV microRNA-like molecules. CONCLUSIONS: Increased sirtuin activity might lead to global histone hypoacetylation signatures, which could contribute to the HBV-induced pathomechanism in nontransformed hepatocytes. Using several techniques to suppress HBV replication, we observed restituted gene expression and chromatin signature patterns reminiscent of noninfected hepatocytes. Importantly, ectopic expression of antiviral short-hairpin RNA, but not microRNA-like molecules, provoked intolerable off-target effects on the gene expression level.

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Hepatitis C virus proteins modulate microRNA expression and chemosensitivity in malignant hepatocytes.

Publications

Hepatitis C virus proteins modulate microRNA expression and chemosensitivity in malignant hepatocytes.

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