The Effect Of Hepatitis C Virus E1Protein On Hepatoma Cells Proliferation And Migration Hcv Core Protein Methylation Modification Of Wnt Inhibitor Sfrps

Objective:Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality in the worldwide. Hepatitis C virus (HCV) infection is the main risk factor for chronic hepatitis, cirrhosis and hepatocellular carcinoma development. Approximately60%to85%adults infected with HCV developed to chronic hepatitis patients, far higher than that of HBV infection (5%-10%). Thus HCV infection is a severe threat to human health. The molecular mechanism of HCV-related cirrhosis and HCC are largely unknown. Recent studies suggest that HCV proteins interact with host cell, and lead to abnormal cell signaling pathway activation, cell proliferation and differentiation.HCV E1protein is a HCV-encoded envelope glycoprotein and an important component of virus particles. C-terminal hydrophobic region of E1protein is located in the endoplasmic reticulum, showing a variety of biological functions.The Mitogen-activated protein kinase is a class of serine/threonine protein kinase, more frequently seen in a range of mammalian cells. MAPKs transduce extracellular signals into cells, thus affecting the process of cell growth, differentiation and apoptosis. The abnormalities of MAPK signaling pathway is involved in tumors, inflammatory, metabolic disorders and a variety of human diseases. ERK1/ERK2signal transduction pathway is a classical MAPK pathway. Sustained MAPKs signal activation is necessary for G1to S phase progression. Studies have shown that HCV Core protein actives a variety of signaling pathways.Such as STAT3, MAPK, Wnt/beta-catenin signal pathway to promote cell proliferation, malignant transformation and other pathological changes, which led to liver injury after HCV infection. But the role of E1protein in HCV-related diseases are not clear. In this study, we used Ad-E1recombinant adenoviral vector to express HCV E1protein in SMMC-7721and Huh7cells, and we investigated the role of E1protein in cell proliferation and migration. Finally, we explored the molecular mechanism of HCV-related pathogenesis.Methods:We have amplified adenovirus Ad-E1, Ad-E2, Ad-NS3and Ad-GFP, which encodes HCV E1, E2, NS3and GFP protein respectively. Over-expression of target gene in the transcriptional or translational level was identified by RT-PCR or Western blot analysis. SMMC-7721and Huh7cells were infected with high titer of adenovirus and the biological function induced by different viral protein was determined by cell proliferation assay. The cell growth, cell cycle analysis and cell migration were detected by MTS, Crystal Violet, Brdu ELISA assay, Flow cytometry and Transwell migration assay. The Wnt/β-catenin signal transduction activity was determined by Western blot assay and the target genes including c-Myc, cyclinDl, c-Jun and c-Fos of MAPK/ERK signal pathway were further determined by Western blot or RT-PCR assay.Results:Ad-E1, Ad-E2, Ad-NS3adenovirus were amplified successfully. Gene transcript products and proteins expression of E1, E2and NS3were identified by RT-PCR and Western blot analysis respectively. Cell proliferation assay indicated over-expression of HCV E1alone resulted in an increased proliferation rate in SMMC-7721and Huh7cells. Cell cycle analysis showed that the S-phase distribution in cells transduced with E1was34.38%, much higher than that of GFP control (27.32%)(p<0.05).Transwell migration assay confirmed that cells migrated through the transwell chamber was significantly higher in Ad-E1group than Ad-GFP group (p<0.05). Moreover, E1protein moderately up-regulated mRNA expression level of transcriptional factors and target genes of MAPK/ERK signal pathway, whereas the molecules related to wnt/β-catenin signal pathway has little change after treatment with E1Protein.Conclusions:Cell growth and migration promotion effects induced by HCV E1Protein in SMMC-7721and Huh7cells were partially correlated with MAPK/ERK signal pathway. Objective: HCV infection is a major risk factor for hepatocellular carcinoma development. Core protein is a HCV-encoded multifunctional protein, which is the major components of the nucleocapsid, Core protein participates in HCV replication and virus particle assemble and release. HCV Core protein can promote a variety of cellular activities including DNA synthesis, cell proliferation, differentiation and transformation, thus involving in the development of hepatocellular carcinoma. HCV Core protein can also activates the canonical Wnt pathway (Wnt/β-catenin) by regulating upstream molecules.SFRP proteins inhibit Wnt/β-catenin pathway by binding to both the frizzled receptor and the Wnt ligand. Methylation down regulating SFRPs expression can lead to abnormal activation of the Wnt signaling pathway. Hypermethylation of SFRPs frequently occurs in human mesothelioma, gastric cancer, lung cancer, breast cancer, bladder cancer, etc. This study was designed to investigate the role of HCV Core protein in the methylation modification of SFRPs. Methods:Adenovirus encoding HCV Core protein (Ad-Core) and control adenovirus (Ad-GFP) were amplified in HEK293cells, and HCV Core gene expression at the transcriptional and translational level were identified by Western blot or RT-PCR. Hepatoma cell line SMMC-7721was infected with Ad-Core to establish an in vitro over-expression model. At36hrs after infection, the mRNA expression level of SFRP genes were detected with RT-PCR analysis. Cells were further demethylated by5-aza-2’-deoxycytidine (DAC) for96hrs, and then infected with Ad-Core or Ad-GFP for48hrs. Methylation modification of CpG islands within SFRPs promoter region were assayed by methylation specific PCR (MSP) and bisulfite sequencing analysis, In addition, the expression level of methyltransferase Dnmtl and Dnmt3a were detected by Western blot analysis.Results:mRNA expression level of SFRP2and SFRPS gene were sharply reduced in Core-expressing SMMC-7721cells. Methylation analysis indicated that HCV Core protein induces hypermethylation of CpG islands within SFRPS promoter regions. Moreover, this hypermethylation modification was partially mediated by DNA methyltransferase Dnmtl and Dnmt3a.Conclusions:HCV Core protein promotes hypermethylation of SFRP genes, which playing an important role in the pathogenesis of hepatocellular carcinoma.