Hepatitis C Virus Core Protein Epigenetically Silences SFRP1and Enhances HCC Aggressiveness

Objective: Hepatocellular carcinoma is one of the most common fatalmalignancy, whereas hepatitis C virus infection is a well known risk factorassociated with HCC. HCV infects approximately200million peopleworldwide, accounting for about3%of the world’s total population. Besides,3million to4million new cases emerge annually. HCV therapy mainlyinvolves treatment with pegylated-interferon and rivabirin. However, thistherapy is not ideal, especially in some patients with high titer HCV-RNAand genotype1. HCV infection severely hazards to human health, thepathogenic mechanism of HCV infection related HCC will supply newprevention and treatment strategies for the HCV infection disease. The Coreprotein is multi-functional structure protein of a molecular weight of21kDathat HCV virus encoded. It has been reported that HCV Core protein isimplicated in virus particle assembly and release, and interacting with sometranscription factors and signaling pathways, which may weaken the body’simmune defensive function and eventually lead to the occurrence of HCVrelated liver cirrhosis and even HCC. HCV Core protein can also bind to several tumor suppressor proteins, such as p53, p73, pRb, which isassociated with cell apoptosis and the occurrence of tumor.The aberrant activation of Wnt/β-catenin signaling pathway plays animportant role in HCC, and this appears in at least50%of HCC. In HCCtissue samples, AXIN gene mutation is relatively rare, β-catenin abnormalactivation caused by mutation only captures20%of these samples,inactivation of APC is not found. These promote that antagonist moleculeSFRPs of pathway will involve in the activation of Wnt/β-catenin signalingpathway.DNA methylation is an important form of epigenetic modification. Ithas been demonstrated that hypermethylation of SFRPs promoter are foundin many solid tumors, such as lung cancer, breast cancer, bladder cancer,gastric cancer, colon cancer, liver cancer and so on. It has been reported thatvirus infection plays a key role in epigenetic regulation to alter genetranscription and expression, leading to uncontrolled proliferation anddifferentiation. It is not seen the report that HCV Core whether regulateSFRPs expression by DNA methylation and histone acetylation. Wepreviously showed that HCV Core protein suppressed the expression level ofSFRP1. In this study, we will further discuss molecular mechanism andstudy the biological behavior of HCC cell lines from both positive andnegative aspects. Methods: We constructed serial deletion mutant of SFRP1promoterregion and each promoter activity was measured using the luciferase assaysystem, then we determined which region is the strongest activity of SFRP1promoter and examined whether HCV Core could inhibit SFRP1promoteractivity. ChIP was further used to determine the levels of Dnmt1andHDAC1bound to the SFRP1promoter region in core-expressing cells.Besides, colony formation, crystal violet, migration and invasion assayswere used to determine whether knock-down of Dnmt1or over-expressionof SFRP1could suppress the proliferation, migration and invasion ability ofhepatoma carcinoma cell. Moreover, we tested the ability that knock-downof Dnmt1or over-expression of SFRP1forms tumors in nude micecompared with control vector-infected cells. DNA methylation status ofSFRP1promoter region was further confirmed by bisulfite sequencing. Thenwe assessed the levels of cell proliferation, invasion markers anddownstream molecular c-Myc in the tumor samples retrieved from miceusing immunohistochemical.Results：We previously showed that HCV Core protein suppressed theexpression levels of of SFRP1and Aza restored SFRP1protein expression ina concentration-dependent manner. In this research, we demonstrated thatthe strongest activity area of SFRP1promoter is-407～-27nt, HCV Corecould inhibit SFRP1promoter activity. ChIP assays further showed thatHCV Core enhanced the recruitment of Dnmt1, HDAC1, MBD1, MBD2, and MeCP2to the SFRP1promoter region in Huh7and SK-Hep1cell linesand inhibited the binding of P300and acetylated histone H3to the SFRP1promoter. Moreover, cell proliferation and colony formation ability of HCCcells stably transduced with Core were shown to increase markedly whencompared with the vector control stable cells. In cell migration assay, theaverage number of HCV Core stable clones migrating through the transwellmembrane was significantly higher compared with that of the control cells.Likewise, in the invasion assay, SK-Hep1-Core cells invading through thematrigel were also higher than in parental cells. Knock-down of Dnmt1orover-expression of SFRP1could also suppress both the migration andinvasion ability of SK-Hep1and Huh7cells ectopically expressing HCVCore protein. We next tested the ability of Huh7cells infected withadenoviruses either expressing siRNA against Dnmt1or over expression ofSFRP1to form tumors in nude mice compared with control vector-infectedcells. We found that tumor growth was inhibited significantly by theknock-down of Dnmt1and restoration of SFRP1. Moreover, PCNAexpression significantly accumulated in the nucleus of control virus-infectedcells, but was almost undetectable in AdsiDnmt1or AdSFRP1infected cells.Accordingly, the enforced expression of SFRP1or knockdown of Dnmt1significantly decreased the intracellular accumulation of β-catenin, theexpression of PCNA, Wnt target gene (c-Myc) and MMP2, MMP9. Takentogether, knockdown of Dnmt1and over expression of SFRP1inhibited in vivo tumor growth through inhibition of β-catenin signaling activity in HCCcells.Conclusion: Our findings suggest that HCV Core can triggerepigenetic silencing of SFRP1by enhancing the recruitment of Dnmt1,HDAC1, MBD1, MBD2, and MeCP2to the SFRP1promoter, resulting inaberrant activation of Wnt/β-catenin and increased proliferation, migrationand invasion ability of hepatoma cells in vitro and tumorigenicity in vivo.