丙型肝炎病毒核心蛋白对Wnt信号抑制因子SFRPs甲基化修饰的影响

Methylation modification of Wnt inhibitor SFRPs by Hepatitis C core protein

目的探讨丙型肝炎病毒核心蛋白是否通过对Wnt信号抑制因子SFRPs的甲基化修饰,从而活化Wnt信号通路。方法将编码HCV核心蛋白的腺病毒(AdCore)或GFP对照腺病毒(AdGFP)感染SMMC-7721细胞,采用逆转录PCR(RT-PCR)方法检测SFRPs基因mRNA的表达;用5-氮杂-2'-脱氧胞苷(DAC)对AdCore或AdGFP腺病毒感染的SMMC-7721细胞进行去甲基化处理,进一步采用甲基化特异性PCR(MSP)和DNA甲基化测序,检测SFRPs基因启动子区CpG岛的甲基化程度,Western blot检测甲基化转移酶Dnmts的表达。结果在HCV核心蛋白过表达的SMMC-7721细胞系中,SFRP2和SFRP5基因mRNA的表达量与对照组相比明显降低(P<0.05);甲基化分析结果表明:SFRP5基因启动子区CpG岛呈现高甲基化修饰;甲基化转移酶Dnmt1、Dnmt3a表达量增加(P<0.05)。结论 HCV核心蛋白可以促进SFRP5基因启动子区CpG岛的甲基化修饰,参与HCV相关疾病的发生。

Objective To determine the effect of hepatitis C core protein on methylation modification of secreted frizzled-related protein（SFRP） genes.Methods Hepatoma cell line SMMC-7721 was infected with AdCore to develop an in vitro over-expression model.At 36 h after infection,the mRNA expression level of SFRP genes were detected with reverse transcriptase PCR in core-expressing SMMC-7721 cells.Cells were further demethylated with 5-aza-2′-deoxycytidine（DAC） for 96 h,and then infected with AdCore or AdGFP for 36 h.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 Dnmt1 and Dnmt3a were detected by Western blotting.Results mRNA expression level of SFRP2 and SFRP5 gene were sharply reduced in core-expressing SMMC-7721 cells.Methylation analysis indicated that HCV core protein induced hypermethylation of CpG islands within SFRP5 promoter regions.Moreover,this modification was partially mediated by DNA methyltransferase Dnmt1 and Dnmt3a.Conclusion HCV core protein can induce hypermethylation in SFRP5 gene,which plays an important role in the pathogenesis of hepatocellular carcinoma.