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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 investigate whether hepatitis C virus core protein activates Wnt signaling through methylation modification of Wnt signaling inhibitor SFRPs. Methods SMMC-7721 cells were infected with AdCore encoding HCV core protein or GFP control adenovirus (AdGFP). The mRNA expression of SFRPs was detected by reverse transcription polymerase chain reaction (RT-PCR) Deoxycytidine (DAC) was used to demethylate SMMC-7721 cells infected with AdCore or AdGFP adenovirus. Methylation-specific PCR (MSP) and DNA methylation sequencing were further used to detect the promoter of SFRPs gene The methylation level of CpG island was detected by Western blot. The expression of methylated transferase Dnmts was detected by Western blot. Results The mRNA expression of SFRP2 and SFRP5 in SMMC-7721 cell line with HCV core protein overexpression was significantly lower than that in the control group (P <0.05). Methylation analysis showed that the expression of SFRP2 and SFRP5 mRNA in CpG island Showed hypermethylation; the expression of methylated transferase Dnmt1 and Dnmt3a increased (P <0.05). Conclusion HCV core protein can promote the methylation of CpG island in the promoter region of SFRP5 and participate in the development of HCV related diseases.