乙型肝炎病毒X蛋白通过DNMT3A诱导肝癌细胞RUNX3基因高甲基化

Hepatitis B virus X protein induced hypermethylation of RUNX3 through DNMT3A in HCC cells

目的研究肝癌细胞中乙型肝炎病毒X蛋白(HBx)对抑癌基因Runt相关转录因子3(RUNX3)表达的影响及相关作用机制,探讨乙肝病毒促肝癌发生的表观遗传调控。方法HBx重组表达载体转染5-aza-CdR处理或未处理的Huh7、HepG2肝癌细胞,Western Blot及RT-qPCR检测RUNX3的表达,分析RUNX3基因启动子CpG岛甲基化水平。合成DNA甲基转移酶3A的小干扰RNA(siRNA_3A)单独转染或与HBx共转染,检测RUNX3的表达。PCR检测21例手术切除的肝细胞癌新鲜组织标本HBx表达情况,比较HBx阴性与HBx阳性组织样本间RUNX3的表达差异以及启动子甲基化程度。结果过表达HBx可导致肝癌细胞内RUNX3表达下调,且甲基转移酶抑制剂5-aza-CdR可完全阻断HBx对RUNX3的抑制作用。HBx诱导RUNX3基因启动子CpG岛发生高甲基化,但不影响DNA甲基转移酶的表达。siRNA_3A转染肝癌细胞后,RUNX3 mRNA与蛋白表达均显著上调;其与HBx共转染后,逆转了单独HBx对RUNX3的抑制作用。HBx阳性的肝癌组织中RUNX3mRNA表达水平较HBx阴性组低,且RUNX3启动子甲基化程度也相对较高。结论 HBx通过促进RUNX3启动子区域发生高甲基化而抑制其表达,且这一过程是由DNMT3A所介导的。

We investigated the effect of hepatitis B virus X protein（HBx）on runt-related transcription factor 3（RUNX3）in hepatocellular carcinoma（HCC）.The expression of RUNX3 was detected by quantitative real-time PCR and western blot as well as the methylation of RUNX3 promoter CpG island was evaluated by methylation specific PCR in HCC cells transfected with HBx recombinant plasmids.The small interfering RNA（siRNA）targeting DNA methyltransferase 3A（DNMT3A）were co-transfected with HBx recombinant plasmids into HCC cells,and then the expression of RUNX3 was measured.In addition,the expression and methylation of RUNX3 were analyzed in 21 HCC tissues.Result showed that ectopic HBx inhibited the expression of RUNX3 and induced hypermethylation of RUNX3 CpG island in HCC cells.Furthermore,this inhibitory effect was abrogated by DNMT3 AsiRNA.The transcription of RUNX3 was higher in HCC tissues with HBx negative than tissues with HBx positive.But the methylation of RUNX3 CpG island exhibited the opposite trend.Our data indicate that HBx may downregulate the expression of RUNX3 through epigenetic mechanism.