Objective To study the relationship between hepatitis B virus (HBV)rtA181T mu-tation and karyopherin α2 (KPNA2)promoter demethylation.Methods KPNA2 mRNA levels and promoter methylation status were measured in LO2 and HepG2 cells using RT-qPCR and BSP-based assay,respectively.Furthermore,levels of KPNA2 mRNA were determined in LO2 and HepG2 cells after transfection with wild-type rtA181T,mutant rtA181T and empty plasmids,re-spectively.Moreover,wild-type rtA181T,mutant rtA181T and empty plasmids were cotransfected into LO2 cells with pGL3-KPNA2 in the methylated status,and the transcriptional activity and methylation status of KPNA2 promoter were examined by dual luciferase reporter and BSP assay. Results Compared with LO2 cells,KPNA2 mRNA levels dramatically increased but the promoter methylation rate remarkably decreased in HepG2 cells.With empty vectors,both wild-type and mutant rtA181T plasmids significantly increased KPNA2 mRNA levels in LO2 cells.rtA181T plasmid was capable of activating KPNA2 promoter by demethylation.Conclusion The mutant HBV rtA181T may increase KPNA2 mRNA levels through promoter demethylation in LO2 cells.%目的 研究乙型肝炎病毒(HBV)rtA181T突变与核质转运蛋白α2(KPNA2)启动子去甲基化之间的关系.方法 采用RT-qPCR和亚硫酸氢盐修饰后克隆测序法(BSP)检测人正常肝细胞株LO2和人肝癌细胞株HepG2细胞中KPNA2 mRNA水平及其启动子甲基化状态;将rtA181T突变型、野生型和空载体质粒转染LO2和HepG2细胞,采用RT-qPCR检测KPNA2 mRNA水平;构建萤光素酶报告基因载体pGL3-KPNA2并使其体外甲基化,分别与rtA181T突变型、野生型和空载质粒共同转染LO2细胞,采用双萤光素酶报告基因试验和BSP法检测其对KPNA2启动子转录活性及其甲基化状态的影响.结果 1)HepG2细胞中KPNA2 mRNA水平显著高于LO2细胞,而其KPNA2启动子甲基化率明显低于LO2细胞;2)转染了rtA181T突变型和野生型的LO2细胞中KPNA2 mRNA表达水平明显高于空载体对照细胞;3)rtA181T突变型能够使高甲基化状态的KPNA2启动子发生去甲基化作用促进其转录表达.结论 HBV rtA181T突变型在LO2细胞中可能通过KPNA2启动子去甲基化提高KP-NA2 mRNA水平.
展开▼
