乙型肝炎病毒rtA181T突变与核质转运蛋白α2启动子去甲基化的关系.docVIP

乙型肝炎病毒rtA181T突变与核质转运蛋白α2启动子去甲基化的关系 吴佳伟 刘玉莹 张桢雪 朱慧 张芳林 南昌大学药学院药理教研室 X 关注成功！ 加关注后您将方便地在 我的关注中得到本文献的被引频次变化的通知！ 新浪微博 腾讯微博 人人网 开心网 豆瓣网 网易微博 摘????要： 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KPNA2mRNA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KPNA2mRNA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KPNA2mRNA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KPNA2mRNA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KPNA2mRNA水平。 关键词： rtA181T; 核质转运蛋白α2; 去甲基化; 作者简介：吴佳伟 (1991—) , 男, 硕士研究生, 主要从事肿瘤基因治疗的研究。 作者简介：张芳林, 副教授, E-mail:zhangfl3@。 收稿日期：2017-02-25 基金：国家自然科学基金 Relationship between Hepatitis B Virus rtA181T Mutation and Karyopherin α2 Promoter Demethylation WU Jia-wei LIU Yu-ying ZHANG Zhen-xue ZHU Hui ZHANG Fang-lin Department of Pharmacology, School of Pharmacy, Nanchang University; Abstract： Objective To study the relationship between hepatitis B virus (HBV) rtA181 Tmutation and karyopherinα2 (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 rtA181 T, mutant rtA181 Tand empty plasmids, respectively.Moreover, wild-type rtA181 T, mutant rtA181 Tand 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 rtA181 T plasmids sig