丙型肝炎病毒核心蛋白抑制E-cadherin基因启动子活性  

Hepatitis C virus core protein inhibits the E-cadherin promoter activity

作　　者：聂丽珠[1] 聂丹[1] 段玉洁[1] 李治[1] 陈震[1] 田玲[1] 唐霓[1] 

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《重庆医科大学学报》2016年第6期547-551,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81572683;81371827;31171307);"十二.五"传染病国家科技重大专项资助项目(编号:2012ZX10002005-003-002;2013ZX10002002-005-003);重庆医科大学科研培育基金资助项目(编号:201401);重庆医科大学附属第二医院"优秀青年人才"资助项目;重庆市研究生科研创新资助项目(编号:CYS14124)

摘　　要：目的:探讨丙型肝炎病毒核心蛋白(hepatitis C virus core protein,HCV core)对E-cadherin启动子活性的调控作用。方法:以Huh7细胞基因组为模板,构建野生型E-cadherin(CDH1)基因启动子荧光素酶报告质粒(pGL3-CDH1),然后设计突变引物序列将人E-cadherin启动子区负性调控关键区域,即位于-80^-75 nt(E-box1),-30^-25 nt(E-box3),和+22^+27 nt(Ebox4)的3个E-box的共识别序列5’-CANNTG-3’突变为5’-AANNTA-3’,分别构建E-box1(pGL3-mut1)、E-box3(pGL3-mut2)、E-box4(pGL3-mut3)、E-box1+3(pGL3-mut1+2)、E-box1+4(pGL3-mut1+3)、E-box3+4(pGL3-mut2+3)、E-box1+3+4(pGL3-mut1+2+3)的荧光素酶报告质粒。将上述质粒与内参质粒p RL-TK分别共转染于过表达核心蛋白的肝癌细胞株SMMC-7721细胞,采用双荧光素酶报告系统检测HCV core对E-cadherin启动子的调控,分析E-cadherin启动子区不同位点的E-box区域在HCV core调控E-cadherin启动子活性中发挥的作用。结果:荧光素酶活性检测结果显示,与GFP对照组相比,HCV core可以明显抑制野生型E-cadherin启动子活性;在E-box单一突变的E-cadherin报告质粒转染细胞中,HCV core对CDH1启动子的抑制作用有不同程度减弱,特别是-80^-75 nt和-30^-25 nt位点的E-box区域作用最为明显;E-box联合突变型质粒转染细胞中,HCV core对CDH1启动子的抑制作用明显减弱。结论:HCV core能明显抑制E-cadherin的转录活性,E-box突变可以部分拮抗HCV core对E-cadherin的转录抑制作用,提示位于-80^-75 nt和-30^-25 nt位点的E-box保守负性调控区域在HCV core对E-cadherin的转录调控中发挥关键作用。Objective：To investigate the regulation of hepatitis C virus core protein（HCV core）on E-cadherin promoter activity.Methods：The CDH1 promoter fragment,amplified from the Huh7 cells,was cloned into pGL3-Basic to construct the luciferase report plasmid（pGL3-CDH1）. The three E-boxes consensus sequences,which located at-80～-75 nt（E-box1）,-30～-25 nt（E-box3）,and＋22～＋27 nt（E-box4）in human E-cadherin promoter region,play important role in E-cadherin transcriptional regulation. Thus,mutagenic primers were designed to induce point mutations in three putative E-box sites in the CDH1 promoter sequence. These mutations changed the sequence of the E-boxes from 5＇-CANNTG-3＇to 5＇-AANNTA-3＇,which resulted in the insertion of a Snailunrecognized site. These reporter constructs were named E-box1（pGL3-mut1）,E-box3（pGL3-mut2）,E-box4（pGL3-mut3）single mutation types and E-box1＋3（pGL3-mut1＋2）,E-box1＋4（pGL3-mut1＋3）,E-box3＋4（pGL3-mut2＋3）,E-box1＋3＋4（pGL3-mut1＋2＋3） mutant constructs respectively. The E-cadherin promoter constructs containing wild-type or mutant E-boxes were co-transfected with p RL-TK（an internal control） into SMMC-7721 cells over expressing HCV core protein respectively,then the luciferase activities were detected. Results：Compared with that of GFP control group,HCV core protein could significantly inhibit wild-type CDH1 promoter activity. In E-box single mutant CDH1 transfection cells,the inhibition of HCV core protein on CDH1 promoter decreased in different degrees. It is most obvious in-80～-75 nt and-30～-25 nt. In compound mutation of CDH1-transfected cells,the HCV core-induced CDH1 inhibition was significantly diminished. Conclusion：HCV Core protein strongly inhibits E-cadherin transcriptional activity. The-80～-75 nt,-30～-25 nt E-boxes consensus sequences within E-cadherin promoter region may play a dominant role in HCV core-induced E-cadherin repression.

关 键 词：肝细胞肝癌 丙型肝炎病毒核心蛋白 E-CADHERIN E-BOX 启动子活性 

分 类 号：R575.1[医药卫生—消化系统]