P53与乙型肝炎病毒增强子Ⅰ上游P53样应答元件结合序列关系研究  

Interaction of p53 with p53 Response Element Like Binding Sequence at Upstream of Hepatitis B Virus Enhancer Ⅰ

作　　者：曲建慧[1] 朱明华[1] 林静[1] 倪灿荣[1] 李芳梅[1] 祝峙[1] 于观贞[1] 

机构地区：[1]第二军医大学长海医院病理科,上海200433

出　　处：《癌症》2004年第5期502-507,共6页Chinese Journal of Cancer

基　　金：国家自然科学基金(No.30070344)~~

摘　　要：背景与目的:在前期工作中,我们经计算机序列分析发现,在乙型肝炎病毒(hepatitisBvirus,HBV)基因组增强子Ⅰ上游1047～1059bp存在P53样应答元件结合序列5'-TGCC(G)TTGCCT-3',体外实验应用凝胶迁移方法(EMSA和EMSSA)发现这段序列与P53蛋白能够进行特异性结合。本实验拟观察肝癌细胞中P53蛋白与HBV基因组P53样应答元件结合序列之间的关系。方法:报告基因质粒pX-CAT上游为X基因增强子和启动子序列,其中含有P53样应答元件结合序列5'-TGCGTTGCCT-3'。首先用PCR方法将pX-CAT质粒中5'-TGCGTTGCCT-3'进行点突变,成为5'-TGTATTGTAT-3',以破坏P53样应答元件结合序列。将pX-CAT和变异后的pX-CAT(mpX-CAT)分别单独或与pCMV-p53质粒共转染HepG2细胞,通过检测报告基因CAT的活化情况,观察P53蛋白与这段DNA序列的作用关系。将HBV基因P53样应答元件结合序列反向构建于真核表达质粒pZeoSV2中(αpZeoXP),转染HepG2.2.15细胞并稳定筛选,以封闭HBV基因上P53样结合位点,观察细胞中P53/P21蛋白表达的改变,以及细胞周期、细胞凋亡的变化。结果:HepG2细胞中,报告基因CAT活性在pCMV-p53与pX-CAT共转染组较pX-CAT单独转染组明显升高(1.353vs0.738,P<0.05),而mpX-CAT单独转染组CAT活性则明显降低(0.304),即使与pCMV-p53共转染CAT活性?BACKGROUND &OBJECTIVE: A p53 response element like binding sequence, 5′ TGCC(G)T TGCCT 3′was found at upstream of hepatitis B virus (HBV) enhancer Ⅰfrom 1047 to 1059 nucleotides after analyzing the HBV genome by a computer program in our previous work. It indicated that the sequence could specifically bind P53 protein in vitro by electrophoretic mobility shift assay (EMSA) and electrophoretic mobility supershift assay (EMSSA). This study was designed to further investigate the interaction between P53 and p53 response element like binding sequence at upstream of HBV enhancer Ⅰ. METHODS: HBV x gene enhancer and promoter which contains p53 response element like binding sequence TGCGT TGCCT was in upstream of CAT enzyme in pX CAT reporter plasmid. Firstly, we designed a point mutation in pX CAT which change TGCGT TGCCT into TGTAT. TGTAT by polymerase chain reaction (PCR) in order to damage the p53 response element like sequence. After pX CAT or mutation pX CAT (mpX CAT) transfected alone or cotransfected with pCMVp53 into HepG2 hepatoma cell, CAT activity was assayed to confirm the correlation of p53 protein with this DNA sequence. In addition, an antisense sequence corresponding to the p53 response element like sequence in HBV was reconstructed into the pZeoSV2 vector (αpZeoXP) and transfected into HepG2.2.15 cell line to block the binding of P53 with this sequence, the stable transfected HepG2.2.15 cell was observed about P53/P21 expression, cell cycle distribution and apoptosis rates. RESULTS: CAT enzyme of HepG2 had an higher expression in cotransfection with pCMVp53 and pX CAT than alone pX CAT (1.353 VS 0.738,P< 0.05), but it was lower in mpX CAT(0.304) and pCMVp53 (0.402). Compared with the control group, P53/P21 expression and cell apoptosis rate decreased greatly in the stable transfected αpZeoXP HepG2.2.15 cell line (0.95%VS 7.84%), the cell number in S phase increased in the same cell line (16.37%VS 9.48%). CONCLUSION: Reporter gene expression of pX CAT in intracellular could be prom

关 键 词：P53 乙型肝炎病毒 增强子I上游 P53样 应答元件 序列关系 

分 类 号：R346[医药卫生—基础医学]