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

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(hepatitis B virus,HBV)基因组增强子I上游l047～l059bp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。

Interaction of p53 with p53 response element like binding sequence at upstream of hepatitis B virus enhancer I

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 I from 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 (alpha pZeoXP) 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 alpha pZeoXP 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 promoted by P53, which further suggest that P53 could bind TGCC(G)T-TGCCT in upstream of HBV enhancer I and induce a prolonged P53 half life. Subsequently, P53 and p21 protein (downstream gene of P53) would have a higher expression and stronger activity.