双月安环己烷草酸铂对肝癌细胞DNA损伤修复基因表达影响及其机制

Effect of Oxaliplatin on expression of growth arrest DNA damage-inducible 45β gene in HepG2 cells and its possible mechanisms

目的初步明确肝癌细胞中特异性表达缺失的DNA损伤修复基因(GADD45β)近端启动子序列,并探索双月安环己烷草酸铂(Oxaliplatin)对人肝癌细胞HepG2中GADD45β基因表达影响及可能机制.方法以30～50个碱基间隔体外人工合成GADD45β近端启动子序列(-547至-436),分别插入pGL3 basic荧光素表达质粒的荧光素基因上游,以电穿孔法转染HepG2,根据启动子活性强度结合TRANSFAC数据库分析可能存在的转录调节因子结合位点;以Northern印迹和实时荧光定量PCR比较Oxaliplatin作用前后HepG2细胞GADD45β表达,并在此基础上进一步比较Oxaliplatin对GADD45β启动子活性的诱导作用.结果GADD45β近端启动子中含有E2F-1(-470/-436)和核因子(NF)-KB(-547/-520)转录调节因子与启动子结合位点,并可能存在'抑制区'.Oxaliplatin能明显诱导HepG2中GADD45β表达,并呈剂量-效应正相关关系,同时Oxaliplatin能相应诱导E2F-1启动子活性.结论Oxaliplatin能明显诱导肝癌细胞中特异性缺失的GADD45β基因表达,增强转录调节因子E2F-1的表达水平是其可能的作用机制.

Objective To identify the proximal promoter of growth arrest DNA damage inducible gene 45β（GADD45β） which was specifically lower-expressed in hepatic carcinoma cell （HCC）, and to evaluate the influence of Oxaliplatin on the expression of GADD45β in HCC cell line HepG2 cells. Methods The proximal promoter fragments （-547/-436） were synthesized in vitro by every 30-50 nucleo tides, cloned into pGL3 basic luciferase expression plasmids respectively, then transfected into HepG2 cells by electroporation. The promoter regions were identified in refers of TRANSFAC database. Before and after Oxaliplatin administration, Northern blot and quantitative real time PCR were performed to validate the expression of GADD45β in HepG2. Results Using the luciferase assay, several transcription factor binding sites were identified in the proximal promoter of GADD45β, which included one E2F 1 （- 470/-436）, one NF-κB （-547/-520） and one putative inhibition region according to the consensus sequences in TRANSFAC datahase. After Oxaliplatin administration, the increasing expression of GADD45β was marked in a dose dependent manner, which was confirmed by Northern blot and quantitarive real-time PCR. The results of luciferase assay also demonstrated that the promoter activity of E2F-1 fragment binding site could be induced by Oxaliplatin. Conclusions Oxaliplatin could induce the expression of GADD45β which is specifically down regulated in HCC. The possible mechanism may be involved in the regulation of transcription factor E2F-1 in GADD45β proximal promoter.