论文部分内容阅读
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pGL3basic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)-κB(-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 determine the promoter region of the GADD45β gene specifically deleted in hepatocellular carcinoma cells and to explore the effect of bimonthly oxaliplatin on the gene expression of GADD45β in human hepatocellular carcinoma cell line HepG2 and its possible mechanism . Methods The GADD45β proximal promoter sequence (-547 to -436) was synthesized in vitro from 30 to 50 base-spacers and inserted into the upstream of the luciferase gene of pGL3basic luciferase expression plasmid respectively. HepG2 was transfected by electroporation, The intensity of binding of TRANSFAC to the transcriptional regulatory factor binding site was analyzed. The expression of GADD45β in HepG2 cells before and after Oxaliplatin treatment was compared by Northern blotting and real-time fluorescence quantitative PCR. Based on these results, the effect of Oxaliplatin on the promoter activity of GADD45β was further compared. Results The proximal promoter of GADD45β contained E2F-1 (-470 / -436) and NF-κB (-547 / -520) transcriptional regulators and promoter binding sites, . Oxaliplatin significantly induced the expression of GADD45β in HepG2 cells, and showed a dose-effect positive correlation. At the same time, Oxaliplatin induced E2F-1 promoter activity. Conclusion Oxaliplatin can obviously induce the gene deletion of GADD45β in hepatocellular carcinoma cells and enhance the expression of transcription factor E2F-1.