S腺苷蛋氨酸对肝癌细胞中GADD45β基因的表达诱导及其机制研究

Induction and mechanism of growth arrest DNA damage-inducible gene 45 β in human hepatoma HepG2 cells by S-adenosylmethionine

目的初步明确肝癌细胞中特异性表达缺失的GADD4513基因近端启动子活性调控中心，并探讨S腺苷蛋氨酸对肝癌细胞HepG2中GADD4513表达的影响及可能机制。方法以30-50个碱基的间隔，于体外人工合成GADD45β近端启动子序列（-618～-520），分别插入pGL3 basic荧光素表达质粒的荧光基因上游，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；实时荧光定量PCR比较S腺苷蛋氨酸作用前后HepG2细胞GADD45β表达，并在此基础上进一步比较S腺苷蛋氨酸对GADD45β启动子活性的诱导作用，探讨其可能作用机制，并为GADD45β近端启动子研究提供功能性证据。结果GADD45β近端启动子中含有3个NF-κB转录调节因子与启动子结合位点（-602／～593、-581／～572、-537／-528）；S腺苷蛋氨酸能明显诱导HepG2中GADD45β的表达，并呈现出剂量-效应的正相关关系，同时其能相应明显诱导NF-κB的启动子活性。结论S腺苷蛋氨酸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NF-κB的活性水平是其可能的作用机制，该研究为S腺苷蛋氨酸的肝脏保护作用提供了新的实验依据。

Objective To identiiy the active region oi proximal promoter growth arrest DINA damage-inducible gene 45β （GADD45/3） and to evaluate the influence of S-adenosylmethionine on HepG2. Methods The proximal promoter fragments （-618/-520） were synthesized in vitro by every 30--50 nucleotides, cloned into pGL3 basic luciferase expression plasmid respectively, then trans fected into HepG2 cells by electroporation. The promoter regions were identified with reference to TRANSFAC database. Following S-adenosylmethionine administration, quantitative real-time PCR was employed to validate the expression of GADD45β in HepG2. The effect of S-adenosylmethionine to promoter activity was further focused on. Results Using the luciferase assay, several transcription factor binding sites were identified in the proximal promoter of GADD45β, which included three NF- κBs （-- 602/-- 593, -- 581/-- 572, and -- 537/-- 528） according to the consensus sequences in TRANSFAC database. After S-adenosylmethionine administration, the increasing expression of GADD4513 was marked in a dose-dependent manner. The lueiferase assay also demonstrated the promoter activity of fragment constructed the first NF-κB binding site could be induced by S-adenosylme- thionine. Conclusion S-adenosylmethionine can induce the expression of GADD45β, which is specifically down-regulated in HCC. The possible mechanism may be involved in the regulation of transcrip tion factor NF-κB in GADD45β proximal promoter.