Triapine对肝癌细胞株HepG2中GADD45β表达影响及其可能机制

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

目的:DNA损伤修复相关基因GADD45β的特异性表达缺失与肝癌的恶性程度密切相关,本研究初步明确肝癌细胞中GADD45β近端启动子序列,探索3-氨基吡啶-2-甲醛硫代缩氨基脲(Triapine)对人肝癌细胞株HepG2的GADD45β表达影响及其可能机制。方法:体外合成GADD45β近端启动子序列(-618至-314),构建荧光素表达质粒,转染HepG2,根据启动子活性强度结合数据库分析存在的转录调节因子结合位点;以实时荧光定量PCR比较Triapine作用前后HepG2细胞GADD45β表达;进一步比较Triapine对GADD45β启动子活性的调控作用,分析Triapine对HepG2的抑制效应;并通过Caspase-8、Caspase-9和Caspase-3的表达变化测定凋亡的发生和发展。结果:GADD45β近端启动子中含有3个NF-κB(-602/-593、-581/-572、-537/-528)和1个E2F-1(-470/-436)转录调节因子与启动子结合位点;2.5μmol/L和5μmol/L的Triapine作用后,GADD45β/GAPDH分别为0.029 3和0.073 9,呈现剂量-诱导效应正相关,同时NF-κB和E2F-1启动子活性分别增强了1.5和0.8倍;高剂量Triapine对HepG2的DNA合成能力和细胞克隆形成能力的抑制率分别为75.25%和60.54%,呈现剂量-抑制效应正相关;Triapine作用后6 h即出现HepG2凋亡高峰。结论:Triapine能通过增强转录调节因子的活性,诱导肝癌细胞中特异性缺失的GADD45β基因表达;进而抑制肝癌细胞的增殖并启动凋亡途径。

Objective To identify the proximal promoter of down-expression of growth arrest DNA damage-inducible gene 45 β（GADD45β） and to evaluate the influence of Triapine to HCC cell lines.Methods The proximal promoter fragments（-618/-314） were synthesized in vitro and cloned into pGL3 basic luciferase expression plasmid,then transfected into the HepG2 cell line by electroporation.The promoter regions were identified in reference to TRANSFAC database.Following Triapine administration,quantitative real-time PCR was employed to validate the expression changes of GADD45β.The effect of Triapine to promoter activity was further focused on.The influence of cell growth and the expression of Caspase-8,Caspase-9 and Caspase-3 were further measured.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,-537/-528） and one E2F-1（-470/-436）.After Triapine administration,the increasing expression of GADD45β was marked in a dose-dependent manner.The luciferase assay results also demonstrated that the promoter activity of fragments constructed by NF-κB and E2F-1 binding sites could be induced by Triapine.The DNA syntheses and colony formation were inhibited apparently and Triapine could trigger the cell line apoptosis rapidly following treatment.Conclusions Triapine could induce the expression of GADD45β,which induces specifically the down-regulation of HCC.The possible mechanism may be involved in the regulation of transcription factors in the GADD45β proximal promoter.