论文部分内容阅读
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: The loss of specific expression of GADD45β, a gene related to DNA damage repair, is closely related to the degree of malignancy of hepatocellular carcinoma. In this study, we initially identified the GADD45β proximal promoter sequence in hepatocellular carcinoma cells and explored the mechanisms of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone Triapine) on the expression of GADD45β in human hepatocellular carcinoma cell line HepG2 and its possible mechanism. Methods: The GADD45β proximal promoter sequence (-618 to -314) was synthesized in vitro. Fluorescein expression plasmids were constructed and transfected into HepG2. The binding sites of transcription regulators were analyzed according to the activity of promoters combined with the database. Real - time quantitative PCR The effect of triapine on the activity of GADD45β promoter was further analyzed, and the inhibitory effect of triapine on HepG2 was analyzed. The apoptosis of HepG2 cells was determined by the changes of Caspase-8, Caspase-9 and Caspase-3 expression And development. Results: The GADD45β promoter contained three NF-κB transcription factors (-602 / -593, -581 / -572 and -537 / -528) and one E2F-1 (-470 / -436) Promoter binding site. After treated with 2.5|Ìmol / L and 5|Ìmol / L Triapine, GADD45|Â / GAPDH were 0.029 3 and 0.073 9, respectively, showing a positive correlation between dose-induction effect and the activities of NF-|ÊB and E2F- Increased by 1.5 and 0.8 times. The inhibitory rates of high dose Triapine on HepG2 DNA synthesis ability and cell clone formation ability were 75.25% and 60.54%, respectively, showing a dose-dependent inhibition effect; HepG2 apoptosis occurred 6 h after Triapine treatment peak. CONCLUSION: Triapine can induce the gene deletion of GADD45β specifically in hepatocellular carcinoma cells by increasing the activity of transcriptional regulators, and then inhibit the proliferation of hepatoma cells and initiate the apoptosis pathway.