Hint1基因抑制HepG2细胞AP-1转录因子活性的实验研究

Hintl over expression inhibits the activity of AP-1 transcription factor in HepaG2 cells

目的探索体外过表达Hint1基因对人肝肿瘤HepG2细胞AP-1转录因子活性的影响。方法分子克隆获得Hint1基因序列，构建pHA—Hint1重组融合基因表达载体。阳离子脂质体介导，pHA—Hint1转导人肝母细胞瘤HepG2细胞，RT—PCR和Western blot检测HepG2细胞中外源HA—Hintl表达。pHA—Hint1质粒、启动子荧光素酶报告质粒AP-1／Luc及巨细胞病毒-β-半乳糖酶报告质粒（β—gel）共转导HepG2细胞。36h后测定荧光素酶活性及β—gel活性。结果重组质粒经EcoR I和BamH I双酶切，获约100bp特异条带，测序证实成功构建pHA—Hint1表达载体。pHA—Hint1及空载体pcDNA3／HA转导HepG2细胞，半定量RT-PCR结果显示pHA—Hint1组Hint1 mRNA表达高于空载体组（t=3．89，P〈0．05）；Western blot结果显示pHA-Hint1组HA标签蛋白表达高于空载体组（t=3．12，P〈0．05）。AP-1转录因子活性检测结果：pHA—Hint1终浓度为0μg/ml，0．5μg/ml，1．0μg/ml，1．5μg/ml，2．0μg/ml时，AP-1转录因子相对活性（10^6）为：5．12±0．25、4．24±0．74、3．43±0．31、2．62±0．48、2．09±0．21。随着pHA—Hint1浓度增加，荧光素酶活性呈明显下降，当pHA—Hint1浓度达到1．5μg及2．0μg时，差异有统计学意义（F=72．009，P〈0．05）。结论过表达Hint1基因可抑制HepG2细胞AP-1转录因子活性。

Objective To study the inhibition of AP-1 transcription factor activity by Hintl gene over expression in HepG2 cell lines. Methods The Hintl gene was amplified, and then was inserted into the pcDNA3/HA eukaryotic expression plasmid. The constructed pHA-Hintl plasmid was confirmed by DNA sequencing. The pHA-Hintl was transfected into the HepG2 human hepatoma cells. Semi-quantitative RT-PCR and Western-blot were used to detecte the expression of HA-Hintl. The HepG2 cells were cotransfected with pHA-Hintl and pAP-1/Luc luciferase reporter. At 36 h after transfection, luciferase assay system was used to detect the AP-1 transcription factor activity. Results The constructed pHA-Hintl was confirmed by DNA sequencing, pHA-Hintl gcne transduction through lipofectine induced over-expression in HA-Hintl mRNA （t = 3.89, P 〈 0. 05 ） and HA-Hintl protein （ t = 3.12, P 〈 0. 05 ）. Co-transfection of Hintl gene inhibits AP-1 luciferase activity. Cotransfection with increased concentration of a pHA-Hintl plasmid （0 μg/ml, 0. 5μg/ml, 1.0 μg/ml, 1.5μg/ml, 2. 0 μg/ml） produced a concentration-dependent inhibition of AP-1 transcription factor activity. At the concentration of 1.5 μg/ml, and 2. 0 μg/ml, the activity inhibition reaches significant difference （ F = 72. 009, P 〈 0. 05 ）. Conclusion Over-expression of Hintl can, at least in part, inhibit the AP-1 transcription factor activity in HepG2 cells.