转录因子E2F-1过表达抑制胃癌细胞MGC-803的增殖

Inhibiton of the proliferation of MGC-803 Cell by over-expression of transcription factor E2F-1

目的了解E2F-1过表达对胃癌细胞MGC-803增殖、生长和细胞周期的影响。方法 PCR扩增E2F-1基因片段,然后经限制性内切酶EcoRⅠ和HindⅢ双酶切后将E2F-1定向克隆到真核表达载体pCMV-HA2中转化筛选。用脂质体Lipofectamine2000将该载体转染MGC-803胃癌细胞,然后用含G418的培养基筛选获得具Genectin抗性的过表达E2F-1的胃癌细胞株。RT-PCR和Western blot技术检测MGC-803/E2F-1细胞内E2F-1表达的情况。对稳定转染E2F-1的胃癌MGC-803/E2F-1细胞(实验组)、转染空载体的MGC-803/EV(阴性对照组)及未转染的MGC-803细胞进行MTT法检测,绘制生长曲线和计算细胞增殖率;流式细胞仪检测各组细胞周期分布。结果 RT-PCR和Western blotting实验证实重组载体pCMV-E2F-1-HA2成功转入胃癌细胞内,并稳定过表达E2F-1。MTT法检测发现,与MGC-803/EV细胞组和MGC-803细胞组相比,胃癌MGC-803/E2F-1细胞的生长受到明显抑制,增殖率下降(26.61±5.19)%vs(93.4±10.29)%、(100±0.00)%(均P<0.01);细胞周期分析显示MGC-803/E2F-1组的细胞在G0/G1期所占比例为70.70%,高于MGC-803/EV组(59.60%,P<0.01)和MGC-803细胞组(1.40%,P<0.01);MGC-803/E2F-1组的细胞在S期所占比例为17.60%,明显低于MGC-803/EV组的30.70%(P<0.01)和MGC-803细胞组的27.30%(P<0.01)。结论 E2F-1基因过表达抑制胃癌细胞的增殖和生长,细胞周期停滞在G0/G1期。

Objective To investigate the effects of E2F-1 over-expression on proliferation of gastric cancer cell line MGC-803.Methods A eukaryotic expression vector containing the E2F-1 gene was contructed and transfected into gastric cancer cell line MGC-803.Total RNA of E2F-1 was reverse transcribed from cDNA from human liver with RT-PCR.E2F-1 was selected via pGEM-Teasy and subsequently recombined into eukaryotic expression vector pCMV-HA2 using restrictive endoculease enzyme Hind Ⅲ and EcoRⅠ.MGC-803 cells transfected with pCMV-E2F-1-HA2 via Lipofectamine 2000 was selected in medium containing G418.RT-PCR and Western blot were used for identification of the E2F-1 expression.Results Successful transfection of pCMV-E2F-1-HA2 was proved with RT-PCR and Western blotting.Cell clones with resistance to Genetecin were obtained and amplified.Proliferation ratio of MGC-803/E2F-1 was 26.61% and significantly lower than those of MGC-803 and MGC-803/EV cells in MTT assay （26.61% vs 100% and 26.61% vs 93.4%,respectively,P0.01）.G0/G1 phase proportion of MGC-803/E2F-1 was significantly higher than those of MGC-803/EV and MGC-803 in cell cycle analysis （70.70% vs 59.60% and 70.70% vs 1.40%,respectively,P0.01）.Cells in S phase was significantly less among MGC-803/E2F-1 cells than those among MGC-803/EV and MGC-803 cells group （17.60% vs 30.70% and 17.60% vs 27.30%,respectively,P0.01）.Conclusion Over-expression of the E2F-1 gene inhibits proliferation and cell circle of MGC-803 cell.