转录因子E2F-1过表达抑制胃癌细胞MGC-803的增殖

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 blotting技术检测MGC-803/E2F-1细胞内E2F-1表达情况。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70.63%，高于MGC-803/EV组（60.03%，P<0.01）和MGC-803细胞组（60.43%，P<0.01）；MGC-803/E2F-1组的细胞在S期所占比例为(11.27%)，明显低于MGC-803/EV组（28.70%，P<0.01）和MGC-803细胞组（29.70，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。

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

Abstract Objective: To investigate the effects of E2F-1 overexpression on proliferation of gastric cancer cell line MGC-803, we constructed a eukaryotic expression vector containing the E2F-1 gene and then transfected it into gastric cancer cell line MGC-803. Methods: Extracted total RNA from the normal of human liver and cloned cDNA of E2F-1 by RT-PCR. The products were firstly cloned into pGEM-Teasy, transfer and select the positive recombinant, and then cloned into eukaryotic expression vector pCMV-HA2 after introducd the sites of restrictive endonuclease enzyme HindIII、EcoRI. Then pCMV-E2F-1-HA2 was transfected into MGC-803 cells with Lipofectamine 2000 and selected in medium containing G418. The genectin-resistant cells were harvested for the coming tests. Methods of RT-PCR and Western blot were used to identify the expression of E2F-1. Results: pCMV-E2F-1-HA2 was digested with Hind III、EcoR I, amplified by PCR, we all obtained prospective fragments, and the result of sequencing was completely consistent with the reported E2F-1 gene sequences, indicating that the E2F-1 gene fragment has been successfully subcloned into the eukaryotic expression vector mRNA. Cell clones with resistance to Genetecin were obtained and amplified. Experimental results of RT-PCR and Western blotting confirmed that the recombinant vector pCMV-E2F-1-HA2 was successfully transfected into gastric cancer cell line MGC-803 and stably overexpressed the E2F-1 gene.In the MTT assay, the proliferation ratio of MGC-803/E2F-1 cells was 26.61% and significantly fewer than MGC-803 cells (100%, P<0.01) and MGC-803/EV cells (93.4%, P<0.01). Cell cycle analysis showed that the G0/G1 phase proportion of MGC-803/E2F-1 group was significantly higher than MGC-803/EV and MGC-803 group (70.63% vs 60.03% and 60.43%, P<0.01); the S phase proportion was significantly lower in MGC-803/E2F-1 cells than MGC-803/EV cells and MGC-803 cells group (11.27% vs 28.70% and 29.70, P<0.01). Conclusion: Eukaryotic expression vector pCMV-E2F-1-HA2 has been successfully constructed. Gastric cancer cell line MGC-803/E2F-1 with stable overexpression of the E2F-1 gene was established. Overexpression of the E2F-1 gene may lead to decreased proliferateion and cell circle inhabition in MGC-803 cell.