靶向EGFR基因RNA干扰对人卵巢癌SKOV_3细胞增殖的抑制作用

RNA interference targeting EGFR on proliferation of human ovarian carcinoma cell line SKOV_3

目的利用RNA干扰技术下调表皮生长因子受体(epidermal growth factor receptor,EGFR)基因的表达,观察其对卵巢癌SKOV3细胞增殖的影响。方法构建针对EGFR基因的siRNA真核表达载体,转染入卵巢癌SKOV3细胞中,并筛选稳定表达siRNA的转化克隆,RT-PCR及Western blot分别检测EGFR mRNA和蛋白表达,流式细胞仪检测细胞周期和凋亡,克隆形成实验及MTT法检测细胞克隆形成率及细胞体外的增殖情况。结果成功构建表达质粒pGenSil-HK、pGenSil-EGFR1和pGenSil-EGFR2,并转染SKOV3细胞,通过G418筛选后获得稳定转染克隆,RT-PCR及Western blot分析表明转染pGenSil-EGFR1、pGenSil-EGFR2后的细胞EGFR mRNA和蛋白表达均受到了明显抑制,EGFR mRNA抑制率分别为41.87%和68.07%,蛋白表达抑制率分别为45.21%和70.25%。与未转染组和pGenSil-HK组相比,pGenSil-EGFR2组细胞凋亡比例显著增加,G1期细胞增多,而S期细胞减少(P<0.05);转染pGenSil-HK组克隆形成率为(0.82±0.03),转染pGenSil-EGFR2组克隆形成率为(0.61±0.04),二者比较差异显著(P<0.05),MTT显示细胞培养36h后,pGenSil-EGFR2组的细胞生长均显著低于未转染组和转染pGenSil-HK组(P<0.05)。结论靶向EGFR基因RNA干扰能够阻抑卵巢癌SKOV3细胞中的EGFR表达,诱导细胞凋亡、调控细胞周期再分布、抑制细胞增殖。

Objective To explore the effect of down-regulating epidermal growth factor receptor （EGFR） expression by RNA interference on the proliferation of ovarian carcinoma SKOV3 cells. Methods The recombinant eukaryotic expression plasmids pGenSil-HK, pGenSil-EGFR1 and pGenSil-EGFR2 were constructed and transfected into SKOV3 cells respectively. The mRNA and protein expressions of EGFR in SKOV3 cells were detected by RT-PCR and Western blotting, respectively. The cell cycle and apoptosis were evaluated by flow cytometry. The proliferation of SKOV3 cells was determined by clone formation assay and MTT assay. Results We successfully constructed the recombinant eukaryotic expression plasmids pGenSil-HK, pGenSil-EGFR1 and pGenSiI-EGFR2 and transfected into SKOV3 cells. Three cell clones were screened by G418. Compared with untransfected SKOV3 cells and SKOV3 cells transfected with pGenSil-HK, the expressions of EGFR in SKOV3 cells transfected with pGenSil-EGFR1, pGenSil-EGFR2 were inhibited significantly at both mRNA and protein levels, with an inhibitory rate of 41.87% and 68.07% for EGFR mRNA and of 45.21% and 70.25% for EGFR protein respectively. Compared with untransfected SKOV3 cells and SKOV3 cells transfected with pGenSilHK, the cell apoptotic rate was significantly increased significantly, the cell cycle was arrested in G~ phase and S phase decreased significantly in pGenSil-EGFR2 SKOV3 cells （P 〈 0.05 ）. The clone formation efficiency was significantly lower in pGenSil-EGFR2 SKOV3 cells than in untransfected SKOV3 cells and SKOV3 cells transfected with pGenSil-HK [ （0.61 ±0.04） vs （0.82 ±0.03）, （P 〈0.05） 1. The proliferative rate was markedly lower in pGenSil-EGFR2 SKOV3 cells than in untransfected SKOV3 cells and SKOV3 cells transfected with pGenSil- HK 36 h after transfection （ P 〈 0.05 ）. Conclusion RNA interference targeting EGFR gene down-regulates the EGFR expression, induces cell apoptosis, regulates cell phase redistribution and inhibits cell proliferation in ovarian carcinoma SKOV3 cells.