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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 down-regulate the expression of epidermal growth factor receptor (EGFR) gene by RNA interference and observe its effect on the proliferation of ovarian cancer SKOV3 cells. Methods The eukaryotic expression vector targeting EGFR gene was constructed and transfected into ovarian cancer cell line SKOV3. Transformed clones stably expressing siRNA were screened. The expression of EGFR mRNA and protein were detected by RT-PCR and Western blot respectively. Cycle and apoptosis, colony formation assay and MTT assay were used to detect the rate of cell clone formation and cell proliferation in vitro. Results The recombinant plasmids pGenSil-HK, pGenSil-EGFR1 and pGenSil-EGFR2 were successfully constructed and transfected into SKOV3 cells. The stable transfected clones were obtained by G418 screening. RT-PCR and Western blot showed that pGenSil-EGFR1 and pGenSil-EGFR2 The expression of EGFR mRNA and protein were significantly inhibited after transfection. The inhibition rate of EGFR mRNA was 41.87% and 68.07% respectively, and the protein expression inhibition rates were 45.21% and 70.25% respectively. Compared with untransfected group and pGenSil-HK group, the percentage of apoptosis in pGenSil-EGFR2 group increased significantly, the number of cells in G1 phase increased and the number of S phase decreased (P <0.05). The clone formation rate in pGenSil-HK group was (0.82 ± 0.03). The rate of colony formation in pGenSil-EGFR2 transfected group was (0.61 ± 0.04), the difference was significant (P <0.05). MTT showed that the cell growth of pGenSil-EGFR2 group was significantly lower after 36h In untransfected group and pGenSil-HK transfected group (P <0.05). Conclusion Targeting EGFR gene RNA interference can inhibit EGFR expression in ovarian cancer SKOV3 cells, induce apoptosis, regulate cell cycle redistribution, and inhibit cell proliferation.