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目的探讨RNA干扰(RNAi)对卵巢癌细胞表皮生长因子受体(EGFR)基因表达的影响及其效应。方法体外合成EGFR的DNA模板序列和pSilencer 2 .1-U6neo质粒构建编码shRNA的表达载体,应用脂质体Lipofectamine 2000将其转染到人卵巢癌Skov-3细胞,采用半定量RT-PCR、免疫细胞化学技术检测转染前后Sk-ov-3细胞EGFR基因的变化;采用Annexin V/PI双染色标记的流式细胞仪检测si RNA诱导的细胞凋亡,PI染色检测细胞周期阻滞。结果成功构建pSilencer-EGFR短发卡状si RNA真核表达载体;靶向EGFR的序列特异性的si RNA可以有效抑制Skov-3细胞EGFR基因的表达;流式细胞分析显示,Skov-3细胞凋亡率明显增加,细胞周期出现明显的G0/G1期阻滞。结论 RNAi沉默EGFR表达可以诱导卵巢癌Skov-3细胞凋亡,并使卵巢癌Skov-3细胞停滞在G0/G1期,RNAi可作为研究卵巢癌发生发展机制和基因治疗的有效工具。
Objective To investigate the effect of RNAi on epidermal growth factor receptor (EGFR) gene expression in ovarian cancer cells and its effect. Methods EGFR DNA template sequence and pSilencer 2 .1-U6neo plasmid were used to construct shRNA expression vector in vitro. The recombinant plasmid was transfected into human Skov-3 cells by Lipofectamine 2000. The expression of shRNA was verified by semi-quantitative RT-PCR. The changes of EGFR gene expression in Sk-ov-3 cells before and after transfection were detected by cytochemistry. The apoptosis induced by si RNA was detected by flow cytometry with Annexin V / PI double staining, and the cell cycle arrest was detected by PI staining. Results The eukaryotic expression vector of pSilencer-EGFR short hairpin RNA was successfully constructed. The sequence-specific siRNA targeting EGFR could effectively inhibit the expression of EGFR gene in Skov-3 cells. Flow cytometry analysis showed that apoptosis of Skov-3 cells The rate was significantly increased, the cell cycle obvious G0 / G1 phase arrest. Conclusion RNAi silencing EGFR expression can induce apoptosis of ovarian cancer Skov-3 cells and arrest the expression of Skov-3 cells in G0 / G1 phase. RNAi can be used as an effective tool to study the mechanism of ovarian cancer development and gene therapy.