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目的:探讨RNA干扰表皮生长因子受体(EGFR)基因对卵巢癌顺铂耐药细胞株SKOV3/DDP凋亡的影响。方法:利用基因重组技术构建携带EGFR小发夹干扰RNA(pEGFR-shRNA)的重组质粒表达载体,脂质体法转染SKOV3/DDP细胞。实验分组:对照组(不进行干扰)、S1组(转染非特异性质粒)、S2组(转染特异性质粒)。用逆转录聚合酶链反应(RT-PCR)和免疫细胞化学法(ICC)检测转染后细胞内EGFRmRNA和蛋白的表达,流式细胞仪(FCM)分析细胞周期和凋亡率。结果:与对照组相比,S2组细胞EGFRmRNA的表达明显受抑制,抑制率达57%(P<0.01)。蛋白质的表达水平显著降低(P<0.01)。流式细胞仪检测结果显示,顺铂作用24h后,特异性转染组细胞周期分布发生明显改变,G0/G1期细胞比例增多,而S期细胞比例减少,凋亡率显著升高(P<0.01)。结论:RNA干扰EGFR基因通过抑制SKOV3/DDP细胞中EGFR基因的表达,恢复细胞对顺铂的敏感性。
Objective: To investigate the effect of epidermal growth factor receptor (EGFR) gene on apoptosis of cisplatin-resistant ovarian cancer cell line SKOV3 / DDP. Methods: Recombinant plasmids containing EGFR small hairpin RNA (shRNA) were constructed by gene recombination technique and transfected into SKOV3 / DDP cells by lipofectamine 2000. The experimental group: control group (without interference), S1 group (transfected with non-specific plasmid), S2 group (transfected with specific plasmid). The expression of EGFR mRNA and protein in transfected cells was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC). Cell cycle and apoptosis rate were analyzed by flow cytometry (FCM). Results: Compared with the control group, the expression of EGFR mRNA was significantly inhibited in S2 group (57%, P <0.01). Protein expression levels were significantly lower (P <0.01). The results of flow cytometry showed that after 24 hours of cisplatin, the cell cycle distribution of the specific transfection group changed obviously, the proportion of cells in G0 / G1 phase increased, the proportion of S phase cells decreased and the apoptosis rate increased significantly (P < 0.01). Conclusion: The RNA interference of EGFR gene restores the sensitivity of cells to cisplatin by inhibiting the expression of EGFR gene in SKOV3 / DDP cells.