RNA干扰技术抑制卵巢上皮性癌细胞IGF1R基因表达及增强顺铂敏感性的实验研究

Suppression of insulin-like growth factor-1 receptor by RNA interference inhibits cell growth in vitro and induces of HO8910PM cell to cisplatin

目的利用RNA干扰（RNAi）技术抑制卵巢上皮性癌（卵巢癌）H08910PM细胞胰岛素样生长因子1型受体（IGF1R）基因的表达，探讨IGF1R的生物学功能及对顺铂敏感性的影响。方法将IGF1R基因的特异性小分子干扰RNA（siRNA）、非特异性siRNA分别转染细胞；转染后48h通过实时PCR、蛋白印迹法（western blot）、流式细胞仪分别测定IGF1R mRNA、蛋白的表达及细胞周期变化；转染后24、48、72、96h通过细胞增殖／毒性检测试剂cell counting kit-8（CCK-8）测定细胞增殖；于转染后24h给予不同浓度的顺铂作用24h，通过CCK-8法测定细胞增殖抑制率；于转染后24h给予10μg／ml顺铂作用24h，通过流式细胞仪及蛋白印迹法分别检测细胞凋亡和B淋巴细胞白血病／淋巴瘤蛋白2（Bcl-2）蛋白的表达。结果（1）转染后48h，IGF1R mRNA和蛋白的表达水平均明显下调，转染后IGF1R mRNA的表达水平下调了85．5％（P〈0．01）。（2）转染后48、72、96h，细胞增殖被明显抑制，细胞增殖活性分别为1．71±0．13、2．32±0．23、2．79±0．28，与未转染及转染非特异性siRNA的对照细胞比较，差异均有统计学意义（P〈0．01）。（3）转染后48h有24．37％的细胞处于G2期（P〈0．05）。（4）转染后24h联合不同浓度的顺铂作用24h，当顺铂浓度为2．5、5、10、20μg／ml时，细胞增殖被明显抑制，细胞增殖抑制率分别为（25．94±0．08）％、（40．25±0．05）％、（59．48±0．03）％、（74．18±0．08）％，差异均有统计学意义（P〈0．01）。（5）在转染siRNA24h后给予10μg／ml顺铂作用24h，可使细胞的凋亡率增加至17．95％（P〈0．05），并使Bcl-2蛋白的表达水平下调。结论通过RNAi技术可有效抑制IGF1R基因在转录和翻译水平的表达，抑制肿瘤细胞的增殖，出现G2期阻滞，明显提高细胞对顺铂化疗的敏感性。

Objective To assess the effect of suppression of insulin-like growth factor-1 receptor （IGF1R） in HO8910PM cell line by small interference RNA （siRNA）. Methods Transfection of siRNA using lipofectamine 2000 was conducted to silence IGF1R gene expression, the expression levels of IGF1R mRNA and protein were evaluated, and the effects on the cell cycles at 48 hours of transfection were assessed by real-time PCR, western blot and flow cytometry （FCM） assay respectively. The cell growth was detected by cell counting kit-8 （CCK-8） at 24, 48, 72, 96 hours of transfection. After 24 hours of transfection, the cells were cultured with different concentrations of cisplatin （DDP） for 24 hours, the cell growth inhibition rate was evaluated by CCK-8. Following incubation with 10 μg/ml DDP for 24 hours after 24 hours of transfection, the apoptosis cells and the protein expression level of apoptosis-related gene, B cell leukemia/ lymphoma 2 （Bcl-2）, were identified by FCM and western blot respectively. Results （1） Expression levels of IGF1R mRNA and protein were markedly decreased respectively at 48 hours of transfection IGF1R siRNA. （2） Suppression of IGF1R accompanied the reduction of cell growth at 48, 72, 96 hours of transfection with IGF1R siRNA, absorbance were 1.71 ± 0. 13, 2. 32 ± 0. 23, 2. 79 ± 0. 28 respectively （P 〈0. 01）. （3） IGF1R siRNA induces arrest of G2 phase, the G2 phase rate of cells were 24. 37% （P 〈 0.05）. （4） Following treatment with 2. 5, 5, 10, 20 μg/ml DDP for 24 hours after 24 hours of transfection, the cell growth inhibition rates were （ 25.94 ± 0.08 ） % , （ 40. 25 ± 0. 05 ） % , （ 59.48 ± 0. 03 ） % and （74. 18 ± 0.08 ） % respectively （ P 〈 0. 01 ）. （5） Treatment with 10 μg/ml DDP for 24 hours after 24 hours of transfection, induces 17.95% of cells apoptosis （ P 〈 0. 05 ）, and decreases Bcl-2 protein level. Conclusion RNA interference of IGF1R gene induces the IGF1R silence in HO8910PM cell line significantly, inhibits cell growth in vitro, arrests the G2 phase, and enhances the chemosensitization to DDP.