Gao Hua, Shi Jun, Ge Sheng-fang, DI Wen
Department of Obstetrics and Gynecology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China.
Zhonghua Fu Chan Ke Za Zhi. 2008 Jan;43(1):45-9.
To assess the effect of suppression of insulin-like growth factor-1 receptor (IGF1R) in HO8910PM cell line by small interference RNA (siRNA).
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 microg/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.
(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 microg/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 microg/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.
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.
评估小干扰RNA（siRNA）对HO8910PM细胞系中胰岛素样生长因子-1受体（IGF1R）的抑制作用。
使用脂质体2000转染siRNA以沉默IGF1R基因表达，分别通过实时荧光定量PCR、蛋白质免疫印迹法和流式细胞术（FCM）检测转染48小时后IGF1R mRNA和蛋白的表达水平，并评估其对细胞周期的影响。在转染后24、48、72、96小时，通过细胞计数试剂盒-8（CCK-8）检测细胞生长情况。转染24小时后，用不同浓度的顺铂（DDP）处理细胞24小时，通过CCK-8评估细胞生长抑制率。转染24小时后，用10μg/ml DDP孵育24小时，分别通过FCM和蛋白质免疫印迹法鉴定凋亡细胞及凋亡相关基因B细胞淋巴瘤/白血病-2（Bcl-2）的蛋白表达水平。
（1）转染IGF1R siRNA 48小时后，IGF1R mRNA和蛋白的表达水平均显著降低。（2）转染IGF1R siRNA后48、72、96小时，IGF1R的抑制伴随着细胞生长的减少，吸光度分别为1.71±0.13、2.32±0.23、2.79±0.28（P<0.01）。（3）IGF1R siRNA诱导细胞G2期阻滞，G2期细胞比例为24.37%（P<0.05）。（4）转染24小时后，用2.5、5、10、20μg/ml DDP处理细胞24小时，细胞生长抑制率分别为（25.94±0.08）%、（40.25±0.05）%、（59.48±0.03）%和（74.18±0.08）%（P<0.01）。（5）转染24小时后，用10μg/ml DDP处理细胞24小时，诱导17.95%的细胞凋亡（P<0.05），并降低Bcl-2蛋白水平。
IGF1R基因的RNA干扰可显著诱导HO8910PM细胞系中IGF1R沉默，体外抑制细胞生长，使细胞阻滞于G2期，并增强对DDP的化疗敏感性。
