慢病毒介导的IGF-1R基因沉默对肝细胞癌的增殖和迁移的影响

Effects of lentivirus mediated IGF-1R gene silencing on proliferation and migration of hepatocellular carcinoma

目的探讨慢病毒介导的胰岛素样生长因子1受体(IGF-1R)基因沉默对肝细胞癌的增殖和迁移的影响。方法设计并筛选高效特异性胰岛素样生长因子1受体(IGF-1R)基因的小干扰RNA,构建稳定遗传的慢病毒载体质粒,观察其转染沉默IGF-1R基因的肝癌细胞株Huh7细胞增殖、迁移以及侵袭能力,进一步观察其对PI3K/Akt及Bax/Bcl-2通路的影响。结果采用倒置相差荧光显微镜观察转染效果,镜下可见较多细胞特异性表达GFP绿色荧光,转染率超过80%。scrambled阴性对照组以及空白对照组IGF-1R mRNA及蛋白的相对表达量显著高于LV-IGF-1R-RNAi转染组(P<0.01)。成功转染后,培养细胞72 h、96 h,LV-IGF-1R-RNAi转染组增值率显著低于scrambled阴性对照组以及空白对照组(P<0.05或P<0.01)。侵袭实验和迁移实验均证实细胞明显受到抑制(P<0.01)。蛋白免疫印迹法显示,LV-IGF-1R-RNAi转染组p-PI3K、p-Akt及Bcl-2相对表达量显著低于scrambled阴性对照组以及空白对照组(P<0.05);而Bax显著高于scrambled阴性对照组以及空白对照组(P<0.05)。结论RNAi技术沉默IGF-1R基因表达能显著抑制肝癌细胞株Huh7细胞的增殖、侵袭作用,而这一作用可能与抑制PI3K/Akt通路蛋白磷酸化及调节Bax/Bcl-2通路蛋白表达有关。

Objective To investigate the effects of lentivirus mediated insulin - like growth factor 1 receptor （IGF - 1R） gene silencing on proliferation and migration of hepatocellular carcinoma. Methods High specificity of insulin - like growth factor 1 receptor （ IGF - 1R） small interfering RNA gene lentiviral vector plasmid, genetic stability were designed and screened, Huh7 cells proliferation of the transfected IGF - 1R gene silencing, migration and invasion ability were observed, its effect on PDK/Akt and Bax/Bcl -2 pathway were further observed. Results The effect of transfection was observed by inverted microscope, GFP expression of green fluorescence microscope showed more cell specificity, the transfection rate of more than 80%. Scrambled negative control group and blank control group IGF - 1R mRNA and protein expression was signifi-cantly higher than that of LV - IGF - 1R - RNAi group （ P〈0.01） . After transfection, cells of 72 h , 96 h , LV - IGF - 1R - RNAi group the value of scrambled was significantly lower than the negative control group and blank control group （ P 〈 0. 05 or P 〈 0 .0 1 ） . Invasion assay and migration assay showed that cell was significantly inhibited （ P 〈0. 01） . Western blotting showed that LV - IGF - 1R - RNAi transfection group p -PI3K, p - Akt and Bel - 2 expression was significantly lower than that of scrambled negative control group and blank control group （ P〈0.05） ; while the Bax was significantly higher than that of scrambled negative control group and blank control group （P〈0.05） . Conclusion RNAi silencing IGF - 1R expression can significantly inhibit Huh7 cell proliferation of hepatocellular carcinoma cell lines, invasion, and this effect may be related to the inhibition of PI3K/Akt pathway protein phosphorylation and regulation of Bax/Bcl -2 pathway protein expression.