shRNA干预IGF-ⅠR活化对肝癌细胞增殖的抑制作用

Short hairpin RNA-mediated silencing of insulin-like growth factor-Ⅰ receptor inhibits proliferation of hepatoma cells

目的:观察干扰胰岛素样生长因子-Ⅰ型受体(insulin-like growth factor-Ⅰreceptor,IGF-ⅠR)表达对肝癌(PLC/PRF/5及Bel-7404)细胞增殖、周期、凋亡的影响及联合抗癌、靶向药物抑制细胞增殖的协同作用.方法:设计与合成多条针对IGF-ⅠR序列的shRNA,插入pGPU6/GFP/Neo载体,构建、转染肝癌细胞株、筛选高效质粒,观察沉默IGF-ⅠR表达对肝癌细胞增殖的抑制作用与机制.结果:4对构建IGF-ⅠR-shRNA经筛选以s h R N A 4干扰效果最佳且具特异性;以shRNA4转染效率PLC/PRF/5细胞为71%和Bel-7404细胞为90%;在mRNA水平上抑制率前者为59.6%±2.8%,后者为54.9%±2.6%;蛋白水平上IGF-ⅠR表达均同步减少;转染72h后,PLC/PRF/5细胞增殖抑制率为63.87%±3.90%(t=19.244,P<0.001)及Bel-7404细胞为61.47%±1.70%(t=5.493,P<0.005),均呈时间依赖性,且增殖周期发生G1期阻滞,细胞周期蛋白(CyclinD1)表达受抑,细胞凋亡增加;shRNA4增加肝癌细胞对靶向药物索拉非尼及化疗药物奥沙利铂的敏感性.结论:下调IGF-ⅠR基因转录可抑制肝癌细胞增殖、诱导凋亡,改善肝癌细胞对靶向药物及化疗药物敏感性,提示IGF-ⅠR有望成为肝癌基因治疗的有效靶点.

AIM： To investigate the effect of short hairpin RNA（shRNA）-mediated silencing of insulinlike growth factor-Ⅰ receptor（IGF-Ⅰ R） gene transcription on cell proliferation,cell cycle progression,apoptosis and sensitivity to targeted therapy and chemotherapy in hepatocellular carcinoma（HCC） cell lines PLC/PRF/5 and Bel-7404.METHODS： Pairs of IGF-ⅠR shRNAs were designed and synthesized based on the IGF-ⅠR se-quence,and inserted into the pGPU6/GFP/Neo vector to screen the most effective one.IGF-ⅠR expression was then down-regulated with the shRNA to observe its inhibitory effect on hepatoma cell proliferation.RESULTS： After screening,the IGF-ⅠR-shRNA4 was found to be the most efficient one for interfering IGF-ⅠR gene transcription among the 4 pairs of successfully constructed plasmids,with a transfection efficiency of 71% in PLC/PRF/5 cells and 90% in Bel-7404 cells.The expression of IGF-ⅠR mRNA was down-regulated by 59.6% ± 2.8% in PLC/PRF/5 cells and 54.9% ± 2.6% in Bel-7404 cells.After the cells was transfected with shRNA4 for 72 h,the reduced rate of cell proliferation was 61.47% ± 1.70% in Bel-7404 cells（t = 5.493,P 0.005） and 63.87 ± 3.90%（t = 19.244,P 0.001） in PLC/PRF/5 cells.Meanwhile,the cell cycle was arrested in the G1 phase,and the expression of Cyclin D1 was significantly down-regulated with increasing cell apoptosis.Besides,the combination of shRNA4 with sorafenib or oxaliplatin showed higher inhibitory effects on cell survival than shRNA4 alone.CONCLUSION： Silencing IGF-ⅠR gene transcription can inhibit hepatoma cell proliferation,induce apoptosis and enhance the sensitivity to targeted therapy and chemotherapy.IGF-ⅠR may be a potential target gene for HCC gene therapy.