下调IGF-ⅠR表达对HBV阳性肝癌细胞的增殖抑制作用

Down-regulation of IGF-ⅠR expression in inhibiting role of HBV-positive hepatocellular carcinoma cell proliferation

目的:观察干预胰岛素样生长因子-Ⅰ受体(insulin-like growth factor-Ⅰreceptor,IGF-ⅠR)表达对乙肝病毒(hepatitis B virus,HBV)阳性肝癌细胞增殖的影响。方法:以有效p GPU6/GFP/Neo IGF-ⅠR-sh RNA4干扰质粒(IGF-ⅠR-sh RNA4),转染肝癌Bel-7404细胞和HBV阳性PLC/PRF/5细胞后,以实时荧光定量逆转录聚合酶链反应(real time fluorescent quantitative reverse transcription polymerase chain reaction,FQ-RT-PCR)和Western Blot分别检测IGF-ⅠR的m RNA转录及蛋白水平;以CCK-8法、流式细胞术和Annexin-V-PE/7-ADD法分别评估细胞增殖、细胞周期与凋亡改变。结果:IGF-ⅠR-sh RNA4转染肝癌PLC/PRF/5和Bel-7404细胞效率为71%和90%;对细胞IGF-ⅠR m RNA抑制率分别为(59.6±2.8)%和(54.9±2.6)%;对IGF-ⅠR蛋白抑制率分别为(49.4±8.4)%和(58.3±9.4)%;转染72 h后,PLC/PRF/5细胞增殖抑制率为(63.9±3.9)%(t=19.244,P<0.001)和Bel-7404细胞为(61.5±1.7)%(t=5.493,P<0.005),伴细胞增殖周期改变、G1期阻滞和Cyclin D1表达受抑;细胞凋亡率增加,PLC/PRF/5为44.8%,Bel-7404细胞为36.0%,均显著高于空白组的12.2%和6.6%(P<0.001)及阴性组的9.4%和4.0%(P<0.001)。结论:下调IGF-ⅠR转录对肝癌细胞增殖具有明显的抑制作用,显示该位点有望成为HBV相关肝癌治疗的靶目标。

Objective： To investigate the effects of silencing insulin-like growth factor-Ⅰ receptor（IGF-ⅠR） on hepatitis B virus（HBV） positive hepatocellular carcinoma（HCC） cell proliferation. Methods： After the effective p GPU6/GFP/Neo IGF-ⅠR-sh RNA4 was transfected into PLC/PRF/5 with HBV positive and Bel-7404 cells, IGF-ⅠR was detected at m RNA level by real time fluorescent quantitative reverse transcription polymerase chain reaction（FQ-RT-PCR）, and at protein level by Western Blot. Cell proliferation, or cell cycle and apoptosis were analyzed by CCK-8, flow cytometry or Annexin-V-PE/7-ADD, respectively. Results： The sh RNA4 transfection efficiency was 71% in PLC/PRF/5 cells and 90% in Bel-7404 cells with inhibited（59.6±2.8）% and（54.9±2.6）% at m RNA level those in accordance with（49.4±8.4）% and（58.3±8.4）% at protein level,respectively. After transfected at 72 h, the cell proliferation inhibiting rate was（63.9 ±3.9）%（t=19.244, P0.001） in PLC/PRF/5 or（61.5±1.7）% in Bel-7404 cells（t=5.493, P0.005） in time dependent manner, with cell cycle alteration, G1 phase arrested,and down-regulated of Cyclin D1 expression. The rates of cell apoptosis were 44.8% in PLC/PRF/5 cells and 36.0% in Bel-7404 cells, with significantly increasing more than those（12.2% and 6.6%, P0.001） in the control group or those（9.4% and4.0%, P0.001） in the negative-sh RNA group. Conclusions： Silencing IGF-ⅠR gene transcription inhibited significantly HCC cell proliferation and the IGF-ⅠR should be an molecular-targeted site for HBV-related HCC therapy.