抑制IGF2基因的siRNA表达载体对肝癌Huh-7细胞增殖的抑制作用

Inhibitory Effect of siRNA Expression Vector Inhibiting IGF2 Gene on the Proliferation of Hepatoma Cell Line Huh-7

目的:研究抑制人胰岛素样生长因子2(IGF2)基因的siRNA表达载体对肝癌Huh-7细胞增殖的抑制作用。方法:将重组人甲胎蛋白(hAFP)和人端粒酶逆转录酶(hTERT)双启动子调控抑制IGF2基因的siRNA表达载体pGL3-h AFP-hTERT-siRNA3(简称siRNA3)转染Huh-7细胞和正常肝L-02细胞,另设阴性对照(载体p GL3-hAFP-hTERT)组和空白对照组。采用实时荧光定量聚合酶链反应法检测各组细胞转染48 h后IGF2 mRNA表达;酶标仪检测转染0、24、48、72 h后的细胞活性;流式细胞仪检测转染48h后细胞周期、细胞凋亡;Western blot法检测细胞IGF2、增殖细胞核抗原(PCNA)、细胞周期蛋白(Cyclin)E2、Cyclin D2、Cdc2、Bcl-2蛋白表达水平。结果:与阴性对照组和空白对照组比较,转染siRNA3的Huh-7细胞中IGF2 mRNA表达明显减弱,转染48、72 h后Huh-7细胞活性明显降低,G1期Huh-7细胞明显增加,S期Huh-7细胞明显减少;Huh-7细胞早期、晚期及总凋亡百分比均明显增加,IGF2、PCNA、Cyclin E2、Cyclin D2、Cdc2和Bcl-2蛋白表达均明显减弱,以上差异具有统计学意义(P<0.01或P<0.05)。转染siRNA3表达载体的L-02细胞上述指标均无明显变化(P>0.05)。结论:重组hAFP和hTERT双启动子调控针对IGF2基因的siRNA可特异性抑制Huh-7细胞IGF2表达及细胞增殖,其可能与下调IGF2 mRNA及蛋白表达,进而引起细胞增殖相关基因PCNA、细胞周期调控相关基因Cyclin E2、Cyclin D2、Cdc2及细胞凋亡调控相关基因Bcl-2蛋白表达下调有关。

OBJECTIVE：To investigate the inhibitory effect of siRNA expression vector inhibiting human insulin-like growthfactor 2（IGF2）gene on the proliferation of hepatoma cell line Huh-7.METHODS：siRNA expression vector pGL3-hAFP-hTERTsiRNA3（＂siRNA3＂）whichinhibited IGF2 gene by dual promoter regulation of recombinant human alpha-foetoprotein（hAFP）and human telomerase reverse transcriptase（hTERT）was transfected into the Huh-7 cell and normal hepatocyte L-02,and then a negative control group（vector p GL3-hAFP-hTERT）and a blank control group were set up.IGF2 m RNA expression was detected by real-time fluorescent quantitative polymerase chain reaction 48 hafter transfection into the cells in all groups;the activity of the cells by the microplate reader 0,24,48 and 72 hthereafter;and the cell cycle and apoptosis by the flow cytometer 48 hthereafter,and the changes in the protein levels of IGF2,PCNA,Cyclin E2,Cyclin D2,Cdc2 and Bcl-2 in the cell were detected by Western blot.RESULTS：Compared withthe negative control group and blank control group,IGF2 m RNA expression in the Huh-7 cell transfected withsiRNA3 was obviously weaker;at 48 and 72 hafter transfection,the activity of Huh-7 cell signigicantly reduced,Huh-7 cells at G1 phase obviously increased and those at S phase markedly decreased;the occurrence of early,late and total apoptosis in Huh-7 cells apparently increased,and the protein expression of IGF2,PCNA,Cyclin E2,Cyclin D2,Cdc2 and Bcl-2 in cells significantly weakened,withstatistically significance（P〈0.01 or P〈0.05）.No obvious change in the above-mentioned indexes could be found in L-02 cells transfected withsiRNA3 expression vector（P〈0.05）.CONCLUSIONS：siRNA whichinhibited IGF2 gene by dual promoter regulation of recombinant hAFP and hTERT can specially inhibit IGF2 gene expression and the proliferation of Huh-7 cells,whichmay be involved withdown-regulated protein expression of cell proliferation-associated gene PCNA,cell cycle control-associated genes Cyclin E2,Cyclin D2 and Cdc2 and apoptosis regulation-associated gene Bcl-2 as a result ofdown-regulated IGF2 mRNA expression and protein expression.