RNA干扰胰岛素生长因子-1受体基因对人肝癌SMMC7721增殖及凋亡蛋白的影响

Effects of RNA interference targeting insulin growth factor-1 receptor gene on proliferation and apoptosis of human hepatocellular carcinoma SMMC7721 cells

目的观察RNA干扰胰岛素生长因子-1(IGF-1)受体基因后对人肝癌细胞株SMMC7721增殖和相关凋亡因子表达的影响。方法构建两个靶向IGF-1R基因的RNA干扰真核表达质粒(IGF-1R-siRNA-1和IGF-1R-siRNA-2)分别转染至SMMC7721细胞,并设立正常对照组、空白对照组、IGF-1R-siRNA-1转染组、IGF-1R-siRNA-2转染组。采用聚合酶链反应(RT-PCR)法和Western blot检测IGF-1R基因表达变化,检测细胞增殖、侵袭、凋亡、凋亡蛋白、骨形态发生蛋白(BMPs)及相关信号通路蛋白的表达水平。结果RT-PCR结果显示,IGF-1R-siRNA-1和IGF-1R-siRNA-2表达载体转染SMMC-7721细胞后,IGF-1R基因的mRNA水平显著下调,IGF-1R-siRNA-1转染组IGF-1R基因的表达低于正常对照组,IGF-1R-siRNA-2转染组IGF-1R基因表达下调77.5%,差异有统计学意义(P<0.05)。Western blot结果显示,IGF-1R-siRNA-1转染组和IGF-1R-siRNA-2转染组IGF-1R蛋白在不同水平显著下调,IGF-1R蛋白表达抑制率分别为73.0%和81.5%,差异有统计学意义(P<0.05)。IGF-1R-siRNA-1转染组和IGF-1R-siRNA-2转染组细胞增殖率明显低于空白对照组,差异有统计学意义(P<0.05)。ImageJ软件分析划痕面积结果显示,与正常对照组和空白对照组相比,IGF-1R-siRNA-1转染组、IGF-1R-siRNA-2转染组修复率下降,差异有统计学意义(P<0.05)。Transwell实验发现,与正常对照组和空白对照组相比,IGF-1R-siRNA-1转染组、IGF-1R-siRNA-2转染组为SMMC-7721细胞侵袭能力明显下降,侵袭细胞数量明显减少,差异有统计意义(P<0.05)。流式细胞检测显示,IGF-1R-siRNA-1转染组和IGF-1R-siRNA-2转染组晚期凋亡率明显高于空白对照组,差异有统计学意义(P<0.05)。Western blot结果显示,IGF-1R-siRNA-1组、IGF-1R-siRNA-2组Bcl-2表达均明显受抑制,Bax、Cleaved Caspase-3蛋白表达量明显高于空白对照组,差异有统计学意义(P<0.05);PI3K、AKT/p-AKT、BMP-2、BMP-7蛋白表达量明显低于空白对照组,差异有统计学意义(P<0.05)。结论由IGF-1R基因沉默对肝癌SMMC7721细胞增殖与侵袭有抑制作用,对细胞凋亡有促进作用,能介导BMP2、BMP-7基因不同水平下调,IGF-1/PI3K/AKT信号通路蛋白可能参与其中。

Objective To observe the effect of silencing insulin growth factor-1(IGF-1)gene by RNA interference on the proliferation of human hepatocellular carcinoma cell line SMMC7721 and the expression of related apoptotic factors.Methods Two RNA interference eukaryotic expres-sion plasmids(IGF-1R-siRNA-1 and IGF-1R-siRNA-2)targeting IGF-1R gene were constructed and transfected into SMMC7721 cells,respectively,the normal control group,blank control group,IGF-1R-siRNA-1 transfection group and IGF-1R-siRNA-2 transfection group were set up.The expres-sion of IGF-1R gene was detected by polymerase chain reaction(RT-PCR)and Western blot,and then the expression levels of cell proliferation,inva-sion,apoptosis,apoptotic protein,bone morphogenetic proteins(BMPs)and related signaling pathway proteins were detected.Results The results of RT-PCR showed that the mRNA level of IGF-1R gene was significantly down-regulated after transfection of IGF-1R-siRNA-1 and IGF-1R-siR-NA2 expression vectors into SMMC-7721 cells,the expression of IGF-1R gene in IGF-1R-siRNA-1 transfection group was lower than that in nor-mal control group,and the expression of IGF-1R gene in IGF-1R-siRNA-2 transfection group was down-regulated by 77.5%,the differences were statistically significant(P<0.05).Western blot results showed that IGF-1R protein was significantly down-regulated at different levels in IGF-1R-siRNA1 transfection group and IGF-1R-siRNA-2 transfection group.The inhibition rates of IGF-1R protein expression were 73.0%and 81.5%,re-spectively,and the differences were statistically significant(P<0.05).The cell proliferation rate in IGF-1R-siRNA-1 transfection group and IGF-1R-siRNA2 transfection group was significantly lower than that in blank control group,and the differences were statistically significant(P<0.05).The analysis of scratch area by ImageJ software showed that compared with the normal control group and the blank control group,the repair rate of IGF 1R-siRNA-1 group and IGF-1R-siRNA-2 group decreased,and the differences were statistically significant(P<0.05).Transwell experiments showed that compared with the normal control group and the blank control group,the invasion ability of SMMC-7721 cells in the IGF-1R-siRNA-1 transfection group and the IGF-1R-siRNA-2 transfection group was significantly decreased,and the number of invasive cells was significantly re-duced,and the differences were statistically significant(P<0.05).Flow cytometry showed that the late apoptosis rate of IGF-1R-siRNA-1 transfec-tion group and IGF-1R-siRNA-2 transfection group was significantly higher than that of the blank control group,and the differences were statistical-ly significant(P<0.05).Western blot results showed that the expression of Bcl-2 in IGF-1R-siRNA-1 group and IGF-1R-siRNA-2 group was signifi-cantly inhibited,and the expression of Bax and Cleaved Caspase-3 protein was significantly highter than those in the blank control group(P<0.05).The expression levels of PI3 K,AKT/p-AKT,BMP-2 and BMP-7 proteins were significantly lower than those in the blank control group,and the dif-ferences were statistically significant(P<0.05).Conclusion The silencing of IGF-1R gene can inhibit the proliferation and invasion of liver cancer SMMC7721 cells,promote cell apoptosis,and mediate the down-regulation of BMP2 and BMP-7 genes at different levels,IGF-1/PI3K/AKT signal-ing pathway proteins may be involved.