SH2B1对肝癌细胞增殖、凋亡及Akt/mTOR通路的影响

Effects of SH2B1 on Proliferation,Apoptosis and Akt/mT OR Pathway of Hepatocellular Carcinoma Cells

目的观察Src同源性2b衔接蛋白1(SH2B1)在肝癌细胞中表达情况,以及对肝癌细胞增殖、凋亡及蛋白激酶B/哺乳动物雷帕霉素靶蛋白(Akt/m TOR)信号通路的影响。方法通过荧光实时定量聚合酶链反应法检测人正常肝细胞株QSG-7701及肝癌细胞株Hep G2中SH2B1 mRNA的表达情况,采用免疫印迹法(Western blot法)检测SH2B1蛋白表达,采用短发夹RNA(shRNA)慢病毒干扰技术构建沉默SH2B1基因的人肝癌细胞Hep G2稳定转染细胞株,通过细胞计数试剂盒-8(CCK-8)法检测Hep G2细胞增殖能力,采用磷脂酰丝氨酸蛋白抗体/碘化丙啶(Annexin V/PI)双染流式细胞术检测Hep G2细胞凋亡,采用Western blot法测定凋亡细胞相关蛋白Bax、Bcl-2、caspase-3以及Akt/m TOR通路蛋白表达变化。结果与正常细胞相比,肝癌细胞中SH2B1 mRNA、SH2B1蛋白相对表达量显著升高(P <0. 05);与空白组、NC shRNA组相比,SH2B1 shRNA组SH2B1 mRNA、SH2B1蛋白表达显著降低(P <0. 05);CCK-8试验显示,与NC shRNA组和空白组相比,SH2B1 shRNA组转染48 h后OD值显著降低(P <0. 05);与空白组、NC shRNA组相比,SH2B1 shRNA组细胞凋亡率显著升高(P <0. 05);与空白组、NC shRNA组相比,SH2B1 shRNA组细胞中Bax、caspase-3蛋白水平显著上升(P <0. 05),Bcl-2蛋白水平下降(P <0. 05);与空白组和NC shRNA组相比,SH2B1 shRNA组p-AKT、m TOR蛋白水平明显降低(P <0. 05)。结论 SH2B1在肝癌细胞株Hep G2中高表达,沉默SH2B1表达可能通过抑制Akt/m TOR信号通路活化,抑制肝癌细胞增殖,促进细胞凋亡。

Objective To observe the expression of Src homology 2 binding protein 1(SH2B1)in hepatocellular carcinoma(HCC)cells,and to explore its effects on the proliferation,apoptosis,and protein kinase B/mammalian target of rapamycin(Akt/m TOR)pathway of HCC cells.Methods The expression of SH2B1 mRNA in human normal hepatic tissue cell line QSG-7701 and HCC cell line Hep G2 was detected by real-time fluorescence quantitative polymerase chain reaction(PCR),the expression of SH2B1 protein was detected by Western blot,the stably transfected human HCC cell line Hep G2 of silenced SH2B1 gene was constructed by shRNA lentivirus interference technique,the proliferation of Hep G2 cells was detected by cell counting Kit-8(CCK8)assay,phosphatidylserine protein antibody/propidium iodide(Annexin V/PI)double staining flow cytometry was used to detect the apoptosis of Hep G2 cells,Western blot was used to detect the changes of apoptotic cell related proteins Bax,Bcl-2,caspase-3,and Akt/m TOR pathway protein.Results Compared with normal cells,the relative expressions of SH2B1 mRNA and SH2B1 protein in HCC cells increased significantly(P<0.05);compared with blank group and NC shRNA group,SH2B1 mRNA expression and SH2B1 protein expression in SH2B1 shRNA group were significantly decreased(P<0.05);CCK-8 test showed that compared with NC shRNA group and blank group,OD value of SH2B1 shRNA group decreased significantly at 48 h after transfection(P<0.05);compared with blank group and NC shRNA group,the apoptotic rate of SH2B1 shRNA group was significantly increased(P<0.05);compared with blank group and NC shRNA group,the levels of Bax and caspase-3 protein in SH2B1 shRNA group increased significantly(P<0.05),while the level of Bcl-2 protein decreased(P<0.05);compared with blank group and NC shRNA group,the levels of p-AKT and m TOR protein in SH2B1 shRNA group were significantly decreased(P<0.05).Conclusion SH2B1 is highly expressed in HCC cell line Hep G2,silencing SH2B1 expression may inhibit the proliferation of HCC cells and promote cell apoptosis by inhibiting the activation of Akt/m TOR signaling pathway.