靶向沉默SSBP1基因对肝癌HepG2细胞增殖、侵袭转移的影响

摘要

Objective To investigate the effects of RNAi-mediated SSBP1 gene silencing on proliferation,invasion and metastasis of human hepatocellular carcinoma HepG2 cells in vitro.Methods The high effective targeting SSBP1 siRNA was obtained by construction and screening,which was transfected into HepG2 cells by liposomes mediated transient transfection.The cells were divided into 3 groups: negative control group,blank transfection group (blank control group) and transfection group.The expression levels of SSBP1 mRNA and protein were detected by Real time-PCR and Western-Blot after the transfection.CCK-8 method was used to detect cell proliferation.Flow cytometry was used to detect the cell cycle,cell apoptosis and mitochondrial trans-membrane potential.The scuffing test and Transwell invasion assay were used to detect invasion and metastasis ability of cells.Moreover the expressions of gene and protein related with cell proliferation,invasion and metastasis were detected by Western Blot.Results The SSBP1 siRNA could obviously inhibit the expressions of SSBP1 mRNA and protein.As compared with negative control group and blank control group,after HepG2 cells were transfected by SSBP1 siRNA,the cell proliferation ability, cell proportion at phase G2 and phase S, mitochondrial trans-membrane potential were obviously decreased,however, the cell proportion at phase G1 and cell apoptosis rate were significantly increased (P<0.05).Meanwhile the expression levels of PCNA,Bcl-2 and MMP-9 proteins were obviously down-regulated,however, the expression levels of Bax proteins were significantly up-regulated (P<0.05).Conclusion The targeting silencing of SSBP1 gene can inhibit proliferation,invasion, metastasis of HepG2 cells,and can induce cell apoptosis via mitochondria pathway.%目的 观察靶向沉默线粒体单链DNA结合蛋白(SSBP1)基因对肝癌HepG2细胞增殖、侵袭转移的影响.方法 设计并构建靶向SSBP1 基因的特异性siRNA,采用脂质体介导瞬时转染肝癌HepG2细胞,细胞分3组:对照组、空白转染组、转染组.Real time-PCR和Western-blot检测靶向干扰后SSBP1 mRNA和蛋白表达变化.CCK-8法检测细胞增殖.流式细胞仪检测细胞周期、凋亡率及线粒体膜电位.划痕实验及Transwell 侵袭实验检测细胞侵袭转移能力.Western-blot检测增殖、侵袭转移相关基因蛋白表达状况.结果 SSBP1 siRNA能够显著抑制SSBP1 mRNA和蛋白表达.与对照组和空白转染组比较,转染SSBP1 siRNA后HepG2细胞增殖能力、G2期和S期细胞比例、线粒体膜电位明显降低,G1期细胞比例、细胞凋亡率明显升高(P<0.05);同时细胞增殖相关基因PCNA、凋亡抑制基因Bcl-2、转移相关基因MMP-9蛋白表达显著下调,凋亡诱导基因Bax蛋白表达显著上调(P<0.05).结论 靶向沉默SSBP1基因能够通过线粒体途径抑制肝癌HepG2细胞增殖、侵袭转移,并诱导其凋亡.