目的:探讨siRNA方法沉默HepG-2细胞中B7-H3基因表达对细胞周期、增殖及迁移能力的影响.方法:设计合成针对B7-H3基因的siRNA及无关干涉siRNA序列,并分别构建干涉载体pSilencer4.1-CMV neo/B7-H3及无关干涉载体pSilencer4.1-CMV neo/NC;采用脂质体LipofectamineTM 2000转染对数生长期肝癌细胞HepG-2,经G418筛选后获得相应细胞模型;利用RT-PCR和Western blot方法分别检测空白对照组(WT)、无关干涉组(si-NC)和B7-H3干涉组(si-B7-H3)细胞中B7-H3 mRNA及蛋白表达水平;利用CCK-8细胞增殖实验及克隆形成实验评价各组细胞增殖情况;利用碘化丙锭(PI)染色流式细胞术检测各组细胞周期分布情况;利用细胞划痕实验检测各组细胞迁移能力.结果:成功构建了pSilencer4.1-CMV neo/B7-H3干涉载体.RT-PCR及Western blot结果显示,B7-H3干涉组较空白对照组、无关干涉组mRNA及蛋白水平明显下降;CCK-8增殖、克隆形成实验、细胞划痕实验及流式结果表明B7-H3干涉组增殖受到明显抑制,克隆较小且克隆数减少,细胞周期阻滞于G0/G1期,细胞迁移能力受抑制.结论:利用siRNA靶向干涉B7-H3表达可抑制HepG-2细胞增殖并诱导细胞周期阻滞,降低细胞迁移能力,实验结果为开发针对B7-H3靶点的肝癌免疫治疗提供了初步实验及理论基础.%Objective:To investigate the effect of small interfering RNA (siRNA) targeting against B7-H3 on cell cycle,proliferation and cell migration in human hepatoma HepG-2 cell.Methods:Two kinds of siRNA,including the B7-H3-targeted and the non-functional,were designed and synthesized.According to the sequence of siRNA,pSilencer4.1-CMV neo/B7-H3 and pSilencer4.1-CMV neo/NC interfering expression vectors were constructed.LipofectamineTM2000 were used to transfect aforementioned vectors into HepG-2 cells,respectively,which were subsequently screened by G418 in order to acquire positive cell model whose mRNA and protein expression level of B7-H3 were detected by RT-PCR and Western blot.Cell counting kit-8 and colony formation assay were also applied to evaluate cell proliferation.Flow cytometry were performed for testing cell cycle distribution.Wound healing assay were carried out to detect the ability of cell migration.Results:pSilencer4.1-CMV neo/B7-H3 was successfully constructed.The results of RT-PCR and Western blot demonstrated that the expression of B7-H3 was largely knocked down.CCK-8,colony formation assay,wound healing assay and flow cytometry showed that the proliferation and migration of cell model with B7-H3 silencing were suppressed,accompanied by smaller and fewer cell colonies,and cell cycle were arrested at G0/G1 phase.Conclusion:B7-H3-siRNA could inhibit the proliferation and migration of HepG-2 cell and drive cell cycle to arrest at G0/G1 phase,which lay good foundation concerning experiment and theory for exploring cancer immune therapy aiming at B7-H3.
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