小干扰RNA靶向抑制DcR3基因对肝癌细胞凋亡和迁移的影响

SiRNA-mediated inhibition of DcR3 gene expression induces apoptosis and reduces migration of hepatocellular carcinoma HepG2 cells

目的:应用RNA干扰技术抑制人肝细胞癌细胞系HepG2细胞DcR3表达,并研究DcR3基因沉默后对HepG2细胞凋亡和迁移能力的影响.方法:设计并合成特异性DcR3-siRNA 4条和阴性对照siRNA 1条,转染HepG2细胞,通过半定量反转录-PCR(RT-PCR)及免疫细胞化学方法检测其对DcR3表达的抑制作用并筛选出干扰效果最强的siRNA,应用流式细胞仪、DNA ladder方法检测细胞的凋亡情况,应用划痕实验检测细胞体外迁移能力的变化.结果:各特异性DcR3-siRNA均能抑制DcR3mRNA表达(P<0.05),其中以DcR3-siRNA4的抑制作用最明显,抑制作用达62.9%;将DcR3-siRNA4转染HepG2细胞能明显抑制HepG2细胞DcR3蛋白的表达(P<0.01).流式细胞仪检测结果显示,特异性DcR3-siRNA转染组细胞的凋亡率(22.97%±2.10%)明显高于空白对照组(1.17%±0.32%)、脂质体转染组(1.44%±0.43%)和非特异性siRNA转染组(1.22%±0.40%)(均P<0.001);DNA ladder实验发现特异性DcR3-siRNA转染组出现DNAladder,而空白对照组、脂质体转染组和非特异性siRNA转染组均未出现DNA ladder.划痕实验结果显示,转染后24、48和72 h,特异性DcR3-siRNA转染组细胞的相对迁移率均低于空白对照组、脂质体转染组和非特异性siRNA转染组.结论:DcR3在肝癌细胞的凋亡及迁移中发挥重要作用,抑制DcR3的表达能诱导肝癌细胞凋亡并降低肝癌细胞的迁移能力.

AIM： To investigate the effect of siRNA-mediat- ed inhibition of DcR3 gene expression on apop- tosis and migratory ability of HepG2 cells.METHODS： Four DcR3-specific siRNAs and one random siRNA were designed, synthesized, and transfected into HepG2 cells using Lipofectami- neTM 2000. Semi-quantitative RT-PCR was used to screen the siRNA that had the best interfering effect. Immunocytochemistry was used to assess the expression of DcR3 protein. Cell apoptosis was assessed by flow cytometry, and cell migratory ability was determined by wound healing assay. RESULTS： All four DcR3-specific siRNAs could decrease the expression of DcR3 mRNA, and siRNA4 had the best interfering effect, which could silence the mRNA expression by 62.9% and inhibit the expression of DcR3 protein. The apoptosis rate was significantly higher in the specific interference group than in the three con- trol groups （22.97% ＋ 2.10% vs 1.17% ＋ 0.32%, 1.44% ＋ 0.43%, 1.22% ＋ 0.40%, all P 〈 0.001）. The relative migratory ability of HepG2 cells in the specific interference group at 24, 48 and 72 h af- ter transfection was significantly lower than that in control groups （all P 〈 0.001）. CONCLUSION： DcR3 plays an important role in the apoptosis and migration of HepG2 cells. In- hibition of expression of DcR3 can induce apop- tosis and repress migration of HepG2 cells.