特异性DcR3-siRNA对肝细胞癌生物学特性的影响

Effect of specific DcR3 siRNA on biological behaviors of hepatocellular carcinoma cell line

目的探讨特异性诱捕受体3(decoy receptor 3,DcR3)的siRNA对HepG2细胞生物学特性的影响。方法设计并合成针对DcR3的siRNA,用脂质体转染HepG2细胞,通过半定量RT-PCR及免疫细胞化学法检测其对DcR3表达的抑制作用;应用MTT法、流式细胞术、划痕实验检测转染后HepG2细胞增殖、凋亡及迁移能力的变化。结果特异性DcR3-siRNA能抑制DcR3 mRNA和蛋白表达(P<0.05);MTT实验结果:转染后24、48和72 h特异性DcR3-siRNA转染组细胞吸光度值低于空白对照组、脂质体转染组及非特异性siRNA转染组(P<0.001),特异性DcR3-siRNA转染组细胞的增殖抑制率在24、48和72 h分别为20.71%、35.00%和34.04%;转染后72 h,特异性DcR3-siRNA转染组细胞与空白对照组、脂质体转染组及非特异性siRNA转染组细胞相比,G1期细胞比例增多而G2/M期细胞比例减少(P<0.01);流式细胞仪检测细胞凋亡结果显示,特异性DcR3-siRNA转染组细胞凋亡率(22.97%±2.10%)高于空白对照组(1.17%±0.32%)、脂质体转染组(1.44%±0.43%)及非特异性siRNA转染组(1.22%±0.40%)(P<0.001);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、脂质体转染组及非特异性siRNA转染组细胞(P<0.001)。结论特异性DcR3-siRNA能有效抑制肝癌细胞系HepG2的DcR3表达,诱导细胞凋亡,并能抑制HepG2细胞的增殖和迁移能力。

Purpose To explore the effect of specific DcR3-siRNA on the proliferation, apoptosis and movement in human hepatocellu-lar carcinoma cell line HepG2. Methods Designed and synthesised specific siRNA targeting DcR3 was transfected into HepG2 ceils by Lipofectamine 2000, Semi-quantitive RT-PCR was used to assess the expression of DcR3 mRNA, immunocytochemical method was used to assess the expression of DcR3 protein. The proliferation of HepG2 cells was tested by MTI＇ assay, the change of periodic situa-tion of cells was analyzed by the flow cytometry （ FCM）, the apoptosis was explored by FCM, and the transferring ability was investiga-ted by the wound healing test. Results The specific siRNA targeting DcR3 decreased the expression of DcR3 mRNA and protein （P 〈 0.05 ）. 24, 48 and 72 hours after the transfection, MTT assay revealed that the value of the absorbance in spesific interfrence group was much lower than other groups （P 〈0. 001 ）, the inhibitory rate of proliferation in 24, 48 and 72 hours was 20. 71%, 35.00% and 34.04%, respectively. Compared with other groups, the rate of the cells in Gl-phase was increased while the rate in the G2/M-phase was decreased in the spesific interfrence group （ P 〈0. 01 ）. FCM showed the apoptosis rate in the spesific interfrence group was higher than other groups （P 〈 0. 001 ）. The relative mobility of the specific interfrence group 24, 48 and 72 hours after the transfection was all lesser than other groups （P 〈 0. 001 ）. Conclusion DcR3 siRNA could efficiently inhibit DcR3 gene expression and the cell prolifera-tion, induce the tumor cell apoptosis and repress the transferring ability in HCC cells.