靶向Cyclin D1基因RNA干扰对肝癌细胞增殖和Mdm2等基因表达影响的研究

Effects of RNA interference of targeting Cyclin D1 gene on hepatoma cell proliferation and Mdm2 gene expression

目的探讨Cyclin D1表达下调对Mdm2等基因表达及人肝癌细胞增殖的影响。方法用脂质体将Cyclin D1-siRNA转染人肝癌细胞株Hep3B细胞。实验分为空白对照组、阴性对照siRNA组、Cyclin D1-siRNA组。采用RT-PCR和Western blot检测Cyclin D1、Mdm2、Mdm4、P53和P21表达,流式细胞仪检测细胞周期,MTT检测细胞活力,TUNEL检测细胞凋亡。结果与空白对照组和阴性对照siRNA组相比,Cyclin D1-siRNA组P53和P21表达上调(P<0.05),Cyclin D1、Mdm2和Mdm4表达下调(P<0.01);3组细胞周期G1、S及G2期均无明显差异(P>0.05);Cyclin D1-siRNA组较其他两组细胞活力明显减弱(P<0.01),细胞凋亡明显增强(P<0.01)。结论 Cyclin D1表达下调能抑制Mdm2和Mdm4的表达,并能上调P53和P21的表达;Cyclin D1表达下调能抑制肝癌细胞的增殖并促进其凋亡。

Objective To investigate the effects of down-regulating Cyclin D1 expression on Mdm2 gene expression and proliferation of human hepatoma cells. Methods Cyclin D1-siRNA was transfected into the human hepatoma cell line Hep3B s with liposome.The experiment was divided into the blank control group,negative control siRNA （NC-siRNA） group and Cyclin D1-siRNA group.RT - PCR and Western blot were used to detect the expressions of Cyclin D1,Mdm2,Mdm4,P53 and P21.The cell cycle was measured by flow cytometer;the cellular activity was tested with MTT;the cell apoptosis was examined by TUNEL. Results Compared with the blank control group and NC-siRNA group,the expressions of P53 and P21 in the Cyclin D1-siRNA group were increased （ P 〈0.05）,while the expressions of Cyclin D1,Mdm2 and Mdm4 were decreased （ P 〈0.01）;there were no significant differences in the G 1,S and G 2 phases among 3 groups（ P 〉 0.05 ）;the cell vitality in the in the Cyclin D1-siRNA group was significantly weakened compared with the other two groups（ P 〈0.01）,while the cell apoptosis was significantly enhanced（ P 〈0.01）. Conclusion The down-regulation of Cyclin D1 gene expression could inhibit the expressions of Mdm2 and Mdm4,and up-regulates the expressions of P53 and P21.Down-regulating Cyclin D1 expression can inhibit the proliferation of liver cancer cells and promotes their apoptosis.