RNA干扰技术沉默CDC25a基因对人肝癌细胞HepG2增殖的影响

Effects of RNA interference targeting CDC25a gene on proliferation of human liver cancer Hep G2 cells

目的:研究细胞分裂周期素25a(cell division cycle 25a,CDC25a)基因沉默后对于人肝癌细胞系Hep G2增殖的影响。同时探讨该影响发生的可能作用机制。方法:使用RNA干扰技术沉默人肝癌Hep G2细胞的CDC25a基因,采用实时荧光定量PCR技术检测肝癌细胞中的CDC25a及其作用基因cyclin E及CDK2的mRNA表达水平,Western blotting检测CDC25a的蛋白表达水平,并采用MTT法、Giemsa染色法及流式细胞技术检测细胞的增殖情况。结果:CDC25a的mRNA及蛋白表达水平在RNA沉默组细胞中的表达低于阴性对照组及正常对照组细胞(P<0.05)。Cyclin E及CDK2的mRNA表达水平在沉默组低于阴性对照及正常对照组(P<0.05)。MTT法、Giemsa染色法结果显示沉默组细胞增殖能力低于阴性对照组及正常对照组细胞(P<0.05),流式细胞技术结果显示沉默组细胞阻滞在G1期。结论:LV-CDC25a-RNAi重组体感染Hep G2细胞可以有效抑制CDC25a基因的表达,使人肝癌Hep G2细胞增殖受到抑制,提示CDC25a基因可能是肝癌治疗的关键靶点。

AIM： To investigate the effect of silencing cell division cycle 25a （CDC25a） gene on the prolifera- tion of human hepatoma HepG2 cells. METHODS： CDC25a gene in human hepatoma HepG2 ceils was silenced by RNA interference. Real-time PCR was applied to detect the expression of CDC25a, cyclin E and CDK2 at mRNA levels in the HepG2 cells. Western blotting was applied to detect the expression of CDC25a at protein level. In addition, MTT assay, Giemsa staining and flow cytometry were used to measure the proliferation of human hepatoma HepG2 cells. RESULTS： The expression of CDC25a at mRNA and protein levels in RNA silence group was lower than those in negative control group and normal control group （ P 〈 0. 05 ）. The mRNA expression of cyclin E and CDK2 in silence group was lower than that in negative control group and normal control group （P 〈 0. 05）. The cell proliferation in silence group was lower than that in negative control group and normal control group （P 〈 0. 05）. The results of flow cytometry revealed that the cells in silence group were blocked in Gl phase. CONCLUSION： Infection of LV-CDC25a-RNAi recombinant to the HepG2 cells effec- tively inhibits the CDC25a gene expression and the proliferation of human hepatoma cells, and arrests the cells in G1 phase, suggesting that CDC25a gene may be a key target for the treatment of liver cancer.