人剪切修复基因XPD对人肝癌SMMC-7721细胞中Ets-1和Cdk6基因的调控作用

摘要

Objective: To observe the expressions of Ets-1 and Cdk6 gene and their effects on the growth and proliferation of SMMC-7721 cells transfected with wild-type XPD gene. Methods:The pEGFP-N2-XPD was transfected into SMMC-7721 cells by Lipofectamine 2000?. There were four groups in this study including SMMC-7721-pEGFP-N2-XPD (XPD) group,SMMC-7721-pEGFP-N2 (N2) group,lpofectamine (Lip) group and blank control group. The expression levels of XPD,Ets-1 and Cdk6 were detected by RT-PCR and Western blot. Flow cytometry (FCM) was used to analyze the cell cycle of SMMC-7721 cells. The cell proliferation was measured by MTT assay. Results: The expression of XPD mRNA and protein was significantly higher in XPD group than that of other three groups (P < 0.001). The expressions of Ets-1 and Cdk6 mRNA and proteins were significantly decreased compared with those of other three groups (P < 0.001). FCM result showed that XPD prevented the hepatoma cells from G1 stage to S stage. The proliferation ability of SMMC-7721 cells was significantly reduced after transfetion with wild-type XPD gene (P < 0.01). Conclusion: XPD gene may regulate the proliferation of hepatoma cells by inhibiting Ets-1 and Cdk6 genes.%目的:观察野生型人剪切修复基因XPD转染入人肝癌细胞SMMC-7721后,细胞内Ets-1和Cdk6基因的表达变化及对SMMC-7721肝癌细胞增殖的影响.方法:将人工合成的pEGFP-N2-XPD重组质粒通过Lipofectamine 2000TM转染SMMC-7721细胞.设重组质粒转染细胞SMMC-7721-pEGFP-N2-XPD (XPD)组、空载质粒转染细胞SMMC-7721-pEGFP-N2(N2)组、脂质体组、无转染空白对照组.分别用逆转录-聚合酶链反应(RTPCR)和蛋白印迹法(Western blot)检测细胞中XPD、Ets-1、Cdk6基因mRNA和蛋白质的表达量,并用流式细胞仪检测细胞周期变化,四甲基偶氮唑盐微量酶反应比色法(MTT)检测各组细胞的增殖活力.结果:XPD组中的XPD的mRNA和蛋白质表达较其他3组明显增高(P＜ 0.001),而Ets-1、Cdk6 mRNA和蛋白质表达较其他3组明显减少(P＜ 0.001).转染pEGFP-N2-XPD重组质粒后细胞停滞在G1期,难于进入S期.转染了野生型XPD的SMMC-7721细胞增殖能力减弱.结论:XPD基因可能通过抑制Ets-1、Cdk6基因的表达影响肝癌细胞的生长.