人剪切修复基因XPD对肝癌细胞生长的抑制作用

Xeroderma Pigmentosum D Gene Inhibits Proliferation of Human Hepatocellular Carcinoma Cell Line SMMC-7721

目的探讨野生型人剪切修复基因着色性干皮病基因D(xeroderma pigmentosum D,XPD)对肝癌细胞SMMC-7721增殖的影响,并探讨其机制。方法用脂质体转染法瞬时转染SMMC-7721细胞,转染重组质粒XPD-N2和空载质粒N2,并用未转染的与XPD-N2、N2具有相同遗传背景和代数的SMMC-7721细胞作为空白对照。荧光显微镜观察绿色荧光蛋白报告基因表达情况,流式细胞仪检测细胞周期,逆转录-聚合酶链反应(RT-PCR)、Western blot法检测细胞中XPD、c-myc、cdc25A、cdK2表达量变化,MTT法观察细胞增殖的活力。结果提取出的重组质粒pEGFP-N2-XPD用酶切鉴定,与Genebank上的相符。在荧光显微镜下,可以在SMMC-7721-pEGFP-N2、SMMC-7721-pEGFP-N2-XPD细胞中观察到绿色荧光蛋白的表达,质粒的转染效率为30%左右。流式细胞仪结果显示,pEGFP-N2-XPD重组质粒转染入细胞后,肝癌细胞进入S期发生阻滞,停滞在G1期。RT-PCR、Western blot检测发现SMMC-7721-pEGFP-N2-XPD细胞与SMMC-7721-pEGFP-N2和SMMC-7721两对照组相比,其XPD表达明显增高(P<0.05)。c-myc、cdc25A、cdK2相对表达量明显减少,差异有统计学意义(P<0.05)。与两对照组相比,SMMC-7721-pEGFP-N2-XPD细胞增殖率明显减弱(P<0.05)。结论野生型XPD基因可以在转录和翻译水平抑制SMMC-7721细胞内c-myc、cdc25A、cdK2的表达。而且野生型XPD基因通过抑制cdK2的表达作用于S期DNA损伤检控点,从而抑制SMMC-7721细胞增殖。

Objective The Xeroderma Pigmentosum D （XPD） gene plays an important role in the path to DNA repair. It has been reported that cells obtained high tumor susceptibility when mutation happened in XPD gene. The purpose of this study is to investigate the influence of XPD gene in the growth and proliferation of hepatoma carcinoma cells （HCC）. Methods pEGFP-N2 and pEGFP-N2-XPD were transfected into SMMC-7721 cell lines by Lipofectamine2000 method. The expression of green fluorescent protein （GFP） was observed through fluorescence microscope. Cell cycle of the plasmid-transfeeted SMMC-7721 cells was analyzed by Flow cytometry （FCM）. The expressions of wild-type XPD, c-myc, cdc25A and cdK2 were detected by RT-PCR and Western blot. Cell proliferation was measured by MTT assay. Resuits The recombinant plasmid pEGFP-N2-XPD was successfully transfected into SMMC-7721 cells and green fluorescence in cells was observed by fluorescent microscopy. Around 30% transfection efficiency was obtained in this experiment. In addition, we have found that wild type XPD blocked the hepatoma cells from S stage to G1 stage The expression of XPD in SMMC-7721-pEGFP-N2-XPD cells was significantly higher than those in control group of SMMC-7721 and SMMC-7721-pEGFP-N2 cells （P〈0.05）. The expressions of c-mye, cdc25A and cdK2 in SMMC-7721-pEGF-N2-XPD cells were significantly lower than those in control cells （all P〈0.05）. Furthermore, SMMC-7721-pEGFP-N2-XPD has reduced proliferation ability than empty plasmid-transfected cells （P^0. 05）. Conclusion Wild-type XPD downregulated the expression of c-myc, cdc25A, and cdK2. Our results suggested that XPD inhibit the proliferation of SMMC-7721 cells probably due to the blockage at S period DNA damage checkpoint in hepatocellular carcinoma cell cycle.