XPD/P44亚复合物对人肝癌细胞周期的调控

目的 探讨着色性干皮病互补基因D(XPD)/P44亚复合物对人肝癌细胞周期的调控机制.方法 重组质粒增强型绿色荧光蛋白(pEGFP)-N2/XPD,空载质粒pEGFP-N2分别通过Lipofectamine 2000转染SMMC-7721细胞,构建稳定表达的细胞株,再应用P44反义寡核苷酸阻断SMMC-7721-pEGFP-N2/XPD中P44的表达.实验分为6组:①空白对照组;②SMMC-7721-pEGFP-N2组;③SMMC-7721-pEGFP-N2/XPD组;④反义SMMC-7721-pEGFP-N2/XPD翻译起始部位组;⑤反义SMMC-7721-pEGFP-N2/XPD翻译终止部位组;⑥反义SMMC-7721-pEGFP-N2/XPD外显子5组.用逆转录聚合酶链反应(RT-PCR)、Western印迹法检测转染各组细胞内P44、XPD以及cdk7、cdk2、c-myc和cdc25A的表达量,并用四甲基偶氮唑盐(MTr)和流式细胞仪检测细胞增殖及其细胞周期的变化.结果 ①、②组中P44、XPD的mRNA表达量均明显低于③组(均P<0.01).P44、XPD的蛋白变化趋势与其mRNA变化趋势一致;而细胞周期调控基因cdk7、cdk2、c-myc和cdc25A的mRNA及蛋白的表达量下调,细胞增殖力减弱,③组与①组、②组相比,停滞在G1期细胞多,进入S期细胞少.阻断P44后XPD的表达量下调,④、⑤、⑥组中XPD的mRNA表达量分别是③组的(0.55±0.09)、(0.65±0.05)、(0.61±0.11)倍,差异均有统计学意义(均P<0.01);④组、⑤组、⑥组中XPD蛋白的表达量分别为③组的(0.75±0.06)、(O.79±0.02)、(0.88±0.07)倍,差异均有统计学意义(均P<0.01).cdk7、cdk2、c-myc和cdc25A的mRNA以及蛋白的表达量上调;细胞增殖明显;与③组相比,④组、⑤组、⑥组的细胞进入S期细胞增多,停滞在G1期细胞减少.结论 XPD基因具有抑制癌细胞生长、促进癌细胞凋亡的功能;XPD的表达受其分子伴侣P44的调节,XPD/P44亚复合物可能是通过DNA损伤检控点来调控细胞周期的.

Mechanism of regulation of hepatoma cell cycle by XPD/P44 subcomplex:an in vitro experiment

Objective To explore the effects of xemderma pigmentosum group D(XPD)/P44 subcomplex on the cell cycle of the hepatoma cells.Methods Human hematoma cells of the line SMMC7721 were cultured and transfected with human XPD gene by LipofectAMINE and 2 strains with stably transfected plasmid pEGFG-N2 and stably transfected recombinant plasmid pEGFG-N2/XPD were selected.After stably transfection,the antisense oligonucleotides of P44 were added to treat the stably transfected cells.The cells were divided into 6 groups:Group①(control group),Group②transfected with the blank plasmid pEGFP-N2,Group③transfected with the recombinant plasmid pEGFP-N2/XPD,Group④transfected with ASODN complementary to the translation initiation site of pEGFP-N2/XPD,Group⑤transfected with antisense oligodeoxynucleotides(ASODN)complementary to the translation terminal site of pEGFP-N2/XPD.and Group⑥transfected with ASODN complementary to the translation exon5 site of pEGFP-N2/XPD.The expression Ievels of wild-type P44,XPD,cdk7,cdk2,c-myc,and cdc25A were detected by RT-PCR and Westem blotting.The cell growth and the cell cycle were examined by MTT and flow cytometry(FCM)Results The P44 and XPD mRNA expression levels of Group③were significantly higher than those of Groups①and②(both P<0.01).Western blotting indicated that the changes of P44 and XPD protein expression levels were consistent with those of their mRNAs respectively:while the tuRNA and protein expression levels of cdk7,cdk2,c-myc,and cdc25A were all decreased.MTT method showed that the hepatoma cells grew slowly,FCM showed that the number of the cells arrested at the G1 stage of Group③were higher than those of Groups①and②.After the blockage of P44 gene expression,the expression levels of XPD mRNA and protein were decreased.The XPD mRNA and protein expression levels of Groups④,⑤,and⑥were significantly higher than those of Group③(all P<0.01).The mRNA and protein expression levels of cdk7,cdk2.c-myc,and cdc25A were upregulated.MTY method indicated that ceils grew fast.FCM showed that the nulnbers of the cells arrested at the G1 stage of Group④,⑤,and⑥were all lower than that of Group③.The expression levels of cell cycle regulatory genes including cdk7,cdk,c-myc,and cdc25A were markedly decreased,the hepatoma cells grew slowly;after the blockage of P44 gene expression the expression levels of XPD mRNA and protein were decreased.whereas the expression levels of the cell cycle regulatory genes mentioned above were enhanced.and the hepatoma cells grew faster.Conclusion XPD gene inhibits the proliferation and promotes the apoptosis of hepatoma ceils.The expression of XPD may be regulated by its molecular partner P44.XPD/P44 subcomplex iB involvod in the regulation of DNA damage checkpoint.