Wang Hong-yun, Xiong Gao-fei, Wu Bo-lin, Zhang Ji-xiang
Department of Gastroenterology, Second Affiliated Hospital of Nanchang University, Jiangxi Provincial Laboratory of Molecular Medicine, Nanchang University, Nanchang 330006, China.
Zhonghua Yi Xue Za Zhi. 2008 Jul 22;88(28):1997-2001.
To explore the effects of xeroderma pigmentosum group D(XPD)/P44 subcomplex on the cell cycle of the hepatoma cells.
Human hematoma cells of the line SMMC-7721 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: Group (1) (control group), Group (2) transfected with the blank plasmid pEGFP-N2, Group (3) transfected with the recombinant plasmid pEGFP-N2/XPD, Group (4) transfected with ASODN complementary to the translation initiation site of pEGFP-N2/XPD, Group (5) transfected with antisense oligodeoxynucleotides (ASODN) complementary to the translation terminal site of pEGFP-N2/XPD, and Group (6) transfected with ASODN complementary to the translation exon5 site of pEGFP-N2/XPD. The expression levels of wild-type P44, XPD, cdk7, cdk2, c-myc, and cdc25A were detected by RT-PCR and Western blotting. The cell growth and the cell cycle were examined by MTT and flow cytometry (FCM).
The P44 and XPD mRNA expression levels of Group (4) were significantly higher than those of Groups (1) and (2) (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 mRNA and protein expression levels of cdk7, cdk2, c-myc, and cdc25A were all decreased. MTF method showed that the hepatoma cells grew slowly, FCM showed that the number of the cells arrested at the G1 stage of Group (3) were higher than those of Groups (1) and (2). 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 Groups (4), (5), and (6) were significantly higher than those of Group (3) (all P < 0.01). The mRNA and protein expression levels of cdk7, cdk2, c-myc, and cdc25A were upregulated. MT method indicated that cells grew fast. FCM showed that the numbers of the cells arrested at the G1 stage of Group (4), (5), and (6) were all lower than that of Group ((3) The expression levels of cell cycle regulatory genes including cdk7, cdk2, 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.
XPD gene inhibits the proliferation and promotes the apoptosis of hepatoma cells. The expression of XPD may be regulated by its molecular partner P44. XPD/P44 subcomplex is involved in the regulation of DNA damage checkpoint.
探讨着色性干皮病D组（XPD）/P44亚复合物对肝癌细胞周期的影响。
培养人肝癌细胞系SMMC-7721，采用脂质体法将人XPD基因转染入细胞，筛选出稳定转染质粒pEGFP-N2和稳定转染重组质粒pEGFP-N2/XPD的2个细胞株。稳定转染后，加入P44反义寡核苷酸处理稳定转染细胞。将细胞分为6组：（1）组（对照组），（2）组转染空质粒pEGFP-N2，（3）组转染重组质粒pEGFP-N2/XPD，（4）组转染与pEGFP-N2/XPD翻译起始位点互补的反义寡脱氧核苷酸（ASODN），（5）组转染与pEGFP-N2/XPD翻译终止位点互补的反义寡脱氧核苷酸，（6）组转染与pEGFP-N2/XPD翻译外显子5位点互补的ASODN。采用逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测野生型P44、XPD、细胞周期蛋白依赖性激酶7（cdk7）、细胞周期蛋白依赖性激酶2（cdk2）、原癌基因c-myc和细胞周期蛋白磷酸酶25A（cdc25A）的表达水平。采用噻唑蓝（MTT）法和流式细胞术（FCM）检测细胞生长及细胞周期。
（4）组P44和XPD mRNA表达水平显著高于（1）组和（2）组（均P<0.01）。蛋白质免疫印迹法显示，P44和XPD蛋白表达水平的变化分别与其mRNA的变化一致；而cdk7、cdk2、c-myc和cdc25A的mRNA和蛋白表达水平均降低。MTT法显示肝癌细胞生长缓慢，FCM显示（3）组停滞于G1期的细胞数高于（1）组和（2）组。P44基因表达被阻断后，XPD mRNA和蛋白表达水平降低。（4）组、（5）组和（6）组XPD mRNA和蛋白表达水平显著高于（3）组（均P<0.01）。cdk7、cdk2、c-myc和cdc25A的mRNA和蛋白表达水平上调。MTT法显示细胞生长加快。FCM显示（4）组、（5）组和（6）组停滞于G1期的细胞数均低于（3）组。细胞周期调节基因cdk7、cdk2、c-myc和cdc25A的表达水平明显降低时，肝癌细胞生长缓慢；P44基因表达被阻断后，XPD mRNA和蛋白表达水平降低，而上述细胞周期调节基因的表达水平增强，肝癌细胞生长加快。
XPD基因抑制肝癌细胞增殖并促进其凋亡。XPD的表达可能受其分子伴侣P44的调控。XPD/P44亚复合物参与DNA损伤检查点调控。
