XPD／P44亚复合物对人肝癌细胞的生物学影响

Biological Effects of Antisense P44 on XPD/P44 Subcomplex and Hepatoma Cell Behavior in Vitro

目的:应用P44反义寡核苷酸转染人肝癌细胞SMMC-7721,探讨XPD/P44亚复合物对人肝癌细胞的生物学影响。方法:应用P44反义寡核苷酸转染人肝癌细胞SMMC-7721,同时用未做处理的SMMC-7721作为空白对照。用RT-PCR、Western blot法检测转染P44反义寡核苷酸后细胞内P44,XPD以及cdk7、cdk2、c-myc和cdc25A表达量的变化,并用四甲基偶氮唑盐(MTT)和流式细胞仪检测细胞增殖及其细胞周期的变化。结果:P44基因反义寡核苷酸转染人肝癌细胞SMMC-7721后,人剪切修复基因XPD mRNA及其蛋白的表达量显著减少,同时细胞周期调控基因cdk7、cdk2、c-myc和cdc25A mRNAs及其蛋白的表达量上调,肝癌细胞增殖明显。结论:XPD基因的表达受其分子伴侣P44的调节,XPD的作用需要有P44的参与;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。

Objective： To investigate the effects of P44 on the cell cycle and its interaction with XPD, cdk7, cdk2, c-myc and cdc25A. MethodS：Antisense oligonueleotides of P44 gene were transfected into SMMC-7721 cells. Cell lines for comparison were matched on the same genetic background and passage. The expression of wild-type XPD, cdk7, cdk2, c-myc and cdc25A were detected by RT-PCR and Western blot. The cell growth and the cell cycle were examined by MTT and flow cytometry. Results： After transfection of SMMC-7721 with antisense oligonucleotides of P44, the expression of XPD mRNA was decreased significantly. The expressions of cell cycle regulatory genes including cdk7, cdk2, c-myc and cdc25A were enhanced, and the inhibitory effects of XPD on cell growth were decreased and the hepatoma cells grew fast. Conclusion： XPD gene is regulated by its upstream regulatory gene P44, and its function requires the interaction of XPD with P44, which can result in the changes of cdk7, cdk2, c-myc and cdc25A genes expression. XPD/P44 subcomplex is involved in the regulation of DNA damage checkpoint.