人剪切修复基因XPD对肝癌细胞中p8/TTDA基因的调控作用

Investiqate of XPD gene regulating expression of p8/TTDA and inhibi lepatoma cell grouth

目的探讨人剪切修复基因XPD对p8/TTDA基因的调控作用。方法用pEGFP-N2/XPD重组质粒、pEGFP-N2空载质粒分别稳定转染人肝癌细胞SMMC-7721,并用具有相同遗传背景和代数的SMMC-7721细胞作为空白对照。用RT-PCR、Western blot法检测转染前后p8、XPD、p53、c-myc表达量的变化,四甲基偶氮唑盐(MTT)法观察细胞增殖活力。结果 (1)RT-PCR检测示:p8 mRNA在人肝癌细胞SMMC-7721是低表达的,稳定转染野生型XPD后,p8 mRNA和XPD mRNA表达量明显增高(P<0.001)。稳定转染重组质粒细胞组p53 mRNA表达量亦明显增高(P<0.05),c-myc mRNA表达量则明显下降(P<0.001)。(2)Western blot法检测示:p8蛋白在人肝癌细胞SMMC-7721是低表达的。稳定转染野生型XPD后,p8蛋白、XPD蛋白、p53蛋白表达量明显增高(P<0.05),c-myc蛋白表达量则明显下降,差异有统计学意义(P<0.001)。(3)MTT检测示:稳定转染重组质粒细胞组细胞增殖率明显减弱(P<0.001)。结论人剪切修复基因XPD可调控p8/TTDA基因的表达。野生型XPD转染后亦可上调p53表达,下调c-myc表达,从而在分子途径上抑制SMMC-7721细胞增殖,促进其凋亡。

Objective To investigate the role of xeroderma pigmentosum D（XPD） gene in regulating the expression of p8/TTDA.Methods The recombinant plasmid pEGFP-N2/XPD and empty plasmid pEGFP-N2 were transfected into SMMC-7721 by lipofectamine and selected in medium containing G-418,800 μg/ml for SMMC-7721-pEGFP-N2/XPD,SMMC-7721-pEGFP-N2;Cell lines for comparison were matched on the same genetic background and passage.The expression of p8,XPD,p53 and c-myc were detected by RT-PCR and Western blot.Cell growth was detected by MTT.Results（1）The p8 mRNA in SMMC-7721 cells were low expressions.The relative expressions of p8 and XPD mRNAs in SMMC-7721-pEGFP-N2/XPD were significantly higher than SMMC-7721-pEGFP-N2,lipofectamine control group and blank control group（P〈0.001）.The relative expressions of p53 mRNA in SMMC-7721-pEGFP-N2/XPD were significantly higher compared with the controls,the difference were statistically significant（P〈0.05）.The relative expression of c-myc mRNA in SMMC-7721-pEGFP-N2/XPD significantly lower than those in the controls（P〈0.001）.（2）The p8 protein in SMMC-7721 cells was low expression;p8 protein,XPD protein and p53 protein in SMMC-7721-pEGFP-N2/XPD significantly were more than those in the controls（P〈0.05）.Compared with the controls,c-myc protein in SMMC-7721-pEGFP-N2/XPD was less with a significant difference（P〈0.001）.（3）Compared with the control groups,cell growth of the SMMC-7721-pEGFP-N2/XPD was inhibited.Conclusion XPD gene can regulate the expression of p8/TTDA.Wild-type XPD could enforce the expression of p8/TTDA and p53 and inhibit the activity of c-myc and cell growth in vitro.