The Role Of Excision Repair Gene XPD In Regulation Of P53and Ets-1of Hepatoma Cells

Objective:We transfected the recombination plasmid with wild-XPD into human hepatomacells (HepG2), then incubated HepG2with Pifithrin-α (p53inhibitor). We detectedthe changes about the expression levels of gene XPD, p53, Ets-1, and the cellularviability.Methods:HepG2cells bought from American Type Culture Collection (ATCC) wererevived and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing10%Fetal bovine serum (FBS). The pEGFP-N2plasmids and pEGFP-N2-XPDplasmids were extracted from the proliferated colibacilluses. Using liposome, wetransfected the two plasmids into HepG2. One of the cells transfected with pEGFP-N2-XPD plasmids were incubated with Pifithrin-α for24h. There were six groupsincluding blank control group, lipidosome group, pEGFP-N2group, pEGFP-N2-XPDgroup, pEGFP-N2-XPD+Pifithrin-α group and Pifithrin-α group. The expressionsmRNA of gene XPD, p53, Ets-1were detected by RT-PCR. The change of XPD, p53,phospho-p53(ser-15), Ets-1protein were observed by Western blot. The cellularproliferation was detected by MTT and the cell cycle was examined with flowcytometry.Results:After transfected with the pEGFP-N2and pEGFP-N2-XPD plasmid, the cellswere observed a lot of green fluorescence in the fluorescence microscope. The mRNAand protein expressions of XPD and p53increased greatly(vs control, P<0.01), andthe level of Ets-1expression down-regulated(vs control, P<0.01). The cellproliferation ability was inhibited, and the cell cycle was stopped in the G1stage.After incubated Pifithrin-α(p53inhibitor), the mRNA and protein expressions of Ets-1up-regulated partly(vs pEGFP-N2-XPD group, P<0.01), as the proteinphospho-p53(ser-15) decreased(vs pEGFP-N2-XPD group, P<0.01). The cellviability partly rose, and the cell cycle entered increasingly S stage. Conclusion:This effect that wild-XPD gene transfected into HepG2improved the expressionof p53and down-regulated the expression of Ets-1, however, was reversed partlyafter added the medicine Pifithrin-α. Therefore XPD gene could down-regulate thelevels of Ets-1via the p53pathway, and restrain the proliferation of HepG2cells.These studies may offer the laboratory evidence of gene therapy of certain tumors inclinic.