Biological Effects Of Wild-type Xeroderma Pigmentosum D Gene On Human Cholangiocarcinoma Cell Line

Objective : The wild-type XPD gene was transfected into human cholangiocarcinoma cell line QBC939 by LipofectAMINE ,to investigate the biological effects of wild-type Xeroderma Pigmentosum D gene on human cholangiocarcinoma.Methods:The recombinant plasmid pEGFP-N2-XPD and the pEGFP-N2 were extracted by alkaline lysis method, then the plasmid were digested by KPNⅠ,BGIⅡand SPHⅠrestriction endonuclease. The experiment were classified four groups: pEGFP-N2-XPD group,pEGFP-N2 group,Lipofectamine(Lip)group , with the same genetic background and the generations of QBC939 cells as blank contron group. Four groups of cells were transfected by LipofectAMINE .The expression of green fluorescent protein(GFP) were observed with fluorescence microscope. Total RNA was extracted from four groups of cells,then this RNA was converted into cDNA. The expression of wild-type XPD, p53, cyclinD1 and c-myc were detected by Reverse Transcription-Polymerase Chain Reaction(RT-PCR). Cell proliferation was detected by MTT. Flow cytometry(FCM)were employed for examining the cell cycle of the transfected QBC939 cells.Results: 1.The relative expressin of XPD mRNA in pEGFP-N2-XPD group,pEGFP-N2 group,Lipofectamine(Lip)group, blank contron group were 0.778±0.018, 0.561±0.039, 0.544±0.035, 0.542±0.034; The relative expression of XPD mRNA in pEGFP-N2-XPD was significantly higher than the control pEGFP-N2 ,Lipofectamine(Lip)group, blank contron group(P<0.01).The relative expression of p53 mRNA in the four cell lines were 0.421±0.019, 0.256±0.014, 0.267±0.015, 0.274±0.018, respectively, those in pEGFP-N2-XPD was significantly higher in compared with the controls ,the difference was statistically significant(P<0.01).The relative expression of cyclinD1 mRNA in the four cell lines was :0.339±0.041, 0.560±0.039, 0.558±0.050, 0.560±0.041, respectively, those in pEGFP-N2-XPD significantly lower than the three controls(P<0.01). The relative expression of c-myc mRNA in the four cell lines was 0.355±0.045, 0.570± 0.075, 0.560±0.041, 0.537±0.050, respectively, those in pEGFP-N2-XPD significantly lower than the three control(sP<0.01). 2. Flow cytometry(FCM)results showed that pEGFP-N2-XPD cells in G1 phase was 81.65%, S phase was 11.83%, three of the control groups Gl phase were 65.54%, 56.61%, 63.26%; S phase were 24.10%, 29.52%, 27.28%, the result was statistically significant (P <0.05).3. MTT test indicated pEGFP-N2-XPD cells growth rate was 0.249±0.02, compared with three control groups, cell proliferation ability was significantly decreased (P <0.01)..Conclusion :The wild-type XPD could inhibit the proliferation of QBC939 cells in vitro. The wild-type XPD could decrease the expression of cyclinD1 ,c-myc , and increase the expression of p53.