Effect of XPG down-regulation gene expression towards the proliferation of epithelial ovarian cancer cells and its chemosensitivity to platinum / 中华妇产科杂志

ABSTRACT

ObjectiveTo investigate the effect of XPG down-regulation gene expression towards the proliferation of epithelial ovarian cancer cells and its chemosensitivity to platinum.Methods The small interference RNA ( siRNA ) -XPG fragments were designed and tranfected into SKOV3/DDP cell lines by lipofectamine transiently for choosing the best siRNA-XPG fragment to silence XPG gene expression.The pGPU6/GFP/Neo vector was used to construct the siRNA-XPG vectors,which was transfected into SKOV3/DDP cell line with expression of XPG gene.Real-time PCR and western blot were employed to confirm the silencing efficacy of siRNA-XPG.The growth curve of cells,cell cycle,the drug-resistance index of cells and intracellular drug concentration were measured by 4-methyl-thiazolyl-tetrazolium (MTT),flow cytometer (FCM) and high performance liguid chromatograph respectively.Results( 1 ) Real-time PCR results showed that XPG mRNA expression copy number in SKOV3/DDP tranfected with siRNA-XPG-733 fragment was 1.050 ± 0.023,which was significantly lower than that in SKOV3/DDP tranfected with other siRNA-XPG fragments(P ＜ 0.05,respectively),and was chosed to construct the siRNA-XPG vectors.The XPG mRNA expression was down-regulated in short hairpin RNA (shRNA)-XPG-733-SKOV3/DDP cell lines that confirmed by western blot.( 2 ) The growth curve showed that growth velocity of shRNA-XPG-733-SKOV3/DDP cell lines was lower than that of shRNA-GAPDH and shRNA-NC cell lines( P ＜ 0.05,respectively).The results of FCM also showed that 34.0％ of cells in shRNA-XPG-733-SKOV3/DDP cell lines were in S + G2/M phase,while only 58.7％ and 51.3％ in shRNA-GAPDH and shRNA-NC cell lines respectively ( P ＜ 0.05,respectively).( 3 ) The drug-resistance index of shRNA-XPG-733-SKOV3/DDP cell lines [ 50％ inhibiting concertration( IC50 )( 13.79 ± 0.06) μg/ml ] was lower than that in shRNA-GAPDH and shRNANC cell lines [ IC50( 27.84 ± 0.34 ) μg/ml and ( 28.32 ± 0.42 ) μg/ml,respectively ] statistically significant (P ＜ 0.05,respectively) ; but there was not statistically significant difference in intracellular drug concentration between shRNA-XPG-733-SKOV3/DDP cell lines [ (0.026 ± 0.005 ) μg/ml ] and shRNAGAPDH [ (0.024 ± 0.003 ) μg/ml ] and shRNA-NC cell lines [ ( 0.025 ± 0.007 ) μg/ml ] after treated by cisplatin in vitro ( P ＞ 0.05,respectively ).Conclusion The down-regulating of XPG gene resulted in slowing growth velocity and descending the drug-resistance index of shRNA-XPG-733-SKOV3/DDP cell lines,which may be related with descending in capability of DNA excision repair in cells.