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Effect of XPG down-regulation gene expression towards the proliferation of epithelial ovarian cancer cells and its chemosensitivity to platinum

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Author:
No author available
Journal Title:
Chinese Journal of Obstetrics and Gynecology
Issue:
4
DOI:
10.3760/cma.j.issn.0529-567x.2012.04.010
Key Word:
卵巢肿瘤;DNA结合蛋白质类;内切核酸酶类;核蛋白质类;转录因子;顺铂;抗药性,肿瘤;基因表达调控,肿瘤;Ovarian neoplasms;DNA-binding proteins;Endonucleases;Nuclear proteins;Transcription factors;Cisplatin;Drug resistance,neoplasm;Gene expression regulation,neoplastic

Abstract: Objective To 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.

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