[Effect of XPG down-regulation gene expression towards the proliferation of epithelial ovarian cancer cells and its chemosensitivity to platinum].
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 + G(2)/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 (IC(50)): (13.79 ± 0.06) µg/ml] was lower than that in shRNA-GAPDH and shRNA-NC cell lines [IC(50): (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 shRNA-GAPDH [(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).


CONCLUSIONS
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.
Department of Gynecology Oncology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China.
Wei
W
Zhang
Fei-Xiang
FX
Wu
Qi
Q
Wang
Li
L
Li
D000077216:Carcinoma, Ovarian Epithelial; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002945:Cisplatin / Q000494:pharmacology*; D004260:DNA Repair / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D009375:Neoplasms, Glandular and Epithelial / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000099250:DNA Excision Repair Protein ERCC-5
D002945:Cisplatin; D004268:DNA-Binding Proteins; D004720:Endonucleases; D009687:Nuclear Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5