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  • Title: [Effect of XPG down-regulation gene expression towards the proliferation of epithelial ovarian cancer cells and its chemosensitivity to platinum].
    Author: Zhang W, Wu FX, Wang Q, Li L.
    Journal: Zhonghua Fu Chan Ke Za Zhi; 2012 Apr; 47(4):286-91. PubMed ID: 22781116.
    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 + 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). 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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