| ObjectiveCisplatin-based chemotherapy is one of the main modalities in the treatment of ovarian malignancies. The mechanism is proved to rely on the generation of DNA damage and the subsequent induction of apoptosis. However, although the tumor cells showed a relatively optimistic response to chemotherapeutic regimens, drug resistance might probably be observed within the progress of malignancies. Cell division cycle 25A (cell division cycle 25A, CDC25A), a dual-specific protein phosphatase, was regarded to regulate cell cycle and along in this way play a role in apoptosis, whereas the function in the DNA damage response and repair pathway in ovarian cancer has been not yet reported. This study aims to investigate the expression of CDC25 A both COC1 and C0C1/DDP cell lines, as well as to demonstrate the correlation of the expression of CDC25A to the sensitivity to chemotherapy between the parental and the daughter strains. Hence, shRNA interference was occupied to knock down the expression of CDC25A for the elucidation of its role in cispiatin resistance. In addition, the potential mechanism in CDC25A-induced cispiatin resistance was investigated and discussed.MethodPart 1.:The survival fractions of ovarian carcinoma cell line CoCl and its induced cisplatin-resistant strain CoC1/DDP cells in the presence of various concentrations of cisplatin were occupied by CCK8 method respectively. The expression of CDC25A, Ku70, BRCA1 and Caspase-3 in COC1/DDP and COC1 cells was detected by real-time fluorescence quantitative PCR. The protein expression of CDC25A, Ku70, BRCA1 and Caspase-3 in COC1/DDP and COC1 cells were detected by Western Blotting assay.Part 2.:The pGPU6/GFP/Neo-CDC25A shRNA expression vector was transfected into COC1/DDP cell line (shCDC25A group) and pGPU6/GFP/Neo plasmid into COC1/DDP cell line (shVector group). Western blotting was used to detect the expression of CDC25A in two cell lines to confirm the transfection efficiency. CCK8 method was carried out to investigate the proliferation ability of the cells to see whether the cell has resistance to reversed cisplatin. Cell apoptosis was analyzed by flow cytometry.Part 3:The expression of Ku70, BRCA1, Rad51 and Caspase-3 were detected by Western Blotting after the transfection of pGPU6/GFP/Neo-CDC25A shRNA expression vector and pGPU6/GFP/Neo plasmid into COC1/DDP cell lines. Cell proliferation, cell cycle and apoptosis were analyzed by flow cytometry.ResultIn the first part of this study:CCK8 cytotoxicity experiment results found that the IC50 of COC1/DDP in cisplatin was (89.8 ± 2.91) μmol/L and the IC50 of COC1 in cisplatin was (53.4 ± 3.0) μmol/L. Compared with COC1/DDP cells, the cell survival rate of COC1 cells decreased obviously after cisplatin incubation, P<0.05. RT-PCR showed that the relative expression of CDC25A mRNA in COC1/DDP cells was (4.07 ± 0.05) folds compared with that in COC1 cells, the relative expression KU70 was (3.35 ± 0.07) folds compared with that in COC1 cells, and the relative expression of BRCA1 mRNA was (3.39 ± 0.08) folds of that of COC1 cells, and the relative expression of Caspase3 mRNA was (0.54 ± 0.01) folds of that of COC1 cells (P<0.01). Compared with COC1 cells, western blotting demonstrated that the protein expression of CDC25A in COC1/DDP increased 1.42-fold, the protein expression of Ku70 increased 1.45-fold, the protein expression of BRCA1 increased 1.38-fold and the protein expression of Caspase3 increased 0.56-fold. Statistical significant (P <0.01) was observed.In the second part of this study:Using CDC25A shRNA and shVector recombinant plasmid transfered to COC1/DDP cells, respectively. Then western blotting method detected CDC25A level. The results showed that the CDC25A protein levels in shCDC25A group decreased 81.5% than shVector group, P< 0.001, the difference was statistically significant. The IC50 of cisplatin in shCDC25A group was (55.26+1.99) mol/L, while the IC50 in empty vector transfection group which was (87.7+2.26) mol/L, P< 0.05, the difference was statistically significant.In the third part of this study:After transfection plasmid, WB method respectively to detect the amount of protein expression of CDC25A, Ku70, BRCA1, RAD1 in shCDC25A group and shVetor group respectively. If the amount of protein expression in shVetor group was 100%, the amount of each protein expression in shCDC25A group was different:CDC25A descent (81.5+7.80)%, Ku70 descent (60.5+3.80)%, BRCA1 descent (63.7+4.9)%, RAD1 descent (61.6+4.20)%, differences were statistically significant (P< 0.01).Transfection CDC25A shRNA cell increment rate was lower than that in group transfection shVector ovarian cancer cells. Cell apoptosis rate of transfection CDC25A shRNA was obviously higher than that of transfection shVector ovarian cancer cells, P< 0.05, the difference was statistically significant. After shRNA transfected into COC1/DDP cell, cell cycle distribution is: G1 phase (66.7+2.4)%, S phase (20.2+1.63)%, G2/M phase (13.1+1.5)%. After sh Vector transfected into COC1/DDP cell, cell cycle distribution is:G1 phase (54.27+1.3)%, S phase (29.16+1.98)%, and G2/M phase (16.57+2.3)%, (P< 0.01).ConclusionThe expression of CDC25A mRNA and protein in COC1/DDP cells were significantly higher than that in COC1 cells; the expression of DSB repair pathway protein Ku70 and BRCA1 in COCl/DDP cells increased significantly than that in COCl cells; the expression of Caspase-3 in COC1/DDP cells was significantly down-regulated than that in COC1 cells. The expression of CDC25A was positively correlated to cisplatin resistance in ovarian cancer. DNA repair protein was positively associated with cisplatin resistance in ovarian cancer, and apoptosis was negatively correlated to cisplatin resistance in ovarian cancer.CDC25A gene silencing could effectively reverse the resistance of COCl/DDP cells to cisplatin.CDC25A gene silencing could down-regulate the expression of DNA damage repair protein. As a consequence, CDC25A gene silencing could compromised the DNA damage repair of ovarian cancer cells. In addition, it could promote cell apoptosis, block cell cycle progression, inhibit proliferation and reduce cell migration. CDC25A gene silencing could hence be remarkable as a potential target in drug discovery for the resistance of cisplatin in ovarian cancer. CDC25 A is considered to play a key role in platinum resistance of ovarian cancer by promoting DNA damage repair, inhibiting apoptosis and promoting cell proliferation. |
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