| Objective: Epithelial ovarian cancer is one of gynecological malignancy with the highest fatality,the five-year survival rate was about 30 percent. The mostchallenging problems were that ovarian cancer resisted to cisplatin that was commonly used as clinical chemotherapeutic agent, which becomes the main cause of recurrence and chemotherapy failure. So it is significant for improving survival rates to increase the sensitivity of ovarian cancer to cisplatin based chemotherapy. Our previous studies have demonstrated that the upregulation of mesothelin gene was the most significant in ovarian malignant tissue and there was a significant difference in changes of proliferation, adhesion, migration and invasion of ovarian cancer cell by overexpressing or silencing mesothelin gene. Cheng WF et al [1] found that ovarian cancer patients with highly expressed mesothelin were more susceptible to chemotherapeutic tolerance and shorter survival than those with low expressed mesothelin. They suggested that the mesothelin correlated with chemotherapy resistance and poor prognosis. The goal of this study is to observe the differential expression levels of mesothelin in cisplatin-resistant ovarian carcinoma cell lines SKOV3/DDP,3AO/DDP and sensitive cell lines SKOV3, 3AO and the proliferation and sensitivity to cisplatin in SKOV3/DDP with stable transfection of mesothelin shRNA by using slow virus mediated RNA interferencing MSLN gene for discussing the relationship MSLN genes and ovarian cancer drug resistance initially.Methods:1 MTT were used to detect the half inhibitory concentration (IC50) of cisplatin in epithelial ovarian cancer cells SKOV3/DDP,3AO/DDP, SKOV3,3AO and then calculate the resistance index(RI ). RI = IC50 (resistant cells) / IC50 (sensitive cells).2 To observe the expression of mesothelin in ovarian carcinoma resistance cell lines SKOV3/DDP, 3AO/DDP and sensitive cell lines SKOV3, 3AO by fluorescent quantitative-PCR and Western blotting.3 RNAi lentivirus aim at MSLN(LV-MSLN-RNAi) and Negative control vector lentivirus(LV-MSLN-neg) were used to transfect SKOV3/DDP cells(SKOV3/DDP-MSLN-RNAi,SKOV3/DDP-MSLN-neg).The transfect efficiency was investigated by immunofluorescence microscope and calculated after 96 hours. SKOV3/DDP cells were conducted as blank control,SKOV3/DDP-MSLN-RNAi as test group and SKOV3/DDP- MSLN-neg as negative group.4 Total protein of the three kinds cells (SKOV3/DDP-MSLN-RNAi, SKOV3/DDP-MSLN-neg,SKOV3/DDP) were extracted and quantitated by Western blotting to evaluate MSLN gene scilencing effect.5 MTT and Plate clone formation test were used to measure the proliferation of the group cells.6 Application of MTT method to detect the changes of survival rate and the half inhibitory rate in cells of SKOV3/DDP-MSLN-RNAi, SKOV3/DDP- MSLN-neg and SKOV3/DDP under the action of different concentrations of cisplatin.7 Using flow cytometry to detect the apoptosis rate of SKOV3/DDP-MSLN- RNAi, SKOV3/DDP-MSLN-neg and SKOV3/DDP cells under the condition of 3mg/l of cisplatin for 48 hours. Analysize the sensitivity to cisplatin in mesothelin silenced ovarian cancer cells.8 Statistical Methods: Data were evaluated using SPSS 13.0 statistical software, each assay was performed at least three times. The measurement data were expressed as mean±standard deviation. For Homogeneity of variance test, single factor analysis of variance (One-Way ANOVA) was used for data analysis.α=0.05 is size of test. When P<0.05, the difference was statistically significant; when P>0.05, There was no statistically significant difference . Results:1 The resistance index of SKOV3/DDP and 3AO/DDP is 4.52 and 4.22 respectively which meet subsequent test requirements.2 The expressions of mesothelin in ovarian carcinoma cisplatin-resistant cell lines were higher than that in sensitive cell lines :2.1 Fluorescent quantitative PCR: Mesothelin mRNA expressions could be detected in SKOV3,SKOV3/DDP,3AO and 3AO/DDP,there was a significant difference in four groups of mRNA expressions (F = 26.4, P<0.01). Mesothelin mRNA expressions levels of SKOV3/DDP and 3AO/DDP cells increased significantly, compared with SKOV3 and 3AO (P<0.01); mRNA expression levels of SKOV3/DDP cells were significantly higher than 3AO/DDP (P<0.01). mRNA expression levels of SKOV3 were significantly higher than 3AO (P<0.05).2.2 Western blotting results showed that: The relative content of MSLN protein in ovarian cancer cell lines of SKOV3, SKOV3/DDP, 3AO and 3AO/DDP was (0.3士0.13),(0.9士0.14),(0.1士0.08),(0.7士0.12) respectively. MSLN protein in SKOV3/DDP and 3AO/DDP was higher than that in SKOV3 and 3AO. The difference was statistically significant (P<0.05). MSLN protein in SKOV3/DDP was significantly higher than that in 3AO/DDP(P<0.05). MSLN protein in SKOV3 was significantly higher than that in 3AO. (P<0.05).3 The transfect efficiency was about 80-90% and was up to standard.4 The expression of MSLN protein in RNAi transfectant group was less than that in the other two groups, which was obviously down-regulated (P<0.05) and the interference efficiency reachs to 95.7%. There were no significant differences in the expression of MSLN protein between SKOV3/DDP -MSLN-neg and SKOV3/DDP (P>0.05).5 Cell proliferation tested by MTT: After 24, 48 and 72 hours of culture respectively, cells grow slowlier in SKOV3/DDP-MSLN-RNAi compare to other two groups (P<0.05).There were no significant differences between SKOV3/DDP-MSLN-neg and SKOV3/DDP (P>0.05). 6 Experimental results showed that colony formation efficiency: Colony formation efficiency in experimental group was (24.6士0.65)%, negative control group was (45.01士1.9)% and blank control group was (45.7士1.52)%.The difference was statistically significant (P<0.01).Colony forming efficiency in the experimental group was significantly lower than the other two groups (P<0.01). there was no significant difference in cloning efficiency between negative control group and blank control group (P>0.05).7 MTT was used to determined the changes in chemosensitivity of SKOV3/DDP cells to anti-tumor drug cisplatin: Along with the concentration of cisplatin increasing, the inhibition rate of ovarian cancer resistant cell with mesothelin gene silencing appeared significantly higher.There were significant differences (P<0.05). IC50 of the experimental group, negative control group and blank control group was (2.7士0.16)ug/ml, (5.3士0.42)ug/ml and (5.4士0.26)ug/ml respectively. The cell inhibition rates in the experimental group was significantly difference with the other two groups (P<0.01). Negative control group and blank control group had no significant difference in cell inhibition rates (P>0.05).8 Flow cytometry was used to detect apoptosis rate:8.1 The apoptosis rate of the experimental group, negative control group and blank control group was (16.4±2.54)%, (1.9±0.9)% and (2.4±0.82)% respectively. the difference was statistically significant(F=78.4,P<0.01). the apoptosis rate of ovarian cancer resistant cells after transfection higner than the other two groups(P<0.01).8.2 The apoptosis rate in experimental group, negative control group and blank control group cells under the action of 3mg/l concentrations of cisplatin continued 48 hours was (39.9士5.46)%,(18.7士7.06)% and (19.7士5.42)% respectively. the difference was statistically significant (F=11.8,P=0.008). The apoptosis rate in the experimental group was significantly difference with the other two groups(P<0.01). There was no significant difference between negative control group and blank control group in apoptosis rate (P=0.85>0.05) Conclusion:1 Mesothelin expression levels of cisplatin-resistant human ovarian cancer cells were significantly higher than sensitive cells,suggesting that overexpression of MSLN was definitely related to increased cisplatin resistance in ovarian cancer cells.2 RNA interferencing MSLN gene in SKOV3/DDP of ovarian cancer resistant cells can decrease the capacity of them growth and proliferation.3 RNA interferencing MSLN gene in SKOV3/DDP of ovarian cancer resistant cells can elevate the chemosensitivity to cisplatin and increase apoptosis rate.Prompted of mesothelin protein down-regulated may be reverse the drug resistance of ovarian cancer cells to cisplatin.
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