| Background:Ovarian cancer is a common malignant tumor in gynaecology, among which the incidence of the epithelial ovarian carcinoma (epithelial ovarian cancer, EOC) is accounted for more than 80%. The late stage EOC patients usually received various combination treatment, among which the cytoreductive surgery combined with platinum plus paclitaxel chemotherapy has been considered a classic effective treatment. however, the platinum resistance is one of the main factors influencing the prognosis. Therefore, the thorough understanding of the platinum resistant mechanisms is the present clinical problem to be solved, that can effectively improve chemotherapy drug resistance and the curative effect of EOC.Possible mechanisms of platinum resistance include decreased uptake/increased efflux of chemotherapeutic drugs, increased metabolism/inactivation of platinum drugs, or increased repair of platinum-induced DNA damage.In human cells, the DNA damage caused by chemical drugs is mainly repaired through nucleotide excision repair way (nucleotide excision repair, NER), the excision repair cross complementary gene 1 (excision repair cross complementing, ERCC1) plays a key role in the repair. ERCC1 is a kind of highly conservative single-strand DNA endonuclease, it plays an important role in DNA damage repair and apoptosis, and its normal expression is an important molecular basis for the repair enzyme function. In recent years studies suggest that ERCC1 is closely related with platinum resistance. During the epithelial ovarian cancer research, some scholars make a Meta analysis by searching the reports about the association of the ERCC1 expression and ovarian cancer platinum chemotherapy reaction on Medline, Pubmed, Web of science and CNKI datas, that all suggest ERCC1 expression was significantly associated with platinum resistance. But at the same time, there are also studies on assessing the association of ERCC1 expression and the clinical symptoms, platinum reaction, progressive stage or lifetime relationship, suggested that there was no significant correlation between ERCC1 expression and the platinum resistance in the initial ovarian cancer patients, there was no significant correlation between ERCC1 expression and the prognosis by testing ERCC1 before treatment in advanced ovarian cancer patients. There are still controversial between ERCC1 with the platinum resistance in EOC or the prognosis, however, so far most of the research focuses on the clinical data analysis, the studies in vitro cell level experiments and mechanism research are rarely reported.The purpose of this study is to investigate the association of ERCC1 and platinum resistance in ovarian epithelial carcinoma, by annalyzing the relationship between the ERCC1 expression and clinicopathological parameters of epithelial ovarian cancer patients, in order to assess the value of ERCC1 in EOC. Detecting the ERCC1 mRNA and protein expression level in ovarian epithelial carcinoma cisplatin resistance cell line SK0V3/DDP and its parent cell line SKOV3 in order to verify the impact of DDP resistance on ERCC1 expression at a cellular level. Using RNA interference(RNA interference, RNAi) technology to build stable ERCC1 silence cell lines, observing the influence of ERCC1 gene silence on the ovarian epithelial cancer cells resisting cisplatin, the cell proliferation and apoptosis at cellular level mediated by lentivirus, exploring the role of ERCC1 in platinum resistance of ovarian epithelial carcinoma, that can provide the experimental and clinical data for taking ERCC1 as the sensitivity index and prognosis prediction index for platinum chemotherapy of ovarian epithelial carcinoma, and provide new targets for the treatment of ovarian epithelial carcinoma resistance.The part one:The expression of ERCC1 in patients with ovarian epithelial carcinoma, analysis of correlation with clinicopathological parameters, prognosis and platinum resistanceObjective:The purpose of the study is to explore the association between the expression of ERCC1 gene and clinical pathology character of EOC or platinum-resistance or survival, by determining the expression of ERCC1 gene in the tissue of EOC.Methods:1、Clinical data:2008 January to 2013 December in the selection of First Affiliated Hospital of Guangzhou medical university hospital, treatment of 92 cases of paraffin blocks of EOC patients as the research object, all the patients had no neoadjuvant chemotherapy, confirmed by operation and pathology, with complete clinical data and follow-up records. The age ranged from 21 to 79, according to the 2009 FIGO clinical staging,29 patients in stage Ⅰ, Ⅱ in 9 cases,35 cases of stage Ⅲ,19 cases of stage Ⅳ. Pathological types:52 cases of serous carcinoma,25 cases of mucinous carcinoma,8 cases of endometrial carcinoma,7 cases of clear cell carcinoma. Pathological classification:16 cases of well differentiated, moderately differentiated in 32 cases, 44 cases of low differentiated.2、Method:ERCC1 expression was determined using immunohistochemistry in 92 patients with EOC, to analyze the association between the expression of ERCC1 gene and clinical pathology character of EOC or platinum-resistance or survival. Chemotherapy efficacy evaluation: ① Chemotherapy sensitive:refers to within 6 months after the end of chemotherapy without recurrence, including normal serum CA125, pelvic examination and imaging examination no new lesions or the original residual tumors to shrink or disappear; ② Clinical resistance:refers to the chemotherapy in the course of disease progression or chemotherapy within 6 months of serum CA125 continued positive (increased or decreased, and then increased again), or imaging findings of new lesions.3、Follow up:the follow-up deadline for 2014 June, followed up for 5～62 months, the median follow-up time was 28 months. Ovarian cancer surgery to disease recurrence or death time is defined for disease progression free survival time (PFS) disease progression-free survival time, to the first appeared as the deadline. Operation date to death or last follow-up time was overallsurvival (overall survival, OS).4、Statistical methods:The association between ERCC1 expression in different clinical pathology character of EOC and platinum-resistance were analyzed by using person x2 test. The survival document was analyzed by Kaplan Meier survival curve and was tested by Log-Rank. The influencing factors for survival time was analyzed by Cox proportional hazards regression models, hazard ratio (HR) and 95%CI was calculated at the same time. P<0.05 said the difference was statistically significant.Results:1、The positive rate expression of ERCC 1 was 89.13% in 92 patients (82/92); that was higher in platinum-resistant patients than in platinum-responding patients, the difference was statistically significant(X2=6.787, P=0.009);There was no significant difference between age, pathology stage, histological type, cell differentiation and clinical stage in ERCC1 expression group.2、The median PFS was 48.0 months in 92 patients,18.0 months in ERCC 1 high expression patients, and loss in low expression group, there was nostatistically significant difference(X2=1.148, P=0.248); The median OS was 30.0 months in total, 25.0 months in ERCC 1 high expression patients and 48.0 months in low expression group; there was no statistically significant difference (X2=2.001, P=0.157)3、Pathologic differentiation and clinical stage is the risk factors affecting the prognosis of patients by COX multiple factors regression analysis (P=0.005; P=0.000); but the expression of ERCC1 proteinum is not (P=0.056)Conclusion:1、The ERCC1 is highly expressed in EOC, the positive expression rate of ERCC 1 was higher in platinum-resistant patients than in platinum-responding patients, that suggests ERCC 1 expression is associated with clinical sensitivity to platinum-based chemotherapy and ERCC1 may be a predictor for sensitivity in platinum-based chemotherapy of EOC.2、Pathologic differentiation and clinical staging are the risk factors affecting the prognosis of patients with EOC.The PFS and OS were both higher in Low ERCC 1 protein expression patients than ERCC 1 protein expression patients, but there were no statistically significant difference, the ERCC 1 protein expression is not the risk factors affecting the prognosis of patients with EOC, prompting that large sample stratification research is needed to verify ERCC1 as a predictor of prognosis in ovarian cancer.The part two:The expression of ERCC1 gene in the cisplatin sensitive/resistant human epithelial ovarian cancer cell lines SKOV3Objective:1、To determine the change of cell multiplication and apoptotic of SKOV3/DDP and SKOV3 cell in different concentration of cisplatin;2、The difference expression of ERCC1 mRNA and protein in SKOV3/DDP and SKOV3 cell were detected by real-time fluorescent quantitative PCR (RT-PCR) and Western blotting.Methods:1、CCK 8 method is used to detect cisplatin resistance of cells, and flow cytometry is used to detect the apoptosis of SKOV3 cells in different cisplatin concentration;2、The difference expression of ERCC1 mRNA and protein in SKOV3/DDP and SKOV3 cell were detected by real-time fluorescent quantitative PCR (RT-PCR) and Western blotting. (RT-PCR adopt 2-delta CT method is adopted to the relative quantitative analysis, accompanied with GAPDH internal genes for correction on sample amount)3、Statistical analysis was performed using SPSS 20.0 software, the results of measurement data represented by x±s， factorial design variance analysis of two independent samples t test or two factors and two sets of data, and compare the interaction effect, main effect, single effect and multiple comparison. Multiple sets of data by using single factor analysis of variance ((One-Way ANOVA)), and the test of homogeneity of variance, homogeneity of variance when using Welch test. Multiple comparisons using LSD method or Dunnett method’s. P<0.05 said the difference was statistically significant. Using GraphPad Prism 5 or SPSS 20.0 statistical graphics.Results:1、Cell viability test results show that SKOV3 cells vigor was significantly lower than that of SKOV3/DDP in different doses of DDP after 24 hours, the difference was statistically significant (F=301.321, P<0.001). There was significantly differences between different doses (F= 158.983, P<0.001). there was interaction between grouping and dose (F= 28.520, P<0.001). Separate effects:fixed dose were compared between groups showed that in addition to 0 Dose, group differences were statistically significant, SKOV3/DDP group were significantly higher than SKOV3 group (P<0.05); comparing different doses in fixed packet, the results show that the difference doses in SKOV3/DDP group was statistically significant (F= 33.850, P <0.001). (N=5)2、Testing the apoptosis of SKOV3 and SKOV3/DDP cell lines in DDP, the results show that the DDP resistance of SKOV3/DDP cells was significantly higher than that of SKOV3, the difference was statistically significant (P< 0.001, N=3). With or not with DDP, there was significant differences in groups (F=28.068,P<0.001). There was interaction between the cells and the group with or not with DDP (F= 27.907, P<0.001). Separate effects:compared the group with or not with DDP in fixed cells, the results show there was no significant differences in SKOV3/DDP groups, that suggests DDP has no effect on apoptosis in SKOV3/DDP groups. There was statistically significant difference in SKOV3 groups (P<0.001), that was significantly more in positive cisplatin group than in negative cisplatin group, suggesting that DDP promote apoptosis in SKOV3 cells. Compared different group regardless of DDP, the results show there was both statistically significant difference in different groups with negative cisplatin (F= 6.295, P= 0.036) or positive cisplatin group (F=85.774, P<0.001), suggesting that the apoptosis rates in SKOV3 cell was higher than in SKOV3/DDP,.regardless of the DDP.(N=3).3、The RT-PCR test results show that:compared with SKOV3/DDP, ERCC1 mRNA content in SKOV3 cell increased obviously, the difference was statistically significant t=-9.856, p= 0.001, N=3.4、Nestern blotting results show that:in SKOV3/DDP cells the expression of ERCC1 protein was obviously higher than that of SKOV3, the difference was statistically significant. t=-33.219, p<0.001, N=3.Conclusion:1. SKOV3/DDP cell lines have obvious resistance to DDP, the follow-up related to cell resistance experiment can be gone ahead.2.The expression of ERCC1 gene in SKOV3/DDP cells is increased significantly, that prompts ERCC1 may be one of the factors conducting cisplatin resistance.The part three:The Construction of ERCC1 gene defect cell linesObjective:Build stable ERCC1 silence cell lines and identify its jamming effectivenessMethods:1、 Directional cloning inserting Supersilencing shRNA plasmids expression vector pLVX-shRNA into the three artificial synthesis specific interference ERCC1 shRNA fragments,3 kinds of ERCC1 shRNA expression vector are obtained.2、The three plasmids were transfected to SKOV3/DDP cells by lentivirus vector, the best silent plasmid was obtained by real-time fluorescent quantitative PCR detecting the silence level of ERCC1, a stable cell lines can be chose, then the ERCC1 expression in stable transfection cell line was detected by real-time fluorescent quantitative PCR and western blotting.3、Statistical analysis was performed using SPSS 20.0 software, the results of measurement data represented by x±s, factorial design variance analysis of two independent samples t test or two factors and two sets of data, and compare the interaction effect, main effect, single effect and multiple comparison. Multiple sets of data by using single factor analysis of variance ((One-Way ANOVA)), and the test of homogeneity of variance, homogeneity of variance when using Welch test. Multiple comparisons using LSD method or Dunnett method’s. P<0.05 said the difference was statistically significant. Using GraphPad Prism 5 statistical graphics.Results:1、Three kinds expression vector of ERCC1 shRNA were successfully be constructed, the real-time fluorescent quantitative PCR results showed that the interference effect of pLVX-ERCC1 shRNAl to ERCC1 is better, the shRNA1 is the best fragments of the gene. F=40.354, p<0.001, N=3.2、Tthe shRNA1 recombinant plasmid was stablely transfected to SKOV3/DDP cells, the expression of protein level of ERCC1 was significantly decreased, the difference was statistically significant. t=4.536, P=0. 011, N=3.3、The stable transfection cell lines was successfully screened for subsequent experiments, and that was named as pLVX-ERCC1 shRNAl.Conclusion:ERCC1 shRNA expression vector was constructed successfully, the stable ERCC1 gene silence SKOV3/DDP cell line was obtained.The part four:the experiment research on reversing epithelial ovarian cancer cells cisplatin resistance with ERCC1 gene silencing mediated by lentivirusObjective:To study the effect of ERCCl gene silencing on the resistance of ovarian carcinoma cisplatin resistance cell line SKOV3/DDP and its possible mechanismMethods:1、PLVX-shRNA lentivirus transfected human ovarian carcinoma cisplatin resistance cell line SKOV3/DDP, a stable transfection cell lines was obtained, CCK 8 method was used to detect the effect on the cell resistance after transfection.2、The influence on apoptosis in control group and transfection group was tested by flow cytometry, before or after that was treated by DDP.3、The influence on cell cycle in control group and transfection group was tested by flow cytometry, before or after that was treated by DDP.4、Statistical analysis was performed using SPSS 20.0 software, the results of measurement data represented by x±s, factorial design variance analysis of two independent samples t test or two factors and two sets of data, and compare the interaction effect, main effect, single effect and multiple comparison. Multiple sets of data by using single factor analysis of variance ((One-Way ANOVA)), and the test of homogeneity of variance, homogeneity of variance when using Welch test. Multiple comparisons using LSD method or Dunnett method’s. P<0.05 said the difference was statistically significant. Using GraphPad Prism 5 or SPSS 20.0 statistical graphics.Results:1、The percentage of cell viability in SKOV3/DDP-shRNAC group and SKOV3/DDP-shRNAl group in different dose of DDP was ananlyzed by analysis of variance of factorial design. There were significantly difference in groups (F= 137.142, P<0.001). and in different dose groups (F=141.914, P<0.001). There was interaction between different groups and different dose groups (F= 10.017, P<0.001). Separate effects:Compared the different groups in fixed dose, that shows there were both statistically significant difference in groups beside 0-dose group, which was significantly higher in SKOV3/DDP-shRNAC group than in SKOV3/ DDP-shRNAl group (P<0.05). Compared the different dose in fixed groups, that shows there were both statistically significant difference in different dose of SKOV3/ DDP-shRNAC groups (F= 46.827, P<0.001) and SKOV3/DDP-shRNAl groups (F= 102.069, P<0.001). There were both statistically significant difference in groups except for 0 dose group and 3 dose group by multiple comparison (P<0.05). That suggest the cisplatin resistance is lower in SKOV3/DDP-shRNAl groups than in control groups,either in different dose or in the same dose of DDP, suggesting the specific silencing ERCC1 gene can increase the sensitivity of SKOV3/DDP cells to cisplatin. (N=6).2、The apoptosis rate in SKOV3/DDP-shRNAC group and SKOV3/DDP-shRNAl group with or not with DDP was detected by Annexin V-PI double staining.That shows there were statistically significant difference in different group (F= 25.950, P <0.001). There were statistically significant difference in groups with or not with DDP (F= 35.193, P<0.001). There was an interaction between the cells and in groups with or not with DDP (F= 35.193, P<0.001). Separate effects:Compared in groups with or not with DDP with fixed cells, that shows the difference in SKOV3/ DDP-shRNAC group was not statistically significant (P=0.758), there was statistically significant difference in SKOV3/DDP-shRNAl groups (P<0.001), that was significantly greater in positive cisplatin group than in negative cisplatin group, suggesting the DDP could promote apoptosis in SKOV3/DDP-shRNAl group but can not effect apoptosis in SKOV3/DDP-shRNAC group. Compared the different groups regardless of, DDP, that show there was no statistically significant difference in negative cisplatin group (F= 0.351, P=0.570), but there were statistically significant difference in positive cisplatin group (F= 60.792, P<0.001), suggesting DDP could promote apoptosis. (N= 3). All that suggests DDP could promote apoptosis of SKOV3/DDP cells after specific silencing ERCC1 gene.3> Cell cycle analysis showed that in different cell Gl group there was statistically significant differences in the rate of apoptosis(F= 161.940, P<0.001). There was statistically significant differences in groups with or not with DDP (F=129.940, P <0.001). There was an interaction between the cells and in groups with or not with DDP (F= 48.495, P<0.001). There was statistically significant differences in the rate of apoptosis in S period (F= 20.441, P=0.002). There was statistically significant differences in groups with or not with DDP (F= 186.402, P<0.001). There was an interaction between the cells and in groups with or not with DDP (F= 12.954, P <0.001).There was statistically significant differences in the rate of apoptosis in G2 period (F=335.035,P<001). There was statistically significant differences in groups with or not with DDP (F= 52.312, P<0.001). There was an interaction between the cells and in groups with or not with DDP (F=141.120, P<0.001). The cell cycle analysis showed the proportion of stable ERCC1 silence cells was descended in G1 and S phase, that was increased in G2 phase, suggesting stable ERCC1 silence cell in DDP may stagnate in G2 phase, ERCC1 silence can inhibit cell proliferation by preventing cell mitosis. (N=3).Conclusion:ERCC1 gene silencing can effectively reverse the resistance of SKOV3/DDP cells to cisplatin, increase the sensitivity of the ovarian epithelial carcinoma cisplatin resistant cells to cisplatin, promote cell apoptosis, make cell stagnating in G2 phase, prevent cell mitosis, that can provide new targets for the epigenetics treatment of resistance of ovarian epithelial carcinoma. |
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