Research On Association Of Folate Binding-protein With Multi-drug Resistance In Ovarian Cancer

Chapter1Research advance on multi-drug resistance in ovarian cancerMalignant ovarian tumors are common in the female reproductive system,and second only to cervical cancer and endometrial cancer among the three gynecologic malignancies of highest incidence.Ovarian cancer.which consists of about80%of malignant ovarian tumors,is now mainly treated by surgery and platinum-based combination chemotherapy. However.most patients are diagnosed late and the chances of surgery for some of them are very little. While70-80%of all patients respond positively to chemotherapy at first,eventually drug resistance will emerge in at least80%of them who have been given the regimen.which leads to the overall5-year survival lower to be20-30%.Especially the efficacy significantly decreases as the result of multi-drug resistance(MDR),and the relative complex mechanisms remain unclear as now.So at this stage it is urgent to prevent and reduce the incidence of MDR as well as reverse it. It has been reported by foreign scholars that folate binding-protein is closely associated with the development of ovarian cancer and also plays an important role in MDR,but the specific mechanisms involved remain to be explored. Chapter2Detection of folate binding protein expression and its clinical significance in ovarian carcinomasObjective:To explore the expression level of folate binding protein (FOLR-1) and its clinical significance in ovarian carcinomas.Methods:Western blot analysis was used to detect the expression levels of FOLR-1in80ovarian carcinomas,50benign ovarian tumors and30normal ovarian tissues, and its association with clinical pathology and multidrug resistance of ovarian carcinomas was analyzed.Results:(1) The expression levels of FOLR-1increased orderly in normal ovarian tissues.benign ovarian tumors and ovarian carcinomas,and the difference was statistically significant (P<0.01).(2) For ovarian carcinomas,the expression levels of FOLR-1in clinical stage Ⅰ-Ⅱ were higher than that of stage Ⅲ-Ⅳ, and the levels of well-differentiated were lower than that of poorly differentiated.The differences were both statistically significant (P <0.05).(3) For ovarian carcinomas, the FOLR-1levels of the platinum-resistant were less than the platinum-sensitive,and the difference was statistically significant (P<0.01). After chemotherapy the FOLR-1levels of progressive ovarian carcinomas were lower than those in remission,and the difference was statistically significant (P<0.01).(4) For ovarian carcinomas,the FOLR-1levels of the mucinous type were lower than that of the serous,and the difference was statistically significant (P<0.05); The FOLR-1levels of ovarian carcinomas with metastasis to lymph nodes and distant organs were higher than those without metastasis, and the difference was statistically significant (P<0.05);However,the FOLR-1levels had no significant association with pathological classification, amounts of ascites and metastasis to the omentum (P>0.05).(5) ROC curve determining the FOLR-1levels associated nature of ovarian tumors demonstrated that the maximum Youden index was3.115.and the FOLR-1levels showed no obvious correlation with median survival time (P>0.05). COX multivariate analysis denied the FOLR-alevel as an independent prognostic factor.Conclusions:The FOLR-1levels of normal ovarian tissues and benign ovarian tumors were lower than that of ovarian carcinomas,which suggested FOLR-1could be a new biomarker for early diagnosis of ovarian carcinomas. Meanwhile,the FOLR-1levels in ovarian carcinomas were low for the platinum-resistant but high for the platinum-sensitive,which suggested that FOLR-1associated with multidrug resistance of ovarian carcinomas and could be a potential target for judging multidrug resistance. Chapter3Construction and Identification of Lentiviral vector carrying FOLR1GeneObjective:To construct a lentiviral vector expressing the gene of folate-binding protein (FOLR1) and identify its expression.Methods:Full-length of FORL1gene was amplified by PCR,then cloned into the plasmid pWPI.and further confirmed by PCR and sequencing.After the recombinant pWPI and its helper plasmid co-transfected the virus packaging293T cells, SKOV3cells were infected with the virus particles and sorted by flow cytometry. Finally protein expression of FOLR1gene was detected by RT-PCR and Western Blotting.Results:FOLR1gene was successfully cloned into the plasmid pWPl.The recombinant expression vector was successfully constructed,and lentiviruses were successfully packaged by the293T cells. SKOV3cells were effectively infected with the lentiviruses carrying pWPI-FOLR1gene. There’s stable expression of FORL1in the infected SKOV3cells.Conclusions:Lentiviral vector carrying FOLR1gene was constructed and could efficiently infect SKOV3cells,and stable expression of FOLR1gene laid foundation for exploring its function. Chapter4Biological effect of the FORL1gene on epithelial ovarian cells acted by cisplatin in experimental study in vitroObjective:To explore the biological effect of upregulated expression of the FORL gene on SKOV3cells and the functional patways.Methods:MTT assay was used to measure the growth rate,the IC50values of cells with cisplatin and inhibition by different cisplatin concentrations at different times in the three groups of SKOV3cells,pWPI-SKOV3cells and pWPI-FOLR1-SKOV3cells.Meanwhile,flow cytometry was used to detect apoptosis and cell cycle distribution of the three groups acted by different cisplatin concentrations at different times.High Performance Liquid Chromatography (HPLC) was used to measure the cisplatin concentration in the three groups of cells acted by same concentration of cisplatin after48hours.Results:Compared with the other two control groups,the experimental group of pWPI-FOLR1-SKOV3cells with cisplatin showed significantly increased growth rate as well as decreased IC50values.Meanwhile growth inhibition of the experimental group increased and was dose-time dependent.Cisplatin-induced apoptosis as the result of cell cycle arrest in S phase also increased in the time-dose dependent manner.The cisplatin concentration in cells was twice as much as before.Conclusion:The FOLR1gene could enhance the growth ability of SKOV3cells,and its upregulated expression led to the increase of inhibition after cisplatin in a dose-time dependent manner.Meanwhile cisplatin-induced apoptosis through cell cycle arrest in S phase also increased significantly in the manner of time-dose dependence,and the cisplatin concentration in cells increased.