A Vivo Study Of RCC2 Contributes To The Cisplatin-resistance In Epithelial Ovarian Cancer

ObjectiveOvarian cancer is insidious because of its occult lesion progression.The majority of patients with ovarian cancer are diagnosed in an advanced disease stage when they present with symptoms.And ovarian cancer is called "silent killer".At present,radical surgical operation followed by chemotherapy with a platinum-based agent plus paclitaxel is a well-established and the first-line option for the treatment of ovarian cancer.In the initial stage,most of the patients were sensitive to chemotherapeutic drugs,and the sensitivity rate reached 60%-80%.Although patients tend to respond initially to therapy,drug resistance inevitably emerges within 6 months after initial treatment in 25%patients.More than 70%of women afflicted with ovarian cancer will recur within the first 5 years.Hence,it is necessary to understand the mechanism of chemo-resistance and to explore novel methods to circumvent cisplatin-resistance.To identify potential factors and genes associated with drug resistance in ovarian cancer is a great significant for improving the clinical outcome of ovarian cancer patients.The research group identified RCC2 as the target gene through the analysis of bioinformatics in the pre-experiments.It was observed from the ovarian cancer tissues that expression of RCC2 was significantly increased in cisplatin resistant tissues relative to cisplatin sensitive tissues.RCC2 may serve as a prognostic indicator in ovarian cancer,the higher the RCC2 expression,the worse the prognosis of the patient.lt is confirmed that RCC2 overexpression significantly promoted cell proliferation,migration and invasion in vitro,which was through the RalA/RalBP1 signaling pathway.And it also induces the ovarian cancer cells cisplatin-resistance.Therefore,this study intends to reveal the contributes of RCC2 in the cisplatin-resistance of epithelial ovarian cancer through the animal model that overexpresses or knocks out the RCC2 gene.Methods1、The A2780/DDP and SK-OV-3/DDP with RCC2 gene knockdown were constructed by liposome-mediated transfection.And the A2780 and SK-OV-3 cells Over expressing RCC2 were constructed respectively by the lentiviral vectors.And the expression of RCC2 levels in the ovarian cancer cells were determined via western blot analysis.2、Divide the ovarian cancer cell suspension into RCC2 Over expression group,RCC2 knockout group,vector group,and negative control group.Tumorigenicity assays in nude mice were performed.Skin tumor xenografts in the nude mice were established by subcutaneous injection of different groups’ ovarian cancer cells with RCC2 Over expression or knockout.Groups were treated with cisplatin.The volume of the tumours were observed and the tumour growth was recorded weekly after the inoculation.Tumor growth was recorded by measuring the tumor’s perpendicular diameter using a caliper estimated by employing the following equation:volume=(tumor length)×(tumor width)2/2.At 4th week after the operation,the mice were sacrificed by rapidly dislocating their cervical spine.After formaldehyde-fixed,tissues were embedded in paraffin,sectioned,and stained for immunohistochemical analysis and research.Results1、The tumor volume of mice which inoculated with siRNA mediated RCC2 gene knockout of ovarian cancer cells or DDP-treated was significantly reduced compared with the corresponding control group.2、The tumor volume and weight of mice which inoculated with the A2780 and SK-OV-3 cells that over expressing RCC2 increased significantly.3、The inhibitory effect of tumor tissue growth is most obvious in the mice which was inoculated with drug-resistant ovarian cancer cells that knocked out RCC2 gene and combined with drug treatment.4、Mice inoculated with ovarian cancer sensitive cells which were overexpressed RCC2 had a significant decrease in tumor tissue growth inhibition after combined treatment with cisplatin.5、The results of immunohistochemistry scores showed that mice injected with RCC2 overexpressing ovarian cancer cells and without DDP treatment had the highest immunohistochemical scores in tumor tissues.6、The mice inoculated with drug-resistant ovarian cancer cells that knocked out RCC2 gene were combined with cisplatin treatment had the lowest immunohistochemical score of RalA in the tumor tissue.ConclusionWe successfully established a subcutaneously implanted tumor model of nude mice by transplanting ovarian cancer cells in the nude mice.The increased expression of RCC2 stimulated cell migration and inhibited apoptosis,while inhibiting RCC2 expression reduced tumor growth in an in vivo animal model.And our results imply that inhibition of RCC2 reverses cisplatin resistance in ovarian cancer.And the signals from RCC2 might promote tumor growth and metastasis through RalA/RalBP1 signaling pathway.It may provide a new possibility of target for the treatment of cisplatin resistance in ovarian cancer.Giving new hope to the patients which sufferd resistance to cisplatin of epithelial ovarian cancer.