The Role And Mechanism Of MIR-378a-3p In Development And Cisplatin Resistance In Ovarian Cancer

Ovarian cancer is one of the most common gynaecologic malignancies in women worldwide,with about 230,000 new cases and almost 140,000 death every year.Ovarian cancer usually occurs in granulosa cells or germ cells,and over 90% of ovarian cancer has epithelial tissue.It is believed to be produced by cells that cover the surface of the ovary or the lower surface of the somatoyst.Inspite of progress in early diagnosis and cytotoxicity as well as targeted drug treatment,only 19% of patients with ovarian cancer are diagnosed at its early stage.Only 30% of patients with cancer diagnosed to be at an advanced stage survive nearly 5 years after initial treatment,while other 70% patients have unfavourable prognosis and a high mortality rate.miRNAs are small,endogenously expressed,single-stranded,non-coding RNAs of almost 19-25 nucleotides in length cleaved from 70 to 100 nucleotide hairpin pre-miRNA precursors.Recently,many research groups have identified and studied altered expression of miRNAs in ovarian tumorigenesis leading to ovarian cancer.The expression profile of miR-378a-3p in ovarian cancer shows that its expression level is closely related to prognosis of disease.It also shows significant association with the survival of patients.According to previous study,we know that miR-378a-3p can enhance tamoxifen's sensitivity to breast cancer cells by targeting GOLT1 A.Moreover,miR-378a-3p is a prognostic factor in colorectal cancer.In treatment of ovarian cancer,miR-378 is considered as a potential biomarker.Nevertheless,the specific biological role and underlying molecular mecha-nism of miR-378a-3p is unclear in the tumor progression and chemosensitivity of ovarian cancer.We still do not know the mechanism of miR-378a-3p in ovarian cancer(OC).The detailed regulation mechanism of miR-378a-3p in OC remains unknown.Therefore,it is necessary to explore its role in epithelial OC and find out new target genes and regulatory mechanisms to provide a potential molecular target for the gene therapy of OC.So,we have designed epidemiological and cytological experiments to explore the role and expression of miR-378a-3p in ovarian cancer,to investigate the mechanism of chemotherapy resistance mechanism and explore the possibility of effectively influencing miRNA for it to become a future clinical treatment.The whole research includes the following three parts:Part 1: Clinical data analysis for 62 patients with ovarian cancer.Part 2: Research on the relationship between the expression of miR-378a-3p in ovarian cancer and ovarian cancer cells and sensitivity to cisplatin of drugresistant cells.Part 3: miR-378a-3p sensitizes ovarian cancer cells to cisplatin through targeting MAPK1/GRB2.Part one Clinical data analysis for 62 patients with ovarian cancerObjective: Epithelial ovarian cancer(EOC)is the leading cause of death in women among all gynecological malignancies,accounting for about 5% of all cancers and 4.2% of all cancer deaths in women worldwide.In recent years,although we have made a progress in cytotoxicity study and treatment,the five year survival rate of ovarian cancer has not been significantly improved.There are many reason for high mortality of ovarian cancer,including lack of obvious characteristic symptoms of ovarian cancer at an early stage and delayed diagnosis,etc.Increased drug resistance to cancer further limits the development of therapies.To study the influence factors to the prognosis of patients with ovarian cancer,we have collected and analyzed clinical data of patients with ovarian cancer in the Fourth Affiliated Hospital of Hebei Medical University,and selected the patients who met the inclusion criteria of this study for follow-up,to obtain the relevant data and analyze the influencing factors of survival time.Methods: Patients with ovarian cancer were selected from this hospital from 2010 to 2012,who accepted pathologic diagnosis.Respondents accepted complete treatment,and complete follow-up data and medical records were kept.We selected 62 respondents according inclusion and exclusion standard,and conducted case review and survival analysis with these data.We adopted the Pearson chi-square,Kaplan Meier survival curve,log rank test and Cox regression to analyze factors that probably affect the prognosis of epithelial ovarian cancer.Results:1.The median survival time of patients whose tumor was bigger than 5 cm and smaller than 5 cm was 35 months and 51 months respectively,showing statistical significance(P = 0.000).2.The median survival time of patients with lymph node metastasis and without lymph node metastasis was 29 months and 47 months respectively,showing statistical significance(P = 0.000).3.The median survival time of patients at clinical stage ?-?,III and IV were 47 months,at stage43 months,and 28 months respectively,showing statistical significance(P = 0.022).4.The median survival time of patients with high/intermediate differentiation and poor differentiation of tumor tissue was 44 months and 31 months respectively,showing statistical significance(P = 0.026).5.The median survival time of patients whose residual tumor diameter was 0 cm,smaller than 1 cm,and bigger than 1 cm was 58 months,40 months,and 32 months respectively,showing statistical significance(P = 0.000).6.The analysis result with the multiple-factor COX regression model showed that the tumor size,lymph node metastasis,pathological stage,pathological differentiation,residual tumor diameter were factors influencing the five year survival rate of patients with ovarian cancer.Conclusions:1.The tumor size,lymph node metastasis,clinical stage,pathological differentiation,and residual tumor diameter are risk factors influencing ovarian cancer prognosis.2.The dangerous degree in descending order for poor prognosis of ovarian cancer are lymph node metastasis,tumor size,residual tumor diameter,pathological differentiation and clinical stage.Part two Research on the relationship between the expression of miR-378a-3p of in ovarian cancer and ovarian cancer cells andsensitivity to cisplatin resistance in ovarian cancer patientsofdrug-resistant cellsObjective: mi-RNA modulates a wide range of gene expression patterns with the process of development and organization steady-state,and performs a variety of functions in the body disease condition.In ovarian cancer,mi-RNA involves in various cellular functions,like cancerization,cell cycle,cell apoptosis,proliferation and development of the chemical resistance of invasion and metastasis.We aim to investigate the biological effects of miR-378a-3p on proliferation and cisplatin sensitivity of OC cells.At first,the relative levels of miR-378a-3p were determined in OC tissues and cell lines.Kaplan Meier analysis was applied to analyze the relevance between miR-378a-3p expression and overall survival rate of OC patients.Subsequ-ently,gain-of-function assays were designed and conducted to evaluate the influence of miR-378a-3p on proliferation,apoptosis and chemosensitivity of OC cell lines.Results:1.Compared with para-carcinoma tissues,the expression of miR-378a-3p was sharply decreased in OC tissues,with significant difference(P<0.01).2.According to the result of Kaplan Meier analysis,low expression of miR-378a-3p was closely associated with unfavorable prognosis of patients with ovarian cancer.3.Compared with the normal ovarian epithelial cell(IOSE80),the expression of miR-378a-3p was sharply decreased in two OC cells(OVCAR3 and SKOV3),with statistical difference(P<0.01).4.MTT and Colony formation assays indicated that miR-378a-3p suppressed cell proliferation,and miR-378a-3p enhanced cisplatin sensitivity of ovarian cancer cells.5.Flow cytometry analysis indicated that overexpression of miR-378a-3p promoted cell apoptosis(OVCAR3 and SKOV3).When ovarian cancer cells were treated with cisplatin,the effect of miR-378a-3p on apoptosis was more significant.Conclusions:1.Compared with normal ovarian tissues,the expression of miR-378a-3p was decreased in epithelial ovarian cancer tissues and cancer cells.2.miR-378a-3p was closely associated with unfavorable prognosis of patients with ovarian cancer.3.miR-378a-3p could suppress cell growth and proliferation,promote cell apoptosis,and enhance cisplatin sensitivity of ovarian cancer cells.Part three miR-378a-3p sensitizes ovarian cancer cells to cisplatinthrough targeting MAPK1/GRB2Objective: Surgical excision and chemotherapy are two main treatment methods for ovarian cancer.Cisplatin-based chemotherapy is commonly used for ovarian cancer patients.However,with the increasing concentration of chemotherapeutic agents,cisplatin-resistance occurs progressively.So far,cisplatin resistance has become an obstacle to the treatment of ovarian cancer.Therefore,it is necessary to explore the specific molecular mechanism of cisplatin resistance.Results:1.Seven potential target mRNAs of miR-378a-3p,i.e.,PAPOLA,MAPK1,KPNA6,GRB2,DYRK1 A,ANKRD52,and HSPA12 A,were preliminarily found out by getting intersection elements from the target gene predicted through 4 different bioinformatics methods,namely,targetScanSites,picTarSites,PITASites and miRandaSites.2.It was discovered that miR-378a-3p mimics efficiently only inhibited the luciferase activities of MAPK1 3'-UTR and GRB2 3'-UTR but that of the other five mRNAs was not affected through dual-luciferase reporter gene report validation result carried out in HEK-293 T cells.3.The real-time fluorescence quantification result showed that,compared with para-carcinoma tissues,the expression levels of MAPK1 and GRB2 were significantly increased in OC tissues,with significant difference(P<0.01),and Spearman's analysis showed the negative expression correlation between miR-378a-3p and MAPK1 or GRB2 in OC tissues.4.There was a negative correlation between the MAPK1 expression or GRB2 expression and the overall survival of OC patients.5.The cell proliferation was significantly suppressed and the cell apoptosis was efficiently accelerated by the knockdown of MAPK1 or GRB2.The more efficient effects of sh-MAPK1 or sh-GRB2 on the proliferation or apoptosis were,the more efficiently cells were treated with cisplatin.6.MAPK1 and GRB2 reversed the effects of miR-378a-3p on chemosensitivity of OC cells,including cell proliferation and apoptosis.Conclusion:1.Mechanism investigations suggested that MAPK1 and GRB2 were two target genes of miR-378a-3p.2.MAPK1 and GRB2 are two oncogenes in OC,which influenced cell proliferation,apoptosis and chemosensitivity to cisplatin.3.Rescue assays revealed that MAPK1 and GRB2 could reverse the effects of miR-378a-3p on chemosensitivity of ovarian cancer cells.