Investigation Of The Role And Molecular Mechanism Of MiR-146b In Epithelial Ovarian Carcinoma

The incidence of ovarian cancer is the third in gynecological malignancies,but the case fatality rate is the first among women.Epithelial ovarian cancer is the most common pathologic types of all ovarian cancers.Although it has been developed rapidly in the clinical therpay strategies of surgery,chemotherapy,molecular targeting therapy and immunotherapy for EOC,the prognosis in patients with advanced EOC is very poor,and the 5-year survival rate falls below 25%.In 2018,about 14070 women are expected to die from ovarian cancer in United States.In China,according to the report made by Academician Jie Hao at the 14th National Annual meeting of radiation Oncology of the Chinese Medical Association in 2017,the morbidity and mortality rates of ovarian cancer patients were 3.06%and 2.24%,which were ranked 10~thh among women’s tumors.Therefore,ovarian cancer has become a serious disease that harms for women’s physical and mental health,and brings heavy economic burden to the country,society and family and great psychological pressure to the patients and their families.The high mortality rate for ovarian cancer is mainly due to the fact that most ovarian cancer patients are in advanced stages when they have clinical symptoms.However,at early stage,the cure rate of ovarian cancer patients can be reach 90%.Another reason of the high mortality rate of ovarian cancer is metastasis and recurrence.Unlike other cancers,ovarian cancer can spread by direct invasion to adjacent organs or by dissemination throughout ascites.Because of the natural diffusion of ovarian cancer,platinum-and taxane-based chemotherapy is necessary after surgical resection.Systemic chemotherapy is initially effective in 80%of patients,however,most of them will develop chemotherapeutic resistance and lead to tumor recurrence.More importantly,the molecular mechanisms of ovarian cancer tumorigenesis and metastasis are still not completely understood.Therefore,there is a pressing need to explore the molecular biology of ovarian cancer for early diagnosis and molecular targeted therapies.MicroRNAs(miRNAs)are approximately 22-nucleotide short noncoding RNAs that regulate gene expression by targeting on the 3’-untranslated regions(UTR),5’-UTR and open reading frame(ORF)of mRNAs,inducing direct mRNA degradation or translation inhibition.MiRNAs have wide-ranging roles in regulating diverse biological processes,including cell growth,differentiation,and apoptosis.In addition,the expression of miRNA has tissue specificity and space-time specificity.Recent studies have shown that miRNAs are dysregulated in most cancers.Moreover,miRNAs can function as oncogenes or tumor suppressors through regulating different targets.Previous studies demonstrated that miR-146b dysregulation was associated with many types of cancer,such as glioma cancer,thyroid cancer,breast cancer and so on.A microarray profiling analysis has revealed that miR-146b was dysregulated in human ovarian cancer.Our preliminary findings declared that miR-146b was decreased in EOC tissues,and its expression level has a negative correlation with the pathological staging.However,the functional roles of miR-146b in EOC have rarely been investigated.Based on the above findings,we further detected the expression of miR-146b in EOC tissues in this study.And then,we explored the function of miR-146b in ovarian cancer cells by miR-146b overexpression.Finally,we predicted and confirmed Fbxl10was a new target of miR-146b in EOC.The further investigation was performed to explore the molecular mechanism of Fbxl10 in EOC.Our findings may supply new potential biomarkers for EOC diagnosis,prognosis or therapy.Part Ⅰ The expression and biological function of miR-146b in EOCObjective:To clarify the expression of miR-146b in EOC and investigate the function of miR-146b in EOC.Methods:1.Quantitative real-time PCR(qPCR)was applied to examine the expression of miR-146b in EOC tissues and EOC cell lines.2.F-actin cytoskeleton staining,wound-healing assay and transwell assay were used to evaluate cell morphology,cell migration and invasion.3.Cell proliferation and chemosensitivity were evaluated by cell count assay,flow cytometry assay,BrdU assay respectively.4.Xenograft mouse models were used to confirm the function of miR-146b in vivo.Results:1.It was found that miR-146b was significantly downregulated in EOC tissues,and negatively correlated with the pathological staging.2.Enforced-expression of miR-146b significantly changed cell morphology,enhanced the intercellular connecting,and significantly inhibited cell migration and invasion.3.Overexpression of miR-146b enhanced cell proliferation and chemosensitivity of ovarian cancer cells in vitro.4.MiR-146b boosted tumor proliferation,sensitized tumors to treatment,but reduced tumor invasion in mouse models.Conclusions:These results suggest that miR-146b was gradually decreased during EOC progression and miR-146b might serve as a potential prognostic biomarker of EOC.Moreover,miR-146b has marked effects on ovarian cancer cells in vitro and vivo,indicating the important role of miR-146b in the progression of EOC.Part Ⅱ Mechanistic studies of miR-146b in regulating epithelial ovarian cancer Objective:To screen and identify the potential targets of miR-146b and further explore the molecular mechanisms of miR-146b in EOC.Methods:1.It was found that Fbxl10 was a potential target of miR-146b via predicting with Target Scan 4.1/5.1 combined with PicTar and miRanda analyses.Dual-luciferase reporter assay was used to validate the target gene of miR-146b in EOC.2.Cell migration,proliferation and chemosensitivity of ovarian cancer cells were evaluated following with Fbxl10 knockdown or overexpression.3.Western blot,Immunofluorescence staining(IFC)and Immunohistochemistry(IHC)were applied to detect MET markers.ChIP assay was used to validate the direct interaction of Fbxl10 and its downstream moleculars.Results:1.MiR-146b directly targeted Fbxl10 in ovarian cancer.2.Knockdown of Fbxl10 enhanced the intercellular connecting,inhibited cell migration,promoted cell proliferation and increased the chemosensitivity.However,the cells with Fbxl10 overexpression were dispersed,and the migration ability increased,cell proliferation was decreased.3.MiR-146b inhibited Fbxl10,increased H3K4me3,and enhanced the expression of VIM and ZO-1.ChIP assay confirmed that Fbxl10 directly acted on the promoters of VIM and ZO-1.Conclusions:In this study,it has been demonstrated that miR-146b downregulated the expression of Fbxl10,upregulated the expression of Cyclin D1,VIM and ZO-1,and further led to cell invasion suppression,but it also promoted cell proliferation and increased the chemosensitivity.Low miR-146b might contribute to EOC progression from primary stage to advanced stage.