The Roles And Mechanism Of MiR-200a In Prostate Cancer Progression And Metastasis

Prostate cancer is a common malignancy in urology.As the population ages,the incidence and mortality of prostate cancer increase year by year.Studies have shown that early androgen deprivation therapy is sensitive to 80% of patients,and most of them are androgen-dependent prostate cancer(ADPC),but eventually develop into castration-resistant prostate cancer.Cancer,CRPC),prostate cancer at this stage is not sensitive to various treatments and is the main cause of death in advanced patients.There are many studies on the development and metastasis mechanism of prostate cancer.In recent years,more and more research has been conducted on the relationship between micro RNAs(miRNAs)and tumors.miRNAs are single-stranded non-coding small RNAs containing 19-25 nucleotides that inhibit or promote target gene expression at the post-transcriptional level by binding to the 3' non-coding region(3'UTR)of the m RNA encoding the gene.A large number of studies have shown that miRNA and cell proliferation and apoptosis are inextricably linked.we found that the expression of miR-200 a in ADPC and CRPC tissues was different,and miR-200 a in CRPC tissues was significantly lower than ADPC tissue.We first used real-time fluorescence quantification.The PCR technique verifies the expression level of miR-200 a in prostate cancer tissues,and explores the biological function of miR-200 a in prostate cancer through an in vitro functional assay of miR-200 a dysregulation.Using the method of combinating bioinformatics and molecular biology,the molecular mechanism of promoting prostate cancer cell apoptosis by miR-200 a.Our fndings indicate that miR-200 a may be a potential and important tumor suppressor gene.Part ?Study of clinical relevance: the differential expression of miR-200 a between androgen-dependent prostate cancer and castration-resistant prostate cancerObjective: Prostate cancer is a complex process involving many different genes.In order to search for miRNAs that may affect the development of prostate cancer,the miRNAs microarray analysis was used to examine and analyze the differentially expressed miRNAs in ADPC patients and CRPC patients.Methods:Early ADPC and advanced CRPC were collected from fresh samples according to the instruction of observing of frozen section and preserved in liquid nitrogen.The data were firstly re-analyzed to compare miR-200 a expression between metastatic prostate cancer and non-metastatic prostate cancer obtained from Memorial Sloan Kettering Cancer Center(MSKCC)prostate cancer database.Then we analyzed the role of miR-200 a in diagnosis of metastasis prostate cancer and the relationship between miR-200 a expression and biochemical recurrence.Results: Both three cases from ADPC and CRPC were detected by micro RNAs chips.Differential expression of miR-200 a in early ADPC and advanced CRPC was detected by q RT-PCR(P<0.05).Gene chip data about the expression of miRNAs were obtained from Memorial Sloan Kettering Cancer Center(MSKCC)prostate cancer database.Kaplan Meier analysis with the log-rank test revealed,after radical prostatectomy,that the biochemical relapse-free survival in the patients with low level of miR-200 a seems to be longer than that in the patients with high level of miR-200a(P > 0.05).Cox regression analysis to confrm the variables of potential prognostic signifcance and the results suggested that the miR-200 a was an independent prognostic factors for biochemical relapse-free survival in patients with PCa.Conclusion: miR-200 a expression in CRPC was much lower than in ADPC,which indicated that miR-200 a decreased along with the progress of prostate cancer,and it may be a potential and important tumor suppressor gene.Part ?Functional analyses of miR-200 a in prostate cancer and study of molecular mechanismObjective:Previous studies have reported that miR-200 a was probably correlated to the development and progression of prostate cancer,and participated the transformation of prostate cancer cells from androgen dependent to castration resistance.We intend to explore the effects of miR-200 a on prostate cancer cell in cell apoptosis,proliferation,migration,invasion.After that we try to find downstream targets of miR-200 a in order to study molecular mechanism of prostate cancer.Methods:The in vitro proliferation of the cell was measured using the MTT assay after they were transformed with miR-200 a mimics or NC.Colony formation assay was performed after miR-200 a mimics or NC transfected by low density cell colony formation experiment,and the cell apoptosis was detected by flow cytometry.The ability of prostate caner cells' penetrating transwell was analyzed by transwell migration and invasion experiment.Through the combination of microarray and miRNA prediction website and western blotting,we try to find downstream targets of miR-200 a.Results:miR-200 a significantly inhibited prostate cancer cell proliferation in the MTT assay in the miR-200 a mimics group than in the NS group(P<0.05),and it also significantly decreased the number and size of cell formation clones in low density cell colony formation assay(P<0.05).Flow cytometry assay revealed that cells transfected with miR-200 a showed higher rates of apoptosis rate than NC.In transwell and invasion assay,the number of cells which passed through transwell chamber significantly reduced in miR-200 a group(P<0.05).Through the combination of microarray and miRNA prediction website and western blotting,we found that BRD4 may be a potential target gene of miR-200 a.Conclusion:miR-200 a can inhibit the proliferation,invasion and migration of prostate cancer cells,suggesting that miR-200 a may be a tumor suppressor gene in prostate cancer,and there is a negative correlation between miR-200 a and BRD4.BRD4 may be miR-200 a potential downstream target genes.