MiR-381 Regulates Development And Progression Of Prostate Caner Cell By Targeting MAP3K2

ObjectiveTo investigate the role of microRNA-381 in prostate cancer cell and find its target gene,so as to provide a scientific basis for finding new targets for prostate cancer treatment.Methods1.Relative mRNA expression of miR-381 was evaluated by quantitative real-time polymerase chain reaction(qRT PCR).MTT assay,colony formation assay and EdU assay were performed to measure cell proliferation ability respectively.Cell migration and invasion capabilities were determined by scratch assay and Transwell assay.Relative expressions of Cleaved-Caspase-3,Cleaved-Caspase-9,MMP-2,MMP-9,E-cadherin,N-cadherin by western blotting.2.Stable cell PC-3 was obtained after transfection of miR-381,and subcutaneously injected into nude mice for prostate cancer xenograft model.The volume and weight of tumor volume were recorded and the growth curve was drawn to determine the effect of miR-139-3p on tumor formation.3.TargetScan was performed to used to evaluate the target of miR-381.In addition,using dual luciferase reporter gene method to verify whether MAP3K2 is the target gene of miR-381.4.QRT-PCR was used to detect the expression level of MAP3K2 in PC-3 and DU145 cells after transfected with pcDNA 3.1-MAP3K2 or pcDNA 3.1-NC.The methods of MTT assay,EdU staining,colony formation assay,Annexin-V/PI double staining,wound healing assay and transwell assay were performed to detect proliferation,apoptosis,migration and invasion of PC-3 and DU145 cells after transfected with pcDNA 3.1-MAP3K2 or pcDNA 3.1-NC,or both pcDNA 3.1-MAP3K2 and miR-381 mimics.Furthermore,western blot was used to detect the level of the proteins involved in cell apoptosis and metastasis.Results1.The results of real time qRT-PCR showed that PC-3 and DU145 cells transfected with miR-381 mimics expressed higher level of miR-381 compared to the negative control,indicating that the transfection of miR-381 succeeded.MTT,colony formation and EdU assay showed that the proliferation ability of cells was obviously enhanced in miR-381 inhibitors group.Flow cytometry assays indicated that the apoptosis capability of cells which transfected with miR-381 inhibitors was reduced.Wound healing test and transwell assay experimental results verified the activities of migration and invasion also showed a downward trend in miR-381 mimics group.The protein expression level of Cleaved-Caspase-3,Cleaved-Caspase-9,and E-cadherin in miR-381 mimics group were increased significantly.However,the expression of MMP-2,MMP-9 and N-cadherin were decreased in miR-381 mimics group compaired with balnk group.2.Overexpression of miR-381 significantly decreased the growth of xenografts in nude mice,induced apoptosis of tumor cells in xenografts.Overexpression of miR-381 significantly increased the expressions of cleaved-caspase-3 and cleaved-caspase-3 in xenograft.3.Targetscan predicted that MAP3K2 was one of the target genes of microRNA-381,and dual luciferase target assay confirmed that MAP3K2 was the target gene of microRNA-381.The expression level of MAP3K2 in prostate cancer decreased with the increase of microRNA-381.4.Proliferation,migration,invasion and EMT capacities were increased,and apoptosis level was decreased in PC-3 and DU145 cells after transfected with pcDNA 3.1-MAP3K2.Compared to pcDNA 3.1-MAP3K2 group,proliferation,migration,and invasion capacities were decreased,and apoptosis level was increased in pcDNA 3.1-MAP3K2+miR-381 mimics group.It was indicated that miR-381 regulated prostate cancer progression by regulating MAP3K2 expression level.ConclusionMiR-381 was low expressed in prostate cancer cells PC-3 and DU145.Overexpression of microRNA381 can significantly reduce cells viability,proliferation,migration,invasion and EMT,and increase the level of apoptosis,while inhibition of miR-381 showed opposite effects.In addition,overexpression of miR-381 significantly reduced the growth of xenografts in nude mice and induced apoptosis of tumor cells in xenografts.MAP3K2 is the target gene of microRNA381,which inhibited the development of prostate cancer cells by reducing the expression of MAP3K2 gene.