Study On MiR-3691-3p Inhibits The Biological Functions Of Prostate Cancer By Target Gene E2F3/PRDM1 Regulation

Objective:To explore the molecular mechanisms of miR-3691-3p and its target gene in regulating the biological behavior of prostate cancer(PCa)cells,to provide a new biomarker for the diagnosis of PCa and to provide a new method to evaluate its treatment and prognoss in clinic.Methods:Part Ⅰ:1.The expression level of 5 miRNAs in PCa cell lines DU145 and PC-3 was detected by real-time PCR-2.The differential expression levels of miRNAs in PCa tissues and benign prostatic hyperplasia(BPH)were detected by real time PCR*3.Based on the above results,we choose the miR-3691-3p as the research object,the synthetic miR-3691-3p mimic(miR-3691-3p group)and the negative control mimic(scramble group)were transiently transfected into DU145 and PC-3 cell lines,and the expression level of miR-3691-3p in transfected cells was detected by real-time PCR.4.The proliferation,apoptosis,migration and invasion ability of DU145 and PC-3 cells after transfected with scramble mimic and miR-3691-3p mimic were detected by MIT assay,flow cytometry,scratch test and Transwell assay.Part Ⅱ:1.The candidate target genes of miR-3691-3p were predicted and screened by Target Scan 6.1,DIANA-MICROT and MIRDB bio informatics analysis software.2.The changes of candidate genes E2F3,PRDM1 mRNA and protein in PCa(DU145 and PC-3)cell lines transfected with different concentrations of miR-3691-3p mimic(10 nM,50 nM,100 nM)were detected by real-time PCR and Western blot.3.The small interfering RNA(E2F3 siRNA,PRDM1 siRNA and scrambled siRNA)were transfected into DU145 and PC-3 cells.The effect of siRNA on the expression of candidate target genes in PCa cells was detected by real-time PCR and western blot.4.The proliferation,migration and invasion ability of DU145 and PC-3 cells transfected with scrambled siRNA,E2F3 siRNA and PRDM1 siRNA were detected by MTT assay,flow cytometry,scratch test and Transwell assay respectively.Part III:1.PCa xenograft model in nude mice was constructed to detect the effect of miR-3691-3p agomir and control agomir on the growth of PCa.2.The expression of tumor proliferation related factors Ki67 protein,tumor invasion related factors vimentin in DU145 cell line and E-cadherin in PC-3 cell line,the expression of apoptosis-related protein caspase-3 were detected by immunohistochemical staining,and the expression level of the target gene was detected by the same method.Result:Part I:1.The expression of miR-3691-3p in 5 candidate miRNAs was the most significant in PCa cell lines,and it was the lowest in PCa cells.2.Compared with scramble group,miR-3691-3p mimic significantly increased the expression of miR-3691-3p in DU145 and PC-3 cells(P<0.001).Compared with scramble group,miR-3691-3p mimic can decrease the cell proliferation ability(DU145,P<0.001;PC-3,P<0.05),decrease migration and invasion ability significantly(P<0.001),and increase the apoptosis level(DU145,P<0.001;PC-3,P<0.01).Part II:1.The candidate genes(E2F3 and PRDM1)of miR-3691-3p were predicted and screened by microRNA target gene prediction software.2.Compared with Scramble group,the candidate gene E2F3,PRDM1 mRNA and protein level decreased gradually with the increasing of miR-3691-3p mimic transfection concentration(P<0.05).3.The expression of E2F3 and PRDM1 in E2F3,PRDM1 siRNA cells was significantly lower than that in transfected Scrambled siRNA cell lines(P<0.05),and low expression of E2F3 and PRDMI could inhibit cell proliferation,invasion and migration significantly(P<0.001).Part Ⅲ:1.The growth of PCa xenografts injected with miR-3691-3p agomir in the tumor-bearing mice model was significantly inhibited compared with the injection of control agomir(P<0.001).2.The expression of Ki67,vimentin,E-cadherin and its target gene in miR-3691-3p agomir treatment group was significantly lower than that in control group,but the expression of caspase-3 was significantly higher than that of control group.Conclusion:The low expression level of miR-3691-3p promotes the development and transformation of PC,which is caused by the increased expression of downstream target genes E2F3 and PRDMI.The expression of miR-3691-3p in PCa cells was restored,and the ability of proliferation,migration and invasion was significantly inhibited,and the ability of apoptosis was significantly increased.In this study,we elucidated the molecular mechanism of miR-3691-3p in regulating the development of PCa,and provided a new way for the clinical diagnosis and tareted therapy of PCa.