MiR-154-5p Inhibits Proliferation, Migration And Invasion Of Prostate Cancer Cells By Regulating E2F5

PartⅠ miR-154-5p inhibits the proliferation,migration,andinvasive in prostate cancer cell linesObjects:Prostate cancer is the second highest cause of cancer mortality after lung tumours.The study of miRNAs’ functions and molecular mechanisms has brought new knowledge in biological processes of cancer.In prostate cancer there is a deregulation of several miRNAs that may function as tumour suppressors or oncogenes.The aim of this study is to analyze the function of miR-154-5p in the proliferation,migration,and invasive in prostate cancer cell liness.Methods:32 primary PCa and 15 nonmalignant tissue samples were collected,and the expression of miR-154-5p levels in these tissue were measured by TaqMan quantitative real-time PCR.Cell migration,invasion assays and flow cytometer analysis were used to evaluate the effects of forced miR-154-5p expression on PCa cells.Results:In primary PCa samples compared with that in nonmalignant samples,miR-154-5p expression was significantly decreased.CCK-8 assay showed that the proliferation rate of PC-3 and DU 145 cells was significantly declined following transfection with miR-154-5p at 96h.Flow cytometer analysis revealed that the percentage of G0/G1 phase was increased in miR-154-5p mimics-transfected group.Taken together,these results demonstrate that miR-154-5p inhibits the proliferation of CaP cells cells.To determine the impact of miR-154-5p is necessary in CaP cell metastasis,cell migration and invasion assays were performed.The results showed that the number of migrated and invaded cells was significantly lower than that of the NC.These results indicate that miR-154-5p can affect migratory and invasive capabilities of PCa cells.Conclusions:Our findings suggest that miR-154-5p inhibits PCa cell migration and invasion.miR-154-5p can act as a tumor suppressor.Part Ⅱ E2F5 promotes the proliferation,migration,andinvasive in prostate cancer cell linesObjects:This study aims to determine the function of E2F5 in the proliferation,migration,and invasive in prostate cancer cell liness.Methods:The effects of E2F5 on PCa cells were evaluated by TaqMan quantitative real-time PCR,cell migration,invasion assays,Western blot,and flow cytometer analysis.Results:To investigate the biological function of E2F5 in PCa carcinogenesis,we transfected PC-3 cells with siRNA molecules targeting E2F5(si-E2F5)and then introduced three E2F5 gene specific siRNA constructs(siE2F5-1,-2,and-3)and control siRNA to the PCa cells.The effect of each siRNA on the E2F5 expression level was analyzed by qRT-PCR and Western blot.After 48 h of transfection,E2F5 mRNA expression levels were significantly reduced in cells with transfected siE2F5-land siE2F5-3 compared with cells which were transfected with control siRNA.SiE2F5-3 efficiently repressed the E2F5 expression than others in the Western blot analysis,.All of the subsequent functional analyses were performed using siE2F5-3(hereafter called siE2F5).To further explore the mechanisms of siE2F5 on cell growth,CCK-8 assay and flow cytometry was performed to analyze the cell cycle distribution in transfected cells.The proliferation rate of PC-3 and DU 145 cells was significantly declined following transfection with siE2F5 at 96h.The percentages of PC3 and DU145 cells in the G0/G1 phase were higher in the siE2F5-transfected group than in the NC group.The numbers of migrated and invaded cells transfected with si-E2F5 were significantly lower than those of cells transfected with NC.Conclusions:E2f5 promotes the proliferation,migration,and invasive in prostate cancer cell lines.Part Ⅲ E2F5 is a direct target gene of miR-154-5pObjects:This study aims to identify the E2F5 is a direct target gene of miR-154-5p.Methods:We used Dual-luciferase reporter assay to identify binding sites between E2F5 3’-UTR and miR-154-5p.Results:Compared with cells treated with control miRNA 72 h post-transfection,the protein levels of E2F5 significantly decreased in cells transfected with the miR-154-5p mimics.To explore whether or not miR-154-5p can directly regulate E2F5 through a putative binding site in PCa cells,we cloned the predicted miR-154-5p binding site to the downstream region of the firefly luciferase gene.After co-transfection of the cells with miR-154-5p mimics or NC,miR-154-5p mimics significantly suppressed the luciferase activity of reporter plasmids compared with NC.In addition,when cells were co-transfected with miR-154-5p inhibitors and NC inhibitors,the miR-154-5p inhibitor significantly increased the luciferase activity of the reporter plasmid compared with the NC inhibitor.These results suggest that E2F5 is a direct target gene of miR-154-5p.Conclusions:E2F5 is a direct target gene of miR-154-5p.