Mechanism of microRNA-20b-5p/E2F transcription factor 5-mediated regulation of epithelial-mesenchymal transition by transforming growth factor-β in prostate cancer cells
Objective To explore the mechanism of microRNA(miR)-20b-5p/E2F transcription factor 5(E2F5)-mediated regulation of epithelial-mesenchymal transition(EMT)by transfonning growth factor(TGF)-β1 in prostate cancer cell line,further promoting dhe occurrence and development of prostate cancer.Methods The database was analyzed,the target gene of miR-20b-5p was predicted,and the tar-geted inhibition of 3'UTR of E2F5 mRNA by miR-20b-5p was detected by double fluorescence reporter gene.Wild type(WT)and mutant(Mut)of E2F5 3'UTR were transfected into PC3 and DU145 cells,re-spectively,and then cells were treated with TGF-β.Double fluorescence reporter gene was used to detect whether E2F5 was the common target of TGF-β and miR-20b-5p.The target regulation of E2F5 by miR-20b-5p was detected by Western blotting.After PC3 and DU145 cells were transfected with miR-20b-5p inhibitor,mimic and control RNA respectively,the target regulation of E2F5 by miR-20b-5p was detected by Western blotting.MRNA gene was sequenced in benign prostatic hyperplasia and prostate cancer tissues of two groups,and the expression of E2F5 was compared between the two groups.miR-20b-5p and E2F5 were knocked down in PC3 cells.The expression of EMT-related genes E-cadherin and Vimentin was de-tected by Western blotting.The experimental data were expressed by mean±standard deviation,and one-way ANOVA was used for comparison between groups.Results A total of 207 target genes were predic-ted,and E2F5 was finally determined as our object of study.Double fluorescence reporter gene test found the miR-20b-5p targeted inhibition of E2F5 mRNA 3'UTR compared with the control group(PC3:103.00±2.00 vs.35.67±2.51,F=1 316.258,P＜0.01;DU145:101.00±2.00 vs.69.33±1.53,F=475,P＜0.01).Compared with the control group,the targeted effect of TGF-β on 3'UTR of E2F5 mRNA was enhanced(PC3:98.67±2.08 vs.155.67±2.08,F=1 124.654,P＜0.01;DU145:97.33±0.58 vs.143.00±2.00,F=1 443.769,P＜0.01).E2F5 was the common target of TGF-β and miR-20b-5p.After PC3 and DU145 cells were transfected with miR-20b-5p inhibitor,mimic and control RNA,the expression of E2F5 in miR-20b-5p inhibitor group was higher than that in control group(PC3:1.23±0.16 vs.0.83±0.02;DU145:0.49±0.2 vs.0.31±0.02,P＜0.01).The expression of E2F5 in miR-20b-5p inhibitor group was lower than that in the control group(PC3:0.63±0.02 vs.0.82± 0.02;DU145:0.14±0.02 vs.0.30±0.02,P＜0.01).MRNA gene sequencing showed that the expres-sion of E2F5 in prostate cancer was higher than that in the control group,and the analysis of TCGA data-base showed that E2F5 was highly expressed in prostate cancer.After knocking down miR-20b-5p,the ex-pression of E-cadherin was lower than that of the control group,while knocking down E2F5 at the same time could reverse this trend(0.43±0.02 and 0.98±0.02 vs.0.95±0.02,P＜0.01).After knocking down the expression of miR-20b-5p,Vimentin was higher than that in the control group,while knocking down E2F5 at the same time could reverse this trend(0.75±0.02 and 0.18±0.02 vs.0.15±0.02,P＜0.01),indicating that E2F5 regulates the expression of EMT phenotypic protein E-cadherin and Vimentin through targeted regulation of E2F5,leading to the occurrence and development of prostate cancer.Conclusion MiR-20b-5p/E2F5 mediates the regulation of TGF-β1 on prostate cancer cell line EMT,which leads to the progression of prostate cancer.
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