Circular RNA transforming growth factor beta receptor Ⅱ promotes the occurrence and development of prostate cancer by regulating polypyrimidine-tractbindingprotein-2/ELAV like protein 1
Objective To explore the regulation of polypyrimidine-tractbindingprotein-2(PTBP2)/ELAV like protein 1(ELAVL1)and activation of SMAD2 signal pathway by Circular RNAtransforming growth factor beta receptor Ⅱ(circTGFBR2)in prostate cancer cells to promote the occurrence and develop-ment of prostate cancer.Methods Bioinformatics methods were used to find the binding sites of microRNA and other related proteins that bind to circTGFBR2,and the RNA-binding proteins that interact with PTBP2.After PC3 was transfected into pcDNA3.1-circTGFBR2,and the expression of circTGFBR2 was detected by RT-qPCR.PC3 cells overexpressed circTGFBR2 and knocked down miR-29b.The expression of ALK5,SMAD2 and p-SMAD2 was detected by Western blotting.After PC3 was transfected into pcDNA3.1circTGFBR2,the interaction between PTBP2 and circTGFBR2 was detected by pulldown with cir-cTGFBR2 specific probe.After overexpression of circTGFBR2 and ELAVL1,the expression of EMT marker gene E-cadherin and Vimentin and ZMYM1,and cell migration were detected by Transwell.The experimen-tal data were expressed by mean±standard deviation((x)±s),and one-way ANOVA was used for comparison between groups.Results There were multiple miR-29b binding sites in circTGFBR2 and miR-29b binding sites in ALK5 mRNA 3'untranslated regions(3'UTR).It was confirmed that PTBP2 interacted with another RNA binding protein ELAVL1.When PC3 cells were stimulated with TGF-β1,the expression of circTGFBR2 was higher than that in the control group(1.83±0.02 vs.1.03±0.022,F=2832.2,P＜0.01).After pcDNA3.1-circTCFBR2 and control plasmid were transfected into PC3 cells respectively,the expression of circTGFBR2 was detected by RT-qPCR.The expression of circTGFBR2 was higher than that in the control group(176.41±1.21 vs.1.04±0.02,F=63426.15,P＜0.01).The protein level of ALK5 in cells trea-ted with miR-29b inhibitor or circTGFBR2 was significantly higher than that in the control group.The expres-sion of ALK5 was further increased after overexpression of circTGFBR2 with miR-29b inhibitor(0.15±0.01,0.48±0.02,0.55±0.01,0.74±0.01,respectively,F=1268.314,P＜0.01).The phosphorylation level of SMAD2(1.14±0.02,1.28±0.02,1.42±0.01,1.5±0.255,F=4.852,P＜0.01)and p-SMAD2(0.18±0.02,0.36±0.01,0.65±0.01,0.94±0.02,F=1663.667,P＜0.01)also showed the same trend.Pulldown-MS assay showed that many proteins could bind to circTGFBR2 and confirmed the interaction between PTBP2 and circTGFBR2.As compared with the control group,the expression of interstitial marker gene Vimentin in prostate cancer cells with PTBP2 knockdown or overexpression of circTGFBR2 was higher,while the expression of epithelioid marker gene E-cadherin was lower than that in the control group.The overexpression of circTGFBR2 and knockdown of PTBP2 simultaneously reversed the above results(Vimentin scores were 0.22±0.02,0.87±0.02,0.85±0.02,0.6±0.01,respectively,F=1319.52,P＜0.01;The E-cadherin scores were 0.8±0.01,0.45±0.03,0.36±0.02,0.9±0.02,respectively,F=614.919,P＜0.01).When circTGFBR2 or ELAVL1 was overexpressed,the interaction between PTBP2 and ELAVL1 was weakened,the expression of EMT marker gene E-cadherin was lower than that in the control group,the ex-pression of Vimentin and ZMYM1 was higher than that in the control group,and the Transwell detection of cell migration was higher than that in the control group.When ELAVL1 and circTGFBR2 were highly ex-pressed at the same time,the above trends were further enhanced(Vimentin:0.22±0.02,0.87±0.02,0.85±0.02,0.6±0.01,respectively,F=1319.52,P＜0.01;E-cadherin:0.93±0.02,0.76±0.02,0.43±0.02,0.27±0.01,F=1 108.46,P＜0.01;ZMYM1:0.24±0.01,0.53±0.02,0.32±0.01,1.04±0.02,respectively,F=2 023.794,P＜0.01).Conclusion circTGFBR2 promotes the carcinogene-sis and development of prostate cancer by regulating PTBP2/ELAVL1-activated SMAD2 signal pathway.
Circular RNA transforming growth factor beta receptor ⅡProstate cancerPoly-pyrimidine-tractbindingprotein-2ELAV like protein 1
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