转化生长因子-β受体Ⅱ环状RNA通过调节核糖核酸结合蛋白2/胚胎致死性异常视觉样蛋白1促进前列腺癌发生发展  

Circular RNA transforming growth factor beta receptorⅡpromotes the occurrence and development of prostate cancer by regulating polypyrimidine-tractbindingprotein-2/ELAV like protein 1

作　　者：杜蕾[1] 李谦 马子越 常学良[1] 齐进春[1] 薛文勇[1] Du Lei;Li Qian;Ma Ziyue;Chang Xueliang;Qi Jinchun;Xue Wenyong(Department of Urology,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,China)

机构地区：[1]河北医科大学第二医院泌尿外科,石家庄050000

出　　处：《中华实验外科杂志》2024年第2期286-290,共5页Chinese Journal of Experimental Surgery

基　　金：河北自然科学基金(H2022206185)。

摘　　要：目的探讨前列腺癌细胞中转化生长因子-β受体Ⅱ环状RNA(circTGFBR2)调节核糖核酸结合蛋白2(PTBP2)/胚胎致死性异常视觉样蛋白1(ELAVL1)激活SMAD2信号途径促进前列腺癌发生发展。方法利用生物信息学方法, 寻找与circTGFBR2结合microRNA以及其他相关蛋白结合位点, 以及与PTBP2存在相互作用的RNA结合蛋白。PC3转染pcDNA3.1-circTGFBR2后, 实时定量反转录聚合酶链反应(RT-qPCR)检测circTGFBR2的表达。PC3细胞过表达circTGFBR2同时敲低微小RNA(microRNA, miR)-29b, 蛋白质印迹法(Western blot)检测ALK5、SMAD2及p-SMAD2表达。PC3分别转染pcDNA3.1-circTGFBR2及对照质粒后, 利用circTGFBR2特异性探针进行pulldown, 检测PTBP2与circTGFBR2的相互作用。过表达circTGFBR2与ELAVL1, 检测细胞上皮-间充质转化(EMT)的标志基因E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)及ZMYM1表达, 以及Transwell检测细胞迁移。组间比较应用单因素方差分析。结果分析结果显示circTGFBR2存在多个miR-29b结合的位点, ALK5(又名TGFBR1)mRNA 3’端非编码区(3’UTR)同样存在miR-29b的结合位点, 并证实PTBP2与另一个RNA结合蛋白ELAVL1存在相互作用。用转化生长因子-β1(TGF-β1)刺激PC3细胞发现, circTGFBR2的表达高于对照组(1.83±0.02比1.03±0.02, F=2 832.200, P<0.01)。PC3分别转染pcDNA3.1-circTGFBR2及对照质粒后, RT-qPCR检测circTGFBR2的表达, 可见circTGFBR2组高于对照组(176.41±1.21比1.04±0.02, F=63 426.15, P<0.01)。应用miR-29b抑制剂或circTGFBR2处理细胞后ALK5的蛋白水平明显高于对照组, 应用miR-29b抑制剂同时过表达circTGFBR2后进一步增加ALK5的表达(分别为0.15±0.01、0.48±0.02、0.55±0.01、0.74±0.01, F=1268.314, P<0.01), 并且SMAD2(分别为1.14±0.02、1.28±0.02、1.42±0.01、1.5±0.25, F=4.852, P<0.01)和p-SMAD2(分别为0.18±0.02、0.36±0.01、0.65±0.01、0.94±0.02, F=1663.667, P<0.01)的磷酸化水平也呈相同趋势。Pulldown-MS实验, 多个蛋白质能�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 receptorⅡ(circTGFBR2)in prostate cancer cells to promote the occurrence and development 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 circTGFBR2 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 experimental data were expressed by mean±standard deviation(±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-circTGFBR2 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 treated with miR-29b inhibitor or circTGFBR2 was significantly higher than that in the control group.The expression of ALK5 was further increased after overexpression of circTGFBR2 with miR-29b inhibitor(0.15�

关 键 词：转化生长因子-β受体Ⅱ环状RNA 前列腺癌 核糖核酸结合蛋白2 胚胎致死性异常视觉样蛋白1 

分 类 号：R737.25[医药卫生—肿瘤]