癌相关成纤维细胞外泌体携带微小RNA-20a靶向CX43调控细胞缝隙连接促进PC-3细胞侵袭和迁移  

MicroRNA-20a secreted by cancer-associated fibroblasts exosomes regulate cell gap junctions to promote PC-3 cell invasion and migration through targeting CX43

作　　者：李晓东[1] 陈烨辉 林婷婷[1] 薛学义[1] 郑清水[1] 陈佳茵 朱君明 柯志滨 陈少豪 陈东宁[1] 许宁[1] Li Xiaodong;Chen Yehui;Lin Tingting;Xue Xueyi;Zheng Qingshui;Chen Jiayin;Zhu Junming;Ke Zhibin;Chen Shaohao;Chen Dongning;Xu Ning(Department of Urology,the First Affiliated Hospital of Fujian Medical University,Fuzhou 350005,China)

机构地区：[1]福建医科大学附属第一医院泌尿外科,福州350005

出　　处：《中华实验外科杂志》2021年第2期301-304,共4页Chinese Journal of Experimental Surgery

基　　金：福建省自然科学基金 (2018J0105);福建医科大学启航基金 (2018QH1067)。

摘　　要：目的探讨癌相关成纤维细胞(CAFs)来源外泌体微小RNA(miR)-20a对前列腺癌细胞间通讯连接(GJIC)、侵袭和转移能力的影响及其机制。方法构建CAFs来源近红外荧光蛋白(IRFPs)标记外泌体,以不同浓度(0、10、100、200 mg/L)与PC-3细胞共培养。蛋白质印迹法检测PC-3细胞中CX43蛋白的相对表达水平;荧光漂白恢复技术测定PC-3细胞间缝隙连接功能;Transwell小室侵袭和迁移实验检测PC-3侵袭能力。构建高表达和低表达miR-20a的CAFs,分别与PC-3细胞共培养,检测缝隙链接蛋白43(Connexin 43,CX43)表达、细胞缝隙连接功能、细胞迁移和侵袭能力。双荧光素酶报告实验验证PC-3细胞中miR-20a与CX43基因是否直接结合。用Student’t检验分析组间差异。结果PC-3细胞的CX43表达水平及细胞缝隙连接功能均随外泌体浓度增加而降低(0.715±0.058、0.544±0.039、0.313±0.028和0.196±0.020,F=19.031,P<0.05),差异有统计学意义。Transwell小室检测结果表明,CAFs来源外泌体促进PC-3细胞迁移[各组每视野穿膜细胞数分别为(176.0±9.2)、(485.0±12.8)、(159.5±9.7)和(198.0±10.5)个,F=11.475,P<0.01]和侵袭[各组每视野穿膜细胞数分别为(150.0±8.4)、(443.0±21.7)、(182.0±11.2)和(153.5.0±10.1)个,F=16.306,P<0.01],差异有统计学意义;增强细胞缝隙连接能抑制此效应(F=14.052,P<0.01;F=14.804,P<0.01)。高表达miR-20a组的PC-3细胞的CX43表达水平(0.126±0.062比0.834±0.103,t=9.430,P<0.05)、荧光恢复率[(25.02±7.85)%比(46.65±6.98)%,t=5.782,P<0.05]均显著低于低表达miR-20a组,但细胞的迁移[(502.5±18.6)个比(128.5.0±9.4)个,t=9.120,P<0.05]和侵袭能力(458.0±16.8比194.5±9.2,t=6.286,P<0.05)均显著高于表达miR-20a组,差异均有统计学意义。双荧光素酶报告实验结果显示,野生型CX43的PC-3细胞中,转染miR-20a的细胞荧光活性显著低于miR-NC组(0.46±0.08比0.99±0.14,t=10.208,P<0.05),差异有统计学意义;而突变型CX43的PC-3细胞中,miR-20a组�Objective To explore the effects and mechanism of exosomes microRNA(miR)-20a secreted by carcinoma-associated fibroblasts(CAFs)on gap junction intercellular communication(GJIC)and the ability to invasion and metastasis of prostate cancer cells.Student′s t test was used to analyze the differences between groups.Methods Exosomes marked by near-infrared fluorescent protein(IRFPs)from CAFs were extracted and were co-cultured with PC-3 cells at a variety of concentrations(0,10,100,200μg/ml).Western blotting was used to detect relative expression level of connexin 43(CX43)protein in PC-3 cells.The function of GJIC in PC-3 cells was measured using fluorescence photobleaching recovery(FRP).Transwell assays were conducted to assess invasive capacity.CAFs with high and low expression level of miR-20a were constructed and were co-cultured with PC-3 cells separately to determine the expression of CX43,the function of GJIC and migratory and invasive abilities.Dual-luciferase reporter assay was performed to verify whether miR-20a was bound to CX43 gene directly.Results Expression level of CX43 and the function of GJIC in PC-3 cells decreased with increasing exosomes concentrations(0.715±0.058,0.544±0.039,0.313±0.028 and 0.196±0.020,P<0.05).Transwell assays revealed that exosomes from CAFs promoted migration(the number of transmembrane cells in each field of vision in each group was 176.0±9.2,485.0±12.8,159.5±9.7 and 198.0±10.5,t=11.475,P<0.01)and invasion(the number of transmembrane cells in each field of vision in each group was 150.0±8.4,443.0±21.7,182.0±11.2 and 153.5.0±10.1,t=16.306,P<0.01)of PC-3 cells.However,the enhancement of GJIC functions could inhibit this effect(t=14.052,P<0.01;t=14.804,P<0.01).The expression level of CX43(0.126±0.062 vs.0.834±0.103,t=9.430,P<0.05),fluorescence recovery[(25.02±7.85)%vs.(46.65±6.98)%,t=5.782,P<0.05]of PC-3 cells in the group treated with exosomes over-expressing miR-20a were significantly reduced,and the migratory(502.5±18.6 vs.128.5.0±9.4,t=9.120,P<0.05)and inv

关 键 词：癌相关成纤维细胞 外泌体 微小RNA 

分 类 号：R737.25[医药卫生—肿瘤]