微小RNA-4318靶向调控外被体蛋白复合物亚基β’在前列腺癌迁移、侵袭转移中的作用  

The role of microRNA-4318 targeting coatomer protein complex subunit beta 2 gene in migration,invasion and metastasis of prostate cancer

作　　者：冯岩岩 潘海燕 万宏源 吴升 孙健[2] 陈雪芬 糜远源 祝黎洁 Feng Yanyan;Pan Haiyan;Wan Hongyan;Wu Sheng;Sun Jian;Chen Xuefen;Mi Yuanyuan;Zhu Lijie(Wuxi Medical College,Jiangnan University,Wuxi 214122,China;Department of Urology,Affiliated Hospital of Jiangnan University,Wuxi 214122,China;Pelvic Floor Rehabilitation Center,Affiliated Hospital of Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学无锡医学院,214122 [2]江南大学附属医院泌尿外科,无锡214122 [3]江南大学附属医院盆底康复中心,无锡214122

出　　处：《中华实验外科杂志》2022年第5期970-974,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81802576);江苏省中管局科研一般项目(YB201827);无锡市卫健委科研课题(T202024、J202012、Z202011、ZM001);无锡市科技局科研项目(WX18IIAN024、N20202021);江南大学无锡医学院研究团队建设经费(1286010242190070);无锡市"太湖人才计划"医疗卫生高端人才,双百中青年医疗卫生拔尖人才(BJ2020061)。

摘　　要：目的探讨微小RNA-4318(miR-4318)靶向调控外被体蛋白复合物亚基β’(COPB2)在前列腺癌侵袭转移中的作用机制。方法通过Target Scan Human 7.0数据库预测COPB23’非编码区(3’UTR)可能结合的miRNA,双荧光素酶报告基因验证miR-4318与COPB2的靶向关系。采用实时定量反转录聚合酶链反应(RT-qPCR)和蛋白印迹法(Western blot)检测转染mimics及inhibitors后的蛋白及RNA表达量,通过Transwell法检测细胞的侵袭和迁移能力。组间比较采用独立样本t检验。结果Target Scan Human 7.0数据库预测COPB23’UTR区可能结合的miRNAs,结果显示miR-4318结合可能性最大。通过临床15对前列腺癌与癌旁组织中miRNAs的表达量比对,结果发现上述3种miRNAs中只有miR-4318在大部分癌组织中表达(10.18±9.32)低于癌旁组织(21.46±36.19)。双荧光素酶报告结果表明,对比mimic-NC和WT-COPB2共转染比较双荧光素酶相对活性(2.31±0.02),miR-4318和WT-COPB2共转染明显降低DU145细胞的双荧光素酶相对活性(0.38±0.12),差异有统计学意义(t=92.870,P<0.05)。RT-qPCR结果显示,在PC3、DU145细胞中转染miR-4318 mimics后,miR-4318的表达水平(3.94±0.34、2.42±0.33)显著高于mimic-NC组(1.09±0.07、0.97±0.05),差异有统计学意义(t=11.630、6.157,P<0.05);转染miR-4318 inhibitors(0.26±0.02、0.30±0.06)后,miR-4318的表达水平显著低于inhibitor-NC组(0.89±0.10、1.28±0.05),差异有统计学意义(t=8.768、18.550,P<0.05)。Western blot结果表明,在PC3、DU145细胞中miR-4318 mimics组和mimic-NC组表达量分别为(0.12±0.01、0.14±0.01),(0.30±0.02、0.30±0.02),miR-4318 mimics组明显低于NC组,上调miR-4318明显降低了COPB2的表达量,差异有统计学意义(t=14.020、12.310,P<0.05)。miR-4318 inhibitors组(0.35±0.02、0.59±0.04)的表达量与inhibitor-NC组(0.31±0.02、0.29±0.02)比较,差异有统计学意义(t=1.893、2.919,P<0.05)。Transwell实验结果显示,miR-4318 mimics组的PC3、DU145细胞迁移数分别为(19.1Objective To investigate the mechanism of microRNA-4318(miR-4318)targeting regulation of coatomer protein complex subunit beta 2(COPB2)gene in invasion and metastasis of prostate cancer.Methods Target Scan Human7.0 was used to predict the possible binding miRNAs in the 3′untranslated region(3′UTR)of COPB2,and the targeting relationship between miR-4318 and COPB2 was verified by luciferase genes assay.The expression of COPB2 protein and RNA after transfection was detected by Western blotting and real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)respectively,and the invasion and migration ability of cells were detected by Transwell assay.Independent sample t test was used for inter-group comparison.Results The Target Scan Human 7.0 database predicted the possible binding miRNAs in COPB23′UTR region,and the results showed that miR-4318 was the most likely to bind.By comparing the expression levels of miRNAs in prostate cancer and tumor-adjacent tissue,it was found that among the above three miRNAs,only miR-4318 expression was lower in most cancer tissues(10.18±9.32)than tumor-adjacent tissue(21.46±36.19).The results of double luciferase report showed that the co-transfection of miR-4318 and WT-COPB2 significantly reduced the relative activity of double luciferase as compared with mimic-NC and WT-COPB2 in DU145 cells(0.38±0.12 vs.2.31±0.02,t=92.870,P<0.05).RT-qPCR results showed that the expression level of miR-4318 in miR-4318 mimics group(3.94±0.34,2.42±0.33)was significantly higher than that in mimic-NC group(1.09±0.07,0.97±0.05)after transfection of miR-4318 mimics in PC3 and DU145 cells.The difference was statistically significant(t=11.630,6.157,P<0.05).After transfection with miR-4318 inhibitors(0.26±0.02,0.30±0.06),the expression level of miR-4318 in miR-4318 inhibitors group was significantly lower than that in inhibitor-NC group(0.89±0.10,1.28±0.05).The difference was statistically significant(t=8.768,18.550,P<0.05).Western blotting results showed that the expre

关 键 词：DU145细胞 靶向调控 双荧光素酶报告基因 PC3 微小RNA 前列腺癌 蛋白复合物 迁移数 

分 类 号：R737.25[医药卫生—肿瘤]