低表达lncRNA RP11-316M1.12通过上调miR-138-5p抑制膀胱癌细胞的侵袭和迁移

Low expression of lncRNA RP11-316M1.12 inhibits invasion and migration of bladder cancer cells by up-regulating miR-138-5p

目的探讨RP11-316M1.12对膀胱癌细胞侵袭、迁移、上皮间充质转化等恶性生物学行为的影响及相关机制。方法lncRNAs from cancer arrays(lnCAR)数据库分析RP11-316M1.12在膀胱癌和癌旁组织中的表达水平。荧光实时定量聚合酶链反应(fluorescence quantitative polymerase chain reaction,qPCR)检测RP11-316M1.12在膀胱癌细胞株UMUC-3、MGH-U3、J82、T24和正常膀胱上皮细胞SV-HUC-1中的表达水平。将MGH-U3细胞分为2组:空载组(转染sh-pSICOR空载质粒)和干扰组(转染sh-pSICOR-RP11-316M1.12质粒)。Transwell实验和细胞划痕实验分别检测干扰RP11-316M1.12对膀胱癌MGH-U3细胞侵袭和迁移的影响。采用lncRNA预测软件lncRNA2Function和双荧光素酶报告基因实验检测RP11-316M1.12与miR-138-5p的靶向调控关系。qPCR检测空载组和干扰组细胞中miR-138-5p的表达,Western blot检测上皮间充质转化蛋白的表达。结果与癌旁组织相比,RP11-316M1.12在膀胱癌组织中高表达(P<0.01)。与SV-HUC-1细胞相比,RP11-316M1.12在膀胱癌细胞UMUC-3、MGH-U3、J82、T24中高表达(P<0.05),且在MGH-U3细胞中表达最高(P<0.01)。与空载组比较,干扰组MGH-U3细胞的侵袭能力下降(P<0.05),MGH-U3细胞迁移能力下降(P<0.01)。双荧光素酶报告基因实验证实RP11-316M1.12靶向结合miR-138-5p(P<0.01)。与空载组相比,干扰组MGH-U3细胞中miR-138-5p表达升高(P<0.01)。与空载组相比,干扰组MGH-U3细胞中间质表型蛋白Zeb2和β-catenin表达降低,上皮表型蛋白Claudin-1和ZO-1表达升高。结论RP11-316M1.12在膀胱癌中呈现高表达,低表达RP11-316M1.12通过靶向上调miR-138-5p抑制膀胱癌细胞MGH-U3的侵袭、迁移和上皮间充质转化进程。

Objective To explore the effect of RP11-316M1.12 on the invasion,migration,epithelial-mesenchymal transition and other malignant biological behaviors of bladder cancer cells and its mechanism.Methods The lncRNAs from cancer arrays(lnCAR)database was used to analyze the expression level of RP11-316M1.12 in bladder cancer and adjacent tissues.Fluorescence quantitative polymerase chain reaction(qPCR)was used to detect the expression level of RP11-316M1.12 in bladder cancer cell lines UMUC-3,MGH-U3,J82,T24 and normal bladder epithelial cells SV-HUC-1.The MGH-U3 cells were divided into two groups:empty group(transfected with sh-pSICOR empty plasmid)and interference group(transfected with sh-pSICOR-RP11-316M1.12 plasmid).Trans-well experiment and cell scratch experiment were used to detect the influence of interference with RP11-316M1.12 expression on the invasion and migration of bladder cancer MGH-U3 cells.The lncRNA prediction software lncRNA2Function and dual luciferase reporter gene experiment were used to detect the target regulation relationship between RP11-316M1.12 and miR-138-5p.The expression of miR-138-5p was detected by qPCR in empty group and interference group.The expression of epithelial mesenchymal transition proteins was detected by Western blot.Results Compared with adjacent tissues,RP11-316M1.12 was highly expressed in bladder cancer tissues(P<0.01).Compared with SV-HUC-1 cells,RP11-316M1.12 was highly expressed in bladder cancer cell lines UMUC-3,MGH-U3,J82,T24(P<0.05),and the expression was the highest in MGH-U3 cells(P<0.01).Compared with empty group,the invasion ability of MGH-U3 cells was decreased in interference group(P<0.05),and the migration ability of MGH-U3 cells was decreased(P<0.01).The dual luciferase reporter gene experiment confirmed that RP11-316M1.12 targets miR-138-5p(P<0.01).Compared with empty group,the expression of miR-138-5p in MGH-U3 cells was increased in interference group(P<0.01).Compared with empty group,the expression of intercellular phenotype proteins Zeb2 andβ-catenin was decreased in MGH-U3 cells in interference group,and the expression of the epithelial phenotype proteins Claudin-1 and ZO-1 was increased.Conclusion RP11-316M1.12 is highly expressed in bladder cancer.The low expression of RP11-316M1.12 can inhibit the invasion,migration and epithelial-mesenchymal transition of bladder cancer cell MGH-U3 by targeting up-regulation of miR-138-5p.