miRNA-20a在膀胱癌中的表达及其机制研究

Expression and potential role of miRNA-20a in bladder cancer

目的:探讨miRNA-20a在膀胱癌组织中的表达及其机制。方法:收集2014年1月至2015年1月昆明医科大学第二附属医院96例患者的组织标本,运用实时定量聚合酶链反应(q RT-PCR)方法检测miRNA-20a在膀胱癌组织和癌旁组织中的表达;生物信息学方法预测miRNA-20a的靶基因并采用双荧光素酶报告基因实验进行验证;q RT-PCR、Western blot以及细胞免疫荧光分别检测人膀胱癌细胞系T24和J82细胞转染miRNA-20a模拟物或阴性对照后对靶基因mRNA和蛋白表达的影响;CCK-8、Transwell侵袭小室和划痕实验检测过表达miRNA-20a后T24细胞体外增殖、迁移和侵袭能力的改变。结果:miRNA-20a在膀胱癌组织中高表达,且与肿瘤的病理分级、临床分期、转移以及复发密切相关(P<0.001);双荧光素酶报告基因实验证实miRNA-20a与人源性长寿保障基因2(Homo sapiens longevity assurance homologue 2,LASS2)的3'-UTR直接靶向结合;转染miRNA-20a模拟物可显著下调膀胱癌细胞中LASS2 m RNA和蛋白的表达(P<0.01),增强膀胱癌细胞的增殖、侵袭和迁移能力(P<0.01)。结论:miRNA-20a在膀胱癌组织中高表达,且miRNA-20a可通过靶向抑制LASS2促进膀胱癌细胞的增殖、侵袭和迁移,与膀胱癌的发生发展相关。

Objective： To investigate micro RNA-20 a（miRNA-20 a） expression in bladder cancer and its potential mechanism. Methods：Mi RNA-20 a expression was examined using quantitative reverse-transcription polymerase chain reaction（q RT-PCR） in human bladder cancer tissues and the paired adjacent non-tumor bladder tissues of 96 patients. The target gene of the miRNA-20 a was predicted and validated using bioinformatics analysis and reporter gene assay, respectively. The m RNA or protein expression of the target gene in bladder cancer T24 and J82 cells transfected with miRNA-20 a mimic or negative control（NC） mimics was detected via q RT-PCR, Western blot analysis, and cell immunofluorescence. CCK-8, Transwell chamber, and wound-healing assays were applied to test the proliferation, migration, and invasion of T24 cells after miRNA-20 a over-expression in vitro. Results： Mi RNA-20 a expression significantly increased in bladder cancer tissues compared with those in corresponding adjacent non-tumor tissues. High miRNA-20 a expression in bladder cancer tissues was closely related to aggressive tumor phenotype, such as high histological grade, poor TNM stage, lymph node invasion, distant metastasis, and tumor recurrence（all P〈0.001）. Dual-luciferase reporter assay confirmed that miRNA-20 a can directly bind to the 3＇-untranslated region（3＇-UTR） of Homo sapiens longevity assurance homologue 2（LASS2）. Transfection with miRNA-20 a mimics significantly inhibited m RNA and protein expression of LASS2 in T24 and J82 cells（all P〈0.01） and promoted T24 cell proliferation, migration, and invasion in vitro. Conclusion： Mi RNA-20 a is highly expressed in bladder cancer tissues. Mi RNA-20 a enhances cell migration as well as proliferation and acts as an oncogene in bladder cancer because of the targeted inhibition of LASS2 expression.