miR-145通过下调PLCε抑制膀胱癌EMT和迁移及其机制研究  

Silence of PLCε Induced by miR-145 Inhibits EMT and Metastasis in Bladder Cancer

作　　者：赵燕[1] 郝燕妮[1] 刘南京 李婷[1] 吴小候[2] 罗春丽[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室重庆市重点实验室,重庆400016 [2]重庆医科大学附属第一医院泌尿外科,重庆400016

出　　处：《中国生物工程杂志》2017年第3期27-36,共10页China Biotechnology

基　　金：国家自然科学基金资助项目(81072086)

摘　　要：目的:探讨miR-145调控PLCε对膀胱癌细胞T24上皮间质转化(EMT)和迁移的影响及可能的分子机制。方法:(1)腺病毒感染T24细胞,划痕实验和Transwell检测细胞的迁移能力;RTPCR、Western blot分别检测PLCε及EMT相关分子的表达;为探究其分子机制,Western blot检测GSK-3β磷酸化(Ser9位点)和Snail的表达情况。(2)利用生物信息学技术预测可能调控PLCε的miRNA,结合文献报道的膀胱癌microRNA表达谱结果筛选出miR-145;转染miR-145 mimics至T24,q PCR检测miR-145、PLCε的表达,Western blot检测PLCε的表达。(3)转染miR-145 mimics,Western blot检测EMT相关分子及p-GSK-3β、Snail;划痕实验、Transwell检测过表达miR-145后细胞的迁移能力。结果:(1)干扰PLCε表达能显著抑制细胞的迁移,同时,使T24细胞中E-cadherin表达上调,N-cadherin和Vimentin表达下调;干扰PLCε后,GSK-3β磷酸化(Ser9位点)水平下降,Snail表达降低。(2)转染miR-145 mimics可使T24细胞中miR-145表达增高,且明显抑制T24细胞中PLCε的表达。(3)在T24中过表达miR-145,细胞迁移能力显著下降,EMT标志分子的表达情况与沉默PLCε结果一致。同时,与阴性对照组相比,转染miR-145 mimics组p-GSK-3β和Snail表达显著减少。结论:PLCε通过GSK-3β/Snail信号通路促进膀胱癌细胞T24发生EMT及迁移,miR-145可以逆转PLCε诱导膀胱癌EMT的发生,从而阻止膀胱癌细胞的迁移。Objective： To study the effect of PLCε regulated by miR-145 on EMT and metastasis in bladder cancer cell T24 and and the potential mechanism. Methods：（1）Using adenovirus infecting T24 cells,the migratory abilities of T24 cells were observed by wound healing and transwell chamber cell migration assay. The expression of EMT related molecules such as E-cadherin,n-cadherin、Vimentin were detected by RT-PCR and Western blot,at genetic and protein level respectively. In order to explore the molecule mechanism,western blot was used to test the protein level of p-GSK-3β and Snail.（2） Using bioinformatic to calculate the possible microRNA regulating PLCε,referenced to the reported microRNA array,miR-145 was chosen for the target microRNA. Using miR-145 mimics transfecting T24,the expression of miR-145 and PLC ε were detected by quantitative Real-timePCR,meantime,the PLCε protein expression was tested by Western blot.（3） After cells transfected with miR-145 mimics,the protein expression of EMT related molecule as well as p-GSK-3β and Snail were detected by Western blot. Wound healing and transwell chamber cell migration assay were adopted to detect he migratory abilities of T24. Results：（1） Wound healing and transwell chamber cell migration assay showed that the migratory ability was remarkably decreased in the Ad-sh PLCε group,compared with the blank contol group and Ad-HK group（ P〈0. 05）. The expressions level of both mRNA and protein of N-cadherin,Vimentin were higher,while E-cadherin was lower in Ad-sh PLCε group than that in blank contol group and Ad-HK group（ P〈0. 05）.Western blot result showed that the protein level of p-GSK-3β and Snail were significantly lower after treated with Ad-sh PLCε（ P〈0. 05）.（2） q PCR result showed that,transfection miR-145 mimics could restore miR-145 expression of bladder cancer cells T24,significantly higher than transfection negative control（ P〈0. 01）,by contrst,the PLCε mRNA expression was lower（ P〈0. 05）. Western blo

关 键 词：PLCε MIR-145 膀胱癌 上皮间质转化 迁移 

分 类 号：Q811.4[生物学—生物工程]