miRNA-99b负调控mTOR抑制脑胶质瘤细胞的侵袭能力  

miRNA-99b inhibits the invasion ability of glioma cells by targeting mTOR

作　　者：马鹏举[1] 汲乾坤 李祥生[1] 常海刚[1] 周文科[1] 金保哲[1] MA Peng-ju;JI Qian-kun;LI Xiang-sheng;CHANG Hai-gang;ZHOU Wen-ke;JIN Bao-zhe(Dept of Neurosurgery,the First Affiliated Hospital of Xinxiang Medical University,Xinxiang Henan　453100,China)

机构地区：[1]新乡医学院第一附属医院神经外科,河南新乡453100

出　　处：《中国药理学通报》2018年第4期473-479,共7页Chinese Pharmacological Bulletin

基　　金：新乡医学院第一附属医院青年基金项目(No QN-2017-B009)

摘　　要：目的研究miRNA-99b及mTOR在胶质瘤中的表达水平及其对脑胶质瘤侵袭能力的影响及机制。方法用real-time PCR法检测脑胶质瘤组织与对照脑组织中miRNA-99b及mTOR的表达。miRNA-99b模拟物与野生型或突变型mTOR 3'UTR重组载体共转染后,应用荧光素酶基因报告系统检测miRNA-99b是否与mTOR基因3'UTR结合。应用real-time PCR法和Western blot检测miRNA-99b模拟物转染U251细胞后,细胞中miRNA-99b、mTOR mRNA和mTOR蛋白的表达,Transwell法检测细胞的侵袭能力。mTOR质粒转染U251细胞后,检测细胞中miRNA-99b的表达及细胞的侵袭能力的变化。统计学分析miRNA-99b表达水平与胶质瘤及预后的相关性。结果脑胶质瘤组织中miRNA-99b表达明显低于对照脑组织,mTOR mRNA表达明显高于对照脑组织;miRNA-99b模拟物和野生型mTOR 3'UTR重组载体共转染的U251细胞的荧光强度明显降低。miRNA-99b模拟物转染后,U251细胞中miRNA-99b的表达水平上升,而mTOR蛋白及mRNA的表达水平下降,U251细胞及侵袭能力下降。改变mTOR表达后,U251细胞中miRNA-99b的表达水平无明显变化,升高mTOR表达能够恢复因miRNA-99b抑制的细胞侵袭能力。Kaplan-Meier生存分析及Log-rank检验显示,低表达miRNA-99b患者的总生存期明显低于高表达组。结论过表达miRNA-99b可能通过靶向调控mTOR抑制脑胶质瘤细胞的侵袭能力,miRNA-99b可作为脑胶质瘤的治疗靶点。Aim To detect the expression of miRNA-99b and mTOR in glioma tissues and to investigate the effect of miRNA-99b on the cell invasion ability of human glioma cell line U251.Methods The expressions of miRNA-99b and mTOR mRNA in glioma tissues and normal brain tissues were detected by real-time PCR.After co-transfection with miRNA-99b mimics and wild or mutation type mTOR 3′UTR recombination vector,the specific binding ability of miRNA-99b to 3′-UTR in mTOR gene was examined by luciferase gene reporter system.The expression levels of miRNA-99b,mTOR mRNA and mTOR protein in glioma cell line U251 after transfected with miRNA-99b mimics were measured by real-time PCR and Western blot,respectively.The cell invasion was measured by Transwell assay.The changes of mTOR and miRNA-99b expression levels in U251 cells after transfection with mTOR PsiCHECK were detected by real-time PCR.The correlation between the expression of miRNA-99b and prognosis was analyzed statistically.Results The expressed level of miRNA-99b was lower,and the expression level of mTOR was higher in the glioma tissues than that in the normal brain tissues.The expression of miRNA-99b was up-regulated,and the expressions of mTOR mRNA and protein were down-regulated in U251 cells after transfection with miRNA-99b mimics.However,the abilities of invasion of U251 cells after transfection with miRNA-99b mimics were inhibited.The relative protein expression levels of mTOR in mTOR PsiCHECK group were significantly different from those in negative control group,but the relative expression levels of miRNA-99b had no significant difference compared with those in negative control group.Over-expression of mTOR restored the abilities of cell invasion in U251 cells,which was reduced by miRNA-99b.The Kaplan-Meier analysis and Log-Rank Test showed that there were significant differences in overall survival(OS)between the miRNA-99b high-expression and low-expression group.Conclusions The expression level of miRNA-99b is low in human glioma tissue.miRNA-99b may inhibi

关 键 词：miRNA-99b 哺乳动物雷帕霉素靶分子 脑胶质瘤 侵袭 预后 治疗靶点 

分 类 号：R342.2[医药卫生—基础医学] R394.2