下调miRNA-99b表达对脑胶质瘤细胞侵袭及mTOR／4E-BP1信号通路调控的影响  

Down-regulation of miRNA-99b expression in mTOR/4E-BP1 signal pathway and invasion in glioma U251 cells

作　　者：汲乾坤 任珂 赵建华[2] 尹志杰[1] 马鹏举[1] 刘瑞华[1] 金保哲[1] Ji Qiankun;Ren Ke;Zhao Jianhua;Yin Zhijie;Ma Pengju;Liu Ruihua;Jin Baozhe(Department of Neurosurgery,First Affiliated Hospital of Xinxiang Medical College,Xinxiang 453100,China;Department of Neurology,First Affiliated Hospital of Xinxiang Medical College,Xinxiang 453100,China)

机构地区：[1]新乡医学院第一附属医院神经外科,新乡453100 [2]新乡医学院第一附属医院神经内科,新乡453100

出　　处：《中华神经医学杂志》2018年第9期886-891,共6页Chinese Journal of Neuromedicine

基　　金：新乡医学院第一附属医院青年基金（QN-2017-B009）

摘　　要：目的探讨下调微小RNA（miRNA）-99b表达对脑胶质瘤细胞侵袭的影响及其作用机制。方法体外常规培养脑胶质瘤U251细胞。（1）将U251细胞分为空白对照组、无义序列对照组和miRNA．99b抑制物组，后2组分别转染无义序列和miRNA-99b抑制物，空白对照组不做任何处理。采用RT-PCR检测U251细胞中miRNA-99b、哺乳动物雷帕霉素靶分子（mTOR）mRNA的表达，采用Westernblotting检测U251细胞中mTOR、真核翻译起始因子4E结合蛋白1（4E-BP1）及磷酸化（p）-4E-BP1蛋白的表达，采用Transwell侵袭实验检测U251细胞的侵袭能力。（2）将U251细胞分为无义序列对照组、mTORsiRNA组，分别转染无义序列和mTORsiRNA。采用RT—PCR检测U251细胞中miRNA-99b、mTORmRNA的表达，采用Westernblotting检测U251细胞中mTOR、p-4E-BPl蛋白的表达。f3）将U251细胞分为miRNA-99b抑制物＋无义序列对照组、miRNA-99b抑制物＋mTORsiRNA组，分别转染miRNA-99b抑制物＋无义序列、miRNA-99b抑制物＋mTORsiRNA。采用Westcrnblotting检测U251细胞中P-4E-BP1蛋白的表达，采用Transwell侵袭实验检测U251细胞的侵袭能力。结果（1）与空白对照组、无义序列对照组比较，miRNA-99b抑制物组U251细胞中miRNA-99bmRNA表达明显降低，mTORmRNA及蛋白表达明显升高．P-4E．BPl蛋白表达明显升高，穿膜细胞数明显增多，差异均有统计学意义（P〈0．05），而4E-BP1蛋白表达差异无统计学意义（P〉0．05）。（2）与无义序列对照组比较，mTORsiRNA组U251细胞中mTORmRNA及蛋白表达明显降低，P-4E-BP1蛋白表达明显降低，差异均有统计学意义（P〈0．05），而miRNA-99bmRNA表达差异无统计学意义（P〉O．05）。（3）与miRNA-99b抑制物＋无义序列对照组比较，miRNA-99b抑制物＋roTORsiRNA组U251细胞中P-4E．BP1蛋白表达明显降低．穿膜细胞数明显减少，差异均有统计学意义（P〈0．05）。结论下调miRNA．99b表达可Objective To detect the down-regulation ofmiRNA-99b expression in cell invasion and its mechanism in human glioma cell line U251. Methods Glioma cell line U251 were routinely cultured in vitro. （1） U251 cells were divided into blank control group, negative control group and miRNA-99b inhibitor group; cells in the latter two groups were transfected with negative control sequences and miRNA-99b inhibitors, respectively; and cells in the blank control group did not give any treatment; mRNA expressions of miRNA-99b and mammalian target of rapamycin （roTOR） in U251 cells were measured by reverse transcription （RT）-PCR; the changes of roTOR, eIF4E-binding protein 1（4E-BP1） and phosphorylated （p）-4E-BP1 protein expressions in U251 cells were detected by Western blotting; cell invasion was evaluated by Transwell assay. （2） U251 cells were divided into negative control group I and mTOR siRNA group, and cells in the two groups were transfected with negative control sequences and roTOR siRNA, respectively; the miRNA -99b and roTOR mRNA expressions in U251 cells were measured by RT-PCR; the mTOR and p-4E-BP1 protein expressions in U251 cells were measured by Western blotting. （3） U251 cells were divided into miRNA-99b inhibitor＋negative control group and miRNA-99b inhibitor＋mTOR siRNA group, and cells in the two groups were transfected with miRNA-99b inhibitor＋negative control sequences and miRNA-99b inhibitor＋mTOR siRNA, respectively; the p-4E-BP1 protein expression in U251 cells was measured by Western blotting; cell invasion was evaluated by Transwell assay. Results （1） As compared with those in the blank control group and negative control group, the miRNA-99b mRNA expression was significantly decreased, the mTOR mRNA and protein expressions and p-4E-BP1 protein expression were significantly increased, and the number of transmembrane cells was significantly larger in U251 cells of miRNA-99b inhibitor group （P〈0.05）; there were no significant differences in 4E-BP1 protein expre

关 键 词：微小RNA-99b 哺乳动物雷帕霉素靶分子 真核细胞翻译起始因子4E结合蛋白1 神经胶质瘤 细胞侵袭 

分 类 号：R730.264[医药卫生—肿瘤]