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

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表达可促进脑胶质瘤细胞侵袭，其作用机制与mYOR／4E-BP1信号通路调控有关。

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 expression among the three groups （P〉0.05）. （2） As compared with those in the negative control group I, the mTOR mRNA and protein expressions and p-4E-BPI protein expression were significantly decreased in U251 cells of mTOR siRNA group （P〈0.05）; there was no significant difference in miRNA-99b mRNA expression between the two groups （P〉0.05）. （3） As compared with those in the miRNA-99b inhibitor＋negative control group, the p-4E-BP1 protein expression and number of transmembrane cells were significantly decreased/smaller in U251 cells ofmiRNA-99b inhibitor＋roTOR siRNA group （P〈0.05）. Conclusions Down-regulation ofmiRNA-99b expression promotes glioma cell invasion, and its mechanism is related to the regulation of mTOR/4E-BP 1 signaling pathway.