Down-regulation of miRNA-99b expression in mTOR/4E-BP1 signal pathway and invasion in glioma U251 cells
10.3760/cma.j.issn.1671-8925.2018.09.004
- VernacularTitle: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
- Author:
Qiankun JI
1
;
Ke REN
;
Jianhua ZHAO
;
Zhijie YIN
;
Pengju MA
;
Ruihua LIU
;
Baozhe JIN
Author Information
1. 新乡医学院第一附属医院神经外科
- Keywords:
Micro RNA-99b;
Mammalian target of rapamycin;
EIF4E-binding protein 1;
Glioma;
Invasion
- From:
Chinese Journal of Neuromedicine
2018;17(9):886-891
- CountryChina
- Language:Chinese
-
Abstract:
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 (mTOR) in U251 cells were measured by reverse transcription (RT)-PCR;the changes of mTOR,eIF4E-binding protein 1 (4E-BP1) andphosphorylated (p)-4E-BPl protein expressions in U251 cells were detected by Westem 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 mTOR siRNA,respectively;the miRNA-99b and mTOR 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 rnRNA 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-BP1 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+mTOR 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-BP1 signaling pathway.
