微小RNA-487a-3p通过抑制磷脂酰肌醇3激酶/蛋白激酶B信号通路调控脑胶质瘤细胞的增殖、迁移和侵袭

MicroRNA-487a-3p regulates the proliferation,migration,and invasion of glioma cells by inhibiting the phosphatidylinositol 3 kinase/protein kinase B signaling pathway

目的探究微小RNA(miRNA,miR)-487a-3p对脑胶质瘤细胞(U-87 MG)的增殖、迁移和侵袭能力的调控作用及信号通路机制。方法利用GEO2R分析工具对基因表达数据库(GEO)的数据集(GSE165937)进行比较分析获得差异表达miRNA。通过定量聚合酶链式反应(qPCR)在HA1800和U-87 MG细胞系中检测miR-487a-3p的表达水平。通过在U-87 MG细胞中转染miR-487a-3p模拟物(miR-mimic)上调其表达并应用qPCR进行验证。分别通过细胞计数试剂盒8(CCK-8)、Transwell迁移实验和Transwell侵袭实验检测各组细胞的增殖、迁移和侵袭能力。通过蛋白质印迹法(Western blot)检测磷酸化蛋白激酶B(p-Akt)和总蛋白激酶B(t-Akt)的表达水平,通过计算比值评价各组细胞中磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路的激活水平。两组间比较采用t检验进行统计学分析。结果共有62个差异表达miRNA(Log2│差异倍数│>2,P<0.05),其中上调9个,下调53个,miR-487a-3p在肿瘤组中表达水平显著下降(Log2差异倍数=-2.527,P<0.01)。与HA1800细胞比较,在U-87 MG细胞中miR-487a-3p的表达显著下调(0.090±0.022比1.000±0.205,t=7.659,P<0.05)。在U-87 MG细胞中转染miR-mimic可以显著上调miR-487a-3p的表达(89.730±16.800比1.000±0.351,t=9.146,P<0.05)。miR-mimc组中U-87 MG细胞的增殖(24 h,0.600±0.034比0.831±0.043,t=7.268,P<0.01;48 h,1.098±0.058比1.624±0.074,t=9.708,P<0.01;72 h,1.615±0.055比2.252±0.096,t=9.959,P<0.01)、迁移(97.750±9.575比226.600±19.360,t=10.330,P<0.01)和侵袭(74.330±9.501比158.600±14.440,t=8.444,P<0.01)能力被显著抑制。miR-mimc组中U-87 MG细胞的PI3K/Akt信号通路水平被显著抑制(0.211±0.384比1.000±0.000,t=35.610,P<0.01)。结论miR-487a-3p可以通过抑制PI3K/Akt信号通路抑制脑胶质瘤细胞的增殖、迁移和侵袭能力。

Objective To explore the role of microRNA(miRNA,miR)-487a-3p in the regulation of the proliferation,migration,and invasion of glioma cells and the associated mechanism of signaling pathway.Methods GEO2R was used to compare and analyze the dataset(GSE165937)of gene expression omnibus(GEO)to obtain differentially expressed miRNAs.The expression levels of miR-487a-3p in HA1800 and U-87 MG cells were detected by quantitative polymerase chain reaction(qPCR).Transfection of miR-mimic was used to up-regulate the expression of miR-487a-3p in U-87 MG cells,and was verified by qPCR.The proliferation,migration,and invasion abilities of cells in each group were measured using cell count kit-8(CCK-8),Transwell migration assay,and Transwell invasion assay,respectively.Western blotting was used to detect the levels of phosphorylated protein kinase B(p-Akt)and total protein kinase B(t-Akt),and ratio of p-Akt/t-Akt was calculated to evaluated the activation level of the phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)signaling pathway.Two-group comparison was analyzed using t test.Results There were a total of 62 differentially expressed miRNAs(Log2│Fold Change│>2,P<0.01),of which 9 was up-regulated and 53 were down-regulated.The expression level of miR-487a-3p was significantly decreased in tumor group(Log2FC=-2.527,P<0.01).The expression level of miR-487a-3p was significantly lower in U-87 MG cells than that in HA 1800 cells(0.090±0.022 vs.1.000±0.205,t=7.659,P<0.05).Transfection of miR-mimic significantly up-regulated the expression of miR-487a-3p in U-87 MG cells(89.730±16.800 vs.1.000±0.351,t=9.146,P<0.05).The proliferation(24 h,0.600±0.034 vs.0.831±0.043,t=7.268,P<0.01;48 h,1.098±0.058 vs.1.624±0.074,t=9.708,P<0.01;72 h,1.615±0.055 vs.2.252±0.096,t=9.959,P<0.01),migration(97.750±9.575 vs.226.600±19.360,t=10.33,P<0.01),and invasion(74.330±9.501 vs.158.600±14.440,t=8.444,P<0.01)of U-87 MG cells from miR-mimic group were significantly inhibited.The level of PI3K/Akt signaling pathway of U-87 MG cells in miR-mimic group was significantly inhibited(0.211±0.384 vs.1.000±0.000,t=35.610,P<0.01).Conclusion MiR-487a-3p inhibits the proliferation,migration,and invasion of glioma cells by inhibiting the PI3K/Akt signaling pathway.