miR-495靶向Notch1对神经胶质瘤细胞增殖、迁移及侵袭的影响

Effect of miR-495 targeting Notch1 on the proliferation,migration and invasion of glioma cells

目的探究miR-495靶向Notch1对神经胶质瘤细胞增殖、迁移及侵袭的影响。方法采用实时荧光定量PCR(qRT-PCR)实验检测神经胶质瘤组织和癌旁组织中miR-495表达水平,然后体外培养人神经胶质瘤细胞U87,随机分为对照组、miR-495 mimics阴性对照组、miR-495 mimics组,分组转染后,CCK-8实验测定各组U87细胞活力,流式细胞术测定各组U87细胞凋亡率;细胞划痕实验、Transwell侵袭实验分别检测各组U87细胞迁移、侵袭力;免疫印迹实验检测各组U87细胞凋亡相关蛋白(Caspase-3和Bax)、上皮间质转化(EMT)相关蛋白(E-cadherin和Vimentin)及Notch1蛋白表达水平;qRT-PCR实验检测各组U87细胞miR-495和Notch1 mRNA表达水平。结果与癌旁组织相比,神经胶质瘤组织中miR-495表达水平显著降低(P<0.05);与对照组相比,miR-495 mimics组U87细胞活力、细胞迁移数、细胞侵袭数、Notch1 mRNA表达及Vimentin、Notch1蛋白表达显著降低,细胞凋亡率、miR-495表达及Caspase-3、Bax、E-cadherin蛋白表达显著升高,差异有统计学意义(P<0.05)。结论miR-495能靶向下调Notch1表达,抑制神经胶质瘤细胞增殖、迁移及侵袭,促进其凋亡。

Objective To investigate the effect of miR-495 targeting Notch1 on the proliferation,migration and invasion of glioma cells.Methods The expression levels of miR-495 in glioma tissues and adjacent tissues were detected by the real-time quantitative PCR (qRT-PCR),and then the human glioma cells U87 were cultured in vitro and randomly divided into a control group,and an miR-495 mimics negative control group and an miR-495 mimics group.After transfection,the activity of U87 cells in each group was determined by the CCK-8 assay,and the apoptosis rate of U87 cells in each group was determined by the flow cytometry.The migration and invasion of U87 cells in each group were detected by the cell scratch assay and Transwell invasion assay.The expression levels of apoptosis-related proteins (caspase-3 and Bax),epithelial-mesenchymal transition (EMT) related proteins (E-cadherin and Vimentin) and Notch1 protein of U87 cells in each group were detected by Western blotting.The expression levels of miR-495 and Notch1 mRNA in U87 cells in each group were detected by the qRT-PCR assay.Results Compared with the adjacent tissues,the expression level of miR-495 in the glioma tissues was significantly decreased (P<0.05).Compared with the control group,the U87 cell viability,cell migration number,cell invasion number,the protein expression levels of Notch1 mRNA and Vimentin and Notch1 were significantly decreased,while the apoptosis rate,miR-495 expression and the protein expression levels of caspase-3,Bax and E-cadherin were significantly increased (P<0.05).Conclusion In a targeted manner,miR-495 can down-regulate the expression of Notch1,inhibit the proliferation,migration and invasion of glioma cells,and promote their apoptosis.