miR-124-3p通过负调控Caveolin-1表达N2a/APPswe细胞凋亡率及细胞内钙离子浓度

miR-124-3p inhibits N2a/APPswe cell apoptosis and cellular calcium ion concentration via down-regulating Caveolin-1

目的:探讨在阿尔茨海默病(Alzheimer’s diseases,AD)细胞模型中miR-124-3p通过调控Caveolin-1的表达对细胞凋亡率及钙离子浓度的影响。方法:体外培养野生型N2a(N2a/WT)和N2a/APPswe细胞,real-time PCR及Western blot分别检测miR-124-3p与β-淀粉样蛋白前体蛋白(β-amyloid precursor protein,APP)m RNA及蛋白的表达。双荧光素酶报告实验分析miR-124-3p与Caveolin-1之间靶向调控关系。N2a/APPswe细胞瞬时转染miR-124-3p模拟物(mimics),real-time PCR、Western blot分别检测Caveolin-1 m RNA和蛋白的表达。N2a/APPswe细胞分别瞬时转染miR-124-3p模拟物(mimics)、pc DNA-Caveolin-1、Caveolin-1-si RNA,流式细胞仪分析细胞凋亡水平,测定细胞内游离钙离子浓度。结果:与WT组相比,APPswe组APP m RNA和蛋白水平表达都明显升高(分别为P=0.000,P=0.000),miR-124-3p表达明显降低(P=0.000)。双荧光素酶报告实验表明,和与突变型载体(p GL3-Caveolin-1 3’UTR MUT)共转染组相比,与野生型载体(p GL3-Caveolin-1 3’UTR WT)共转染组的相对荧光素酶活性和明显减弱(P=0.004);转染miR-124-3p mimics后,与空载体组比较,miR-124-3p mimcs转染组的Caveolin-1 m RNA和蛋白的表达明显下调(分别为P=0.000,P=0.000);分别转染miR-124-3p mimics、Caveolin-1-si RNA后,与空载体组比较,细胞凋亡率降低(分别为P=0.000,P=0.000),游离钙离子浓度降低(分别为P=0.000,P=0.000);转染pc DNA-Caveolin-1后,得到与上述相反的结果(分别为P=0.000,P=0.000)。结论:miR-124-3p可以通过靶向下调Caveolin-1的表达,抑制AD细胞凋亡,降低细胞中游离钙离子浓度,从而发挥神经保护作用,为AD的防治提供新的思路和靶点。

Objective：To investigate the effect of miR-124-3p on the apoptosis and the concentration of free calcium in cell by regulating the expression of Caveolin-1 in Alzheimer＇s diseases（AD）cell model of N2a/APPswe cells. Methods：N2a/APPswe and wild type N2a（N2a/WT）cells were cultured in vitro;real-time fluorescence quantitative PCR（real-time PCR）and Western blot（WB）were respectively used to detect the expression levels of miR-124-3p and amyloid precursor protein（APP）m RNA and protein. Dual luciferase report experiments were used to detect targeting regulation relationship between miR-124-3p and Caveolin-1. N2a/APPswe cells were transiently transfected by miR-124-3p mimics;real-time PCR and WB were respectively used to detect the expression levels of Caveolin-1 m RNA and protein. N2a/APPswe cells were transiently transfected by miR-124-3p mimics,pc DNA-Caveolin-1,Caveolin-1-si RNA and the rate of cell apoptosis was analyzed by flow cytometry. The intracellular free calcium concentration was measured.Results：Compared with those of WT group,APP m RNA and protein levels were significantly increased（P=0.000,P=0.000）in APPswe group,miR-124-3p expression was obviously decreased（P=0.000）. Dual luciferase report experiment showed that compared with those of co- transfection with the mutant vector（p GL3-Caveolin-1 3＇UTR MUT）group,relative luciferase activities in co-transfection with the wild type vector（p GL3-Caveolin-1 3＇UTR WT）group were significantly decreased（P=0.004）. After the transfection of miR-124-3p mimics,Caveolin-1 m RNA and protein levels were significantly decreased（P=0.000,P=0.000）in transfection group than in control group. After the transfection of miR-124-3p mimics and Caveolin-1-si RNA,the cell apoptosis rate and free calcium concentration were decreased（P=0.000）in transfection group than in control group（P=0.000）. After the transfection of pc DNA-Caveolin-1,the opposite results were obtained（P=0.000,P=0.000）. Conclusion：miR-124-3p can inhibit the apoptosis of cell and reduce the concentration of free calcium in cell by targeted down-regulation of Caveolin-1 expression,which plays a neuroprotective role in prevention and cure of AD and provides new ideas and targets for AD.