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

摘要:

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）mRNA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 mRNA和蛋白的表达。N2a/APPswe细胞分别瞬时转染miR-124-3p模拟物（mimics）、pcDNA-Caveolin-1、Caveolin-1-siRNA，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 mRNA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（pGL3-Caveolin-1 3’UTR MUT）共转染组相比，与野生型载体（pGL3-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 mRNA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-siRNA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pcDNA-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。

Abstract:

Objective：To investigate the effect of miR-124-3p on the apoptosis and the concentration of free calcium in cell by regu－lating 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 re－spectively used to detect the expression levels of miR-124-3p and amyloid precursor protein（APP） mRNA 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 mRNA and protein. N2a/APPswe cells were transiently transfected by miR-124-3p mimics，pcDNA-Caveolin-1，Caveolin-1-siRNA 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 mRNA and protein levels were significantly increased（P=0.000，P=0.000） in APP－swe 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（pGL3- Caveolin-1 3’UTR MUT） group，relative luciferase activities in co-transfection with the wild type vector（pGL3-Caveolin-1 3’UTR WT） group were significantly decreased（P=0.004）. After the transfection of miR-124-3p mimics，Caveolin-1 mRNA 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-siRNA，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 pcDNA-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 neuro－protective role in prevention and cure of AD and provides new ideas and targets for AD.