The Molecular Mechanism Of Neuroprotection Of MiR-124-3P Via Down-Regulating Caveolin-1 In Alzheimer’s Disease

Objective:Alzheimer’s disease (AD) is the most common type of dementia, its pathogenesis is still not clear so far. The role of microRNA(miRNA) has been increasing concerned and even becomes a hot spot in neurodegenerative diseases. Caveolin-1 is a marker for caveole protein of cell membrane; studies found that it is closely related to the AD. In our study, it is aimed to confirm the inhibition of targeting Caveolin-1 by miR-124-3p in AD cell model. Then, the aim is to investigate the regulation of phosphorylation levels of Tau protein and the effect of cell apoptosis rate by miR-124-3p and its potential mechanism in vitro, with expecting to shed light on pathogenesis and therapeutic targets of AD.Methods:N2a/APPswe and wild type N2a (N2a/WT) cells were cultured in vitro, real-time fluorescence quantitative PCR (Realtime-PCR) and Western blot(WB) was respectively used to detect the expression of miR-124-3p,APP and Caveolin-1. Dual luciferase report experiments were used to detect targeting regulation relationship between miR-124-3p and Caveolin-1. RT-PCR was performed to detect the RNA expression of Caveolin-1 and Tau, the rate of cell apoptosis was analyzed by flow cytometry,WB was carried out to detect the changes of Caveolin-1, Tau, Tau-Ser404, GSK-3β、GSK-3β-Ser9, PI3K, Akt, Akt-Ser473 at protein level after respective transient transfectin of miR-124-3p mimics, pcDNA Caveolin-1 and Caveolin-siRNA in N2a/APPswe cells.Results:The expression of miR-124-3p was significantly decreased in APPswe group in comparison to Wild Type (WT) group (P<0.01), while the mRNA and protein of APP was increased (P<0.01), and Caveolin-1 was increased (P<0.001;P<0.05); dual luciferase report experiment showed that the relative luciferase activity in co-transfection with the wild type vector (pGL3-Caveolin-1 3’UTR WT) group were significantly decreased, compared with co-transfection with the mutant vector (pGL3-Caveolin-1 3’ UTR MUT) group (P<0.01); the expression of Caveolin-1 deceased (P< 0.001); the rate of cell apoptosis decreased (P<0.001),the expression of Tau-Ser404/Tau decreased (P<0.01), the expression levels of PI3K, Akt-Ser473/Akt increased (P<0.01), GSK-3β-Ser9/GSK-3β increased (P <0.001); in N2a/APPswe cells after respective transfection of miR-124-3p mimics and Caveolin-1-siRNA; the rate of cell apoptosis increased (P< 0.001),the expression of Tau-Ser404/Tau increased (P< 0.01), the expression of PI3K, Akt-Ser473/Akt,GSK-3β-Ser9/GSK-3β decreased (P <0.001) in N2a/APPswe cells after transfection pcDNA-Caveolin-1.Conclusion:In AD cell model, Caveolin-1 was targeted for negative regulation by miR-124-3p, and Caveolin-1 can regulate the phosphorylation level of Tau protein via PI3K/AKT/GSK3β signaling pathway. Therefore, we conclude that the regulation pathway, miR-124-3β-Caveolin-1-PI3K/Akt/GSK3β can inhibit abnormal hyperphosphorylation of Tau protein to show neuroprotection (inhibition of cell apoptosis) in AD, which may provide new ideas for prevention and treatment of AD.