| Objective:The mechanisms of Alzheimer's disease(AD)is complex,and not clear completely until now.Recently many studies focus on the blood-brain barrier and angiogenesis.The permeability of blood-brain barrier in AD patients is open Vasculopathy exists in the onset and progression of AD,which also ahead of the pathological change and Clinical symptoms.MicroRNAs(miRNAs)are a class of small non-coding RNAs,approximately 22-24 nucleotides in length,playing important roles in cell development,differentiation,multiplication and apoptosis.miR-424-5p is the member of the miR-15/107 group.The miR-15/107 was reported in relation to human tumor,angiocardiopathy and neural degeneration disease including AD.miR-424-5p upregulated in anoxic vascular endothelial cell,and promoted the angiogenesis in vitro Endophilin-1 is a multifunction protein which located on cell membrane and cytoplasm of central nervous system,participates in the regulation of endocytosis and cell signal transduction.In our previous study,we proved that endophilin-1 regulates the permeability of BBB by altering the expression of ZO-1 and occludin via the EGFR-ERK1/2 and EGFR-JNK path-way.Moreover,the recombinant expression plasmid endophilin-1 was constructed successfully and over-expression of endophilin-1 suppressed endothelial cell tube formation in vitro.In our study,we established an vitro BBB model of AD with hCMEC/D3 cells Abeta-induced pre-transfected with overepression or silencing plasmids of miR-424-5p,then we investigated the effects of miR-424-5p on the permeability of BBB and angiogenesis in ADMethods:1.Cell cultures of human brain microvascular endothelial cell line hCMEC/D3 cells,hCMEC/D3 cells with Abeta-induced and human embryonic kidney cell line HEK293T cells.2.Establishment of transient transfection endothelial cell lines of overexpression or silencing of miR-424-5p,establishment of vitro BBB model of above-mentioned cells.3.Establishment of stable transfected enothelial cell lines of overexpression or silencing of Endophilin-1.4.Cotransfection with miR-424-5p and Endophilin-1 and establishment of vitro BBB model of above-mentioned cells.5.The endogenous expression level of miR-424-5p in hCMEC/D3 cells and hCMEC/D3 cells with Abeta-induced was detected by real-time quantitative PCR.6.The permeability of vitro BBB model was detected ty transendothelial electric resistance(TEER)and horseradish peroxidase(HRP)flux assays.7.The protein expression of Endophilin-1,tight junction related proteins Zonula occluden-1(ZO-1)and occludin in hCMEC/D3 cells with Abeta-induced were respectively determined by Western blot and immunofluorescence assays.8.The interaction of miR-424-5p and Endophilin-1 was evaluated by dual fluorescence reporter gene analysis.9.The proliferation ability of endothelial cells was detected by CCK-8 assay.10.The migration ability of endothelial cells was detected by Transwell inserts assay.11.The angiogenesis of ability of endothelial cells was detected by tube formation assay.Results:1.The transient transfection endothelial cell lines of overexpression or silencing of miR-424-5p were established successfully,also the vitro BBB model of above-mentioned cells.2.The stable transfection endothelial cell lines of overexpression or silencing of Endophilin-1 were established successfully.3.The endogenous expression level of miR-424-5p in hCMEC/D3 cells with Abeta-induced was higher than that in hCMEC/D3 cells.4.The overexpression of miR-424-5p significantly increased the permeability of BBB,the silencing of miR-424-5p significantly decreased the permeability of BBB.5.The overexpression of miR-424-5p significantly down-regulated the expression of tight junction related proteins ZO-1 and occludin in hCMEC/D3 cells with Abeta-induced,the silencing of miR-424-5p up-regulated the expression of ZO-1 and occludin in hCMEC/D3 cells with Abeta-induced.6.The binding site of miR-424-5p and Endophilin-1 was found.7.The overexpression of miR-424-5p significantly up-regulated the expression of Endophilin-1 in hCMEC/D3 cells with Abeta-induced,the silencing of miR-424-5p significantly down-regulated the expression of of Endophilin-1 in hCMEC/D3 cells with Abeta-induced.8.The permeability of BBB was regulated by combined action of miR-424-5p and Endophilin-1.9.The expression levels of ZO-1 and occludin were co-regulated by miR-424-5p and Endophilin-1.10.The cell proliferation of hCMEC/D3 cells with Abeta-induced was inhibited by overexpression of miR-424-5p,while promoted by silencing of miR-424-5p.11.The cell migration of hCMEC/D3 cells with Abeta-induced was inhibited by overexpression of miR-424-5p,while promoted by silencing of miR-424-5p.12.The tube formation of hCMEC/D3 cells with Abeta-induced was inhibited by overexpression of miR-424-5p,while promoted by silencing of miR-424-5p.13.The cell proliferation of hCMEC/D3 cells with Abeta-induced was inhibited by overexpression of Endophilin-1,while promoted by silencing of Endophilin-1.14.The cell migration of hCMEC/D3 cells with Abeta-induced was inhibited by overexpression of Endophilin-1,while promoted by silencing of Endophilin-1.15.The tube formation of hCMEC/D3 cells with Abeta-induced was inhibited by overexpression of Endophilin-1,while promoted by silencing of Endophilin-1.16.The cell proliferation,migration and tube formation of hCMEC/D3 cells with Abeta-induced were effected by the combined action of miR-424-5p and Endophilin-1.Conclusions:1.The endogenous expression level of miR-424-5p in hCMEC/D3 cells with Abeta-induced was higher than that in hCMEC/D3 cells.2.MiR-424-5p regulates the permeability of vitro BBB model with Abeta-induced by Endophilin-1,modulating the expression of tight junction related proteins ZO-1 and occludin.3.Endophilin-1 regulates the cell proliferation,migration and tube formation of hCMEC/D3 cells with Abeta-induced.4.MiR-424-5p effcets the cell proliferation,migration and formation of hCMEC/D3 cells with Abeta-induced by Endophilin-1. |
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