Mechanism Of MiR-218 In EndMT-mediated Cardiac Fibrosis

Objective:Endothelial-to-mesenchymal transition(EndMT)is one major origin of fibroblasts in cardiac fibrosis.In this work,we investigated the role and mechanism of miR-218 in EndMT-mediated cardiac fibrosis.This study may provide novel biomarkers and therapeutic targets for cardiac fibrosis.Methods:1.Expression of miR-218 in EndMT:Endothelial progenitor cells(EPCs)were isolated from human umbilical cord blood and cultured in endothelial cell growth medium.EPCs were treated with TGF-?1 to induce EndMT.Inverted microscope was used to observe the alteration of cellular morphology.The cellular markers were detected by qPCR,Western blot and Immunofluorescent staining.Cell migration was assessed using transwell chambers.Angiogenic function was assessed by matrigel assay.qPCR was used to measure the expression of miR-218.2.Effects and Mechanisms of miR-218 on EndMT:miR-218 mimic and inhibitor were used to overexpress or knockdown miR-218.After transfected with miR-218 mimic and inhibitor,the cellular markers,migration and angiogenic function were detected.The expression of MeCP2 was detected after miR-218 overexpression,miR-218 knockdown,and TGF-?1 treatment.Luciferase reporter assays were used to detected binding site of miR-218 and MeCP2.EPCs were transfected with adenoviral vectors harboring wild type or shRNA MeCP2 to overexpress or knockdown MeCP2.The cellular markers,migration and angiogenic function were detected after transfection with MeCP2 and co-transfection with miR-218.3.Effects of miR-218 on cardiac fibrosis:Cardiac fibrosis was induced by osmotic mini-pump with Ang II infusion.EPCs transfected or co-transfected with miR-218 and MeCP2 were injected through tail vein.Amount of fibrosis was determined by Masson trichrome staining.Results:1.Aberrant expression of miR-218 in TGF-?1-induced EndMT:Primary EPCs showed a typical cobblestone appearance,and combined with Ac-LDL and UEA-I.Cell morphology was changed to a spindle,fibroblast-like shape after TGF-?1 treatment for 48h.Cells that underwent EndMT displayed significantly reduced levels of the endothelial markers CD31 and vWF along with increased levels of the myofibroblastic markers VIM and ?-SMA.Migration ability increased and angiogenic function declined.Moreover,miR-218 was decreased significantly in TGF-?1-treated EPCs.2.miR-218 inhibits EndMT by targeting MeCP2:miR-218 overexpression significantly promoted endothelial markers expression and angiogenesis,while reduced myofibroblastic markers expression and migration.miR-218 knockdown had opposite effects.miR-218 overexpression suppressed MeCP2 expression,while TGF-?1 and knockdown of miR-218 increased MeCP2 expression.miR-218 directly combined with MeCP2 in 3'UTR.MeCP2 promoted EndMT and decreased the effects of miR-218 on EndMT.3.miR-218 inhibits EndMT-mediated cardiac fibrosis:Compared to control,higher amounts of collagen were established after 28-day infusion of Ang ?.Injection of EPCs overexpressing miR-218 attenuated cardiac fibrosis.The effects of miR-218 on cardiac fibrosis decreased after treatment with EPCs transfected miR-218 and MeCP2.Conclusions:miR-218 inhibits EndMT-mediated cardiac fibrosis in EPCs by targeting MeCP2.The inhibition of MECP2 by miR-218 will be a potential therapeutic strategy for cardiac fibrosis.