The Mechanism Of MECP2/SIRT1 Mediated Regulation Of Senescent EPCs

Objective:EPC senescence is the initiate change and critical progression of atherosclerosis forming and developing.To clarify the mechanism of EPCs senescence and find the target is effective to prevent EPC senescence.MeCP2 is a transcription factor expressed in various tissues and cells.MeCP2 expressed imbalancedly in many pathological states.The effect of MeCP2 on EPCs is unclear.This article aims to study whether MeCP2 regulates senescent EPCs dysfunction through depressing SIRT1 and the mechanism.Methods:EPCs were isolated and cultured from human umbilical cord blood,and identified by surface marker and the ability of uptaking acetylated low density lipoprotein?Ac-LDL?and combining ulex europaeus agglutinin 1?UEA-1?.Continuously subcultured EPCs were used as replicative senescence model,H₂O₂treated EPCs were identified as stress-induced premature cellular senescence model.CCK-8 was used to test cell survival,cell cycle experiments were performed to detect cell proliferation ability,Transwell assay and scratch test were used to detect cell migration ability,matrigel assay was performed to observe angiogenesis,flow apoptosis was performed to test cell apoptosis.Immunofluorescence was performed to observe cellular localization of MeCP2.Western blot and qPCR were respectively used to detect SIRT1 and MeCP2 protein and mRNA expression level.EPCs transfected with adenovirus vector containing wild-type MeCP2 or short hairpin RNA MeCP2 was used to overexpress or silence MeCP2.Bisulfite sequencing PCR was used to test SIRT1 promoter methylation level.Chromatin immunoprecipitation was performed to observe the DNA-protein interaction between SIRT1 promoter and MeCP2 or H3K9me2.Results:Endothelial progenitor cells grew in colonies and showed a cobblestone morphology,they were double-positive stained for Dil-Ac-LDL and FITC-UEA-1,and were high expressed with CD34,CD133 and VEGFR-2.Cells repeatedly subcultured till the 25th passage appeared irregular in shape and growth arrest,cells that were incubated with 40?M H₂O₂ for 24 h and then replaced fresh medium for another 3 days'culture also exhibited the same condition as P25.The survival rate of H₂O₂ treated cells was reduced and dose dependent.EPC function declined and impaired with senescence,MeCP2 protein and mRNA expression levels increased and positivly related with replicative passages,while SIRT1 protein and mRNA expression levels lowered and negatively connected with replicative passages.EPCs function declined with MeCP2 overexpressed,SIRT1 protein and mRNA expression were down-regulated in MeCP2 overexpressed EPCs.SIRT1 protein and mRNA expression were up-regulated with MeCP2 silence.SIRT1 DNA promoter was hyper-methylated in MeCP2 overexpressed EPCs,and more MeCP2 proteins and H3K9me2 were rich in SIRT1 promoter region than control.Conclusion:MeCP2 mediated senescent EPCs dysfunction,the mechanism might be that MeCP2 inhibit SIRT1 transcription through SIRT1 DNA methylation and H3K9dimethylation.