Roles Of MicroRNA In Vascular Damage And Repair

Part IDown regulation of microRNA-126in endothelial progenitor cells from diabetespatients, impairs their functional properties, via target gene Spred-1Objectives:Diabetes mellitus (DM) adversely affects the number and function of circulatingendothelial progenitor cells (EPCs). Consequently, there is also a reduction in therepair mechanism of these cells, which is a critical and initiating factor in thedevelopment of diabetic vascular disease, such as atherosclerosis. The aim of thepresent study was to analyze the expression profiles of microRNA in EPCs frompatients with DM and choose the most significantly regulated microRNA to study itspossible role in the dysfunction of EPCs and elucidate its mechanism.Methods:EPCs were collected from subjects with Type II DM and non-diabetic controlsubjects which were gender,age-matched. Total RNA was harvested from EPCs, and atotal of5candidate miRNAs were identified through microarray screening and werequantified by TaqMan real-time PCR. Lentiviral vectors expressing miR-126andmiR-126inhibitor (anti-miR-126) were transfected into EPCs in order to test thefunction of EPCs and the expression level of the target gene of miR-126,Spred-1. Weused the colone formation assay to detect the colony-forming capacity of EPCs; MTTassay was used to testify the proliferation activity; migratory activity of EPCs wasdetermined by improved Boyden Chamber invasion assay; flow cytometry wasutilited to test the differentiation capacity and the apoptotic susceptibility.WesternBlotting and mRNA real-time PCR analyses were performed to assess the expressionof Spred-1. To study the precise mechanisms, Lentiviral vectors expressing Spred-1and a short interfering RNA (siRNA) targeting Spred-1were prepared to measure thecapacity of colony-forming, proliferation, apoptosis, differention and migratory ofEPCs.Real-time PCR and Western Blotting experiments were done to determine the gene expression level of Ras/ERK/VEGF and PI3K/Akt/eNOS.Results:Five miRs were aberrantly downregulated in EPCs from DM patients. ThesemiRs included miR-126, miR-21, miR-27a, miR-27b and miR-130a,miR-107.Downregulation of miR-126in EPCs inhibited the capacity of colone formation,proliferation, migration，however the apoptosis was enhanced in EPCs. RestoredmiR-126expression in EPCs from DM promoted EPC colony-forming, proliferation,migration, and inhibited EPC apoptosis ability. Therefore, the inbibition of miR-126in EPCs shown the absolutely outcomes. Despite this, we found out that miR-126hadno effect on EPC differentiation. miR-126overexpression significantlydownregulated Spred-1in EPCs. The knockdown of Spred-1expression in EPCs fromDM promoted proliferation, migration, and inhibited apoptosis of the cells. Theoverexprseeion of miR-126in EPCs could up-regulate the level of p-ERK1/2/VEGFand Akt/eNOS, however the target gene Spred-1showed the opposite function.Conclusions: This study provides the first evidence that five microRNAs were founddown-regulated in EPCs with diabetes mellitus, including miR-21, miR-27a，miR27b，miR-126and miR-130a.T overexpression of miR-126in EPCs can reduce the level ofSpred-1which can improve the capacity of proliferation, migration and inhibitcellular apoptosis, so that could repair the function of EPCs in diabetes. PARTIIDownregulation of microRNA-130a contributes to endothelial progenitor celldysfunction in diabetic patients via its target Runx3Objectives:Dysfunction of endothelial progenitor cells (EPCs) contributes to diabeticvascular disease. MicroRNAs (miRs) have emerged as key regulators of diversecellular processes including angiogenesis. We recently reported that miR-126, miR-130a, miR-21, miR-27a, miR-27b and miR-107were downregulated in EPCsfrom type II diabetes mellitus (DM) patients, and downregulation of miR-126impairsEPC function. The present study further explored whether dysregulated miR-130awere also related to EPC dysfunction.Methods:EPCs were cultured from peripheral blood mononuclear cells of diabetic patientsand healthy controls. Assays on EPC function (proliferation, migration, differentiation,apoptosis, and colony and tubule formation) were performed. Bioinformatics analyseswere used to identify the potential targets of miR-130a in EPCs. Gene expression ofmiR-103a and Runx3was measured by real-time PCR, and protein expression ofRunx3, extracellular signal-regulated kinase (ERK), vascular endothelial growthfactor (VEGF) and Akt was measured by Western blotting. Runx3promoter activitywas measured by luciferase reporter assay. A miR-130a inhibitor or mimic andlentiviral vectors expressing miR-130a, or Runx3, or a short hairpin RNA targetingRunx3were transfected into EPCs to manipulate miR-130a and Runx3levels.Results:MiR-130a was decreased in EPCs from DM patients. Anti-miR-130a inhibitedwhereas miR-130a overexpression promoted EPC function.MiR-130a promoted EPCscolony formation, migration, differentiation and EPCs tube formation.MiR-130anegatively regulated Runx3(mRNA, protein and promoter activity) in EPCs.Knockdown of Runx3expression enhanced EPC colony formation, migration,differentiation. MiR-130a also upregulated protein expression of ERK/VEGF and Aktin EPCs.Conclusions:Down-regulate of miR-130a in EPCs from DM impaired EPC function.MiR-130a enhance EPCs colony formation, migration, differentiation and EPCs tubeformation,and likely via its target Runx3and through ERK/VEGF and Akt pathways. PART IIIMicroRNA107partly inhibits endothelial progenitor cells differentiation via HIF-1βObjectives:Endothelial progenitor cells (EPCs) play an important role in tissue repair afterischemic heart disease. In particular, the recovery of endothelial function is reliant onthe ability and rate of EPCs differentiate into mature endothelial cells. The presentstudy evaluated the effect of microRNA107(miR-107) on the mechanism of EPCsdifferentiation.Methods:EPCs were isolated from rats’ bone marrow and miR-107expression of EPCs inhypoxic and normoxic conditions were measured by real-time qualitative PCR. CD31was analyzed by flow cytometry and eNOS was examined by real-time qualitativePCR and western blotting and these were used as markers of EPC differentiation. Inorder to reveal the mechanism, we used miR107inhibitor and lentiviral vectorexpressing a short hairpin RNA (shRNA) that targets miR-107and hypoxia-induciblefactor-1β (HIF-1β) to alter miR107and HIF-1β expression.Results:MiR-107expression were increased in EPCs under hypoxic conditions. Up-regulationof miR-107partly suppressed the EPCs differentiation induced in hypoxia, whiledown-regulation of miR-107promoted EPC differentiation.Up-regulation of miR-107in EPCs substantially decreased HIF-1β expression, and vice versa.up-regulation ofmiR-107would inhibit EPCs differentiation via inhibiting HIF-1β.Down-regulation ofmiR-107would promote EPCs differentiation via up-regulating HIF-1βConclusions:This study indicated that miR-107was up-regulated in hypoxia to prevent EPCsdifferentiation via its target HIF-1β. The physiological mechanisms of miR-107mustbe evaluated if it is to be used as a potential anti-ischemia therapeutic regime.