Differential Expression Of MicroRNAs In Cardiac Stem Cells Under Hypoxia And Bioinformatics Analysis

BackgroundPlaque rupture,thrombotic occlusion,or coronary artery spasms on the basis of coronary atherosclerosis which resulting in the cardiac tissue becomes ischemic and hypoxic are fundamental to the development of ST-elevation myocardial infarction(STEMI).Oxidative stress and energy metabolism caused by continuous hypoxia seriously affect cardiac function.STEMI patients are associated with poor prognosis.Our previous study found that non-high-density lipoprotein cholesterol is a predictor of non fatal recurrent myocardial infarction and the correlation between elevated liver transaminase and high mortality in patients with STEMI.Traditional drug therapy can prevent heart failure partly,but it cannot replace the necrotic tissue.Loss of functional cardiomyocytes result in diminished cardiac function and heart failure.In order to make the damaged myocardial tissue regenerate in different levels,different sources of stem cells transplantation,especially fetal or embryonic cardiac stem cells,have made great achievement in some experiments.The therapeutic use of stem cell therapy has become an important strategy for recovery of cardiac function and promoting endogenous repair.Although stem cell transplantation is a promising strategy for the treatment of heart disease,there is still a major challenge for regeneration and repairment of adult heart.Stem cell transplantation is associated with a lower relapse rate but a higher procedure-related mortality rate,which is one of the most urgent problems to be solved.It has been reported that cardiac stem cells can recover better under hypoxia conditions(5% oxygen)than under normoxia conditions(20% oxygen)after transplantation.Stem cells are more tolerant of oxidative stress in three-dimensional myocardial cell aggregation cultures than in monolayer transplanted stem cells.c-Kit –positive cardiac stem cells are particularly sensitive to oxidative stress at the time of differentiation,and are resistant to peroxidative induced cell death by expressing high levels of superoxide dismutase.However,hypoxic preconditioning enhanced survival and homing ability of c-Kit-positive cardiac stem cells during transplantation in infarcted myocardium.The mechanisms are not entirely clear.Maybe hypoxia condition in vitro is mimicking the vivo microenvironment.Inhibition of mitochondrial oxidative phosphorylation by hypoxia can also activate the expression of transcription factor HIF.Up-regulation of glucose transporters and glycolytic enzymes decrease mitochondrial enzymes and improve the efficiency of transport in turn affecting the stem cells fate.However,the current researches on microRNAs(miRNAs)expression of cardiac stem cells are not sufficient and the molecular mechanisms of miRNAs envoled in hypoxic environments are unclear,which become the focus of our research.The widespread regulation of miRNAs biogenesis,function and decay have been atracting increasing attention in recent years.It controls almost all gene expression and biological processes,including growth,proliferation,differentiation and apoptosis.The number of miRNA profiling studies have grown rapidly in recent years.How to interpret the enormous data information is as well as challenges in applying of gene chip technology.Bioinformatics is a researching tool,which is based on matematics,statistics,and computer programs.It is suited for massive high throughput screening applications by sequence comparisons,cluster analysis and path analysis.It can enrich the understanding of diseases from the molecular level.However,there are several issues that have to be considered,namely the inter-lab reproducibility of the results are often problematic due to the small sample sizes,technological variations between the studies,inconsistent annotation and ongoing discovery of new miRNAs,different detection methods used by different technological platforms,various methods for data processing and analysis.To overcome the limitations in current researches,we performed a meta-analysis applying the robust rank aggregation method,which combines the results of several individual studies in order to increase the statistical power,followed by pathway analysis.This research also adopts the new biological research model,that is,using the existing data information for theoretical conjecture,and then conducts experimental verification.AimsThe aims of the research were to find differential expression of miRNAs induced by hypoxia in c-Kit-positive cardiac stem cells,prediction of target gene and bioinformatics analysis of differentially expressed miRNAs.Methodsc-Kit-positive cardiac stem cells were isolated by immunomagnetic cell sorting and microbeads conjugated anti-rat c-Kit antibody.The expressions profiles of miRNAs in different time of hypoxia were obtained from Gene Expression Omnibus(GEO)database.We carried out a comprehensive meta-analysis of published studies that compared miRNA expression profiles between hypoxia and normal condition.Robust rank aggregation approach had been specifically designed for comparison of several ranked gene lists and identification of commonly overlapping genes.The results were tested in cardiac stem cells by RT-PCR validation method,followed by enrichment analyses of Gene oncology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)pathway to identify miRNAs deregulation in hypoxia and the pathways that key miRNAs may impact.The STRING database and Cytoscape software tool were used to provide a critical assessment and integration of protein–protein interactions.Resultsc-Kit-positive cardiac stem cells were cultured,isolated and evaluated.According to search criteria,4 hypoxia miRNAs expression profiling datasets were retrieved from GEO database.Three gene expression profiles(GSE47532,GSE60431,and GSE56870)contained miRNA expression profles of 12h-24 h hypoxia condition.We found the 3 most significantly up-regulated(miR-210,miR-574-3p and miR-193b*)and 2 down-regulated miRNAs(miR-29b-1* and miR-503).Three gene expression profiles(GSE47532,GSE52743 and GSE60431)contained miRNA expression profiles of 32h-48 h hypoxia condition.Among them,miR-210 was the most significantly up-regulated miRNAs with the prolongation of hypoxia,followed by miR-193b* and miR-574-3p.miR-503 was the most significant down-regulated miRNAs,followed by miR-29b-1*.The different miRNAs were verified using RT-PCR sequencing in c-Kit-positive cardiac stem cells in hypoxia condition(1% oxygen,24h).miR-210,miR-574-3p and miR-193b* were up-regulated and miR-29b-1* and miR-503 were down-regulated(P<0.05).Bioinformatics analysis was used for prediction and analysis of up-regulated and down-regulated miRNAs target genes.The target genes of different of miRNAs play a role in intracellular components and cell connections,cell processes and signaling pathways in GO enrichment analysis.Associated with protein autophosphorylation(GO:0046777),negative regulation of transcription from RNA polymerase II promoter(GO:0000122),receptor signaling protein serine/threonine kinase activity(GO:0004702),GTPase activator activity(GO:0005096),cell junction(GO:0030054),channel activity(GO:0015267),passive ransmembrane transporter activity(GO:0022803),mRNA catabolic process and RNA catabolic process(GO:0006402,GO:0006401),positive regulation of apoptosis(GO:0043065),positive regulation of programmed cell death(GO:0043068),ubiquitin protein ligase binding(GO:0031625),modification-dependent protein catabolic process(GO:001994),modification-dependent macromolecule catabolic process(GO:0043632),protein serine/threonine kinase activity(GO:0004674),mRNA binding(GO:0003729),golgi apparatus(GO:0005794)and endoplasmic reticulum(GO:0005783).KEGG signal pathway analysis indicated that the functions of differential miRNAs were mainly focused on the signal pathway and the interaction of cytokines,such as PPAR signaling pathway(KEGG:03320),TGF-beta signaling pathway(KEGG: 04350),phosphatidylinositol signaling system(KEGG: 04070),signaling pathways regulating pluripotency of stem cells(KEGG:04550),HIF-1 signaling pathway(KEGG:04066),ErbB signaling pathway(KEGG: 04012),metabolic pathways(KEGG: 01100),Jak-STAT signaling pathway(KEGG: 04630),Ras signaling pathway(KEGG:04014),apoptosis(KEGG:04210),PI3K-Akt signaling pathway(KEGG:04151),Hippo signaling pathway-multiple species(KEGG:04392),NF-kappa B signaling pathway(KEGG:04064).ConclusionsThe expression of miRNAs at hypoxia in c-Kit-positive cardiac stem cells contained miR-210,miR-574-3p,miR-193b*,miR-29b-1* and miR-503,which played an important role in regulation of growth factors and transcription factors,cell cycle and its associated downstream signaling pathways,ultimately affecting metabolism and gene expression.