Bioinformatics-based Screening Potential Biomarkers And Regulation Targets Of Acute Myocardial Infarction

Acute myocardial infarction(AMI)is one of the most severe types of coronary heart diseases.Partially or completely occluded coronary arteries caused part of myocardial tissue necrosis as a result of continuous ischemia and hypoxia.The 2012 WHO report showed that a total of 17,500,000 people in the world died of cardiovascular disease,and among the 3,500,000 people who died of cardiovascular disease in 2013 in China,2,500,000(71%)people were attacked and died of myocardial infarction.Therefore,myocardial infarction has become the leading cause of death from cardiovascular disease.ObjectiveOver the recent decades,biomarkers have made important contributions to the diagnosis,treatment and prognosis of AMI.However,AMI is still a critical disease with high fatality rate.Due to the unconspicuous early symptoms,abruptness and high fatality rate of AMI,great difficulty exists in the prevention,diagnosis and treatment of the diseas.An ideal biomarker is needed for rapid diagnosis or exclusion of AMI,guiding treatment and judgment of prognosis.With the development of microarray technology,the microarray data volume is steadily on the increase.By analysis and utilization of this big data,the occurrence and development of the AMI can be more accurately understood at the molecular level.Using microarray expression profiling analysis in the early diagnosis can effectively understand the underlying cause of the disease,learn the genetic pathogenesis of AMI and provide a new idea strategy for the treatment of the disease.In this study,a variety of bioinformatics analytical methods were adopted to conduct data mining on the microarray expression profiling of AMI peripheral blood.These methods include analysis of differential expression gene,functional enrichment analysis,pathway enrichment analysis,and analysis of gene interaction,network relation between pathways,protein-protein interaction network and weighted gene co-expression network.The experiment will be the basis of further study the diagnosis and the possible regulation mechanism through screening of potential biomarkers of AMI.Trichostatin A(TSA)is a broad-spectrum reversible histone deacetylase(HDAC)inhibitors,and the currently known most effective HDAC inhibitor.The preliminary work of the research group has confirmed that TSA can inhibit the apoptosis of myocardial cells and the generation of multiple inflammatory factors and has presented significant curative effect on various myocardial disease models.The early results gotten by the research group showed that in AMI rat models,TSA can reduce myocardial infarction size,improve cardiac function,alleviate the degree of inflammation,inhibit the apoptosis of myocardial cells and protect the structure of myocardial tissues,but the specific regulatory mechanism remains unclear.Methods1 ? Microarray data GSE48060 was downloaded from the GEO database,including peripheral blood samples of 31 cases of AMI patients and 21 cases of healthy control samples,and subsequent bioinformatics analysis was conducted on the samples.Limma package in R software was used to calculate the differentially expressed genes(DEGs),and P-value<0.05 and Fold change>1.2 were selected as the significant threshold values to screen the DEGs.GO database was used to carry out GO functional annotation on the DEGs;based on Kyoto Encyclopedia Genes and Genomes(KEGG),pathway enrichment analysis was conducted on the DEGs.Gene function regulation network was built based on the GO hierarchical structure;the mutually regulative relations between the pathways were sorted into a database to build the signaling pathway regulatory network;then the transcriptional regulatory network of DEGs was built based on JASPAR database.2?Affy package and limma package in R software were used to screen the DEGs,and the Pearson correlation and K-core value between two arbitrary genes were calculated to act as the screening condition for differentially co-expression genes.Subsequently,weighted gene co-expression network analysis(WGCNA)was used to obtain gene modules,weight gene co-expression network,hub genes and the results of GO and pathway enrichment analysis of gene modules.3?A total of 38 cases of patients with AMI and 25 cases of healthy controls were selected in the study.Elisa kit was used to test the levels of IFN-? and IL-4 in blood serum.RT-PCR was adopted to detect the mRNA levels of TBX21,GATA-3,IFN-? and IL-4 in PBMCs.The clinical and biochemical reports of the patients were collected and relevant indexes were sorted.Gensini's degree integral was used to evaluate the degree of coronary artery stenosis.Statistical methods were used to process the data and correlation research was conducted on the level of TBX21,so as to assess the possibility of TBX21 as potential biomarkers.4?In this study,Wistar rats were used as experimental subjects,and AMI rat models were built by ligating the left anterior descending branch of coronary artery,and the time points were 1 day,3 days and 7 days.TTC staining method was used to de termine the degree of myocardial infarction and to observe the protective effect of TSA on rats with myocardial infarction;the effect of TSA on cardiac morphology was observed by HE staining method;RT-PCR was used to detect the level of mononuclear cells in peripheral blood of rats,and levels of TBX21 and GATA-3 in infarcted myocardial tissues of rats,so as to preliminarily observe the effect of TSA on the levels of mRNA of TBX21 and GATA-3.Western-blot was used to detect the expression of TBX21 in myocardial tissues of rats as well as the effect of TSA on the level of TBX21protein;Elisa kit was adopted to detect the expression of IFN-? and IL-4 in AMI rat serum.Results1?AMI microarray expression profiling data was downloaded from the GEO database,including peripheral blood samples of 31 cases of AMI patients and 21 healthy control samples.Bioinformatics analysis was conducted on the AMI DEGs using packages of affy,limmar,clusterProfiler and pathview in R Bioconductor and Genesis,GO database,KEGG and other tools.A total of 551 DEGs were obtained,including 164 up-regulated genes and 387 down-regulated genes.GO and pathway enrichment analysis were conducted on the DEGs,and gene-act-net,GO-tree and pathway-act-net.when AMI occurs,the main accompanied biological process is inflammatory response,including Inflammatory Response(GO:0006954),Chemotaxis(GO:0006935)and Immune Response(GO:0006955).The core pathways mainly contain chemotactic factor signaling pathway(PATH:04062),Toll-like receptor signaling pathway(PAHT:04620)and JAK/STAT signaling pathway(PATH:04630).2?The DEGs in peripheral blood of AMI patients and healthy controls were taken as data source to conduct WGCNA,and a total of 4 gene modules(blue?brown?yellow and turquoise module)closely related to GO classifications such as inflammatory response,immune response,cellular defensive response,platelet activation and blood coagulation were obtained.Through the correlation analysis between the modules and the disease,it can be known that the blue and brown modules are inflammation modules.Subsequently,pathway analysis was carried out on each pair of modules to obtain the pathway enrichment of the modules.The gene co-expression network of each pair of modules was constructed and the GS,MM and IC values of the modules were synthesized.The results of RT-PCR were used to detect 20 patients with acute myocardial infarction and 15 healthy controls,and the expression of 8 hub genes in PBMCs of acute myocardial infarction was consistent with that of bioinformatics analysis,and TBX21,AQP9,PRF1 and NFIL3.Finally,according to the transcription factor of differentially expressed genes and its target regulatory network,TBX21 is the core transcription factor in transcriptional regulation network.It is speculated that TBX21 is closely related to the development of acute myocardial infarction and may become acute myocardium Potential biomarkers of infarcts and possible regulatory targets.3?The results of 38 patients with acute myocardial infarction and 25 healthy controls showed that TBX21 and GATA-3 levels were significantly decreased in PBMCs of patients with acute myocardial infarction compared with the control group.Elisa results showed that compared with the control group,the expression of IFN-? was up-regulated and the expression of IL-4 was down-regulated in acute myocardial infarction.The clinical significance of TBX21 suggests that TBX21 levels are negatively correlated with coronary artery stenosis(Gensini score)and are closely related to serum creatinine,fibrin eclay and low-density lipoprotein in risk factors for acute myocardial infarction.4?AMI rat model was built.RT-PCR was used to detect the expression of TBX21 in myocardial tissue and PBMCs.The result showed that the expression of TBX21 decreased;Western-blot result showed that TBX21 decreased firstly on the first dayand then increased in myocardial tissue;Elisa kit test results showed that the expression of IFN-? was negatively correlated with that of IL-4 in serum of rats of AMI.The above analysis results are consistent with the clinical sample test results.5?TSA can up-regulate the level of TBX21 mRNA in myocardial tissue and PBMCs of rats with AMI and up-regulate the expression of protein TBX21.TSA inhibits the abnormal expression of IFN-? and IL-4 in serum of rats with AMI,reduces the area of myocardial infarction and alleviates the morphologic change of myocardial tissues induced by ischemic injury.Conclusion 1.The clinical samples verified that the expression levels of the eight hub genes wereconsistent with those analyzed by bioinformatics.The key transcription factorTBX21 with the most significant difference may be a potential biomarker and apossible regulating target for AMI.2.The analysis results of clinical samples showed that the level of TBX21 issignificantly correlated with the myocardial injury marker,the risk factors of AMIand the degree of coronary artery stenosis.3.In the AMI rat model,the levels of mRNA and proteins of TBX21 significantlydecreased,and TSA can restore the abnormal expression of TBX21.In summary,TBX21 may be a potential biomarker and regulating target of AMI.