Correlation Between Plasma Exosomal MiR-223、miR-146a Expression Levels And Coronary Thrombus Burden In Patients With ST-segment Elevation Myocardial Infarction

Objective:To investigate the correlation between plasma exosome miR-223 and miR-146 a levels and infarct-related vascular thrombus burden in patients with ST-segment elevation myocardial infarction(STEMI),and to evaluate the possibility of plasma exosome miR-223 and miR-146 a as predictive biomarkers of thrombus burden in STEMI patients,in addition to using bioinformatics methods to explore their potential target genes,and to further analyse the potential mechanisms and possible signaling pathways involved in plasma exosome miR-223 and miR-146 a in the process of STEMI thrombosis.Methods:In this study,45 patients with STEMI who were admitted to Taizhou People’s Hospital for emergency percutaneous coronary intervention(PCI)from December 2021 to September 2022 were selected,and the thrombus burden of the infarct-related vessels(IRA)was assessed according to the TIMI thrombosis classification criteria,which included 25 patients with low thrombus burden and 20 patients with high thrombus burden,while 25 patients with normal coronary angiography or coronary CTA examination in the same period were selected as the control group.Clinical data including gender,age,body mass index(BMI),smoking history,previous medical history including history of diabetes mellitus,history of hypertension and relevant laboratory findings including troponin I(c Tn I)levels,routine blood count,blood biochemistry,and index of hemostasis and coagulation were collected.All participants enrolled in the study were required to take venous blood before PCI for the subsequent extraction of exosomes.Exosomes were identified by transmission electron microscope and nanoparticle tracking analysis.Total miRNA of exosomes was extracted by miRNeasy kit,and the Ct values of exosomal miR-223 and miR-146 a were detected by Quantitative Real-time PCR,to calculate their relative expression.SPSS26.0 software was used for statistical analysis to compare patients’ general data,laboratory indices and plasma exosomal miR-223 and miR-146 a expression levels for any statistical differences.The predictive value of plasma exosomal miR-223 and miR-146 a on thrombotic burden in STEMI patients was assessed by Receiver operating characteristic(ROC)curves.The miRDB,Target Scan Human 7.2and miRTar Base databases were used to predict miR-223,miR-146 a target genes,and R4.2.1 software was used for GO biological annotation,KEGG signaling pathway enrichment analysis and visualization,using the STRING database to analyse the target gene expression protein interaction network.Result:1.Extraction and identification of exosomes: under transmission electron microscope,the exosomes showed a round or circular structure with a double layer of lipid molecules,with a non-uniform size and distribution,their specific diameters were30-150 nm,the nanoparticle tracking analysis indicated that the peak particle size of exosomes was 101.2 nm and the average particle size was 93.5 nm,which was similar to the basic characteristics of exosomes.2.Relative expression of plasma exosomal miR-223 and miR-146a: plasma exosomal miR-223 and miR-146 a expressions were higher in STEMI patients than in the control group,with statistical differences(P < 0.05).The exosomal miR-223 and miR-146 a expressions were higher in the high thrombotic burden group than in the low thrombotic burden group in STEMI patients,with statistically different(P < 0.05).3.ROC curves: ROC curves showed that plasma exosome-derived miR-223 expression in the low and high thrombotic burden groups were compared when the area under the ROC curve AUC=0.877(95%CI: 0.758-1.000)(sensitivity 0.882,specificity0.81),and plasma exosome-derived miR-146 a expression ROC curve AUC=0.911(95%CI:0.802-1.000)(sensitivity 0.756,specificity 0.95),both of which were statistically different(p<0.05).4.Exosomal miR-223 and miR-146 a target gene prediction and bioinformatics analysis:(1)miR-223-3p and miR-146a-5p target gene prediction by miRDB,Target Scan Human 7.2 and miRTarbase database.miR-223-3p obtained 415,519 and 113 target genes,respectively,and a total of 27 target genes after taking intersection between the above three.miR-146a-5p obtained 283,488 and 221 target genes,and a total of 11 target genes after taking intersection between the above three.(2)GO biological analysis showed that miR-223-3p and miR-416a-5p target genes were mainly enriched in positive regulation of kinase activity,endothelial cell migration,and epithelial cell differentiation,etc.;in terms of cellular components,they were mainly enriched in cell cortex,sarcoplasm,plasma membrane rafts,etc.;in terms of molecular functions,they were mainly enriched in DNA/RNA synthesis,transcriptional regulation,etc.;(3)KEGG signaling pathway enrichment analysis showed that: miR-223-3p and miR-416a-5p target genes were mainly involved in MAPK signaling pathway,NF-κB signaling pathway,Toll-like receptorrelated pathway and lipid and atherosclerosis;(4)STRING database analysis of ProteinProtein Interaction Networks showed that 30 key target genes encode proteins which play key roles.Conclusions:1.Plasma exosomal miR-223 and miR-146 a are expressed higher in patients with high thrombotic burden in STEMI compared to those with low thrombotic burden,and have certain predictive value for thrombotic burden in STEMI patients.2.GO analysis of exosomal miR-223 and miR-146 a target genes was mainly enriched in biological processes and functions such as endothelial cell migration,epithelial cell differentiation,DNA/RNA synthesis and transcriptional regulation.3.KEGG enrichment analysis of exosomal miR-223 and miR-146 a target genes resulted in MAPK signaling pathway,NF-κB signaling pathway,Toll-like receptorrelated pathway and lipid and atherosclerosis.