Screening Of Differentially Expressed Micrornas In Serum Exosomes Associated With Acute Myocardial Infarction Based On A New Generation Of Sequencing

Objective: In this study,we used a new generation of sequencing technology to obtain the differentially expressed microRNAs in serum exosomes of acute myocardial infarction,and to identify the differentially expressed microRNAs;to study the role of differentially expressed microRNAs in the diagnosis of acute myocardial infarction;to identify the target genes related to them,and to elucidate their role in acute myocardial infarction.Methods: In this study,20 serum samples from patients with acute myocardial infarction and 20 healthy adults were collected to extract exosome RNA.The expression changes of exosome markers CD63 and CD81 were detected by Western blot.The differential expression profiles of microRNAs in serum exosomes of patients with acute myocardial infarction were obtained by new generation sequencing technology combined with bioinformatics analysis.The differential exosome microRNAs were screened by RT-PCR.To validate the relationship between serum exogenous microRNAs and the diagnosis,treatment and prognosis of acute myocardial infarction,and to predict the target genes of secondary screened microRNAs,and to reveal the possible mechanism of theirrole in acute myocardial infarction.Results:1.The body mass index of patients with acute myocardial infarction was higher than that of normal subjects,indicating that patients with myocardial infarction had a tendency to obesity;troponin T(cTnT),myoglobin,creatine kinase(CK)and creatine kinase isoenzymes in patients with acute myocardial infarction.The expression of CK-MB in acute myocardial infarction was higher than that in normal subjects(P < 0.05).2.The expression of CD63 and CD81 could be detected in serum exosomes of patients with AMI and normal persons,but the expression of internal reference beta-actin could not be detected.The expression of CD63 and CD81 in leukocytes of patients with AMI and normal persons is very low,while the expression of beta-actin is very low.These results suggest that CD63 and CD81 can be used as marker proteins of exosomes.3.Differential expression profiles of microRNAs: 89 differentially expressed microRNAs were obtained in patients with acute myocardial infarction and healthy adults,including 63 up-regulation and 26 down-regulation.4.Real-time fluorescence quantitative PCR was used to validate the differential expression profiles.Three up-regulated miR-1,miR-133 a and miR-208 a were selected to detect the expression of miR-1 in patients with AMI and healthy adults.The results showed that the up-regulated trend of mir-NA gene was consistent with the sequencing results.5.Bioinformatics analysis: The target genes regulated by microRNA-1 were945 and 883 in microRNA DB and Targetscan respectively,and there were 32 common overlapping genes.The target genes regulated by microRNA-133 a were654 and 572 in microRNADB and Targetscan respectively,and there were 23 common intersecting genes among them.The target genes regulated by microRNA-208 a were 210 and 201 in microRNADB and Targetscan respectively,and there were 15 common crossing genes among them.Conclusion:1.Differential expression profiles of microRNAs associated with acute myocardial infarction were obtained.2.Three microRNAs(miR-1,miR-133 a and miR-208a)affect the occurrence and development of acute myocardial infarction through related signaling pathways.3.Three microRNAs(miR-1,miR-133 a and miR-208a)can be used in the diagnosis of acute myocardial infarction and may become potential markers of acute myocardial infarction.