| Objective:To investigate whether the regulation of miRNA from plasma and plasma-derived exosome in patients with unstable angina is consistent,and analyze the circulating miRNA profile of patients with unstable angina to identify disease-associated miRNAs and study their regulatory pathways.Methods:A total of 121 patients with chest pain who were admitted to the General Hospital of the Northern Theater from October 2017 to May 2018 were enrolled,including 35 patients in the control group and 86 patients in the coronary heart disease(CHD)group[37 patients with unstable angina(UA)subgroup,49 cases of acute myocardial infarction(AMI)subgroup].Three patients with unstable angina and another 3 healthy subjects were selected for miRNA sequencing.One aliquot plasma was directly used for RNA extraction and another aliquot plasma from the same people was used for exosome isolation and subsequent RNA extraction.The exosomes were identified by particle size,electron microscopy and Western Blot.A cDNA library of Small RNA was constructed and high-throughput sequencing was performed to analyze the sequencing results.The relative expression of miR-92a in the plasma of the remaining 118 patients with chest pain was detected.The miRNA database was used to predict the relevant target genes.The signal pathways related to coronary heart disease were predicted by GO analysis and KEGG pathway analysis.The platelet aggregation rate of the subjects was examined to determine whether there was aspirin resistance(AR)and clopidogrel resistance(CR).Results:1.The exosome sample has a peak particle size of 104.2 nm and a particle concentration of 3.4E+12 particles/mL.In the Western Blot experiment,the two exosome surface marker proteins CD63 and TSG101 were positive.Electron microscopy showed that a circular or round-shaped vesicle with a diameter of about 100 nm was visible in the field of view,and the outer layer was a lipid bilayer membrane.In healthy people,469 known miRNAs were detected in plasma,and 380 miRNAs were detected in exosomes,of which 350 miRNAs were expressed in plasma and exosomes.In patients with unstable angina,447 miRNAs were detected in plasma,and 362 miRNAs were detected in exosomes,of which 346 were expressed in plasma and exosomes.Co-expressed miRNA plasma was highly correlated with exosomal expression levels(r1=0.972,r2=0.973,P<0.05).Compared with healthy people,there were significant differences in the expression of 66 miRNAs in plasma between patients with unstable angina.There were significant differences in the expression of 78 miRNAs in plasma-derived exosomes.Kappa analysis revealed exosome miRNAs from plasma and plasma.The adjustment was inconsistent(kappa=0.078,P>0.05).Differentially expressed miRNAs in plasma are mainly related to mucin O-glycan biosynthesis,cytochrome P450 metabolism to xenobiotics,steroid hormone biosynthesis,etc.Differentially expressed miRNAs in exosomes are primarily involved in the ECM-receptor interaction pathway.2.The plasma level of mir-92a in the control group was significantly higher than that in the UA subgroup and AMI subgroup(P<0.05).There was no significant difference in mir-92a level between UA subgroup and AMI subgroup(P>0.05).A total of 118 target genes of mir-92a were predicted by the database.GO analysis and KEGG pathway analysis showed that the most significant signal pathway related to coronary heart disease was platelet activation.In the control group,the level of mir-92a in AR patients was significantly lower than that in NAR patients(P<0.05).Mir-92a level in CR patients was significantly lower than that in NCR patients(P<0.05)In the CHD group,mir-92a levels of AR patients and NAR patients,CR patients and NCR patients were compared(P<0.05).Conclusion:Differential changes of miRNAs can be detected in both exosomal and plasma samples.In addition,our results demonstrate that plasma miRNA and exosome miRNA may reflect different biological processes of the disease,and the two measurements may not be replaced by one another.Compared with CHD patients,miR-92a is highly expressed in plasma of non-CHD patients,which may delay the progression of CHD by inhibiting platelet activation. |
PDF Full Text Request