Studies On Relationship Between Alteration Of Plasma Exosomal MiRNA Expression And Atrial Fibrillation

BackgroundAtrial fibrillation(AF)is the most common arrhythmia.Studies have shown that AF is an important risk factor of thromboembolic events and ischemic stroke.The pathogenesis of AF is complex,and it is not fully understood Previous researches on atrial fibrillation have focused on atrial structural remodeling,ion channel remodeling,and calcium ion handling in cardiac cells.In recent years,exosomes,as information media in their pathological functions in the cardiovascular system have attracted more and more attention.Exosomes are lipid bilayer membrane vesicles with a diameter of about 30-150 nm secreted by cells,which play a role in transmitting information between cells.Eukaryotic cells of protozoa,fungi,plants,and animals can secrete exosomes.Inward buddings of the cell membrane are processed in the endoplasmic reticulum and Golgi apparatus to form multivesicular bodies containing multiple small membrane vesicles that selectively enrich proteins,RNA and DNA molecules and other biologically active substances.After the multivesicular bodies fuse with the cell membrane,the inner membrane vesicles are released outside the cell and become exosomes.Proteins,RNA or DNA derived from specific cells are loaded into exosomes,and the contents of exosomes secreted by cells variate in different physiological or pathological states.Researches reveal that some exosomal contents can serve as biomarkers of cancer diseases.As one of the most important contents of exosomes,micro RNA(miRNA)is a single-stranded non-coding small molecule RNA encoded by an endogenous gene.It contains approximately 22 nucleotides and its main function is to regulate gene expression at the post-transcriptional level.The miRNA binds to the complementary sequence of the 3 'untranslated region of the mRNA to inhibit translation or induce degradation of the mRNA.Previous studies have shown that miRNAs play an important role in the pathogenesis of the occurrence and development of atrial fibrillation,but there are few studies on the relationship between exosomal miRNAs and atrial fibrillation.As in many cancer diseases,miRNAs in exosomes may play a role as biomarkers in the treatment and prognosis of AF.Objectives1.In this study,we try to detect the differential expression of miRNA in plasma exosomes between patients with non-valvular persistent AF and those with sinus rhythm(SR)control.2.We then analyze the potential functions of the differentially expressed miRNAs(DE miRNAs)in AF.3.In the end,we detect which miRNAs are risk factors of AF.MethodsNon-valvular persistent AF patients(n=44)and SR patients(n=24)with chest tightness who were hospitalized in the Department of Cardiothoracic Surgery,Second Affiliated Hospital of Naval Military Medical University from March 2016 to July 2018.Morning fasting peripheral venous blood specimens were obtained.1.Extraction and identification of plasma exosomesBlood samples from patients with AF and patients with SR underwent twice of highspeed centrifugations to remove cells and cell debris,and washed with PBS to obtain exosome suspension.In order to identify the isolated particles as exosomes,the size,concentration and morphology of the particles were analyzed by Nanosight system and electron microscope.Exosome positive markers CD63,CD9 and negative marker GAPDH were detected by Western blot.2.RNA high-throughput sequencing and qRT-PCR validationThe plasma exosomal RNAs of AF patients(n=4)and SR patients(n=4)were performed high-throughput sequencing and analyzed to find DE miRNAs.Some of the DE miRNAs in the sequencing results were performed qRT-PCR validation on AF patients(n=40)and SR patients(n=20).3.Bioinformatics analysisThe DESeq2.0 algorithm was used to analyze the DE miRNA.When the false discovery rate(FDR)<0.05,the difference in miRNA expression was considered statistically significant.Miranda and RNAhybrid algorithms were used to predict the target genes of the DE miRNAs.Kyoto Encyclopedia of Genes and Genomes(KEGG)and the Fisher's exact test were used to analyze the target genes for gene ontology(GO)analysis and signal pathway analysis.P <0.05 was thought to be statistical different.4.Statistical analysisThis study used SPSS21.0 and Graphpad to analyze the data.The measurement data was expressed as mean ± standard deviation,and when the measurement data met the normal distribution,the difference between groups was compared using t test.The count data was expressed as n(%)and compared using chi-square test or Fisher's exact method.Univariate and multivariate logistic regression analysis were used to analyze the risk factors related to AF.P <0.05 was considered statistically significant.Results1.Identification of plasma exosomesThe isolated particles were identified by Nanosight LM10 system,electron microscope and Western blot.The particle size of the separated particles is 30-150 nm,which meets the size range of exosomes,and there is no significant difference in the concentration of plasma exosomes between the atrial fibrillation group and the control group.The exosomal markers CD63 and CD9 were positive in the isolated particles in both groups,while the intracellular protein GAPDH was negative.These results proved that the particles we isolated were exosomes.2.High-throughput sequencing of miRNAsBy performing high-throughput sequencing,39 DE miRNAs were detected in the plasma exosomes between patients with atrial fibrillation and sinus rhythm,including 21 upregulated miRNAs(miR-4786-3p,miR-4640-5p,miR-3663-3p,miR-6891-3p,miR-4690-5p,miR-6768-5p,miR-3916,miR-4654,miR-483-5p,miR-3929,miR-4800-5p,miR-7110-3p,miR-6124,miR-3611,miR-320 b,miR-455-5p,miR-320 a,miR-4758-5p,miR-4429,miR-34a-5p,miR-1181)and 18 down-regulated miRNAs(miR-184,miR-3174,miR-1185-1-3p,miR-548ah-3p,miR-6513-5p,miR-376c-3p,miR-548 p,miR-3170,miR-4504,miR-6516-5p,miR-1296-5p,miR-223-3p,miR-30d-3p,miR-335-5p,miR-142-5p,miR-223-5p,miR-4326,miR-425-3p).3.qRT-PCR validation of DE miRNAsqRT-PCR was performed on 6 of the DE miRNAs(miR-320 a,miR-320 b,miR-483-5p,miR-142-5p,miR-223-3p,miR-223-5p)which were reported to be related to cardiovascular diseases.The qRT-PCR results showed that in the AF patients,the expression of miR-483-5p(2.11 ± 1.13 vs.1.11 ± 0.50,P <0.001)in plasma exosomes was significantly increased,while miR-142-5p(0.84 ± 0.34 vs.1.06 ± 0.37,P =0.026),miR-223-3p(0.81 ± 0.43 vs.1.14 ± 0.53,P =0.011),miR-223-5p(0.77 ± 0.48 vs.1.11 ± 0.69,(P =0.028)expression level was significantly lower than the control group.4.Bioinformatics analysisBioinformatics analysis showed that the target genes of the DE miRNAs were related to pathways of myocardial remodeling(PI3K-Akt signaling pathway,adrenergic signaling in cardiomyocytes,focal adhesion,Wnt signaling pathway,calcium signaling pathway)and oxidative stress(MAPK signaling pathway,oxytocin signaling pathway).5.Logistic regression analysis of risk factors of AFUnivariate logistic regression analysis was performed on gender,age,body mass index,coronary heart disease,hypertension,diabetes,hyperlipidemia,left ventricular ejection fraction,left atrial diameter,plasma exosmal miR-483-5p,miR-142-5p,miR-223-3p and miR-223-5p level.The analysis revealed that the increased left atrium diameter,the increased expression level of miR-483-5p in plasma exosomes,the decreased expression level of miR-142-5p in plasma exosomes and the decreased expression of miR-223-3p in plasma exosomes are risk factors for AF.While multivariate logistic regression analysis showed that the increased left atrial diameter and the increased miR-483-5p expression in plasma exosomes were independent risk factors for AF.Conclusions1.There are 39 DE miRNAs in the plasma exosomes between non-valvular persistent AF and SR control.qRT-PCR showed plasma exosomal miR-483-5p was upregulated while miR-483-5p,miR-142-5p,miR-223-3p,and miR-223-5p were downregulated in nonvalvular persistent AF.2.The target genes of the DE miRNAs are related to signaling pathways of myocardial remodeling and oxidative stress.3.The increased left atrial diameter and the increased miR-483-5p expression in plasma exosomes were independent risk factors for non-valvular persistent AF.DE miRNAs in plasma exosomes may exert potential of acting as biomarkers in assessing severity or prognostic of non-valvular persistent AF.