MicroRNAs Sequencing Of Plasma Exosomes Derived From Patients With Atrial Fibrillation: MiR-124-3p Promotes Atrial Fibrosis By Regulating AXIN1

IntroductionAtrial fibrillation(AF)is the most frequent arrhythmia in clinical practice,and its morbidity gradually increases with age.AF is associated with an increased risk of stroke,heart failure,dementia,and mortality.As a result,AF has become a major public health burden.However,current available medical treatments for AF have unsatisfactory efficacy.The main reason is that the mechanism of AF is not yet very clear,and it is difficult to implement early preventive measures and precise targeted therapy.The principle pathophysiological mechanisms of AF include electrical remodeling,structural remodeling and autonomic nervous system remodeling.Studies have found that microRNAs(miRNAs)are involved in the remodeling mechanism of AF.As endogenous and highly conserved non-coding small RNA molecules,miRNAs negatively regulate the expression of target genes by base pairing with target mRNAs.Various miRNAs have emerged to play significant roles in cardiovascular diseases such as cardiomyopathy,heart failure and atherosclerosis.MiR-124-3p has been shown to be abnormally up regulated in acute myocardial infarction,inhibiting inflammatory responses and apoptosis of cardiomyocytes.Several miRNAs including miR-21,miR-26,miR-29b,miR-30,miR-133 and miR-590 have been implicated in atrial remodeling and fibrosis.Therefore,these novel findings provide a theoretical basis for miRNAs in the pathogenesis and treatment of AF.Exosomes are membrane-derived vesicles that are actively secreted by cells.Exosomes carry various molecular constituents,including proteins,lipids,mRNAs,and miRNAs.MiRNA-containing exosomes are secreted into the circulation and play an important role in cell-to-cell communication and influence both physiological and pathological processes.Previous studies have found differential expression of exosomal miRNAs between patients with AF and sinus rhythm(SR),and these miRNAs were predicted to regulate genes to involve in oxidative stress(e.g.,MAPK),fibrosis(e.g.,WNT),and other pathways.The WNT/β-catenin signaling pathway is currently known to play a key role in the development of organ fibrosis and to be targeted by some differentially expressed miRNAs detected in fibrosis diseases.These findings provided further evidence that exosomal miRNAs might be involved in the atrial fibrosis and AF.However,the specific mechanism by which exosomal miRNAs participate in the pathophysiology of AF is still unclear.This study was designed to screen differential expression of exosomal miRNAs of plasma,and explore how the miRNAs regulate target gene to influence the molecular biological mechanism of AF.This research was divided into two parts.In the first part,patients with AF and SR were screened from the clinic,exosomes were extracted from their plasma,and the significantly differentially expressed exosomal miRNAs were analyzed by high-throughput sequencing.The sample size was further expanded to validate the differentially expressed miRNAs by qRT-PCR.Through target gene prediction,biological function and signal pathway analysis,the target miRNAs that could regulate AF and the possible mechanisms were further predicted.In the second part,the effects of the target miRNA on atrial fibrosis were explored in vivo and vitro respectively.This study would provide new clues and evidences for the molecular pathogenesis of AF.Methods1.Patients recruitment and plasma specimensPatients with AF and SR were recruited from according to pre-established inclusion and exclusion criteria.Ten milliliter fasting whole blood was collected and then centrifuged at 3000 g at 4℃ for 10 minutes to remove the cellular components.The upper clarification solution containing plasma was collected and stored at-80℃.2.Plasma exosomes isolationExosomes were isolated by serial ultracentrifugation:3000 g for 15 minutes to remove debris,then 16500 g for 30 min at 4℃,and thereafter two centrifugations at 110000 g at 4℃for 70 min to pellet the exosomes(Beckman Coulter Optima XPN 100 with Ti 70 rotor;Beckman Coulter,Fullerton,Calif).The final precipitates containing exosomes were frozen at-80℃ for RNA or protein analysis or co-culture with rat fibroblasts.3.Identification of exosomesThe morphology of exosomes was observed by transmission electron microscopy.Particle size concentration and distribution were then analyzed using nanoparticle-tracking analysis(NTA).Western blot was used to analyze the expression of CD63,Hsp70,TSG101 and Calnexin.RNA was extracted from exosomes,and the concentration and purity of RNA samples were detected.4.High-throughput sequencingThree patients with AF and three patients with SR were selected to perform secondgeneration high-throughput sequencing of plasma exosomal miRNAs to detect the differentially expressed miRNAs in the two groups.Furthermore,real-time quantitative PCR(qRT-PCR)was performed to verify the accuracy of the sequencing results.5.Bioinformatics analysis to predict miRNA related to AF and fibrosisTarget genes and signaling pathways of differentially expressed miRNAs were predicted using miRanda software.The biological functions of differentially expressed miRNAs were analyzed and predicted through Gene Ontology(GO)analysis.The signaling pathway of differential expression of miRNAs was analyzed through Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.On this basis,all the results were combined to screen out the significantly differentially expressed target miRNA related to atrial fibrosis.6.Isolation and culture of rat primary myocardial fibroblastsPrimary myocardial fibroblasts were isolated from rats.Primary cardiac fibroblasts were cultured in DMEM medium containing 10%fetal bovine serum and fluid was changed every 2-3 days.7.Co-culture of exosomes and myocardial fibroblasts(1)Exosomes isolated from patients with AF were labeled with PKH67 green fluorescent dye,and then were co-incubated with rat primary myocardial fibroblasts at 37℃ for Oh,8h and 16h in a 5%CO2 incubator.At the end of incubation,cell slides were dyed with DAPI.Cell slides were observed under a confocal laser-scanning microscope.(2)Exosomes isolated from patients with AF and SR were co-incubated with rat primary myocardial fibroblasts,respectively.After the myocardial fibroblasts were co-incubated for 24h,RNA was extracted,and the expression level of miR-124-3p was detected by qRT-PCR.8.Cell transfectionPrimary rat cardiac fibroblasts were transfected with plasmids containing miR-124-3p,miR-124-3p inhibitor and negative control(NC)plasmids,respectively.After transfection,the proliferation activity of cells was measured by EDU staining.After 48 hours of transfection,cell viability was determined by CCK8 assay.Total RNA and protein were extracted 48 hours after transfection.9.Infection of rats with adenovirusEighteen adult healthy male Wistar rats were selected and randomly divided into three groups:(1)miR-124-3p up-regulated group;(2)miR-124-3p up-regulated+Wnt/β-catenin pathway inhibitor MASB group;(3)negative control group.After 14 days of virus infection and pathway inhibitor treatment for 14 days,electrophysiological examinations were performed on the rats in each group to determine atrial effective refractory period and the induction rate of AF.After the examination,the rats were sacrificed,and the left atrial tissue was collected at low temperature for future use.10.Detection of molecular biological indicatorsThe total RNA of transfected cells was extracted by TRIzol method,and the expression of miR-124-3p was detected by qRT-PCR method.Proteins from cells and atrial tissues were extracted,and Western blot was used to determine the changes in the expression of target genes,signaling pathway proteins and fibrosis indicators.11.Histological examinationLeft atrial tissue was isolated and fixed with 4%paraformaldehyde.The samples were embedded in paraffin and cut into 4 μm slices.These slices were subjected to Masson staining to evaluate the morphological changes of atrial muscle tissues and the degree of atrial fibrosis.12.Dual luciferase reporting validates gene targetsThe putative miR-124-3 p binding sites in the 3’UTR of AXIN1(both wild-type and mutant forms)were cloned into pmir-GLO luciferase vectors.The luciferase vectors were cotransfected with miR-NC or miR-124-3p mimics into 293T cells.Forty-eight hours after transfectio1,cells were lysed and assayed using the Dual-Luciferase Assay Kit on a GloMax TM 20/20 Luminometer.The relative luciferase signal values were normalized according to the matched Renilla values.Results1.A total of 43 patients with AF and 43 patients with SR were recruited in this study.The left atrial diameter in AF group was significantly larger than that in SR group(P<0.01).The level of BNP in AF group was higher than that in SR group(P<0.05).Other indicators,such as age,weight,height,BMI and LVEF,showed no statistical significance.In addition,there was no statistically significant difference in the above indicators between the 3 patients with AF and the 3 patients with SR used for exosome sequencing.2.The size and shape of exosomes were observed by TEM,and the plate-like vesicles with diameter of 30-150 nm were regarded as exosomes.NTA revealed a similar size distribution of the exosomes,from SR controls and from AF patients.Western blot showed that plasma exosomes from AF and SR were positive for exosomal markers CD63,HSP70 and TSG101,and negative for Calnexin compared with cells.3.The heat map showed that the expression of multiple miRNAs was significantly different in AF group compared with the SR group.A total of 852 miRNAs were consistently expressed,among which 13 miRNAs were up-regulated and 27 miRNAs were down-regulated(P<0.05).Of which,9 miRNAs were reported to have effects on cardiac diseases.4.Eight miRNAs(5 up-regulated and 3 down-regulated miRNAs)were chosen to validate the sequencing outcomes by qRT-PCR.The results showed that miR-124-3p,miR-378d,miR2110,miR-3180-3p were remarkably up-regulated miRNAs,while miR-223-5p,miR-125a-3p and miR-1299 were down regulated.MiR-574-3p was aslo down regulated by validation,which did not conform to the sequencing result.Compared with the SR control,the target genes of miRNAs in patients with AF are involved in cellular component,molecular function,and biological process(e.g.,cell adhesion,cell junction and metal ion binding).KEGG categorization of target genes of miRNAs differentially expressed is shown in Figure 5.A total of 144 pathways were predicted,in which signaling pathways including Calcium signaling pathway(hsa04020),Wnt signaling pathway(hsa04310),Insulin secretion(hsa04911)and cGMP-PKG signaling pathway(hsa04022)were among top 20.Of which,the Wnt signaling pathway was reported to be associated with myocardial fibrosis.Therefore,the differently expressed miRNAs might influence the Wnt signaling pathway in atrial fibrosis and AF.Combining KEGG pathway analysis and TargetScan method,we predicted miR-124-3p may regulate AXIN1 to influence the expression of β-catenin protein,which is the crucial factor of Wnt signaling pathway.5.Exosomes were labeled with PKH67 and co-incubated with cardiac fibroblasts.Fluorescence microscope analysis showed that the rat myocardial fibroblasts absorbed fluorescence,and the number of exosomes entering the cells gradually increased with time.The results showed that exosomes could be taken up by cardiac fibroblasts.6.Compared with exosomes from patients with SR,miR-124-3p was significantly upregulated in myocardial fibroblasts of rats after plasma exosomes from patients with AF were co-cultured with myocardial fibroblasts for 24h.The results showed that exosomes from AF patients could be taken up by fibroblasts and affect the expression of miRR-124-3p.7.Primary rat cardiac fibroblasts were transfected with miR-124-3p overexpression plasmid,miR-124-3p suppressor plasmid and negative plasmid,respectively.When miR-1243p was up regulated,EDU-labeled fluorescent staining and CCK-8 results showed a significant increase in cell proliferation and viability.Meanwhile,when miR-124-3p was downregulated,the proliferation and viability of cardiac fibroblasts were significantly decreased.Compared with the miR-124-3p inhibition group,AXIN1 in the miR-124-3p overexpression group was significantly down regulated at both mRNA and protein levels.The expression levels of βcatenin,Collagen I and α-SMA proteins were up-regulated in the miR-124-3p overexpression group,while the expression levels of these proteins were down-regulated in the miR-124-3p inhibition group.8.Rats were infected with adeno-associated virus containing up-regulated miR-124-3p,and Wnt pathway was inhibited with Wnt pathway inhibitor.The electrophysiological examination found that compared with the negative control group and the miR-124-3p upregulated+Wnt pathway inhibitor group,the inducible rate of AF increased,and the atrial effective refractory period was shortened in the miR-124-3p up-regulated group.The protein expression of Collagen I in the miR-124-3p up-regulated group was significantly higher than that in the negative control group and the miR-124-3p up-regulated+Wnt pathway inhibitor group.The obtained left atrial tissue samples were stained with Masson.The staining results showed that the degree of myocardial fibrosis in the miR-124-3p up-regulated group was significantly higher than the other two groups.9.After co-transfection of miR-124-3p mimic and wild-type luciferase reporter plasmid,luciferase activity decreased significantly compared with that of the control group,but the activity of mutant luciferase did not change.Luciferase reporter gene assay showed that AXIN13’UTR was a direct target gene of miR-124-3p.Conclusions1.There are significant differentially expressed miRNAs between patients with AF and patients with SR.Bioinformatics analysis predicted that miR-124-3p promotes atrial fibrosis by regulating AXIN 1.2.MiR-124-3p can regulate Wnt/β-catenin signaling pathway to promote the proliferation and activity of myocardial fibroblasts,which provides a theoretical basis for atrial fibrosis.3.Through adeno-associated virus infection in rats and Wnt pathway inhibitor application,the results showed that miR-124-3p affects Wnt/β-catenin pathway via AXIN 1 to promote atrial fibrosis.