| BackgroundAtrial fibrillation(AF)is the most common persistent tachyarrhythmia in clinical practice.AF is easy to cause hemodynamic instability,blood hypercoagulability,easy to lead to stroke,thromboembolism,cardiac dysfunction and other complications,and has the characteristics of high disability rate,high mortality and poor treatment effect.Studies have shown that AF increases the risk of heart failure by up to three times and exacerbates the symptoms of heart failure.Patients with AF have an annual incidence of myocardial infarction of between 0.4%and 2.5%,and their risk of developing myocardial infarction is two times higher than that of patients without AF.Although the drug and interventional treatment of AF has made great progress in recent years,the antiarrhythmic drugs currently used have different degrees of limitations due to poor efficacy and toxicity of the drug itself.While invasive catheter ablation has a lower complication rate,it still has a higher recurrence rate over time.This is related to the fact that the complex pathogenesis of AF has not been fully elucidated.Therefore,it is particularly important to clarify the pathogenesis of AF and seek intervention targets for the precise treatment of AF.It is well known that transcriptome is not only regulated by the upstream genome,but also affected by factors such as tissue distribution,environment and diseases,and can directly affect the expression of downstream proteins,so it plays an important role in the occurrence and development of diseases.Nowadays,the sequencing work of the Human Genome Project has been completed.With the continuous development of sequencing technology,gene chip technology has been widely used as a powerful tool in the study of various human diseases.The microarray data of related diseases were downloaded from public databases,and the differentially expressed genes were screened through bioinformatics analysis,and then the function and pathway enrichment analysis were performed.This provides a new method for us to study the molecular mechanism of disease occurrence and development.AimsThis study aims to explore the role and mechanism of transcriptome in the development of AF.The study consisted of two parts.In the first part,transcriptomic datasets were retrieved from the Gene Expression Omnibus(GEO)database,and bioinformatics analysis was performed to screen mRNAs associated with AF.Cell and animal models were constructed to verify the bioinformatic findings and the target mRNA was selected.In the second part,GO function and KEGG pathway enrichment analysis were performed on the target mRNA to predict its possible mechanism.The mechanism by which it plays a role in AF was further studied through in vitro functional acquisition experiments,in order to provide new ideas for the prevention and treatment of AF.Method1.Microarray data related to AF transcriptome were downloaded and analyzedTranscriptomic data sets were retrieved on the Gene Expression Omnibus(GEO)database,and bioinformatics analysis was performed to screen mRNAs associated with AF.Differential expression analysis was investigated with limma package in R software.Robudt Rank Aggreg package in R software was made use of constructing the integrate analysis.Gene Ontology(GO)annotations along with Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis was performed on account of DAVID database(https://david.ncifcrf.gov/)and protein-protein interaction(PPI)analysis was performed on String online platform(https://string-db.org).2.Establishment of cell and animal models of AF and verification of bio informatics analysis resultsThe primary atrial myocytes were isolated from hearts of Wistar rats(1 day old)and stimulated by rapid electric field(10Hz,1.5V/cm).RNA and protein were extracted 72 hours after cell stimulation to detect the expression changes of differentially expressed mRNA.Twelve adult male Wistar rats weighing about 300-350g were randomly distributed to pacing group(n=6)and control group(n=6).In the pacing group,high right atrial rapid pacing was performed at fixed frequency(900bpm)for 14 consecutive days(6h/day)to establish the rat model of AF.The control group was not undergo pacing after sham operation.RNA and protein were extracted from atrial tissues of the two groups after 14 days,and the expression changes of differentially expressed mRNAs were detected.3.Prediction of potential mechanisms of target mRNAGo and KEGG analysis were performed on the target mRNA to screen the biological functions and signaling pathways related to AF.Further explore potential target sites and analyze the molecular mechanism of target mRNA acting on AF.4.Function acquisition experiment of target mRNAHuman atrial fibroblasts(HCF)were taken as the research object,and exogenous SPP1,PI3K/Akt pathway inhibitor LY294002 and mitomycin(MMC)were used as treatment factors.The cells were randomly divided into control group,SPP1 treatment group,MMC treatment group,LY294002 treatment group,SPP1+MMC co-treatment group,and SPP1+LY294002 co-treatment group.After 24 hours of treatment,proteins were extracted and the expression levels of relative proteins were detected by Western Blotting.Results1.Five AF-related transcriptome data sets(GSE14975,GSE31821,GSE79768,GSE115574,and GSE128188)were eventually included in this study.2.Limma package and RRA package in R software were used to conduct standardization,differential expression analysis and multi-dataset integration analysis of each chip data.As a result,147 differentially expressed mRNAs were screened from AF-left atrial appendage,of which 71 were up-regulated and 76 were down-regulated.In the AF-right atrial appendage,a total of 85 differentially expressed mRNAs were obtained,among which 34 mRNAs were up-regulated and 51 mRNAs were down-regulated.3.GO analysis showed that differentially expressed gene transcripts were mainly involved in cardiac conduction regulation,extracellular space and apoptosis.KEGG analysis was mainly concentrated in calcium signaling pathway,and the differentially expressed mRNAs concentrated in this pathway included AGTR1,HTR2B,CD38 and BDKRB1.EGFR,ACTA1,SPP1,and FHL2 were identified as central gene transcripts in the PPI analysis.4.The primary atrial myocytes AF model and rat AF model were established to verify the screened differentially expressed mRNAs.The results of qRT-PCR and Western Blotting showed that the expression trends of HTR2B and SPP1 were consistent with the results of bioinformatics analysis,and SPP1 was selected as the target mRNA for further mechanism study.5.The experiment proved that the exogenous increase of SPP1 at the cellular level could up-regulate the expression of fibrosis related genes,including collagen I and α-SMA.Through further analysis of GO and KEGG of SPP 1,it was found that SPP 1 is involved in extracellular matrix,inflammatory response and other biological processes,and plays an important role in PI3K/Akt/p27 signaling pathway.6.Compared with the control group,overexpression of p27 could reduce the pro-fibrosis effect induced by SPP1,while exogenous increase of SPP1 could inhibit the expression of p27 and induce Akt phosphorylation.However,the PI3K/Akt pathway inhibitor LY294002 can block the downstream regulation effect of SPP1,suggesting that SPP1 regulates the expression of p27 through the PI3K/Akt signaling pathway,and then plays a role in promoting atrial fibrosis in AF.Conclusion1.According to the integrated bioinformatic analysis of transcriptome sequencing data of AF,it is concluded that mRNA of AGTR1,HTR2B,CD38,BDKRB1,EGFR,ACTA1,SPP1,FHL2,HPCAL4,APOA1,SPHKAP and LTBP1 may be involved in the occurrence and development of AF.Meanwhile,calcium signaling pathway may play an important role.2.Cell and animal level experiments confirm that SPP1 and HTR2B expression trends are consistent with the results of bioinformatic analysis.3.SPP1 can up-regulate the protein expression of collagen I and α-SMA in HCF,and play a role in promoting fibrosis.Moreover,by activating the PI3K/Akt signaling pathway,SPP1 can play an inhibitory role on p27 and up-regulate the expression of collagen I,thereby promoting myocardial fibrosis and participating in the occurrence and development of AF. |
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