Studies On Plasma Circular RNA In Predicting Atrial Fibrillation Recurrence After Surgical Ablation

BackgroundAtrial fibrillation（AF）is the most common sustained arrhythmia.It leads to high disability rate,high mortality and expensive medical costs,which poses significant burden to patients,physicians,and healthcare systems globally.The initiation and maintenance mechanism of AF is not completely understood,but can include complex re-entrant mechanisms as well as localized trigger.The ideal management for AF is still cardioversion and long-term maintenance of sinus rhythm（SR）,so as to relieve symptoms,improve hemodynamics and reduce adverse events such as thromboembolism.At present,there are three main methods of AF cardioversion:pharmacological/electrical cardioversion,catheter ablation and surgical intervention.For patients with AF complicated with organic heart disease,such as valvular disease,coronary heart disease or other heart diseases requiring cardiac surgery,it is considered as the"gold standard"of AF treatment.However,there are large individual differences in the success rate of cardioversion to SR after surgical ablation,and there is no special biomarker to predict the AF recurrence after surgical ablation,which makes it difficult to effectively identify the high-risk population with a high incidence of AF recurrence after surgical ablation.Circular RNA（circ RNA）is a kind of circular single stranded RNA produced from pre-m RNA back-splicing events,which plays an important role in the regulation of gene expression.Circ RNA has been identified to exhibit cell type,tissue,and developmental stage specific expression and resistant to RNase R making it ideal candidates for future diagnostic biomarkers and therapeutic interventions.In the cardiovascular field,many studies have shown that circ RNA is specifically expressed in the myocardium of many cardiovascular diseases,such as aneurysm,vascular endothelial hyperplasia,myocardial fibrosis,cardiac aging and so on,and participates in the initiation and development of these diseases.However,there are few reports about the expression profile of circ RNA in patients with AF and its function in the initiation and development of AF.Moreover,no study has explored the clinical application value of circ RNA as a biomarker for patients after surgical ablation.Objectives1.To obtain differentially expressed profiles of circ RNA in atrial appendage tissue of AF patients along with non-AF patients.2.To perform functional prediction and analysis of differentially expressed circ RNA3.To investigate the predictive value of plasma circ RNA level for recurrence of AF after surgical ablation.MethodsIn this study,patients diagnosed with AF in Department of cardiothoracic surgery,Second Affiliated Hospital of Naval Medical University from August 2018 to February2020 were enrolled.At the same time,patients who underwent mitral valve replacement or valvuloplasty due to mitral valve disease were included as control group.Among them,15cases（AF:n=9;SR:n=6）of atrial appendage tissue samples were used for high-throughput transcriptome sequencing;30 cases（AF:n=15;SR:n=15）of atrial appendage tissue samples and peripheral venous blood samples were used for q RT-PCR validation;104 AF cases of peripheral venous blood samples were used to explore the predictive value of circ RNA a in recurrent AF after surgical ablation.1.Study the expression profile of circ RNA in atrial appendage tissue of AF patients.After obtaining the atrial appendage tissue of AF and SR patients,we obtained the expression profile of circ RNA in these tissues by high-throughput transcriptome sequencing technology,and screened out the differentially expressed circ RNA.The sequencing results were validated by q RT-PCR.2.Bioinformatics analysis of differentially expressed circ RNA.Through the Kyoto Encyclopedia of Gene and genome（said 49-year-old kyoko Encyclopedia of Genes and Genomes,KEGG）database and Fisher’s exact test to Gene ontology learning target Genes（Gene ontology,GO）analysis and signal path analysis,to explore whether the host Genes differentially expressed circ RNA may participate in the occurrence of AF,and predict the circ RNA presence of corresponding micrornas binding sites.3.Study on the correlation between circ RNA in plasma and recurrence of AF after MIS ablation in patients with AF.The expression of preoperative circ RNA in plasma was measured.The predictive value of circ RNA in recurrent AF after surgical ablation was analyzed by univariate and multivariate Cox regression models.Results1.Through high-throughput transcriptome sequencing,we found 147 differentially expressed circ RNA（|log₂ FC|>1,P<0.05）in 15 patients（AF:n=9;SR:n=6）,of which102 circ RNA were up-regulated and 45 circ RNA were down-regulated.We then analyzed the expression profiles of circ RNA in the left atrial appendage of 7 patients（AF:n=4;SR:n=3）.We found 86 differentially expressed circ RNA（|log₂ FC|>1,P<0.05）,of which63 circ RNA were up-regulated and 23 circ RNA were down-regulated.We also analyzed the expression profile of circ RNA in right atrial appendage of 8 patients（AF:n=5;SR:n=3）.We found 577 differentially expressed circ RNA（|log₂ FC|>1,P<0.05）,of which366 circ RNA were up-regulated and 211 circ RNA were down-regulated.Finally,we constructed the Wayne map and found 6 differentially expressed circ RNA in three comparision（hsa＿circ＿0005643、novcl＿circ＿0026284、novel＿circ＿0077334、novel＿circ＿0046852、novel＿circ＿0068696 and novel＿circ＿0055884）。The 6 differentially expressed circ RNA were verified by q RT-PCR using the atrial appendage tissue sample and plasma samples of 30 patients（AF:n=15;SR:n=15）.Finally,we found novcl＿circ＿0026284 was highly expressed in the tissues and plasma samples of AF patients,and the validation results were consistent with the sequencing results.2.Bioinformatics analysis indicated that the target genes of differentially expressed circ RNA were mainly involved in viral myocarditis,glutamatergic synapse,ECM receptor interaction and nicotinic acid and nicotinamide metabolism related signaling pathways.At the same time,these circ RNA had multiple binding sites with corresponding mi RNA.3.Univariate Cox regression analysis showed that age（HR:1.08,95%CI:1.02-1.14,P=0.013）、type of AF（HR:3.98,95%CI:1.64-9.68,P=0.002）、CHA₂DS₂-VASc（HR:6.78,95%CI:3.11-14.77,P<0.001）、LAD（HR:1.43,95%CI:1.29-1.59,P<0.001）、lymphocyte count（HR:0.49,95%CI:0.28-0.85,P=0.011）、creatinine（HR:1.37,95%CI:1.05-1.77,P=0.018）、BUN（HR:1.03,95%CI:1.01-1.05,P=0.002）and novcl＿circ＿0026284（HR:4.94,95%CI:2.03-12.02,P<0.001）were associated with AF recurrence after MIS.Multivariate Cox regression analysis showed that LAD（HR=1.26,95%CI:1.14-1.38,P<0.001）,CHA₂DS₂-VASc（HR=4.09,95%CI:1.90-8.80,P<0.001）,and novcl＿circ＿0026284（HR=3.22,95%CI:1.29-8.06,P=0.012）were independent indictors for predicitng AF recurrence after surgical ablation.Conclusions1.A total of 147 circ RNA expression levels were significantly changed in atrial appendage tissues of AF patients,and the expression level of novcl＿circ＿0026284 was significantly increased in the atrial appendage tissues and plasma of AF patients after q RT-PCR verification.2.The preoperative plasma level of novel＿circ＿0026284 was an independent predictor of AF recurrence after surgical ablation.