Identification Of Atrial Fibrillation-related CircRNA And Constructing The Integrative Regulatory Network Of CircRNA,miRNA,and MRNA By Bioinformatics Analysis

Atrial fibrillation(AF)is one of the main risk factors for stroke and more than half of the patients with paroxysmal AF will develop into persistent AF within 10 years.Radiofrequency catheter ablation is an effective strategy to cure AF,which can significantly reduce the risk of stroke and death,but there is still a risk of recurrence from 20%to 40%after the first RFCA for patients with persistent AF.An array of pathophysiological mechanisms has been linked to AF progression and development The outcome of AF is also variable due to patient heterogeneity.Moreover,current treatment strategies used for AF are not dependent upon the specific mechanism of AF,with infrequent exceptions being observed.Therefore,further investigation of the mechanisms underlying AF could identify alternatives to current therapy,and clinicians might be better able to tailor therapy in each specific patientIt is well known that atrial structural remodeling and atrial electrical remodeling play an important role in the occurrence and progression of atrial fibrillation.However,the molecular mechanisms leading to atrial structural remodeling and electrical remodeling are still unclear.Recent studies have shown that non-coding RNA(ncRNA)is closely associated with atrial structural remodeling and electrical remodeling in patients with atrial fibrillation.Abnormal levels of non-coding RNA in atrial tissues can significantly increase the susceptibility of patients to atrial fibrillation and are associated with the risk of long-term recurrence of atrial fibrillation after catheter ablation in patients with atrial fibrillation.Circular RNA(circRNA)is a class of non-coding RNA newly discovered in recent years.Its closed and stable circular structure is closely related to the maintenance of cardiac function,thus opening up a new research idea for further research on the molecular mechanism of atrial fibrillation.CircRNAs are important regulators of microRNA(miRNA)function,and the interaction between them can promote the progression of atrial fibrillation disease.In addition,the circRNA-miRNA-mRNA regulatory network has been shown to be associated with myocardial infarction,heart failure,cardiomyopathy and other cardiovascular diseases,highlighting its potential in the treatment of heart disease.Because of the presence of binding sites of a variety of microRNAs(miRNAs),circRNAs can adsorb microRNAs sponge-like,and then regulate the expression of miRNAs and downstream target genes,that is,influencing the disease process through the mechanism of competing endogenous RNAs(cerRNAs).However,the studies with regard to indicating the relationship between the functions of circular RNAs and atrial fibrillation are less at present.In addition,its functions related to the occurrence and development of atrial fibrillation have not yet been fully elucidated,so more studies need to be conducted to open a new research field demonstrating the molecular mechanisms of atrial fibrillation,which may provide novel diagnostic and treatment targets for atrial fibrillation in clinic in the future.Therefore,we collected 10 samples of the right atrial appendages isolated from patients with persistent AF or sinus rhythm undergoing open-heart surgery.Bioinformatics was used to screen circRNAs associated with persistent AF and to evaluate their cell function,enriched signaling pathways,and downstream targets to further understand the role of circRNAs in the pathological mechanism of persistent AF.Moreover,we constructed a circRNAs-miRNAs-mRNAs integrative regulatory network related to persistent AF,and used quantitative Real-time PCR(qRT-PCR)and double luciferase reporter gene assay to validate it.We hope that our work may help to clarify the important role of circRNAs in the pathogenesis of persistent AF.Part ? Identification of Atrial Fibrillation-Related Circular RNAs in Atrial Tissue of Patients with Persistent Atrial FibrillationObjective:High throughput sequencing was used to identify atrial fibrillation-related circular RNAs in the right atrial appendages of patients with persistent atrial fibrillation.Methods:1.Clinical specimen acquisition Specimens of heart tissues were obtained from 10 patients with rheumatic mitral valvular disease undergoing cardiac mitral valve replacement surgery in our hosptial.The data of demographic characteristics,medical history,coronary angiography results,electrocardiogram,echocardiography and so on were collected for each patient.According to the electrocardiogram results,specimens were divided into AF group or SR group.High throughput sequencing was used to identify atrial fibrillation-related circular RNAs in the right atrial appendages of patients with persistent atrial fibrillation.2.Quality control of specimens Agilent 2200 Bioanalyzer accurately detected the integrity of RNA.The RNA integrity index(RIN value)was calculated according to the RNA peak graph,and the threshold of the RIN value was greater than 7.0.3.Library construction CircRNA library was not constructed until rRNA was removed by probe and linear RNA was removed by RNase R from Total RNA.4.High-throughput circRNA sequencing Raw Data filtering was performed and joint sequences and low-quality reads were removed to obtain high-quality Data.Then,the ribosomal RNA sequence was removed and effective reads was obtained by comparing with the ribosomal database.Effective reads were compared with the reference genome,and the comparison results were used for the next identification of circRNAs.Two kinds of software were used for comprehensive identification of circRNAs to obtain high-confidence circRNAs.Sequence prediction,expression value calculation and expression difference analysis of identified circRNAs were further carried out.5.Correlation analysis between specimens Pearson correlation coefficient r was used to measure the linear correlation between two variables.The closer the absolute value of r was to 1,the higher the similarity of expression patterns between samples was.It is recommended that r for repeatable samples be at least greater than 0.8.6.Analysis of expression differences of circRNAs between two groups DESeq2 method was used to calculate the differences of circRNAs between two groups.P-value<0.05 and |log2FoldChange|>1 were used as the defining criteria for differential circRNAs.Results:1.Specimens of heart tissues were obtained from 10 patients undergoing surgery in our hospital,including 5 samples in the AF group and 5 samples in the SR group.2.The RNA integrity index(RIN value)was 8.5,which was calculated according to the RNA peak graph,and the quality control results of the specimens met the requirements of high-throughput sequencing.3.Pearson correlation coefficient r was 0.847,indicating that there was a high similarity in expression patterns between samples within the AF group or the SR group,which to some extent measured the reliability of sequencing results.4.Compared with the SR group,a total of 600 significantly differentially expressed circRNAs were identified in the persistent AF group,of which 340 were up-regulated and 260 were down-regulated.Conclusions:1.The quality control results of the 10 specimens met the requirements of high-throughput sequencing.2.There was a high similarity in expression patterns between samples within the AF group or the SR group,which to some extent measured the reliability of sequencing results.3.A total of 600 circRNAs that were closely related to persistent AF were identified,of which 340 were up-regulated and 260 were down-regulated.Part ? Constructing the Integrative Regulatory Network of circRNA,miRNA,and mRNA by Bioinformatics AnalysisObjective:To construct an integrative regulatory network of circRNAs,miRNAs and mRNAs for persistent AF,and to explore the function and the enriched biological signaling pathways of the downstream regulatory target genes of persistent AF associated circRNAs.Methods:1.The dataset of miRNA microarray GSE68475 was downloaded from the Gene Expression Omnibus(GEO)database,in which 10 samples were from AF group and 11 samples from SR group,and normalized analysis was performed on the data to evaluate the quality of the dataset.2.The statistical significance of differential expression between two groups was also estimated with t-test using the R software limma package and further filtered with fold change.microRNAs with P values<0.05 and |logFC|>1 were identified as significantly differentially expressed miRNAs.3.To facilitate the investigation,the interactions between circRNAs and miRNAs were predicted by Targetscan,miRanda,and RNAhybrid.circRNAs of which target miRNAs were simultaneously predicted by three tools mentioned above were identified as candidate circRNAs.The candidate circRNAs,which were annotated in circBase database,with an absolute value of fold change>2.0 were selected for further analysis.We screened overlapped miRNAs based on differentially expressed miRNAs in microarray and target miRNAs using Draw Venn Diagram(http://bioinformatics.psb.ugent.be/webtools/Venn/).4.The target genes of overlapped miRNAs were predicted by using DIANA-TarBase v8,which is a decade-long collection of experimentally supported miRNA-gene interactions database,and facilitates the extraction of miRNA interactions derived from>33 experimental methodologies,applied to about 600 distinct cell types/tissues under approximately 451 experimental conditions.5.Significantly expressed circRNAs with an absolute value of fold change>2.0,overlapped miRNAs,and target genes related to overlapped miRNAs were superimposed onto the circRNAs-miRNAs-mRNAs network by using Cytoscape(version 3.4.0)and the network topology was analyzed by utilizing CentiScaPe app.6.We utilized DAVID(http://david.abcc.ncif-crf.gov/)tool to perform Gene ontology(GO)enrichment analysis of miRNA target genes.GO terms,including molecular function(MF),biological processes(BP),and cellular components(CC),with P value<0.05 were considered significantly enriched by target genes.KEGG enrichment analysis of target genes was conducted with Kyoto Encyclopedia of Genes and Genomes(KEGG),which is a database resource for further understanding high-level functions and effects of the biological system.Results:1.The box plot showed the median of different samples was almost on the same line after Background Correction and Quantile Normalization of the miRNA expression profile(GSE68475),indicating the high quality of the dataset.2.The differentially expressed miRNAs of the miRNA expression profile of GSE68475 was generated by utilizing the R software limma package.For the same criteria,60 differentially expressed miRNAs were identified from the miRNA expression profile,including 31 up-regulated and 29 down-regulated miRNAs.Based on the differential expression of miRNAs,we mapped the volcano plot to visualize the differential expression.3.We utilized three online tools,including Targetscan,miRanda,and RNAhybrid,to predict the interactions between circRNAs and miRNAs.The candidate circRNAs annotated in circBase database with P values<0.05 and|logFC|>2 were selected for further analysis.A total of 15482 interactions between 133 circRNAs and 2403miRNAs were identified.Based on differentially expressed miRNAs in microarray and target miRNAs of 133 circRNAs,31 overlapped miRNAs were screened by using Draw Venn Diagram.4.The target miRNAs of the 31 overlapped miRNAs were predicted by using Tarbase V8.0 and 293 interactions between 278 genes and 31 overlapped miRNAs.What's more,9 miRNAs,all of 130 target mRNAs of the 9 miRNAs with a prediction score>0.4,and 30 circRNAs which function as the sponge of the 9 miRNAs were utilized to construct the circRNA-miRNA-mRNA integrative regulation network.5.In the network of 30 circRNAs,including 8 upregulated circRNAs and 22 downregulated circRNAs respectively,9 miRNAs ranked relatively higher,of which 6 are upregulated miRNAs and 3 are downregulated miRNAs,and 130 target mRNAs of these miRNAs were collected.hsa-miR-339-5p had the most interactions with circRNAs and mRNAs,which indicated that hsa-miR-339-5p was the hub miRNAs in the regulation network.6.GO functional enrichment analysis,which involved BP,MF and CC categories,was conducted to investigate the biological function of the target mRNAs of over-lapped miRNAs.All of the results of GO analysis were ranked by enrichment score(-log(P value))and top ten of every category were showed in histogram.KEGG pathway enrichment analysis was performed for a further understanding of the target genes.In KEGG pathway analysis,the results were ranked by enrichment score and 19 pathways associated with the target mRNAs were displayed in bubble diagramConclusions:The high-throughput sequencing data of 10 tissue samples were analyzed by using bioinformatic technologies,and a circRNA-miRNA-mRNA comprehensive regulation network for persistent atrial fibrillation was constructed.The GO function enrichment and KEGG signaling pathway analysis of mRNAs in the comprehensive regulatory network indicated that the circRNA-miRNA-mRNA integrative regulatory network maight play an important role in the pathogenesis of persistent AFPart ? Verification the circRNA-miRNA-mRNA regulatory axis for persistent AF at the cellular levelObjective:To validate the interactions of circRNA-miRNA-mRNA targeting regulatory axis by using qRT-PCR and dual luciferase reporter gene assayMethods:1.According to the absolute value of log2FC and the abundance value of the persistent AF associated circRNAs,the top 5 differentially expressed up-regulated circRNAs and down-regulated circRNAs were selected from the integrative regulatory network respectively,and the 10 circRNAs above and their target miRNAs were selected for further experimental validation2.Quantitative Real-time PCR(qRT-PCR)was used to detect the relative mRNA expression levels of the above 10 circRNAs and their target miRNAs in specimens,and the targeted regulatory axis was selected for further experimental validation3.Double luciferase reporter gene assay was used to verify the interaction of the targeted regulation axisResults:1.According to the absolute value of log2FC and the abundance value of persistent AF related circRNAs,the top 5 differentially up-regulated and down-regulated circRNAs were selected from the integrative regulatory network,such as hsa circ₀004255,hsa_circ 0005637,hsa_circ₀003777,hsa circ₀087564,hsa circ 0082689,has_circ 0001850,hsa_circ₀086414,hsa_circ 0008900,hsa_circ₀000666,and hsa_circ₀085362,and the target miRNAs of the 10 circRNAs above were also selected,including hsa-miR-339-5p,hsa-miR-3667-5p,hsa-miR-3176,hsa-miR-331-5p and hsa-miR-4279,all of which were validated by subsequent experiments.2.The results of Quantitative Real-time PCR(qRT-PCR)showed that compared with the SR group,the mRNA levels of hsa_circ₈6414,hsa_circ₀000666*,hsa_circ₀082689,h s a_circ₀003777,hsa-miR-3176**,hsa-miR-331-5p**,hsa-miR-339-5p and hsa-miR-4279 were relatively up-regulated in AF group Compared with SR group,the mRNA levels of hsa_circ₀008900*,hsa_circ₀004255,hsa_circ₀005637,hsa_circ₀085362,hsa circ₀087564 and hsa-miR-3667-5p**.*represents SR group vs AF group p<0.05;**represents SR group vs AF group p<0.013.The results of double luciferase reporter gene assay showed that co-transfection with hsa-miRNA-3176 mimics,but not hsa-miRNA-3176 mutant,sig-nificantly mitigated the hsa_circ₀008900-regulated luciferase activity in HEK293T cells(P<0.05),indicating that hsa_circ₀008900 may sponge hsa-miRNA-3176 in HEK293T cells.Furthermore,co-transfection with hsa-miRNA-3176 mimics,but not hsa-miRNA-3176 mutant,significantly mitigated the BCL2L2-regulated luciferase activity in HEK293T cells(P<0.05),indicating that hsa-miRNA-3176 may target BCL2L2 mRNAConclusions:1.The results of qRT-PCR showed that the relative mRNA levels of hsa_circ₀008900,hsa-miR-339-5P and hsa-miR-3176 in AF group were consistent with the predicted results of bioinformation technology and the differences between two groups were statistical.hsa_circ₀008900,hsa-miR-3176 and their target genes could be further validated by double luciferase reporter gene assay.2.According to the results of previous studys and the dual-luciferase reporter gene assay,there were hsa_circ₀008900-hsa-miR-3176-BCL2L2 targeted regulatory axes in cardiomyocytes.