The Roles And Mechanisms Of Non-coding RNAs In The Canine Model Of Rapid Atrial Pacing

Objective: Atrial fibrillation（AF）is one of the most common arrhythmias in the clinical medicine,the high prevalence and recurrence rate of AF has been a serious threat to human health.In recent years,with the development of biological sequencing technology and bioinformatics,the roles of non-coding RNAs in cardiovascular diseases have been gradually discovered.In order to study the roles of non-coding RNAs in AF,the differentially expressed lnc RNAs,circ RNAs and micro RNAs in the animal model of AF were analyzed.Methods:（1）Twelve healthy adult mongrel dogs were randomly divided into the control group（C,n = 6）and the rapid pacing group（P,n = 6）.A programmable atrial pacemaker was placed at the right atrium of the dog.Atrial pacing stimulation was performed at a high frequency of five hundred beats one minute for fourteen consecutive days in the atrial pacing group,while the pacemaker did not work in the control group.HE and Masson staining were used to detect the morphological changes of atrial tissue,and electrophysiological tests were performed to evaluate the effective refractory period and atrial fibrillation induction rate.（2）Total RNA extraction was performed on dog atrial tissues and was used for high-throughput sequencing of non-coding RNAs,like lnc RNAs,circ RNAs and micro RNAs.Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis was used to analyze the differentially expressed lnc RNAs,circ RNAs and micro RNAs.（3）Establishment of fibrosis model in vitro by stimulating canine fibroblasts with Ang II.RT-PCR was used to detect the expression of cfa＿circ RNA₀09305 both in vivo and in vitro fibrosis models.The immunofluorescence,CCK8,and wound healing technique were used to examine the effect of cfa＿circ RNA₀09305 on the differentiation,proliferation and migration of fibroblasts after inhibition or overexperssion of cfa＿circ RNA₀09305.Western blot was used to detect the effect of cfa＿circ RNA₀09305 on fibrosis-related proteins.Bioinformatics predicted that cfa＿circ RNA₀09305 had an interaction relationship with miR-433,and the expression of fibrosis-related proteins in fibroblasts after transfection of miR-433 mimics were also detected.Spearman’s correlation analysis was used to examine the correlation between cfa＿circ RNA₀09305 and miR-433.Dual luciferase reporter assay was performed to detect the binding of cfa＿circ RNA₀09305 and miR-433.Results:（1）Compared with the control group,the atrial cells in pacing group had obvious morphological changes,collagen deposition,and increased atrial fibrosis（C vs P: 7.90% vs 27.37%）.Electrophysiological results showed that in the pacing proup,the effective refractory period was shortened,the dispersion of the refractory period increased,and the induction rate of atrial fibrillation was also increased significantly（C vs P: 9.6% vs 32.7%）,suggesting that electrical and structural remodeling might have occurred in rapid pacing atria.（2）A total of 33 differentially expressed lnc RNAs,146 differentially expressed circ RNAs,101 differentially expressed micro RNAs,1154 differentially expressed m RNAs were screened.GO classification,KEGG analysis,and interaction networks showed that AF-related lnc RNAs,circ RNAs and micro RNAs participated in the regulation of AF in diverse biological processes,cellular components,molecular functions,signalling pathways,and complex interactions between lnc RNAs and circ RNAs with miRNAs.（3）RT-PCR revealed that cfa＿circ RNA₀09305 was down-regulated in the atrium of pacing group.In the Ang II-induced fibrosis cell model,the expression of fibrosis-related proteins were down-regulated,and the expression of cfa＿circ RNA₀09305 was decreased as well.Inhibition of cfa＿circ RNA₀09305 promoted the ability of fibroblasts proliferation,migration and differentiation into myofibroblasts,increased the expression of fibrosis-related proteins,including Collagen I,Collage III,MMP2,MMP9,α-SMA.Overexpression of cfa＿circ RNA₀09305 could reverse these changes.Bioinformatics analysis speculated that there were some complementary base pairs between cfa＿circ RNA₀09305 and miR-433.The expression of miR-433 were increased both in the atrial tissues of pacing dogs and Ang II-stimulated cardiac fibroblasts.Western blot showed that overexpression of miR-433 promoted the expression of the above fibrosis-related proteins.Spearman’s correlation showed that cfa＿circ RNA₀09305 was negatively correlated to miR-433 in Ang II-stimulated cardiac fibroblasts.Dual luciferase reporter assay and RT-PCR suggested that there was a targeting relationship between cfa＿circ RNA₀09305 and miR-433,cfa＿circ RNA₀09305 inhibited the expression of miR-433.Therefore,it was speculated that up-regulation of cfa＿circ RNA₀09305 could inhibit the pro-fibrosis effect of miR-433.Conclusions:（1）Atrial fibrilation can be induced after the rapid right atrial pacing stimulation.No-coding RNAs are involved in various biological processes in the regulation of the occurrence of AF.（2）Cfa_circ RNA₀09305 participates in the regulation of atrial fibrosis,via working as a ce RNA of miR-433.