| BACKGROUND and OBJECTIVE Atrial fibrillation(AF)is the most common form of tachyarrhythmia in the clinic with high morbidity and mortality,and the incidence increases with age.Atrial fibrillation can lead to stroke,induce or aggravate heart failure,cause death,high disability,and seriously threaten human life and health.At present,the pathogenesis of atrial fibrillation is still not fully defined.Efforts to explore the potential molecular mechanisms of atrial fibrillation and to find new and effective intervention targets will play a positive role in the treatment of atrial fibrillation.Studies have shown that atrial electrical remodeling is one of the main causes of atrial fibrillation.It is mainly characterized by changes in the charac-teristics of ion channels and connexin(Cx),resulting in delayed depolarization and increased trigger activity,atrial motion.The potential duration and effective refractory period are shortened,the refractory period is increased,the conduc-tion velocity is slowed down,and the conduction heterogeneity is increased.The above-mentioned atrial electrophysiological changes caused by electrical re-modeling provide the necessary matrix for the formation of ectopic activation and local micro-reentry,thereby promoting the occurrence and maintenance of atrial fibrillation.The gap junction protein composed of Cx is an important channel for intercellular electrical signal and chemical signal transmission,and plays an important role in maintaining normal electrical conduction of cardi-omyocytes and achieving synchronous atrial or ventricular contraction.Cx43 is encoded by the GJA1 gene,which is one of the most important Cx in mamma-lian atrium.A large amount of research evidence indicates that atrial Cx43 re-modeling due to abnormal number,function and spatial distribution of Cx43 is closely related to the occurrence of atrial fibrillation.MicroRNAs(miRNAs)are small RNAs of about 18-22 nucleotides in length that bind to the 3'-untranslated region(3'-UTR)of the target gene mRNA,and regulate the level of gene expression by Post-transcription.Multiple miR-NAs,such as miRNA-206 and miRNA-208,have been shown to be closely re-lated to the occurrence of atrial fibrillation.According to bio informatics predic-tion,miRNA-613 and miRNA-206 belong to the same family,have the same seed sequence,and may have similar biological functions.More interestingly,GJA1 encoding Cx43 is a potential target gene for miRNA-613,suggesting that miRNA-613 may be involved in the regulation of Cx43 remodeling in AF.Long non-coding RNAs(lncRNAs)are a class of non-coding RNAs that are more than 200 nucleotides in length and were originally considered to be"transcriptional waste",but with further research,researchers have discovered their structure.Similar to mRNA,it is involved in cell differentiation,apoptosis,and various systemic diseases,and can participate in complex regulatory func-tions.Studies have found that lncRNAs are associated with cardiovascular dis-eases such as heart failure,dilated cardiomyopathy,and myocardial infarction.Recent studies have shown that lncRNAs may also play an important role in the pathogenesis of atrial fibrillation.Hox transcript antisense RNA(HOTAIR)is a recently discovered lncRNA associated with cardiovascular diseases such as heart development and myocardial infarction,but is HOTAIR related to the oc-currence of atrial fibrillation?No reports have been reported yet.Bioinformatics predictions also suggest that HOTAIR is a potential target gene for miRNA-613.LncRNAs,as miRNA "molecular sponges",regulate the expression of target genes by competitively binding miRNAs to miRNA target genes,which is one of the important mechanisms for their biological regulation.A number of studies have reported that HOTAIR,as a "molecular sponge" of miRNA-613,competes with target genes for binding to miRNA-613,thereby regulating the expression of target genes.Is HOTAIR a competing endogenous RNA(ceRNA)that regu-lates the expression of Cx43 by binding to GJA1 and binding to miRNA-613?No reports have been reported yet.This project explores whether HOTAIR par-ticipates in the occurrence of atrial fibrillation by regulating Cx43.And its poss-ible mechanisms involved in the regulation of Cx43,in order to provide a new target for the intervention of atrial fibrillation.Methods(1)The study was divided into atrial fibrillation group(AF)and sinus rhythm(SR)group by collecting right atrial tissue specimens from patients with valvular heart disease undergoing heart valve repair or replacement in the First Affiliated Hospital of Guangxi Medical University.The expression levels of HOTAIR,miRNA-613 and Cx43 mRNA and protein in the two groups were detected by real-time quantitative polymerase chain reaction(PCR)and West-ern-blot.(2)Transfected mouse atrial myocytes(HL-1)with HOTAIR over-expression lentiviral vector,and constructed HL-1 cell model with overexpres-sion of HOTAIR.The expression of Cx43 mRNA and protein in HL-1 cells was detected by using RT-qPCR,Western-bolt,and the effect of HOTAIR on the ex-pression of Cx43 in mouse atrial myocytes was observed.(3)Using miRNA-613 mimics to transfect HOTAIR to overexpress HL-1 cells,and using Western-blot to detect the change of Cx43 protein expression level in HL-1 cells miRNA-613 mimics,miRNA-613 mimics+HOTAIR.The lentiviral vector was transfected into HL-1 cells,and the expression of Cx43 protein in HL-1 cells was detected by Western-blot.Thus,it was confirmed that HOTAIR regulates Cx43 expres-sion through miRNA-613.(4)Finally,the dual luciferase reporter assay system was used to verify that GJA1 encoding Cx43 is a direct target of miRNA-613,further confirming that HOTAIR acts as a ceRNA and regulates mouse atrial muscle by competitively binding to miRNA-613 with GJA1.Results(1)Analysis of clinical data of patients with atrial fibrillation(23 cases)and sinus rhythm group(22 cases),gender,age,disease composition,left ventricular end-systolic diameter and end-diastolic diameter,left There was no significant difference in the ejection fraction of the room(P>0.05).The left atri-al diameter(53.43 mm±11.21 mm)in the atrial fibrillation group was signifi-cantly greater than that in the sinus rhythm group(44.45 mm±7.58 mm)(P<0.01).The expression level of HOTAIR in right atrial appendage of patients with atrial fibrillation was significantly lower than that of sinus rhythm group(2.5396±1.0202)(P<0.05).However,the relative expression level of miR-NA-613 in the right atrial tissue of the atrial fibrillation group(1.9010±0.75)was not significantly different from that of the sinus group(1.8201±0.8384)(P>0.05).The expression of Cx43 mRNA in the right atrium of patients with atrial fibrillation(1.9844±0.4707)was significantly lower than that of sinus rhythm group(2.4179±0.5498)(P<0.05).The expression of Cx43 protein in the right atrium of patients with atrial fibrillation(0.266±0.151)was also signifi-cantly lower than that of patients with sinus rhythm(0.422±0.179)(P<0.05).(2)The expression level of Cx43 mRNA in HL-1 cells of HUTAIR overexpression group(4.8882±1.0126)was significantly higher than the blank virus transfection group(0.4872+0.0542)(P<0.05).Meanwhile,HOTAIR had expression level of Cx43 protein in the expression group HL-1 cells(1.6111±0.1620)was signifi-cantly higher than the blank virus transfection group(0.4761±0.0931)(P<0.05).(3)Increased experimental data of HOTAIR low expression group(4)Compared with blank virus vector transfection group(0.4299±0.0880),the expression level of Cx43 protein in HL-1 cells of HOTAIR+miRNA-613 negative control group(2.3297±0.3116)was significantly increased.(P<0.0001),while the expression level of Cx43 protein in HL-1 cells of HOTAIRR+miRNA-613 group(0.3834±0.0723)was significantly lower than that in HOTAIR+miRNA-613 negative control group(P<0.0001),but transfected with blank virus vector.There was no difference in the group(P>0.05).(5)Compared with the blank control group(0.8007±10.0547)and the miRNA-613 negative control group(0.8491±0.0508),the expression level of Cx43 protein in HL-1 cells of miR-NA-613 group decreased significantly(0.135±0.0028)(P<0.0001).The expres-sion level of Cx43 protein in HL-1 cells of miRNA-613+HOTAIR group(0.0853±0.0074)was significantly higher than that of miRNA-613 group,but it was still significantly lower than that of blank control group and miRNA-613 negative control group.(6)Compared with miRNA-613 negative control group+ GJA1 3'-UTR wild type group(4.9664±2.0499),miRNA-613+GJA1 3'-UTR wild type group firefly/Renila fluorescein The enzyme ratio(2.5562±0.7087)decreased significantly(P<0.05);miRNA-613+GJAl 3'-UTR mutant group firefly/Renila value(4.7680±1.2353)and miRNA-613 negative control group+GJA1 3'-UTR mutation There was no significant difference between the type group(5.8508±2.839)(P>0.05),but it was significantly higher than the miRNA-613+ GJA1 3'-UTR wild type group(P<0.05).The above results suggest that:(1)the expression of HOTAIR in the right atrial tissue of patients with valvular atrial fibrillation is down-regulated,and the abnormal expression of HOTAIR may be involved in the occurrence of atrial fi-brillation;(2)HOTAIR positively regulates the expression of Cx43 in mouse atrial myocytes;(3)miRNA-613 significantly attenuated the positive regulation of HOTAIR on Cx43 in mouse atrial myocytes,while HOTAIR partially rescued miRNA-613-induced downregulation of Cx43 in mouse atrial myocytes,sug-gesting that HOTAIR regulates mouse atrial myocytes via miRNA-613.Expres-sion of Cx43;(4)GJA1 encoding Cx43 is a direct target gene of miRNA-613,further confirming that HOTAIR participates in the regulation of Cx43 expres-sion in mouse atrial myocytes by competing with GJA1 for binding to miR-NA-613.Conclusions(1)Downregulation of HOTAIR may play an important role in the occurrence of atrial fibrillation;(2)HOTAIR acts as a ceRNA and regu-lates the expression of Cx43 in mouse atrial myocytes by competitively binding to GJA1 to bind miRNA-613;(3)HOTAIR Down-regulation,decreased binding ability to miRNA-613,increased binding of miRNA-613 to GJA1,and enhanced negative regulation of GJA1,leading to down-regulation of Cx43 expression,which may be one of the important mechanisms of HOTAIR involvement in atrial fibrillation. |
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