| Atrial fibrillation(AF)is the most common arrhythmia in clinical practice.The pathogenesis of AF is complex.Especially,atrial remodeling,which includes atrial electrical and structural remodeling,is central to AF.It’s more difficult for reversion of atrial structural remodeling than atrial electrical remodeling.Atrial fibrosis is a hallmark of structural remodeling.The exploration of mechanism of atrial fibrosis may provide new insights for intervention for atrial structural remolding.Platelet derived growth factors(PDGF)are important profibrotic cytokines.PDGFs,which activate the downstream signal pathway by binding to Platelet derived growth factor receptors(PDGFRs)and promote extracellular matrix secretion of cardiac fibroblasts,play an important role in atrial fibrosis.microRNAs(miRNAs)is a kind of endogenous non coding RNAs.They inhibit the translation of mRNA by binding to complementary sequences that are usually located in the 3’-untranslated region(UTR)of target mRNA and suppress the expression of proteins at the post transcriptional level.Many miRNAs have been proved to contribute to atrial fibrosis.PDGFB is the target gene of miRNA-590-3p by bioinformatics prediction.But wether miRNA-590-3p contributes to atrial fibrosis by targeting PDGF-B/PDGFR-β signal pathway is unknown in atrial fibrillation.This study was aimed to investigate the role of miRNA-590-3p and the association between miRNA-590-3p and PDGF-B/PDGFR-β signal pathway in atrial fibrosis.Firstly,the tissues of right atrial appendage(RAA)of patients with sinus rhythm(SR)or AF were obtained.The expression of miRNA-590-3p and the mRNA and protein expression of PDGF-B,PDGFR-β and Collagen Type I(COL1)were measured by real time quantity polymerase chain reaction(RT-qPCR),Western-blot and immune histochemical(IHC),respectively.Collagen fibers deposition was observed by Masson staining.Secondly,sustained AF canine model was made by rapid atrial pacing.The tissues in left atrial appendage(LAA),left atrium(LA),RAA and right atrium(RA)of sustained AF canines were obtained at week 1,2 and 4,respectively.The expression of miRNA-590-3p and the mRNA and protein expression of PDGF-B,PDGFR-βand COL1 were measured by RT-qPCR,Western-blot and IHC,respectively.Collagen fibers deposition was observed by Masson staining.Finally,wether PDGFB is the target gene of miRNA-590-3p was verified by Luciferase reporter assay and the atrial fibrobalsts wered intervened with PDGF-BB or PDGF-BB+AG1295(PDGFR tyrosine kinase inhibitor).The results of this study show:1.(1)The expression of miRNA-590-3p in RAAs of patients with AF was lower than that of patients with SR but the expression of COL1 and collagen fibers deposition were higher in patients with AF.(2)The expression of miRNA-590-3p in LAA,LA,RAA and RA of sustained AF canines declined gradually with the extension of time and maintained at a low expression level 2 weeks after AF.The expression of COL1 in LAA,LA,RAA and RA of sustained AF canines increased gradually with the extension of time.(3)Spatial distribution of collagen fibers was anisotropic.The collagen fibers deposition was more in RAA and RA than that in LAA and LA.2.The expression of PDGF-B and PDGFR-β in RAA.of patients with AF was higher than that of patients with SR.The expression of PDGF-B in LAA,LA,RAA and RA of sustained AF canines increased gradually with the extension of time.The expression of PDGFR-β increased gradually 2 weeks since AF.3.(1)PDGFB was proved to be a direct target gene of miRNA-590-3p by Luciferase assay.(2)PDGF-BB promoted the secretion of COL1 by atrial fibroblasts at a manner depending on both concentration and time of treatment.This effect can be attenuated partially by AG 1295.All of the above results indicate the down-regulation of miRNA-590-3p isan important risk factor in atrial fibrosis,which activates atrial fibroblasts and promotes the secretion of COL1 by atrial fibrobalsts through the upregulation of PDGF-B/PDGFR-β signaling pathway.Part Ⅰ The effects of miRNA-590-3p on atrial fibrosis in atrial fibrillationObjiective:To investigate the role of miRNA-590-3p in atrial fibrosis.Methods:72 patients undergoing thoracotomy surgery(39 AF and 33 SR cases)were included into the study.Tissues in RAA of patients with AF or SR weighted about 150 mg were obtained before cardiopulmonary bypass.The miRNA-590-3p expression and the mRNA and protein expression of COL 1 were measured by RT-qPCR,Western-blot and IHC respectively.Collagen fibers deposition was observed by Masson staining.20 canines were divided into 5groups inclouding Control group,Sham group,AF lasting lweek group(1W group),AF lasting 2weeks group(2W group),AF lasting 4weeks group(4W group).There were 4 canines in each group.Sustained AF canine model was made by rapid atrial pacing.The tissues in LA and RA were obtained from canines in each group.The expression of miRNA-590-3p and the mRNA and protein expression of COL1 were measured by RT-qPCR,Western-blot and IHC respectively.Collagen fibers deposition was observed by Masson staining.Results:1.The expression of miRNA-590-3p in RAA of patients with AF was lower than that of patients with SR,respectively(0.7981±0.5422 vs.1.6123±1.1132)(P<0.05).2.Compared with patintes with SR,the mRNA expression of COL1 was higher inpatients with AF,respectively(3.2719±2.0143 vs 1.7813±0.8255)(P<0.05).The protein expression of COL1 was higher inpatients with AF,respectively(0.6524±0.2097 vs 0.3123±0.1218)(P<0.05).3.The collagen fibers deposition was more in pateints with AF than patients with SR.The collagen volume fraction in RAA of patients with AF was more than that of patients with SR,respectively(32.83%±8.07%vs 11.07%±4.02%)(P<0.05).4.The expression of miRNA-590-3p in LAA,LA,RAA and RA of sustained AF canines declined gradually with the extension of time and maintained at low expression level 2 weeks after AF.The expression of miRNA-590-3p in RAA and RA was lower than that in LAA and LA 2 weeks after AF.5.The mRNA and protein expression of COL 1 in LA and RA of sustained AF canines increased gradually with the extension of time.The mRNA and protein expression of COL1 in RAA and RA was higher than that in LAA and LA 2 weeks after AF.6.The marked collagen fibers deposition was observed and the collagen fibers deposition in RAA and RA was more than that in LAA and LA 2 weeks after AF.Conclusion:(1)Atrial fibrosis was more obvious and the expression of miRNA-590-3p was lower in patients and canines with AF,compared with patients and canines with SR.These evidences indicate the down-regulation of miRNA-590-3p may promote atrial fibrosis in AF.(2)Spatial distribution of collagen fibers was anisotropic in AF canine atriums.Part Ⅱ The effects of PDGF-B/PDGFR-P signal pathway on atrial fibrosis in atrial fibrillationObjiective:To investigate the role of PDGF-B/PDGFR-β signal pathway in atrial fibrosis.Methods:72 patients undergoing thoracotomy surgery(39 AF and 33 SR cases)were included into the study.Tissues in RAA of patients with AF or SR weighted about 150 mg were obtained before cardiopulmonary bypass.The mRNA and protein expression of PDGF-B and PDGFR-β were measured by RT-qPCR,Western-blot and IHC respectively.20 canines were divided into 5groups inclouding Control group,Sham group,1W group,2W group,4W group.There were 4 canines in each group.Sustained AF canine model was made by rapid atrial pacing.The tissues in LA and RA were obtained from canines in each group.The mRNA and protein expression of PDGF-B and PDGFR-β were measured by RT-qPCR,Western-blot and IHC respectively.Results:1.The mRNA expression of PDGF-B in RAA of patients with AF was higher than that of patients with SR,respectively(2.5675±2.3481 vs 1.5674±0.8314)(P<0.05).The protein expression of PDGF-B was higher in patients with AF,respectively(0.8074±0.2407 vs 0.3806±0.1049)(p<0.05).2.Compared with patintes with SR,the mRNA expression of PDGFR-β was higher in patients with AF,respectively(2.0112±1.6320 vs 1.3566±0.7931)(P<0.05).The protein expression of PDGFR-β was higher in patients with AF,respectively(0.4044±0.1818 vs 0.1950±0.0841)(P<0.05).3.The mRNA and protein expression of PDGF-B in LAA,LA,RAA and RA of sustained AF canines increased gradually with the extension of time.4.The mRNA and protein expression of PDGFR-P in LAA,LA,RAA and RA of sustained AF canines increased gradually 2 weeks since AF.Conclusion:The mRNA and protein expression of PDGF-B and PDGFR-βwas higher in patients and canines with AF,compared with patients and canines with SR.The PDGF-B/PDGFR-β signal pathway plays an important role in atrial fibrosis.Part Ⅲ The effects of miRNA-590-3p on atrial fibrosis in atrial fibrillation by targeting PDGF-B/PDGFR-β signal pathwayObjiective:To investigate the role of miRAN-590-3p-PDGF-B-PDGFR-βsignal pathway in atrial fibrosis.Methods:The pri-miRNA-590 plasmid vector,pri-miRNA negative control plasmid vector,3’-UTR of PDGF-B luciferase reporter plasmid vector and mutant-3’-UTR of PDGF-B luciferase reporter plasmid vector were constructed.293T cells were divided into 4 groups including miRNA-590 group(293T cells were transfected with pri-miRNA-590 plasmid vector and 3 ’-UTR of PDGF-B luciferase reporter plasmid vector),miRNA negative control group(293T cells were transfected with pri-miRNA negative control plasmid vector and 3’-UTR of PDGF-B luciferase reporter plasmid vector),mutant-3’-UTR group(293T cells were transfected with pri-miRNA-590 plasmid vector and mutant-3’-UTR of PDGF-B luciferase reporter plasmid vector)and mutant-3’-UTR control group(293T cells were transfected with pri-miRNA negative control plasmid vector and mutant-3’-UTR of PDGF-B luciferase reporter plasmid vector).The activity of Gaussia Luciferase(Gluc)and SEAP in each group was detected 48h after being transfected.The atrial fibroblasts were extracted from Wistar rat’s heart.The proliferation and activity of atrial fibroblasts cultured with PDGF-BB were measured by MTT test.Atrial fibroblasts were treated with 0,10,50,100ng/ml PDGF-BB or 50ng/ml PDGF-BB+AG 1295(10μmol/L)for 24 h respectively.Then,atrial fibroblasts were treated with 50ng/ml PDGF-BB for 24 h,48 h or 72 h.The mRNA and protein expression of COL1 in each group was measured by RT-qPCR and Western-blot respectively.Results:1.Compared with miRNA negative control group,the Gluc/SEAP in miRNA-590 group decreased by 20.96%(P<0.05).However,There weren’t significant difference on Gluc/SEAP between mutant-3’-UTR group and mutant-3’-UTR control group(P>0.05).2.MTT-test showed PDGF-BB can promote proliferation and activity of atrial fibroblasts.3.The mRNA and protein expression of COL1 secreted by atrial fibroblasts increased gradually,with the increasing concentration of treatment with PDGF-BB(P<0.01).4.The mRNA and protein expression of COL1 secreted by cardiac fibroblasts increased gradually,with the increasing time of treatment with PDGF-BB(P<0.05).5.The effect that PDGF-BB promotes COL1 secretion of atrial fibroblasts can be attenuated partially by AG1295.Conclusion:PDGFB was proved to be a direct target gene of miRNA-590-3p by Luciferase reporter assay.The inhibitory effect of miRNA-590-3p on the PDGF-B/PDGFR-β signal pathway is weaken,because of the down-regulation of miRNA-590-3p.The up-regulation of PDGF-B/PDGFR-β signal pathway stimulates that atrial fibroblasts secrete COL1 and other extracellular matrix and promotes atrial fibrosis in AF. |
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