| Backgrand:Atrial fibrillation(AF)is the most common arrhythmia in clinical practice.With the increase of cardiovascular disease and chronic diseases,and the incidence is rising.Due to atrial fibrillation has a high morbidity and disability,therefore,the treatment of atrial fibrillation has become the burden of patients and society.The current mainstream view is that the occurrence and maintaining of atrial fibrillation associated with atrial remodeling.Gap junction is electricity and biochemical coupling between myocardial cells,which is consisting of connexin(Cx).Connexin channels ensure molecular and electrical signals propagation and hence are crucial in myocardial synchronization and heart function.Cx remodling includes expression quantity,function,spatial distribution of abnormalities,lead to anisotropy conduction and form micro reentry caused by atrial fibrillation.Cx43 is one of the main Cx in mammals' atrial,Cx43 remodling is an important part of atrial electrical remodeling and pathological basis which occurs and maintains atrial fibrillation.Explore the mechanism of atrial remodling,blocking atrial remodeling,will provide a new and effective for the treatment of atrial fibrillation.micro RNAs(miRNAs)is a kind of endogenous non coding RNAs.They inhibit the translation of m RNA by binding to complementary sequences that are usually located in the 3'-untranslated region(UTR)of target m RNA and suppress the expression of proteins at the post transcriptional level.Mi RNA-590-3p has the potential of promoting myocardial cells proliferation.Bioinformatics prediction is that the coding gene PDGFB of PDGF-B is one of the target genes which regulated by miRNA-590-3p.But whether the miRNA-590-3p involving in the Cx43 reconstruction in atrial fibrillation by regulating PDGF-B,have not been reported.Objective: To explore effect of microrna-590-3p on connexin43 through regulation of platelet derived growth factor-B in atrial fibrillationMethod:1.One hundred and fourteen patients who were enrolled in the First affiliated hospital of Guang Xi medical university undergoing thoracotomy surgery(59 AF and 55 SR cases)were included into the study.Tissues in RAA of patients with AF or SR were obtained before cardiopulmonary bypass.HE staining was used to observe histomorphology of the tissues in RAA.The spatial distribution of miRNA-590-3p was evaluated by In Situ Hybridizationrespectively.The spatial distribution of Cx43 was evaluated by Immunofluorescence technique.The spatial distribution of PDGF-B was evaluated by Immunohistochemical.The miRNA-590-3p expression and the m RNA expression of Cx43 and PDGF-B were measured by RT-q PCR,protein expression of Cx43 and PDGF-B measured by Western-blot.2.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.18 canines were divided into 3 groups inclouding Control group,Sham group,1W group.There were 6 canines in each group.HE staining was used to observe histomorphology.The spatial distribution of Cx43 was evaluated by Immunofluorescence technique.The expression of miRNA-590-3p and the m RNA and protein expression of Cx43 were measured by RT-q PCR,protein expression of Cx43 and PDGF-B measured by Western-blot.3.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.293 T 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 48 h after being transfected.4.Human adult cardiomyocytes(AC16 cells)were transient transfected with inhibitor hsa-590-3p and Target Side Blocker hsa-590-3p,Negetive Control,were divided into control group,Mock group(only added the same volume of transfect reagent),then The protein expression of PDGF-B were measured by Western-blot.5.AC16 cells were treated with 0,10,50,100ng/ml PDGF-BB respectively.Then,AC16 cells were treated with 50ng/ml PDGF-BB for 24 h,48 h or 72 h.The protein expression of Cx43 in each group was measured by Western-blot respectively.Resulte:1.(1)The expression of miRNA-590-3p in RAA of patients with AF was lower than that of patients with SR,respectively(0.7718±0.3495)vs(1.9538±0.7575)(P<0.05).(2)Compared with patintes with SR,the m RNA expression of Cx43 was lower in patients with AF,respectively(1.4758±0.5666 vs 2.1964±0.8887)(P<0.05).The protein expression of Cx43 was lower in patients with AF,respectively(0.4703±0.2388 vs0.9762±0.5564)(P<0.05).(3)Compared with patintes with SR,the m RNA expression of PDGF-B was higher in patients with AF,respectively(2.4222±1.0551 vs1.4844±0.5823)(P<0.05).The protein expression of PDGF-B was higher in patients with AF,respectively(0.7604±0.3661 vs 0.5728±0.2799)(P<0.05).2.(1)The expression of miRNA-590-3p in LAA of caines in AF 1 week group(3.0759±0.345)was lower than in the control and sham group(2.7713±0.4543,1.9326±0.2827,P<0.05).The expression of miRNA-590-3p in LA of caines in AF 1 week group(2.8483±0.3539)was lower than in the control and sham group.(3.0087±0.4177,1.9487±0.2259,P<0.05).The expression of miRNA-590-3p in RAA of caines in AF 1 week group(2.7999±0.3390)was lower than in the control and sham group(3.0492±0.4550,1.6454±0.3121,P<0.05).The expression of miRNA-590-3p in RA of caines in AF 1 week group(3.0726±0.4553)was lower than in the control and sham group(2.9270±0.3166,1.6074±0.3644,P<0.05).The expression m RNA of Cx43 in LAA of caines in AF 1 week group(1.5513±0.422)was lower than in the control and sham group(1.2343±0.2702,0.8032±0.1282,P<0.05).The expression m RNA of Cx43 in LA of caines in AF 1 week group(1.2868±0.2086)was lower than in the control and sham group(1.1004±0.1886,0.7716±0.1023,P<0.05).The expression m RNA of Cx43 in RAA of caines in AF 1 week group(1.196±0.1593)was lower than in the control and sham group(1.2139±0.2704,0.696±0.1111,P<0.05).The expression m RNA of Cx43 in RA of caines in AF 1 week group(1.2405±0.2296)was lower than in the control and sham group(1.192±0.2421,0.6499±0.1182,P<0.05).(3)The protein expression of Cx43 in in LAA of caines in AF 1 week group(0.7078±0.1500)was lower than in the control and sham group(0.7245±0.1598,0.471±0.1539,P<0.05).The protein expression of Cx43 in LA of caines in AF 1 week group(0.7528±0.1238)was lower than in the control and sham group(0.7064±0.1238,0.3990±0.1809,P<0.05).The protein expression of Cx43 in in RAA of caines in AF 1 week group(0.8625±0.1349)was lower than in the control and sham group(0.7626±0.2665,0.4729±0.056,P<0.05).The protein expression of Cx43 in in RA of caines in AF 1 week group(0.9161±0.2450)was lower than in the control and sham group(0.8809±0.1896,0.467±0.2147,P<0.05).3.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).4.The expression of miRNA-590-3p in AC16 cells which were transient transfected with inhibitor hsa-590-3p and Target Side Blocker hsa-590-3p(0.3113±0.1738)and(0.2643±0.1358)were lower than that control group(1.44±0.3766)?Mock group(1.317±0.5008)?NC group(1.065±0.1958)(P<0.05).Compared with control group(0.2913±0.0847),Mock group(0.2445±0.0687),NC group(0.3071±0.1306),the protein expression of PDGF-B were higher respectively in Inhibitor group(0.6380 ± 0.1794)and TSB group(0.5933±0.1068,<0.05).5.Compared with 0 ng/ml group(1.3233±0.3795),the protein expression of Cx43 were lower in 10ng/ml group(0.6643±0.1419),50ng/ml group(0.4645±0.1498),100ng/ml group(0.2226±0.0996)respectively(P<0.05).Compared with 0 hour group(0.98±0.1625),the protein expression of Cx43 were lower in 10ng/ml group(0.6131±0.2038),50ng/ml group(0.3156±0.0987),100 ng/ml group(0.1733±0.0775)respectively(P<0.05).Conclusion: 1.The Down-expression of miRNA-590-3p in atria take part in the occurrence and maintained of atrial fibrillation.2.The Down-expression of Cx43 is important in the occurrence and maintained of atrial fibrillation.3.PDGF-B is a target gene which directly regulated by miRNA-590-3p.4.The Down-regulation of miRNA-590-3p promotes the expression of PDGF-B,and reduces the expression of Cx43,which maybe one of the mechanisms of electrical remodeling of atria,and possibly causes the occurrence and maintained of atrial fibrillation. |
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