The Regulation Roles Of MiR199a-3p In Rat Atrial Fibrillation By MMP16/CollagenⅠ Pathway

Atrial fibrillation（AF）is a kind of common arrhythmia.Patients have heart failure,ischemic stroke and other complications,even death.The etiology of AF is complex,and the exact mechanism is not clear.It is generally believed that AF mainly includes atrial myoelectric remodeling and structural remodeling.Structural remodeling includes apoptosis,proliferation of cardiac fibroblasts,excessive deposition of extracellular matrix and fibrosis.Atrial fibrosis is an important factor in the occurrence and maintenance of AF.MicroRNA（miRNA）is a kind of noncodingRNA with a length of about22 nucleotides,which can degrade mRNA or inhibit the translation of protein encoded by mRNA and regulate the expression of target genes.Matrix metalloproteinase（MMP）plays an important role in degradation of protein components in extracellular matrix（ECM）,including collagen Ⅰ,the main fibrogenic component.If the degradation of MMP is lower than the production of ECM,ECM degradation will be reduced,which will lead to excessive deposition of ECM and tissue fibrosis.Therefore,we hypothesized that mir-199a-3p might regulate the occurrence and maintenance of AF by regulating the expression of MMP16.The purpose of this study was to investigate the role and mechanism of miR-199a-3p in atrial fibrillation.There are three parts in the experiment.The first part is to establish the rat atrial fibrillation model,and to explore whether miR-199a-3p is related to collagen volume fraction（CVF）.The second part uses the bioinformatics database targetscan to find the potential target gene of mir-199a-3p.In the third part,the adeno-associated virus was constructed and transfected into the rat atrium.The expression of miR-199a-3p in the atrium of the rats was intervened to verify whether MMP16 was the target gene of miR-199a-3p.PartⅠ:The role of miR-199a-3p in the rat model of atrial fibrillationObjective and significance:To explore the role of mir-199a-3p in the occurrence and development of atrial fibrillation in rats.Methods:The atrial fibrillation model of SD rats was established by tail vein injection of CaCl₂-ACh complex.The model was divided into sinus rhythm（SR）group and atrial fibrillation（AF）group.RT-qPCR was used to detect the expression of miR-199a-3p,RT-qPCR was used to detect the expression of collagen Ⅰ mRNA,Masson staining was used to detect the CVF,WB was used to detect the expression of collagen Ⅰ protein.Results:miR-199a-3p（2.11±0.44）and CVF（14.09±0.65）in AF group were higher than those in SR group（1.00±0.19）（P<0.05）,CVF（11.63±0.66）（P<0.05）,collagen Ⅰ protein expression in AF group（0.91±0.04）was higher than that in SR group（0.73±0.04）（P<0.05）.There was a positive correlation between the expression of mir-199a-3p and CVF（r=0.78 P<0.05）.Conclusion:miR-199a-3p may play a role in the occurrence and maintenance of AF,which may be caused by promoting AF.PartⅡ: Prediction of MMP 16 as a potential target gene of mir-199a-3p by bioinformatics softwareObjective and significance: To predict the target gene and binding site of miR-199a-3p.Methods: The target gene and binding site of miR-199a-3p were detected by targetscan bioinformatics website.Results: The prediction results of targetscan website showed that there was a binding site of MMP 16-3’UTR with miR-199a-3p.Conclusion: MMP 16 may be the target gene of miR-199a-3p.PartⅢ: The role of mir-199a-3p in the regulation of MMP 16 / collagen Ⅰ pathway in rat atrial fibrosisObjective and significance: To investigate the effect of mir-199a-3p on MMP16 and collagen Ⅰ in AF model of SD rats by constructing and using mir-199a-3p low expression and over expression adeno-associated virus.Method:In the first section,we constructed miR-199a-3p low expression and over expression genes,and integrated them into the viral gene sequence with adeno-associated virus as the vector.The constructed virus vector was transfected into AAV-293 cells（virus packaging）,the virus was purified and the titer of the virus was detected.In the second section,30 male SD rats were randomly divided into three groups according to different treatments:（1）Negative control（NC）group（n = 10）: negative control adeno-associated virus（HBAAV2 / 9-TNT-GFP NC control,dose of 200 UL /per rat）was injected into tail vein after routine feeding,and tail vein was injected with CaCl₂-ACh compound solution two weeks later;（2）Low expression group of miR-199a-3p（n = 10）: tail intravenous injection The low expression of miR-199a-3p adeno-associated virus（HBAAV2 /9-mir30-rno-miR-199a-3p-GFP,200 UL /per rat）was injected into the vein,and then the rats were fed regularly.After 2 weeks,the rats were injected intravenously with CaCl₂-ACh compound solution.（3）The overexpression group of miR-199a-3p（n = 10）: the rats were injected intravenously with the overexpression of miR-199a-3p adeno-associated virus(HBAAV2 /9-TNT-rno-miR-199a-GFP,200 UL / per rat.After 2 weeks,they were injected by tail vein with CaCl₂-ACh compound solution.RT-qPCR was used to detect the expression of miR-199a-3p,MMP16 and collagen Ⅰ mRNA,Masson staining was used to detect CVF,WB was used to detect the expression of MMP-16 and collagen Ⅰ protein,and electrocardiograph（ECG）was used to record the induction rate and duration of AF.Results: Compared with NC group（1 ± 0.45）,the expression of mir-199a-3p in the low expression group（0.22 ± 0.13）decreased（P < 0.05）,and that in the over expression group（2.58 ± 1.52）increased（P < 0.05）.There was no significant difference in the mRNA expression of MMP 16 and collagen Ⅰ among the three groups（P > 0.05）.Comparison of relative expression of MMP16 protein: compared with NC group（0.48 ± 0.11）,the expression of MMP16 in the low expression group（0.63 ± 0.12）increased（P < 0.05）,indicating that the low expression of miR-199a-3p can increase the expression of MMP16 protein in rat myocardial tissue;compared with NC group,the expression of MMP16 in the over expression group（0.32 ± 0.11）decreased（P < 0.05）,showing that the overexpression of miR-199a-3p can reduce the expression of MMP-16 protein in rat myocardium.Comparison of collagen Ⅰ protein expression: compared with NC group（0.62 ± 0.15）,the expression of collagen Ⅰ in low expression group（0.46 ± 0.14）decreased（P < 0.05）,showing that miR-199a-3p low expression can reduce the expression of collagen Ⅰ protein in rat myocardial tissue;compared with NC group,the expression of collagen Ⅰ protein in over expression group（0.81 ± 0.12）increased(The low expression of mir-199a-3p could increase the expression of collagen Ⅰ protein in rat myocardium.Masson results:Compared with NC group（14.90 ± 1.29）,CVF in low expression group（12.84 ± 1.32）decreased（P < 0.05）,showing that low expression of mir-199a-3p could reduce the degree of myocardial fibrosis;compared with NC group,CVF in over expression group（16.77 ±1.35）increased（P < 0.05）,showing that over expression of mir-199a-3p could increase the degree of myocardial fibrosis.ECG results:Compared with NC group（7 / 10）,the induction rate of atrial fibrillation in the low expression group（4 / 10）decreased without statistical difference;compared with NC group,the induction rate of atrial fibrillation in the over expression group（9 /10）increased without statistical difference.Comparison of AF duration in each group: compared with NC group（14.0 ± 3.9）,AF duration in low expression group（6.8 ± 2.5）was shortened（P < 0.05）,indicating that low expression of miR-199a-3p could reduce AF duration;compared with NC group,AF duration in over expression group（26.7 ± 5.1）was increased（P < 0.05）,indicating that over expression of mir-199a-3p could increase AF duration.Conclusion: By constructing the method of miR-199a-3p low expression and over expression adeno-associated virus transfection in SD rats,we can effectively achieve the purpose of low expression and over expression of miR-199a-3p in rat myocardium.miR-199a-3p may play a role in the occurrence and development of AF in rats by targeting and regulating MMP 16/ collagen Ⅰ signal pathway.