| With the aging of the population,the incidence of chronic heart failure is increasing year by year.The main cause of death was sudden death caused by malignant arrhythmia.Therefore,effective prevention and treatment must be carried out.Therefore,the study of the pathogenic mechanism of malignant arrhythmia in chronic heart failure is very helpful to find the target of prevention and treatment.The pathogenesis of malignant arrhythmia is complex,which is related to electrophysiological ion channels with prolonged action potential.In previous experiments,the establishment of canine chronic heart failure animal model suggested that the abnormal elevation of INaL in the heart tissues of failure led to a significant prolongation of action potential plateau,which eventually led to early or late depolarization of the heart failure,and then to malignant arrhythmia.Late sodium current(INaL)is a persistent influx of sodium current during the plateau phase of action potential duration.Its abnormal increase is one of the important molecular mechanisms leading to malignant arrhythmia.INaL is a voltage-dependent current,which is mainly composed of protein alpha subunit encoded by SCN5 A.It is believed that in the myocardium of patients with cardiovascular disease(ischemic myocardium,failure myocardium and hypertrophic myocardium),the expression of SCN5 A gene is up-regulated,which results in INaL.MicroRNA(miRNA)is a kind of non-coding RNA,which is about 22 nt in size.It can affect gene expression.In recent years,studies at home and abroad have found that the expression of a specific microRNAs in the blood or myocardium of cardiovascular patients is abnormal through gene chip screening.Some studies have further shown that the role of microRNAs in cardiovascular diseases can be down-regulated or up-regulated by negatively regulating the downstream genes of related genes.It further leads to abnormal ion channels and malignant arrhythmias.Previous experiments established canine models of chronic heart failure,screened abnormal expression of microRNA-microRNA-29 a in myocardial tissue by gene chip,and predicted SCN5 A regulated by microRNA-29 a by double luciferase report.Objective: To explore the regulatory mechanism of microRNA29 a on Nav1.5 in human cardiac myocytes(HCM)and provide a new target for early diagnosis and late monitoring of arrhythmia.Methods: First,design and construct a chronic virus that encapsulates miRNA-29 a,use software to query and obtain the base sequence of miR-29 a,obtain a linear vector by enzymatic cleavage technique,and adhere the target sequence to the vector for in vitro cyclization.This was subjected to purification analysis to determine the construction sequence as required,and then to measure the titer.The human cardiomyocyte model was then established by transient transfection of HCM with lentivirus.After 72 hours of culture,the transfected virus HCM was screened with puromycin to construct a stably transfected cell model,followed by extraction of total RNA and protein,using Quantitative Real-time polymerase chain reaction(qRT-PCR)detection of miRNA-29 a mimics expression in HCM,Western blot(WB)to verify the regulation of Nav-15 protein by miRNA-29 a mimics,using cell proliferation assay(CCK-8)detects HCM proliferation ability after transfection of miRNA-29 a mimics.Results: Chronic viruses of microRNA-29 a were constructed.Enzyme digestion results showed that the construction of chronic viruses of microRNA-29 a was successful.Detection of titers showed that the purified viruses belonged to the scope of use.When microRNA-29 a mimics were transfected into HCM,the expression of SCN5 A was decreased by qRT-PCR.Cell proliferation experiment showed that the proliferation rate of HCM transfected by microRNA-29 a mimics was lower than that of blank control group,and WB experiment showed that the expression of Nav1.5 protein was decreased.Conclusion: In the human cardiomyocyte model,miR-29 a reduces the expression of Nav1.5 protein by negatively regulating the downstream gene SCN5A... |
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