| Atrial fibrillation(AF)is one of the most common arrhythmias in clinic,which has attracted more and more attention all over the world.The prevalence and mortality of atrial fibrillation show age dependence.The older the age,the higher the incidence rate.Meanwhile,it can cause serious complications such as arterial embolism and stroke,which has seriously threatened human life and health.Although the current research on atrial fibrillation continues to deepen,but the effect of treatment and cure is not ideal.If we can deeply explore the occurrence and development of atrial fibrillation,study its pathogenesis from the molecular level,and find the appropriate intervention targets,it will be of great significance for the diagnosis and treatment of atrial fibrillation.With the development of science and technology,the emergence of high-throughput sequencing and the application of bioinformatics,more and more gene information is mined and known by people.In recent years,the emergence of non coding RNA(nc RNA)has attracted the attention of researchers.Studies have shown that only 2%of human transcripts encode proteins,and the remaining 98%are non coding RNAs,including r RNA,t RNA,mi RNA,Lncrna,etc.Their common feature is that they are transcribed from the genome,but not translated into proteins,and only perform corresponding biological functions at the RNA level.Generally,nc RNA with nucleotide sequence larger than 200 nt is defined as long non coding RNA(lncrna),and micro RNA(mi RNA)with nucleotide sequence size between19-25.More and more studies have confirmed that Lncrna and mi RNA are involved in various life activities of the body,including individual growth and development,cell damage repair,tumor migration and invasion,as well as various cardiovascular and cerebrovascular diseases,nervous system diseases,digestive system diseases,endocrine system diseases and so on.In recent years,a large number of studies have found that the abnormal expression of non coding RNA plays an important role in the pathogenesis of cardiovascular diseases,including atrial fibrillation,and has gradually become a new hotspot and focus of cardiovascular basic and clinical research.Myocardial infarction associated transcript(MIAT)of Lncrna was first found and named in patients with myocardial infarction.Some scholars found that the expression of Lnc RNA MIAT was closely related to myocardial hypertrophy,myocardial fibrosis,ventricular remodeling and ejection fraction.A large number of studies have confirmed that atrial fibrosis and atrial remodeling are one of the important mechanisms of the occurrence and maintenance of atrial fibrillation.Therefore,we speculate that l Lnc RNA MIAT may play a role in atrial fibrillation.mi R-133a is one of the most abundant mi RNAs in the heart.Previous studies have shown that mi R-133a-3p plays a regulatory role in cardiovascular diseases such as atrial fibrillation.Because previous studies have confirmed that there is a targeted binding relationship between Lnc RNA MIAT and mi R-133a-3p,we chose Lnc RNA MIAT and mi R-133a-3p as the research objects to detect their expression in the rat model of atrial fibrillation,By regulating their expression levels,we analyzed the effect of Lnc RNA MIAT/mi R-133a-3p signal axis on the occurrence and development of atrial fibrillation and atrial remodeling,hoping to provide new theoretical basis and practical significance for molecular targeted therapy of atrial fibrillation.At the same time,this study analyzed the expression of Lnc RNA MIAT and mi R-133a-3p in the blood of patients with atrial fibrillation,and combined with the clinical pathological characteristics and echocardiographic indexes of patients with atrial fibrillation,to explore the relationship between the two RNAs and atrial fibrillation and cardiac function,in order to provide new ideas for the prediction,early diagnosis and treatment of atrial fibrillation.This study was divided into three parts:Part I:Study on the mechanism of Lnc RNA-MIAT/mi R-133a-3p on rats with atrial fibrillation.Part II:Study on the effect of Lnc RNA-MIAT/mi R-133a-3p on atrial remodeling in rats with atrial fibrillation.Part III:Expression analysis of Lnc RNA-MIAT and mi R-133a-3p in patients with atrial fibrillation and its clinical significance.Part one The mechanism of Lnc RNA-MIAT/mi R-133a-3p on atrialfibrillation in ratsObjective:Atrial fibrillation is one of the most common arrhythmias in clinical practice.With the increase of age,the incidence of atrial fibrillation is increasing.Atrial fibrillation can increase the incidence and mortality of thromboembolic diseases,stroke,and heart failure,and seriously affect the quality of life of patients.Studies have suggested that non-coding RNAs may be involved in the pathogenesis of atrial fibrillation.Animal models of rats with atrial fibrillation were induced,and lentiviruses targeting Lnc RNA-MIAT and mi R-133a-3p were constructed.The effects of Lnc RNA-MIAT/mi R-133a-3p axis on atrial fibrillation and possible mechanisms were observed by injecting lentivirus into the right atrium of rats with AF.Methods:1.The experimental group was divided into six groups:sham,AF,AF+shcontrol,AF+sh MIAT、AF+sh MIAT+anti-mi R-Control、AF+sh MIAT+anti-mi R-133a-3p.In addition to Sham group and AF group,the other groups were injected with corresponding lentivirus and control 10μl respectively,with a titer was 10~8TU,Sham group and AF group were injected with the same volume of solvent.After 48 hours of injection,the expression of Lnc RNA-MIAT/mi R-133a-3p genes in each group was detected by q RT-PCR to verify the effect of the virus.2.After the successful transfection of lentivirus,the occurrence of atrial fibrillation in rats was induced by electric shock.P wave disappeared in ECG,stable f wave appeared and RR interval was unequal,which was regarded as the model success.The electrocardiogram of rats in each group was tested once a week after continuous induction for 5 weeks.The right atrium of 3 rats in each Sham group and AF group was taken every weekend and stored in a refrigerator at-80℃after quick-freezing of liquid nitrogen.After 5 weeks,the expression levels of Lnc RNA-MIAT and mi R-133a-3p genes in the two groups were detected by q RT-PCR at 5 time points,and the expression levels of Sham group rats detected in the first test were used as a benchmark to draw the gene expression-time curve.3.Five weeks after the induction of AF,the electrocardiogram indexes of rats of different groups were detected to analyze atrial fibrillation,and analyze the influence of Lnc RNA-MIAT and mi R-133a-3p on the the duration of atrial fibrillation.4.q RT-PCR was used to detect the expression of Lnc RNA-MIAT and mi R-133a-3p in each group after 5 weeks of AF induction.5.TUNEL staining was used to analyze the effects of Lnc RNA-MIAT and mi R-133a-3p on cardiomyocyte apoptosis.6.Double luciferase experiment was used to verify the targeting relationship between Lnc RNA-MIAT and mi R-133a-3p.Results:1.After 48h of lentivirus injection,Lenti-sh MIAT could significantly reduce the expression level of MIAT,and Lenti-anti-mi R-133a-3p could significantly inhibit the decline of MIAT,playing a salvage role,with statistical significance(P<0.05).Lenti-sh MIAT injection significantly increased the expression level of mi R-133a-3p,and anti-mi R-133a-3p injection inhibited the expression level of mi R-133a-3p(P<0.05).Lentivirus transfection was effective.2.During 5-weeks of AF induction,the ECG of Sham was normal,while F wave,P wave disappeared and RR interval were different in AF group.The gene expression-time curve showed that the relative expression of MIAT increased significantly after AF induction compared with Sham group,and the expression of MIAT decreased with time.On the contrary,the relative expression of mi R-133a-3p decreased significantly after AF induction,while the relative expression of mi R-133a-3p increased gradually.However,there was significant difference in their expression between Sham and AF groups(P<0.05).3.After 5 weeks of AF induction,ECG of rats in each group were analyzed.The results showed that sinus rhythm occurred in Sham group,and typical F-wave and P-wave disappearance and absolutely irregular RR interval occurred in AF group.The duration of atrial fibrillation can be effectively shorten in rats with knockdown MIAT,and injection of anti-mi R-133a-3p lentivirus could significantly reverse the effect of MIAT gene knockout(P<0.05).4.After 5 weeks of AF induction,q RT-PCR results showed that MIAT expression level in AF group was significantly increased compared with Sham group(P<0.05).Lenti-sh MIAT could significantly inhibit MIAT gene expression(P<0.05).MIAT expression was significantly increased after the injection of anti-mi R-133a-3p(P<0.05).The expression trend of mi R-133a-3p in each group was just opposite to that of MIAT,and the expression level of AF group was significantly lower than that of Sham group.Lenti-sh MIAT injection significantly increased its expression,and anti-mi R-133a-3p could reduce its expression in addition(P<0.05).5.TUNEL staining results showed that compared with Sham group,apoptosis rate of myocardial cells in AF group was significantly increased(P<0.05),while sh MIAT intervention significantly inhibited apoptosis of myocardial cells(P<0.05).However,anti-mi R-133a-3p could reverse the inhibitory effect of sh MIAT on myocardial cell apoptosis(P<0.05).6.Double luciferase assay showed that there were two binding sites on MIAT that could target anti-mi R-133a-3p,but the two binding sequences had no synergistic effect on the interaction between MIAT and mi R-133a-3p.Summary:1.There is a target binding relationship between MIAT and mi R-133a-3p,which plays an important role in the process of AF.After MIAT knockdown,atrial fibrillation can be significantly alleviated,ECG parameters can be improved,duration of atrial fibrillation can be shortened.Injection of anti-mi R-133a-3p reversed the effect of sh MIAT.2.MIAT knockdown can significantly inhibit the apoptosis of myocardial cells in rats with atrial fibrillation;anti-mi R-133a-3p could significantly reverse the inhibitory effect of sh MIAT on myocardial cell apoptosis.Part Two Effect of Lnc RNA-MIAT/mi R-133a-3p on atrial remodelingin rats with atrial fibrillationObjective:The effects of Lnc RNA-MIAT/mi R-133a-3p signal axis on atrial remodeling in rats with atrial fibrillation was analyzed by detecting the expression of relevant marker genes and proteins in atrial electrical remodeling and structural remodelingMethods:1.Western blot was used to detect the expressions of sodium channel protein Nav1.5 and potassium channel protein Kv1.5.2.Masson staining was used to detect the atrial tissue fibrosis of rats in each group.3.q RT-PCR and Western blot were used to detect the expression changes of TGF-β1、CTGF、Collagen I and III genes and proteins respectively.Results:1.WB results showed that Nav1.5 and Kv1.5 protein expressions in AF group were significantly decreased compared with Sham group(P<0.05).Sh MIAT significantly increased the expression levels of the two channel proteins(P<0.05).The intervention of anti-mi R-133a-3p could significantly reverse the effect of knockdown MIAT(P<0.05).2.Masson staining results showed that compared with Sham group,the collagen content in the right atrium of AF group rats was significantly increased,and knockdown of MIAT gene significantly alleviated atrial fibrillation fibrosis.In addition,rescue experiments showed that anti-mi R-133a-3p could significantly reverse fibrosis alleviated by knockdown MIAT.3.q RT-PCR results showed that the expression levels of TGF-β1、CTGF、Collagen I and III were significantly increased in the right atrium tissues of rats in AF group(P<0.05),and the expression levels of their m RNA were significantly reduced after MIAT gene expression was inhibited(P<0.05).In addition,after the intervention of anti-mi R-133a-3p,the expression levels of these genes were significantly increased in atrial fibrillation rats with MIAT gene knockdown(P<0.05).The results of WB and PCR were consistent.Summary:1.The change of Lnc RNA-MIAT/mi R-133a-3p gene expression in atrial fibrillation rats can affect the relative proteins expression of Nav1.5 and Kv1.5.2.Lnc RNA-MIAT/mi R-133a-3p signaling axis can affect atrial remodeling in rats with atrial fibrillation.Part Three Expression and clinical significance of Lnc RNA-MIAT andmi R-133a-3p in patients with atrial fibrillationObjective:This study aims to detect the expression changes of lnc RNA-MIAT and mi R-133a-3p in blood white blood cells of patients with atrial fibrillation and volunteers,and to analyze the relationship between lnc RNA-MIAT and mi R-133a-3p and atrial fibrillation and heart function in combination with clinicopathological characteristics and cardiac ultrasound indicators of patients with atrial fibrillation,so as to explore their predictive and diagnostic value in atrial fibrillation.Methods:1.A total of 80 patients with atrial fibrillation(40 cases of persistent atrial fibrillation,40 cases of paroxysmal atrial fibrillation)and 47 volunteers were collected.The expression levels of Lnc RNA-MIAT and mi R-133a-3p in white blood cells of the subjects were detected by q RT-PCR.2.Echococardiography was performed for all subjects,and LAD,LAVmax,LAVmin,LAEF,LVEF and E/e’were determined.The correlation between atrial fibrillation and Lnc RNA-MIAT,mi R-133a-3p and other factors were analyzed based on the clinicopathological characteristics of subjects in each group,and regression analysis was conducted for each related factor and atrial fibrillation.Two-class Logistic regression analysis was used to established biomarkers for the diagnosis of AF.Results:1.Expression analysis of Lnc RNA-MIAT and mi R-133a-3p in persistent atrial fibrillation,paroxysmal atrial fibrillation and white blood cells in the control groupThe results of q RT-PCR showed that the expression level of Lnc RNA-MIAT in blood white blood cells of patients with persistent atrial fibrillation and paroxysmal atrial fibrillation was significantly higher than that of the control group(P<0.05),there is no statistically significant difference between the persistent atrial fibrillation group and the paroxysmal atrial fibrillation group(P>0.05),However,the expression trend of mi R-133a-3p was opposite to that of Lnc RNA-MIAT.The control group is the highest,the paroxysmal atrial fibrillation group is lower than the control group,and the persistent atrial fibrillation group has the lowest expression level.There are significant differences among the three groups(P<0.05).2.Correlation between clinical information characteristics of patients with atrial fibrillation or healthy volunteers and atrial fibrillationCorrelation analysis results showed that gender,age,BMI,history of hypertension,systolic blood pressure level,lnc RNA-MIAT and mi R-133a-3p expression level,LAD,LAVmax,LAVmin,LAEF,E/e’were all significantly correlated with the occurrence of atrial fibrillation(P<0.05),while diastolic blood pressure level and LVEF were not significantly correlated with atrial fibrillation(P>0.05).Lnc RNA-MIAT and mi R-133a-3p were significantly correlated with LAD,LAVmax,LAVmin and LAEF(P<0.05),while LVEF and E/e’were not significantly correlated with Lnc RNA-MIAT and mi R-133a-3p(P>0.05).3.Regression analysis of atrial fibrillation and related factorsRegression analysis results showed that mi R-133a-3p,LA and LAVmin had significant significance in the logistic regression diagnostic equation of atrial fibrillation(P<0.05),while lnc RNA-MIAT had no significant significance in the logistic regression diagnostic equation of atrial fibrillation(P>0.05).Summary:1.Lnc RNA-MIAT was highly expressed in patients with AF,while mi R-133a-3p was low expressed in patients with AF.Both expression levels are closely related to the occurrence of atrial fibrillation and left atrial function.2.mi R-133a-3p can be used as a potential novel marker for the prediction and diagnosis of atrial fibrillation.Conclusions:1.There is a target binding relationship between MIAT and mi R-133a-3p,which plays an important role in the process of AF.After MIAT knockdown,atrial fibrillation can be significantly alleviated,duration of atrial fibrillation can be shortened.MIAT knockdown can significantly inhibit the apoptosis of myocardial cells in rats with atrial fibrillation.2.Lnc RNA-MIAT/mi R-133a-3p signaling axis can affect atrial remodeling in rats with atrial fibrillation.3.Lnc RNA-MIAT was highly expressed in patients with AF,while mi R-133a-3p was low expressed in patients with AF.Both expression levels are closely related to the occurrence of atrial fibrillation and left atrial function.mi R-133a-3p can be used as a potential novel marker for the prediction and diagnosis of atrial fibrillation. |
PDF Full Text Request