The Role And Molecular Mechanism Of Long Non-coding RNA NEAT1 In Heart Failure

Background:Heart failure(HF)is a complex clinical syndrome characterized by the inability of the heart to pump out enough blood to meet the body’s needs.The occurrence of heart failure is usually related to various diseases,including but not limited to myocardial infarction,hypertension,valve disease,and cardiomyopathy[1].Although modern medicine has made remarkable progress in the treatment and management of heart failure,its incidence rate and mortality remain high posing a serious threat to global public health.This current situation prompts us to continuously explore its pathogenesis,search for new treatment strategies and methods,in order to more effectively address this challenge.In the study of heart failure,the role of long non-coding RNAs is gradually receiving attention.Long non-coding RNAs(lncRNA)are a class of RNA molecule with transcript length exceeding 200 nucleotides and no protein coding ability.They play important regulatory roles in various biological processes,NEAT1(nuclear enriched abundant transcript 1),as an lncRNA located in the nucleus,has received widespread attention in recent years.An increasing number of studies indicate that lncRNA plays an indispensable role in the occurrence and development of cardiovascular diseases.They participate in regulating gene transcription,mRNA splicing,histone modification microRNA expression,cell proliferation,differentiation,and apoptosis through various methods such as cis and trans regulation,and are involved in the development,functional maintenance,and disease progression of the heart.Some studies have found that certain specific lncRNAs are abnormally expressed in the heart tissue or blood of patients with heart failure,and these abnormal expressions may be closely related to the occurrence,development,and prognosis of heart failure.Therefore,in-depth research on the mechanism of lncRNA in heart failure is of great significance for revealing the pathological and physiological processes of heart failure,searching for new therapeutic targets,and improving the prognosis of patients.NEAT1(nuclear enriched abundant transcript 1),as an lncRNA located in the nucleus,has received widespread attention in recent years.NEAT1 is a long chain non coding RNA located in the nucleus,which has received widespread research attention due to its regulatory role in various cancers.In hepatocellular carcinoma,it has been found that NEAT1 promotes cell proliferation "by regulating the miR-129-5p-VCP-I κ B axis[10].Although there are still few studies directly investigating the role of NEAT1 in heart failure,this regulatory mechanism suggests that NEAT 1 may play a similar role in the regulation of heart failure.Based on its known regulatory functions in other cardiovascular diseases and considering the pathophysiological mechanisms of heart failure,studying the role of lncRNA NEAT1 in heart failure and its possible molecular mechanisms may be of great significance for exploring the pathophysiological processes of heart failure,finding new therapeutic targets,and improving patient prognosis.Based on the above background,the main contents of this study include:1.By searching databases and using bioinformatics analysis techniques,screening and speculating on miRNAs that may participate in myocardial cell apoptosis together with NEAT1,and selecting them as research objects to deeply explore the impact of related signaling pathways on the degree of myocardial cell apoptosis with NEAT1;2.Exploring the relationship between NEAT1 and myocardial cell apoptosis by detecting the expression of NEAT 1 in the serum of heart failure,acute myocardial infarction,angina pectoris,and healthy individuals.3.Establish an in vitro myocardial cell model and use various cell experimental techniques to evaluate the effects of NEAT1 on myocardial cell migration,proliferation,apoptosis,and protein deposition,and reveal its mechanism of regulating microRNA expression.Motivation:Firstly,it is hoped that through in-depth research on the mechanism of lncRNA in heart failure,its relationship with the occurrence and development of heart failure can be revealed.By understanding the expression changes,regulatory networks,and interactions with other molecules of lncRNA in the process of heart failure,we aim to explore the pathological and physiological processes of heart failure and provide theoretical basis for finding new diagnostic and therapeutic targets.Secondly,lncRNA has the characteristics of tissue-specific and spatiotemporal specific expression,making it a potential biomarker and therapeutic target.By studying the function and regulatory mechanism of specific IncRNAs in heart failure,we can explore their potential as biomarkers for early diagnosis,prognostic evaluation,and treatment monitoring of heart failure.Meanwhile,targeted therapy strategies targeting specific lncRNAs may also provide new avenues for the treatment of heart failure.Finally,with the rapid development of high-throughput sequencing technology and bioinformatics,we can more efficiently identify and analyze lncRNAs in heart failure.The application of these technologies will provide us with more new information and discoveries about lncRNA in heart failure,which will help us to gain a deeper understanding of the complexity and diversity of heart failure and provide strong support for personalized treatment.In summary,our research motivation is to provide new ideas and methods for the prevention and treatment of heart failure by in-depth studying the mechanism and functional role of lncRNA in hear failure.We hope that through this research,more effective and safer treatment methods can be brought to patients with heart failure,improving their quality of life and life expectancy.Meanwhile,we also hope to provide new perspectives and strategies for the prevention and control of cardiovascular diseases through this research.Objectives:The aim of this study is to investigate the role of lncRNA in cardiomyocyte apoptosis in heart failure and explore its possible molecular mechanisms.Methods:The serum samples for this study were collected from 135 patients with heart failure,acute myocardial infarction,and angina who were hospitalized in the Cardiovascular Department of the Lequn Campus of the First Hospital of Jilin University from January 2018 to January 2019,as well as healthy individuals who underwent physical examinations at the examination center of the First Hospital of Jilin University during the same period.They were divided into four groups(see Chapter 2 for details).The study population was all from Northeast China,and there was no blood relationship between individuals.Conduct in vitro experiments using H9c2(2-1)cell lines obtained and subcloned from DB1X rat embryonic heart tissue.These cells were cultured in specific media and induced apoptosis by H2O2 to simulate the in vivo myocardial injury environment.The experimental methods applied in the research process include:1.Bioinformatics analysis:Using bioinformatics databases to predict miRNAs and their binding regions targeting NEAT1 can aid in subsequent experimental design and mechanism exploration.2.Analyze the expression levels of NEAT1 and miR-129-5p in human serum and H9c2(2-1)cells using real-time fluorescence quantitative PCR(qRT PCR).This step is crucial for understanding the expression patterns of these genes in disease states.3.Cell transfection:In order to further investigate the functions of NEAT1 and miR-129-5p,we constructed corresponding knockdown and overexpression plasmids and introduced these plasmids into H9c2(2-1)cells through transfection technology.4.Apoptosis detection;Use flow cytometry and Caspase-3 activity detection kit to evaluate the apoptosis of H9c2(2-1)cells.These experiments can quantify the proportion of apoptotic cells and the activity of Caspase-3,thereby evaluating the impact of NEAT1 and miR-129-5p on the apoptosis process.5.Western blot:Analyze the expression levels of apoptosis related proteins such as Bcl-2,Bax,and cleared Caspase-3 to further verify the regulatory effects of NEAT1 and miR-129-5p on the apoptosis pathway.6.Construction of reporting vector and luciferase reporting experiment:Construct a NEAT1 reporting vector containing predicted miR-129-5p binding sites,and verify the direct regulatory effect of miR-129-5p on NEAT1 through luciferase reporting experiment.7.Biotin coupled miRNA capture:Through pull-down experiments,capture the RNA complex bound to biotin coupled miR-129-5p,and analyze the level of NEATl using qRT PCR to verify the interaction between the two.Results:1.NEATl has the highest expression in the heart failure group,followed by the myocardial infarction group,lower expression in the angina group,and lowest expression in the healthy population group.2.There is an inhibitory interaction between miR-129-5p and NEATl.3.NEAT1 enhances cell apoptosis,while miR-129-5p inhibits this apoptosis.4.Inhibiting NEAT1 can reduce cell apoptosis and promote cell proliferation.Conclusion:1.NEAT1 promotes myocardial cell apoptosis and exacerbates heart failure by downregulating miR129-5p expression.2.The NEAT1-miR129-5p axis may provide new ideas or targets for the diagnosis and treatment of heart failure.