Study On The Heart Specific Transcription Factors Related To The Arrhythmia Regulation

Background and Objective: Cardiovascular disease is a common diseases which threat human beings seriously, especially to those person over 50 years old, and it’s the disease with the highest mortality. In cardiovascular system diseases, arrhythmia is one of the most serious illness. Studies have shown that arrhythmia is associated with mutations of ion channeles on the membrane of myocardial cell. However, most clinical patients without substantial disease still developed arrhythmia, maybe due to variation of the regulatory region on transcription factors inhibited the transcription of ion channel genes downstream, and decreased the expression of ion channel. Although there are many studies about the functions of transcription factors in arrhythmia, the regulatory mechanism is still unclear because the regulatory network is complex. Recent studies showed that GWAS can help to provid prediction and research basis for the regulatory mechanism of disease. O ur study aims at:1. Analyse GWAS data related with arrhythmia, looking for arrhythmia susceptible gene, especially those cardiac specific transcription factors. 2. Analyse C hIP- seq data of myocardial cells HL-1by bioinformatics, looking for the target site of SCN5 A, a common arrhythmia regulatory gene, based on both the GWAS and the enhancer signal. 3. Study mainly on the target site of TBX5 o n SCN5 A, illuminate the relationship between these target and the expression of SCN5 A. Our results will provide new content for the regulatory mechanisms of arrhythmia related ion channel protein Nav1.5, and will provide theoretical foundation for the regulatory of arrhythmia related gene SCN5 A.Methods:1. Looking for arrhythmia susceptible gene by analyse GWAS data related with arrhythmia, and speculate the effects of genomic regulator in arrhythmia. 2. Analyse ChIP- seq data by bioinformatics, looking for the target site of transcription factors TBX3, TBX5, TBX20, NKX2-5 and GATA4 on SCN5 A gene. 3. Detect the promoter/enhancer activity of the target sites with luciferase reporter gene system: Design the primers for TBX5 binding sites on SCN5 a, constructed in the pGL3 vectors individually and transfected to 293 T cells, then detected the dual luciferase activity. 4. sgRNA on the binding sites was design with crispr/cas9 system, repression model for SCN5 A gene of mice myocardial cell was set up, and the repression effects was detected by realtime PCR.Results:1. 1. There are 137 arrhythmia related genes in the heart and myocardial cell of mice, they binding xites are TBX3(72), TBX5(112), TBX20(50), NKX2-5(68) and GATA4(25)individually. MYH6, MYH7, PLN, CDKN1 A, SCN5 A, GOT2, CASQ2, PRKCD, ATP1B1 and FADS1gene are regulated by TBX20, SCN5 A, CASQ2, KCNH2, PLN, SCN10 A and KCNQ1 gene are regulated by TBX5. 2. There are 4, 1, 1 and 6 binding sites on SCN5 A gene for TBX3, TBX20, GATA4 and NKX2-5 respectivly. There are 12 binding sites on SCN5 A gene for TBX5, and one of them is 9kb from the upstream(chr9:119498070-119498519), others are located at the downstream(chr9:119378730-119379479, etal)of the gene. 3. Recombinant of pGL3-enhancer and pGL3-promoter of SCN5 A have been constracted successfully. 4. The binding site of TBX5 on SCN5 A have both promoter/enhancer activity. 5. sgRNA of SCN5 A gene have been constracted successfully, and SCN5 A gene in HL-1 cell was scilenced by dCas9 system.Conclusion: chr9: 119498070-119498519 and chr9: 119378730-119379479 maybe the important targets for the transcription factors TBX5 on arrhythmia related gene SCN5 A.