心律失常调控相关的心脏特异性转录因子探讨
发布时间：2019-03-29 12:34
【摘要】：背景与目的:心血管疾病是严重威胁人类,特别是50岁以上中老年人健康的常见病,居各种死因之首;且心血管系统疾病中,心律失常是最为严重的病症之一。研究表明,心律失常与心肌细胞膜上离子通道的突变有关;但大部分离子通道并未发生实质性病变的临床病人,仍然发生了心律失常,可能与转录因子的调控区发生变异,抑制下游离子通道基因的转录,导致离子通道表达量减少相关。虽然转录因子在心律失常中的作用研究得较多,但心律失常的调控网络复杂且机制尚不清楚。而近年来研究表明GWAS分析可为疾病的调控机制提供预测和研究基础,因此,本研究旨在1.分析心律失常相关GWAS数据,找到心律失常易感基因,特别是心脏特异性转录因子;2.通过生物信息学手段,分析心肌细胞HL-1的ChIP-seq数据,结合GWAS和增强子信号,寻找转录因子调控心律失常常见基因SCN5A的靶点;;3.重点研究TBX5调控SCN5A的靶点,阐明这些靶点与目的基因SCN5A表达的相关性;研究结果将为心律失常相关的离子通道蛋白Nav1.5的调控机制提供新的内容,为心律失常相关基因SCN5A的调控提供理论依据。方法:1.结合心律失常的GWAS数据,找到心律失常易感基因,推测基因组调控区在心律失常中的作用。2.运用生物信息学手段分析小鼠心肌细胞HL-1的C hIP-seq数据,找到转录因子TBX3、TBX5、TBX20、NKX2-5、GATA4对SCN5A基因的结合位点;3.用Luciferase报告基因系统验证结合位点的启动子/增强子活性:设计TBX5对SCN5A基因结合位点区的引物,分别构建入pGL3载体中,将质粒转染293T细胞,进行双荧光素酶活性测定;4.运用crispr/cas9技术在结合位点区域设计sgRNA,建立针对小鼠心肌细胞SCN5A基因的阻遏模型,通过荧光定量PCR技术检测阻遏模型对SCN5A基因转录的影响;结果:1.小鼠心脏或小鼠心肌细胞HL-1中与心律失常相关的基因共137个,分别与转录因子TBX3(72个)、TBX5(112个)、TBX20(50个)、NKX2-5(68个)、GATA4(25个)结合;受TBX20调控的主要心律失常基因有MYH6、MYH7、PLN、CDKN1A、SCN5A、GOT2、CASQ2、PRKCD、ATP1B1和FADS1;受TBX5调控的主要心律失常基因有SCN5A、CASQ2、KCNH2、PLN、SCN10A、KCNQ1;2.TBX3、TBX20、GATA4、NKX2-5分别在靶基因SCN5A上有4个、1个、1个、6个结合位点;TBX5在靶基因SCN5A上有12个结合位点,其中1个距基因上游9kb左右(chr9:119498070-119498519),其他11个都位于基因下游(chr9:119378730-119379479,等);3.SCN5A基因的增强子(pGL3-enhancer)及启动子(pGL3-promoter)重组质粒已成功构建;4.TBX5在靶基因SCN5A上的结合位点有增强子和启动子双重活性;5.成功构建了SCN5A基因的sgRNA,并通过dCas9系统进行了HL-1细胞的SCN5A基因沉默;结论:chr9:119498070-119498519和chr9:119378730-119379479可能是转录因子TBX5调控心律失常相关基因SCN5A的重要靶点。
[Abstract]:Background & objective: cardiovascular disease is a common disease which seriously threatens the health of human beings, especially the middle-aged and aged over 50 years old and ranks first in all kinds of causes of death, and arrhythmia is one of the most serious diseases in cardiovascular diseases. The results showed that arrhythmias were related to the mutation of ion channels in myocardial cell membrane. However, most of the clinical patients with no substantial pathological changes of ion channels still have arrhythmias, which may be related to the variation of the regulatory region of transcription factors, inhibit the transcription of downstream ion channel genes, and lead to the reduction of ion channel expression. Although the role of transcription factors in arrhythmias has been studied, the regulatory network of arrhythmias is complex and the mechanism is still unclear. In recent years, it has been shown that GWAS analysis can provide a basis for predicting and studying the mechanism of disease regulation. Therefore, the purpose of this study is 1. Analysis of arrhythmia-related GWAS data to identify arrhythmias susceptible genes, especially cardiac-specific transcription factors; 2. By means of bioinformatics, the ChIP-seq data of cardiac myocyte HL-1 were analyzed, and combined with GWAS and enhancer signals, the target of transcription factor to regulate arrhythmia was found to be the common gene SCN5A; 3. The target of SCN5A regulated by TBX5 was studied, and the correlation between these targets and the expression of target gene SCN5A was elucidated. The results will provide a new content for the regulation mechanism of arrhythmias-related ionic channel protein Nav1.5 and provide theoretical basis for the regulation of arrhythmias-related gene SCN5A. Methods: 1. Combined with the GWAS data of arrhythmias, the arrhythmias susceptible genes were found, and the role of genomic regulatory regions in arrhythmias was speculated. 2. The C-hIP-seq data of mouse cardiomyocyte HL-1 were analyzed by bioinformatics, and the binding site of transcription factor TBX3,TBX5,TBX20,NKX2-5,GATA4 to SCN5A gene was found. The promoter / enhancer activity of the binding site was verified by Luciferase reporter gene system. The primers of TBX5 to the binding site of SCN5A gene were designed and constructed into the pGL3 vector respectively. The plasmid was transfected into 293T cells and the double luciferase activity was determined; 4. the promoter / enhancer activity of the binding site was determined by double luciferase assay. The suppression model of SCN5A gene in mouse cardiomyocytes was established by designing sgRNA, in binding site region by crispr/cas9 technique, and the effect of repression model on transcription of SCN5A gene was detected by fluorescence quantitative PCR. Results: 1. There were 137 arrhythmias-related genes in mouse heart or mouse cardiomyocyte HL-1, which combined with transcription factors TBX3 (72), TBX5 (112), TBX20 (50), NKX2-5 (68) and GATA4 (25). The main arrhythmia genes regulated by TBX20 are MYH6,MYH7,PLN,CDKN1A,SCN5A,GOT2,CASQ2,PRKCD,ATP1B1 and FADS1;. The major arrhythmia genes regulated by TBX5 are SCN5A,CASQ2,KCNH2,PLN,SCN10A,KCNQ1;. 2. There were 4, 1, 1, 6 binding sites in the target gene SCN5A of TBX3, TBX20, GATA4, NKX2, respectively. TBX5 has 12 binding sites on the target gene SCN5A, one of which is about 9kb upstream of the gene (chr9:119498070-119498519), and the other 11 are located downstream of the gene (chr9:119378730-119379479, et al.). The recombinant plasmids of enhancer (pGL3-enhancer) and promoter (pGL3-promoter) of 3.SCN5A gene have been successfully constructed, and the binding sites of 4.TBX5 to the target gene SCN5A have double activities of enhancer and promoter; 5. The sgRNA, of SCN5A gene was successfully constructed and the SCN5A gene of HL-1 cells was silenced by dCas9 system conclusion: chr9:119498070-119498519 and chr9:119378730-119379479 may be important targets of transcription factor TBX5 to regulate arrhythmias-related gene SCN5A.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R541.7
本文编号：2449548
[Abstract]:Background & objective: cardiovascular disease is a common disease which seriously threatens the health of human beings, especially the middle-aged and aged over 50 years old and ranks first in all kinds of causes of death, and arrhythmia is one of the most serious diseases in cardiovascular diseases. The results showed that arrhythmias were related to the mutation of ion channels in myocardial cell membrane. However, most of the clinical patients with no substantial pathological changes of ion channels still have arrhythmias, which may be related to the variation of the regulatory region of transcription factors, inhibit the transcription of downstream ion channel genes, and lead to the reduction of ion channel expression. Although the role of transcription factors in arrhythmias has been studied, the regulatory network of arrhythmias is complex and the mechanism is still unclear. In recent years, it has been shown that GWAS analysis can provide a basis for predicting and studying the mechanism of disease regulation. Therefore, the purpose of this study is 1. Analysis of arrhythmia-related GWAS data to identify arrhythmias susceptible genes, especially cardiac-specific transcription factors; 2. By means of bioinformatics, the ChIP-seq data of cardiac myocyte HL-1 were analyzed, and combined with GWAS and enhancer signals, the target of transcription factor to regulate arrhythmia was found to be the common gene SCN5A; 3. The target of SCN5A regulated by TBX5 was studied, and the correlation between these targets and the expression of target gene SCN5A was elucidated. The results will provide a new content for the regulation mechanism of arrhythmias-related ionic channel protein Nav1.5 and provide theoretical basis for the regulation of arrhythmias-related gene SCN5A. Methods: 1. Combined with the GWAS data of arrhythmias, the arrhythmias susceptible genes were found, and the role of genomic regulatory regions in arrhythmias was speculated. 2. The C-hIP-seq data of mouse cardiomyocyte HL-1 were analyzed by bioinformatics, and the binding site of transcription factor TBX3,TBX5,TBX20,NKX2-5,GATA4 to SCN5A gene was found. The promoter / enhancer activity of the binding site was verified by Luciferase reporter gene system. The primers of TBX5 to the binding site of SCN5A gene were designed and constructed into the pGL3 vector respectively. The plasmid was transfected into 293T cells and the double luciferase activity was determined; 4. the promoter / enhancer activity of the binding site was determined by double luciferase assay. The suppression model of SCN5A gene in mouse cardiomyocytes was established by designing sgRNA, in binding site region by crispr/cas9 technique, and the effect of repression model on transcription of SCN5A gene was detected by fluorescence quantitative PCR. Results: 1. There were 137 arrhythmias-related genes in mouse heart or mouse cardiomyocyte HL-1, which combined with transcription factors TBX3 (72), TBX5 (112), TBX20 (50), NKX2-5 (68) and GATA4 (25). The main arrhythmia genes regulated by TBX20 are MYH6,MYH7,PLN,CDKN1A,SCN5A,GOT2,CASQ2,PRKCD,ATP1B1 and FADS1;. The major arrhythmia genes regulated by TBX5 are SCN5A,CASQ2,KCNH2,PLN,SCN10A,KCNQ1;. 2. There were 4, 1, 1, 6 binding sites in the target gene SCN5A of TBX3, TBX20, GATA4, NKX2, respectively. TBX5 has 12 binding sites on the target gene SCN5A, one of which is about 9kb upstream of the gene (chr9:119498070-119498519), and the other 11 are located downstream of the gene (chr9:119378730-119379479, et al.). The recombinant plasmids of enhancer (pGL3-enhancer) and promoter (pGL3-promoter) of 3.SCN5A gene have been successfully constructed, and the binding sites of 4.TBX5 to the target gene SCN5A have double activities of enhancer and promoter; 5. The sgRNA, of SCN5A gene was successfully constructed and the SCN5A gene of HL-1 cells was silenced by dCas9 system conclusion: chr9:119498070-119498519 and chr9:119378730-119379479 may be important targets of transcription factor TBX5 to regulate arrhythmias-related gene SCN5A.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R541.7
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相关期刊论文 前2条
1 李勇;李宾公;;Tbx3在生物起搏中的研究进展[J];生物医学工程学杂志;2014年04期
2 邓立彬;王立;熊招平;匡渤海;赵会安;;遗传性心律失常猝死的分子解剖研究现状及展望[J];中国法医学杂志;2013年06期
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