心律失常調(diào)控相關(guān)的心臟特異性轉(zhuǎn)錄因子探討
發(fā)布時(shí)間：2019-03-29 12:34
【摘要】：背景與目的:心血管疾病是嚴(yán)重威脅人類,特別是50歲以上中老年人健康的常見病,居各種死因之首;且心血管系統(tǒng)疾病中,心律失常是最為嚴(yán)重的病癥之一。研究表明,心律失常與心肌細(xì)胞膜上離子通道的突變有關(guān);但大部分離子通道并未發(fā)生實(shí)質(zhì)性病變的臨床病人,仍然發(fā)生了心律失常,可能與轉(zhuǎn)錄因子的調(diào)控區(qū)發(fā)生變異,抑制下游離子通道基因的轉(zhuǎn)錄,導(dǎo)致離子通道表達(dá)量減少相關(guān)。雖然轉(zhuǎn)錄因子在心律失常中的作用研究得較多,但心律失常的調(diào)控網(wǎng)絡(luò)復(fù)雜且機(jī)制尚不清楚。而近年來研究表明GWAS分析可為疾病的調(diào)控機(jī)制提供預(yù)測和研究基礎(chǔ),因此,本研究旨在1.分析心律失常相關(guān)GWAS數(shù)據(jù),找到心律失常易感基因,特別是心臟特異性轉(zhuǎn)錄因子;2.通過生物信息學(xué)手段,分析心肌細(xì)胞HL-1的ChIP-seq數(shù)據(jù),結(jié)合GWAS和增強(qiáng)子信號(hào),尋找轉(zhuǎn)錄因子調(diào)控心律失常常見基因SCN5A的靶點(diǎn);;3.重點(diǎn)研究TBX5調(diào)控SCN5A的靶點(diǎn),闡明這些靶點(diǎn)與目的基因SCN5A表達(dá)的相關(guān)性;研究結(jié)果將為心律失常相關(guān)的離子通道蛋白Nav1.5的調(diào)控機(jī)制提供新的內(nèi)容,為心律失常相關(guān)基因SCN5A的調(diào)控提供理論依據(jù)。方法:1.結(jié)合心律失常的GWAS數(shù)據(jù),找到心律失常易感基因,推測基因組調(diào)控區(qū)在心律失常中的作用。2.運(yùn)用生物信息學(xué)手段分析小鼠心肌細(xì)胞HL-1的C hIP-seq數(shù)據(jù),找到轉(zhuǎn)錄因子TBX3、TBX5、TBX20、NKX2-5、GATA4對(duì)SCN5A基因的結(jié)合位點(diǎn);3.用Luciferase報(bào)告基因系統(tǒng)驗(yàn)證結(jié)合位點(diǎn)的啟動(dòng)子/增強(qiáng)子活性:設(shè)計(jì)TBX5對(duì)SCN5A基因結(jié)合位點(diǎn)區(qū)的引物,分別構(gòu)建入pGL3載體中,將質(zhì)粒轉(zhuǎn)染293T細(xì)胞,進(jìn)行雙熒光素酶活性測定;4.運(yùn)用crispr/cas9技術(shù)在結(jié)合位點(diǎn)區(qū)域設(shè)計(jì)sgRNA,建立針對(duì)小鼠心肌細(xì)胞SCN5A基因的阻遏模型,通過熒光定量PCR技術(shù)檢測阻遏模型對(duì)SCN5A基因轉(zhuǎn)錄的影響;結(jié)果:1.小鼠心臟或小鼠心肌細(xì)胞HL-1中與心律失常相關(guān)的基因共137個(gè),分別與轉(zhuǎn)錄因子TBX3(72個(gè))、TBX5(112個(gè))、TBX20(50個(gè))、NKX2-5(68個(gè))、GATA4(25個(gè))結(jié)合;受TBX20調(diào)控的主要心律失�；蛴蠱YH6、MYH7、PLN、CDKN1A、SCN5A、GOT2、CASQ2、PRKCD、ATP1B1和FADS1;受TBX5調(diào)控的主要心律失�；蛴蠸CN5A、CASQ2、KCNH2、PLN、SCN10A、KCNQ1;2.TBX3、TBX20、GATA4、NKX2-5分別在靶基因SCN5A上有4個(gè)、1個(gè)、1個(gè)、6個(gè)結(jié)合位點(diǎn);TBX5在靶基因SCN5A上有12個(gè)結(jié)合位點(diǎn),其中1個(gè)距基因上游9kb左右(chr9:119498070-119498519),其他11個(gè)都位于基因下游(chr9:119378730-119379479,等);3.SCN5A基因的增強(qiáng)子(pGL3-enhancer)及啟動(dòng)子(pGL3-promoter)重組質(zhì)粒已成功構(gòu)建;4.TBX5在靶基因SCN5A上的結(jié)合位點(diǎn)有增強(qiáng)子和啟動(dòng)子雙重活性;5.成功構(gòu)建了SCN5A基因的sgRNA,并通過dCas9系統(tǒng)進(jìn)行了HL-1細(xì)胞的SCN5A基因沉默;結(jié)論:chr9:119498070-119498519和chr9:119378730-119379479可能是轉(zhuǎn)錄因子TBX5調(diào)控心律失常相關(guān)基因SCN5A的重要靶點(diǎn)。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R541.7
本文編號(hào)：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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R541.7
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相關(guān)期刊論文 前2條
1 李勇;李賓公;;Tbx3在生物起搏中的研究進(jìn)展[J];生物醫(yī)學(xué)工程學(xué)雜志;2014年04期
2 鄧立彬;王立;熊招平;匡渤海;趙會(huì)安;;遺傳性心律失常猝死的分子解剖研究現(xiàn)狀及展望[J];中國法醫(yī)學(xué)雜志;2013年06期
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