本文選題：GATA4 + GATA6　； 參考：《鄭州大學(xué)》2012年博士論文

【摘要】：研究背景與目的： 心臟具有生成ATP的巨大潛能以滿足對(duì)能量的高度需求。在胚胎時(shí)期及心肌肥大發(fā)生時(shí),心臟主要依賴葡萄糖和乳糖作為底物產(chǎn)生ATP,而在出生后及成人心臟則主要依賴脂肪酸作為底物產(chǎn)生ATP而獲得能量。由于心肌的脂質(zhì)和葡萄糖儲(chǔ)存能力有限,因此,它們作為底物的選擇及ATP的生成是受到快速調(diào)節(jié)的,以迅速適應(yīng)心臟的能量需求。 這一調(diào)節(jié)過程涉及到多種基因不同時(shí)間的先后表達(dá)和相互作用,它們形成一個(gè)有序的網(wǎng)絡(luò)。該調(diào)節(jié)網(wǎng)絡(luò)包括大量的,在時(shí)間和空間上極為精確的一系列分子事件。在這個(gè)過程中,哪怕是極微小的錯(cuò)誤都將導(dǎo)致心肌能量代謝障礙的發(fā)生,而心肌能量代謝障礙,如線粒體脂肪酸氧化活性的降低,造成ATP產(chǎn)量減少,將會(huì)導(dǎo)致心泵功能、收縮功能及鈣離子的轉(zhuǎn)運(yùn)功能等失調(diào),從而誘發(fā)心肌肥大和心力衰竭等。因此,深入研究心肌能量代謝關(guān)鍵基因的調(diào)控機(jī)制,將有助于從分子水平闡明心血管疾病的分子機(jī)理和病理學(xué)基礎(chǔ),同時(shí)也可為心血管疾病的臨床防治和新藥設(shè)計(jì)提供更多線索。 那么,涉及心肌能量代謝的關(guān)鍵基因有什么呢?Glut4是調(diào)節(jié)心肌能量代謝的一個(gè)基因,它是葡萄糖轉(zhuǎn)運(yùn)蛋白家族的成員,維持著機(jī)體葡萄糖的內(nèi)穩(wěn)態(tài)平衡。Glut4的表達(dá)具有組織特異性并受到激素調(diào)節(jié),Glut4基因敲除小鼠出現(xiàn)心肌肥大癥狀。因此,GIut4是調(diào)節(jié)心肌能量代謝的關(guān)鍵基因之一。鑒于此,GIut4作為心肌能量代謝的關(guān)鍵基因,其表達(dá)的降低或上調(diào),對(duì)于心肌能量代謝的調(diào)節(jié)具有重要的生理或病理意義,所以研究其調(diào)控機(jī)制,是心血管分子生物學(xué)領(lǐng)域的重要課題。 那么,參與心肌能量代謝的轉(zhuǎn)錄因子有哪些呢?過氧化物酶體增殖物激活受體(peroxisome proliferator activated receptors, PPARs)是調(diào)節(jié)編碼脂肪酸β-氧化相關(guān)酶類基因的重要轉(zhuǎn)錄因子,它包含三種異形體：PPARa、PPARγ和PPARδ/1β。這三種異形體都在心臟中表達(dá),與PPARy相比,PPARa和PPARδ/β在心肌細(xì)胞中的表達(dá)更為豐富。研究表明,心臟特異性過表達(dá)PPARa將誘導(dǎo)與脂肪酸利用有關(guān)的基因表達(dá),進(jìn)而增強(qiáng)心臟脂肪酸的利用和氧化；PPARδ/β基因敲除小鼠嚴(yán)重地?fù)p害心肌脂肪酸氧化相關(guān)基因的表達(dá),增加心臟脂肪酸堆積,從而誘發(fā)脂毒性。因此,PPARs在調(diào)節(jié)心肌能量代謝中發(fā)揮著重要作用。 GATA蛋白是具有鋅指結(jié)構(gòu)的轉(zhuǎn)錄因子,因其識(shí)別序列(A/T) GATA (A/G)而得名。在該家族6個(gè)成員中,GATA4和GATA6是近年來(lái)心血管領(lǐng)域的研究熱點(diǎn)。大量的研究證明GATA4和GATA6是調(diào)控心臟發(fā)育以及心肌細(xì)胞增殖、分化和凋亡的重要轉(zhuǎn)錄因子。但它們是否在心肌能量代謝中發(fā)揮調(diào)控作用,目前知之甚少。 MicroRNAs (miRNAs)是進(jìn)化上高度保守的內(nèi)源性單鏈非編碼小RNA,通過轉(zhuǎn)錄后機(jī)制調(diào)控靶基因的表達(dá)。近年來(lái)的研究表明miRNAs是心臟基因轉(zhuǎn)錄調(diào)控網(wǎng)絡(luò)的一個(gè)重要組成部分,但miRNAs是否作用于GATA蛋白與PPARs,進(jìn)而對(duì)細(xì)胞的表型產(chǎn)生影響,目前還鮮有報(bào)道。 因此,本課題以GATA轉(zhuǎn)錄因子為中心,采用多種方法深入研究了GATA6、 PPARa、miR-200b以及GATA4對(duì)心肌能量代謝和細(xì)胞增殖的調(diào)控機(jī)制。 研究方法與結(jié)果： 第一部分：轉(zhuǎn)錄因子GATA6招募PPARa協(xié)同激活GIut4基因的表達(dá) 為進(jìn)一步了解GATA家族對(duì)心肌能量代謝的調(diào)控作用,采用了瞬時(shí)轉(zhuǎn)染、Q-PCR、熒光素酶報(bào)告基因、Western Blot、CoIP、GST pull-down和ChIP等實(shí)驗(yàn)方法,證實(shí)了在PPARa激動(dòng)劑fenofibrate刺激的心肌細(xì)胞中,轉(zhuǎn)錄因子GATA6能夠招募核受體PPARa至葡萄糖轉(zhuǎn)運(yùn)蛋白GIut4啟動(dòng)子的GATA元件上,這種招募能夠進(jìn)一步激活Glut4基因的表達(dá),并且伴隨著線粒體功能的促進(jìn)及葡萄糖利用的增加。 Western Blot及Q-PCR實(shí)驗(yàn)顯示GATA6或PPARa及其激動(dòng)劑fenofibrate都能夠刺激Glut4基因的表達(dá),若二者共表達(dá)則可以產(chǎn)生更強(qiáng)烈的激活效應(yīng),說明GATA6和PPARa協(xié)同激活Glut4基因轉(zhuǎn)錄。CoIP實(shí)驗(yàn)證實(shí)GATA6和PPARa相互作用。GST pull-down實(shí)驗(yàn)顯示GATA6的C端鋅指和PPARa的N端鋅指介導(dǎo)二者間的相互作用。ChIP及熒光素酶報(bào)告基因?qū)嶒?yàn)進(jìn)一步證實(shí)GATA6通過與PPARa蛋白的N端TAD和鋅指結(jié)構(gòu)域相互作用,招募PPARa至Glut4啟動(dòng)子從而協(xié)同激活Glut4基因的表達(dá),促進(jìn)線粒體的功能及葡萄糖的利用。 該部分的研究證實(shí)了轉(zhuǎn)錄因子GATA6是心肌能量代謝的重要調(diào)節(jié)因子,核受體PPARa是GATA6調(diào)節(jié)基因轉(zhuǎn)錄過程中新的相互作用因子,這為理解GATA6在心臟發(fā)育和心臟疾病的作用機(jī)制提供了新的分子基礎(chǔ)。 第二部分：fenofibrate和Dox對(duì)小鼠NADH氧化酶及檸檬酸脫氫酶活性的影響 多柔比星(doxorubicin, Dox)是一種常用的抗腫瘤藥物,由于它的嚴(yán)重毒性大大限制了它的臨床使用。線粒體功能障礙是其潛在的致毒機(jī)制之一,但具體機(jī)制未知。由于在第一部分研究中觀察到PPARa對(duì)細(xì)胞線粒體功能具有保護(hù)作用,所以我們進(jìn)一步研究了PPARa激動(dòng)劑fenofibrate在體內(nèi)能否保護(hù)線粒體功能并逆轉(zhuǎn)Dox的毒性。 將8周大的小鼠隨機(jī)分為四組并給予不同的處理：對(duì)照組：每天0.5%羧甲基纖維素鈉灌胃一次,連續(xù)14天；fenofibrate處理組：每天fenofibrate(100mg/kg)灌胃一次,連續(xù)14天；Dox處理組：第12、13和14天,每天腹腔注射Dox(15mg/kg)一次；聯(lián)合組：同時(shí)接受fenofibrate和Dox處理。14天后處死小鼠,分離提取心室、心房、肝臟、腎臟、肺和脾臟組織的線粒體,檢測(cè)線粒體功能的標(biāo)志酶檸檬酸合酶及NADH氧化酶活性。結(jié)果顯示：fenofibrate可在多個(gè)組織中保護(hù)線粒體功能,相反,Dox則在多數(shù)組織中抑制線粒體的功能,而且,fenofibrate在心室和腎臟中能夠逆轉(zhuǎn)Dox的毒性。 這一研究證實(shí)fenofibrate對(duì)線粒體檸檬酸合酶和NADH氧化酶具有保護(hù)作用,并且在心室和腎臟中能夠逆轉(zhuǎn)Dox的毒性作用,為臨床上如何減輕Dox的化療副作用提供了理論依據(jù)。 第三部分：miR-200b作用于GATA4對(duì)細(xì)胞增殖、周期和凋亡的調(diào)控 為了尋找GATA家族的上游調(diào)控分子,經(jīng)生物信息學(xué)分析,發(fā)現(xiàn)GATA4可能是miR-200b的靶基因。miR-200b參與調(diào)控上皮間質(zhì)轉(zhuǎn)化(EMT)過程,是腫瘤細(xì)胞形成的重要調(diào)控因子。由于miRNAs調(diào)控細(xì)胞增殖、分化和凋亡等多個(gè)生理學(xué)和病理學(xué)過程,因此,我們采用穩(wěn)定轉(zhuǎn)染、RNAi、熒光素酶報(bào)告基因、MTT、流式細(xì)胞術(shù)、DAPI染色RT-qPCR等實(shí)驗(yàn)方法研究了miR-200b對(duì)細(xì)胞增殖、周期和凋亡的影響。 結(jié)果顯示：過表達(dá)GATA4和rniR-200b分別促進(jìn)和抑制C2C12及P19細(xì)胞的增殖。而且,過表達(dá)(?)niR-200b及siRNA抑制GATA4表達(dá)均誘導(dǎo)細(xì)胞發(fā)生G0/G1期阻滯,S期和G2/M期細(xì)胞百分比降低。DAPI染色證實(shí)miR-200b過表達(dá)還能誘導(dǎo)細(xì)胞核發(fā)生凋亡。熒光素酶報(bào)告基因?qū)嶒?yàn)表明miR-200b可與GATA43'非翻譯區(qū)(3’-UTR)特異性結(jié)合。Western Blot實(shí)驗(yàn)進(jìn)一步驗(yàn)證了miR-200b可以在蛋白水平上抑制GATA4的表達(dá),表明GATA4確系：-niR-200b的靶基因。 該研究證實(shí)了GATA4是miR-200b的靶基因,miR-200b通過轉(zhuǎn)錄后機(jī)制調(diào)控GATA4的蛋白表達(dá),進(jìn)而抑制細(xì)胞的增殖,致使細(xì)胞發(fā)生G0/G1期阻滯,并誘導(dǎo)細(xì)胞發(fā)生凋亡。這些結(jié)果表明miR-200b可能通過靶向GATA4在心臟發(fā)育、增殖與凋亡等方面發(fā)揮重要的調(diào)控作用。 結(jié)論： 1、在PPARa激動(dòng)劑fenofibrate刺激的心肌細(xì)胞中,轉(zhuǎn)錄因子GATA6招募核受體PPARa至葡萄糖轉(zhuǎn)運(yùn)蛋白Glut4啟動(dòng)子的GATA元件上,這種招募能夠進(jìn)一步激活Glut4的表達(dá),并且伴隨著線粒體功能的促進(jìn)及葡萄糖利用的增加。2、PPARa激動(dòng)劑fenofibrate對(duì)線粒體檸檬酸合酶和NADH氧化酶具有保護(hù)作用,可減輕抗癌藥物Dox誘導(dǎo)的心、腎毒性。3、miR-200b作用于轉(zhuǎn)錄因子GATA4,抑制C2C12細(xì)胞和P19細(xì)胞的增殖,致使細(xì)胞發(fā)生G1/GO期阻滯并誘導(dǎo)細(xì)胞凋亡。本研究證實(shí)了GATA6是心肌能量代謝調(diào)控網(wǎng)絡(luò)中的重要組成成分,并且作為PPARα的輔助激活因子協(xié)同PPARα調(diào)控心肌能量代謝。這為理解GATA6在心臟發(fā)育和心臟疾病的作用機(jī)制提供了新的分子基礎(chǔ)。這一研究還揭示了miR-200b在細(xì)胞增殖與凋亡中的作用機(jī)制,為進(jìn)一步理解GATA家族轉(zhuǎn)錄因子在心肌中的調(diào)控作用奠定了基礎(chǔ)。
[Abstract]:Research background and purpose:
The heart has a great potential to generate ATP to meet the high demand for energy. At the time of embryonic and cardiac hypertrophy, the heart mainly relies on glucose and lactose as a substrate for producing ATP, while at birth and in adults, the heart is mainly dependent on fatty acids as a substrate to produce ATP. Because of their limited capacity, they are rapidly adjusted to substrate selection and ATP generation to rapidly adapt to the energy requirements of the heart.
This regulation involves the successive expression and interaction of various genes at different times, and they form an orderly network. The regulatory network includes a large number of molecular events that are extremely accurate in time and space. In this process, even tiny errors will lead to the occurrence of energy metabolic disorders in the heart. The decrease of mitochondrial fatty acid oxidation activity, such as the decrease of mitochondrial fatty acid oxidation activity, causes the decrease of ATP production, which will lead to the dysfunction of cardiac pump function, contraction function and calcium ion transport, and induce cardiac hypertrophy and heart failure. Therefore, it will be helpful to study the regulation mechanism of the key gene of cardiac energy metabolism, which will help from the molecular water. Ping elucidate the molecular mechanism and pathophysiology of cardiovascular disease, and provide more clues for the clinical prevention and treatment of cardiovascular diseases and the design of new drugs.
So, what are the key genes involved in myocardial energy metabolism? Glut4 is a gene that regulates myocardial energy metabolism. It is a member of the glucose transporter family. It maintains the homeostasis.Glut4 expression of the body's glucose, which is tissue specific and is regulated by hormone, and the Glut4 gene knockout mice have cardiac hypertrophy symptoms. Therefore, GIut4 is one of the key genes regulating the energy metabolism of myocardium. As a key gene of myocardial energy metabolism, GIut4 is a key gene of cardiac energy metabolism. The decrease or up-regulation of its expression is of important physiological or pathological significance for the regulation of myocardial energy metabolism. So it is an important subject in the field of cardiovascular molecular biology to study its regulatory mechanism.
So, what are the transcription factors involved in cardiac energy metabolism? The peroxisome proliferator activated receptors (PPARs) is an important transcription factor regulating the gene for the encoded fatty acid beta oxidation related enzymes, which contains three different forms: PPARa, PPAR gamma and PPAR Delta /1 beta. These three heteromorphs are all Expressed in the heart, the expression of PPARa and PPAR Delta / beta in cardiac myocytes is more abundant than PPARy. Studies have shown that cardiac specific overexpression of PPARa induces gene expression related to fatty acid utilization, thereby enhancing the use and oxidation of cardiac fatty acids; PPAR Delta / beta knockout mice seriously damage the correlation of myocardial fatty acid oxidation. Gene expression increases cardiac fatty acid accumulation and induces lipotoxicity. Therefore, PPARs plays an important role in regulating myocardial energy metabolism.
GATA protein is a transcription factor with zinc finger structure, named after its identification sequence (A/T) GATA (A/G). Among the 6 members of the family, GATA4 and GATA6 are the research hot spots in the cardiovascular field in recent years. A large number of studies have shown that GATA4 and GATA6 are important transcription factors regulating cardiac development and myocardial cell proliferation, differentiation and apoptosis. Little is known about whether it plays a regulatory role in myocardial energy metabolism.
MicroRNAs (miRNAs) is a highly conserved endogenous single strand non coding small RNA, which regulates the expression of target genes through post transcriptional mechanism. Recent studies have shown that miRNAs is an important part of the transcription regulatory network of the heart gene, but whether miRNAs acts on GATA protein and PPARs, and then affects the phenotype of the cells, and is present at present. There are few reports.
Therefore, based on the GATA transcriptional factor, we have studied the regulation mechanism of GATA6, PPARa, miR-200b and GATA4 on the energy metabolism and cell proliferation of myocardium by a variety of methods.
Research methods and results:
Part I: transcription factor GATA6 recruited PPARa to co activate the expression of GIut4 gene.
In order to further understand the regulatory role of the GATA family on myocardial energy metabolism, transient transfection, Q-PCR, luciferase reporter gene, Western Blot, CoIP, GST pull-down and ChIP have been used to confirm that the conversion factor GATA6 can recruit nuclear receptor PPARa to glucose transporter eggs in the cardiomyocytes stimulated by PPARa agonist fenofibrate. On the GATA element of the white GIut4 promoter, this recruitment can further activate the expression of the Glut4 gene, which is accompanied by the promotion of mitochondrial function and the increase of glucose utilization.
Western Blot and Q-PCR experiments showed that both GATA6 or PPARa and their agonist fenofibrate could stimulate the expression of Glut4 gene. If co expression of the two was more intense, it was proved that GATA6 and PPARa co activated Glut4 gene transcription.CoIP experiment demonstrated that GATA6 and interaction experiments showed the zinc finger. The interaction of.ChIP and luciferase reporter gene between the two N terminal zinc finger mediated.ChIP and the luciferase reporter gene experiment further confirmed that GATA6 could recruit PPARa to Glut4 promoter by interacting with the N terminal TAD and zinc finger domain of PPARa protein to activate the expression of Glut4 gene and promote the function of the grain body and the use of glucose.
This part of the study confirms that the transcription factor GATA6 is an important regulator of cardiac energy metabolism, and the nuclear receptor PPARa is a new interaction factor in the transcription of GATA6 regulating genes, which provides a new molecular basis for understanding the mechanism of GATA6 in heart development and heart disease.
The second part: the effects of fenofibrate and Dox on NADH oxidase and citric acid dehydrogenase activity in mice.
Doxorubicin (Dox) is a common antitumor drug, because its severe toxicity greatly restricts its clinical use. Mitochondrial dysfunction is one of its potential toxic mechanisms, but the specific mechanism is unknown. Because of the protective effect of PPARa on cell mitochondrial function in the first part of the study, we have observed that We further studied whether PPARa agonist fenofibrate could protect mitochondrial function and reverse Dox toxicity in vivo.
The 8 week old mice were randomly divided into four groups and treated with different treatments: the control group was given 0.5% carboxymethyl cellulose sodium every day for 14 days for a continuous period of 14 days; fenofibrate treatment group: every day, fenofibrate (100mg/kg) was gavage for 14 days for 14 days; Dox treatment group: 12,13 and 14 days, daily intraperitoneal injection of 15mg/kg (15mg/kg); the joint group: same The mice were treated with fenofibrate and Dox after.14 days. The mitochondria of the ventricles, atrium, liver, kidney, lung and spleen were isolated and extracted. The activity of citrate synthase and NADH oxidase, a marker of mitochondrial function, was detected. The results showed that fenofibrate could protect mitochondrial function in many tissues. On the contrary, Dox was inhibited in most tissues. The function of mitochondria is also made, and fenofibrate can reverse the toxicity of Dox in the ventricles and kidneys.
This study confirms that fenofibrate has a protective effect on mitochondrial synthase and NADH oxidase, and can reverse the toxic effect of Dox in the ventricles and kidneys, which provides a theoretical basis for how to reduce the side effects of Dox in the clinic.
The third part: the regulation of miR-200b on GATA4 proliferation, cycle and apoptosis.
In order to find the upstream regulator of the GATA family, it is found that GATA4 may be the target gene of miR-200b, which may be the target gene.MiR-200b involved in the regulation of epithelial mesenchymal transformation (EMT), and is an important regulatory factor in the formation of tumor cells. As a result of miRNAs regulation of cell proliferation, differentiation and apoptosis, many physiological and pathological processes, therefore, we The effects of miR-200b on cell proliferation, cycle and apoptosis were studied by stable transfection, RNAi, luciferase reporter gene, MTT, flow cytometry, and DAPI staining RT-qPCR.
The results showed that overexpression of GATA4 and rniR-200b promoted and inhibited the proliferation of C2C12 and P19 cells. Moreover, overexpression (?) niR-200b and siRNA inhibited GATA4 expression to induce G0/G1 phase block, S phase and G2/M phase cell percentage decreased.DAPI staining and confirmed that miR-200b over table could induce nuclear apoptosis. Luciferase Report Gene experiments show that miR-200b can specifically bind to the GATA43'non translation region (3' -UTR) specific binding.Western Blot experiment to further verify that miR-200b can inhibit the expression of GATA4 at the protein level, indicating that GATA4 is the target gene of -niR-200b.
The study confirmed that GATA4 is the target gene of miR-200b. MiR-200b regulates the protein expression of GATA4 through the post transcriptional mechanism and inhibits cell proliferation, causing G0/G1 phase block and inducing cell apoptosis. These results suggest that miR-200b may play an important role in cardiac development, proliferation and apoptosis by targeting GATA4. Control.
Conclusion:
1, in the cardiac myocytes stimulated by the PPARa agonist fenofibrate, the transcription factor GATA6 recruits the GATA element of the nuclear receptor PPARa to the glucose transporter Glut4 promoter. This recruitment can further activate the expression of Glut4, and is accompanied by the promotion of mitochondrial function and the increase of.2 for glucose utilization, PPARa agonist fenofibrate to the grain. The body citric acid synthase and NADH oxidase have protective effect, which can reduce the heart of Dox induced by anticancer drug, renal toxicity.3, miR-200b acting on the transcription factor GATA4, inhibiting the proliferation of C2C12 cells and P19 cells, causing G1/GO phase arrest and inducing cell apoptosis. This study confirms that GATA6 is important in the control network of cardiac energy metabolism. This study provides a new molecular basis for understanding the mechanism of GATA6 in cardiac development and heart disease. This study also reveals the mechanism of the role of miR-200b in cell proliferation and apoptosis in order to further understand the GATA family transcriptional factors in PPAR. The regulatory role of the myocardium has laid the foundation.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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相關(guān)期刊論文 前1條

1 姜升陽(yáng);徐明;張幼怡;;GATA結(jié)合蛋白4在心臟發(fā)育及心肌重塑中的作用[J];生理科學(xué)進(jìn)展;2008年04期

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本文編號(hào)：1932476