GATA转录因子对心肌能量代谢及细胞增殖的调控机制研究

发布时间：2018-05-25 06:43

本文选题：GATA4 + GATA6　；参考：《郑州大学》2012年博士论文

【摘要】：研究背景与目的：心脏具有生成ATP的巨大潜能以满足对能量的高度需求。在胚胎时期及心肌肥大发生时,心脏主要依赖葡萄糖和乳糖作为底物产生ATP,而在出生后及成人心脏则主要依赖脂肪酸作为底物产生ATP而获得能量。由于心肌的脂质和葡萄糖储存能力有限,因此,它们作为底物的选择及ATP的生成是受到快速调节的,以迅速适应心脏的能量需求。这一调节过程涉及到多种基因不同时间的先后表达和相互作用,它们形成一个有序的网络。该调节网络包括大量的,在时间和空间上极为精确的一系列分子事件。在这个过程中,哪怕是极微小的错误都将导致心肌能量代谢障碍的发生,而心肌能量代谢障碍,如线粒体脂肪酸氧化活性的降低,造成ATP产量减少,将会导致心泵功能、收缩功能及钙离子的转运功能等失调,从而诱发心肌肥大和心力衰竭等。因此,深入研究心肌能量代谢关键基因的调控机制,将有助于从分子水平阐明心血管疾病的分子机理和病理学基础,同时也可为心血管疾病的临床防治和新药设计提供更多线索。那么,涉及心肌能量代谢的关键基因有什么呢?Glut4是调节心肌能量代谢的一个基因,它是葡萄糖转运蛋白家族的成员,维持着机体葡萄糖的内稳态平衡。Glut4的表达具有组织特异性并受到激素调节,Glut4基因敲除小鼠出现心肌肥大症状。因此,GIut4是调节心肌能量代谢的关键基因之一。鉴于此,GIut4作为心肌能量代谢的关键基因,其表达的降低或上调,对于心肌能量代谢的调节具有重要的生理或病理意义,所以研究其调控机制,是心血管分子生物学领域的重要课题。那么,参与心肌能量代谢的转录因子有哪些呢?过氧化物酶体增殖物激活受体(peroxisome proliferator activated receptors, PPARs)是调节编码脂肪酸β-氧化相关酶类基因的重要转录因子,它包含三种异形体：PPARa、PPARγ和PPARδ/1β。这三种异形体都在心脏中表达,与PPARy相比,PPARa和PPARδ/β在心肌细胞中的表达更为丰富。研究表明,心脏特异性过表达PPARa将诱导与脂肪酸利用有关的基因表达,进而增强心脏脂肪酸的利用和氧化；PPARδ/β基因敲除小鼠严重地损害心肌脂肪酸氧化相关基因的表达,增加心脏脂肪酸堆积,从而诱发脂毒性。因此,PPARs在调节心肌能量代谢中发挥着重要作用。 GATA蛋白是具有锌指结构的转录因子,因其识别序列(A/T) GATA (A/G)而得名。在该家族6个成员中,GATA4和GATA6是近年来心血管领域的研究热点。大量的研究证明GATA4和GATA6是调控心脏发育以及心肌细胞增殖、分化和凋亡的重要转录因子。但它们是否在心肌能量代谢中发挥调控作用,目前知之甚少。 MicroRNAs (miRNAs)是进化上高度保守的内源性单链非编码小RNA,通过转录后机制调控靶基因的表达。近年来的研究表明miRNAs是心脏基因转录调控网络的一个重要组成部分,但miRNAs是否作用于GATA蛋白与PPARs,进而对细胞的表型产生影响,目前还鲜有报道。因此,本课题以GATA转录因子为中心,采用多种方法深入研究了GATA6、 PPARa、miR-200b以及GATA4对心肌能量代谢和细胞增殖的调控机制。研究方法与结果：第一部分：转录因子GATA6招募PPARa协同激活GIut4基因的表达为进一步了解GATA家族对心肌能量代谢的调控作用,采用了瞬时转染、Q-PCR、荧光素酶报告基因、Western Blot、CoIP、GST pull-down和ChIP等实验方法,证实了在PPARa激动剂fenofibrate刺激的心肌细胞中,转录因子GATA6能够招募核受体PPARa至葡萄糖转运蛋白GIut4启动子的GATA元件上,这种招募能够进一步激活Glut4基因的表达,并且伴随着线粒体功能的促进及葡萄糖利用的增加。 Western Blot及Q-PCR实验显示GATA6或PPARa及其激动剂fenofibrate都能够刺激Glut4基因的表达,若二者共表达则可以产生更强烈的激活效应,说明GATA6和PPARa协同激活Glut4基因转录。CoIP实验证实GATA6和PPARa相互作用。GST pull-down实验显示GATA6的C端锌指和PPARa的N端锌指介导二者间的相互作用。ChIP及荧光素酶报告基因实验进一步证实GATA6通过与PPARa蛋白的N端TAD和锌指结构域相互作用,招募PPARa至Glut4启动子从而协同激活Glut4基因的表达,促进线粒体的功能及葡萄糖的利用。该部分的研究证实了转录因子GATA6是心肌能量代谢的重要调节因子,核受体PPARa是GATA6调节基因转录过程中新的相互作用因子,这为理解GATA6在心脏发育和心脏疾病的作用机制提供了新的分子基础。第二部分：fenofibrate和Dox对小鼠NADH氧化酶及柠檬酸脱氢酶活性的影响多柔比星(doxorubicin, Dox)是一种常用的抗肿瘤药物,由于它的严重毒性大大限制了它的临床使用。线粒体功能障碍是其潜在的致毒机制之一,但具体机制未知。由于在第一部分研究中观察到PPARa对细胞线粒体功能具有保护作用,所以我们进一步研究了PPARa激动剂fenofibrate在体内能否保护线粒体功能并逆转Dox的毒性。将8周大的小鼠随机分为四组并给予不同的处理：对照组：每天0.5%羧甲基纤维素钠灌胃一次,连续14天；fenofibrate处理组：每天fenofibrate(100mg/kg)灌胃一次,连续14天；Dox处理组：第12、13和14天,每天腹腔注射Dox(15mg/kg)一次；联合组：同时接受fenofibrate和Dox处理。14天后处死小鼠,分离提取心室、心房、肝脏、肾脏、肺和脾脏组织的线粒体,检测线粒体功能的标志酶柠檬酸合酶及NADH氧化酶活性。结果显示：fenofibrate可在多个组织中保护线粒体功能,相反,Dox则在多数组织中抑制线粒体的功能,而且,fenofibrate在心室和肾脏中能够逆转Dox的毒性。这一研究证实fenofibrate对线粒体柠檬酸合酶和NADH氧化酶具有保护作用,并且在心室和肾脏中能够逆转Dox的毒性作用,为临床上如何减轻Dox的化疗副作用提供了理论依据。第三部分：miR-200b作用于GATA4对细胞增殖、周期和凋亡的调控为了寻找GATA家族的上游调控分子,经生物信息学分析,发现GATA4可能是miR-200b的靶基因。miR-200b参与调控上皮间质转化(EMT)过程,是肿瘤细胞形成的重要调控因子。由于miRNAs调控细胞增殖、分化和凋亡等多个生理学和病理学过程,因此,我们采用稳定转染、RNAi、荧光素酶报告基因、MTT、流式细胞术、DAPI染色RT-qPCR等实验方法研究了miR-200b对细胞增殖、周期和凋亡的影响。结果显示：过表达GATA4和rniR-200b分别促进和抑制C2C12及P19细胞的增殖。而且,过表达(?)niR-200b及siRNA抑制GATA4表达均诱导细胞发生G0/G1期阻滞,S期和G2/M期细胞百分比降低。DAPI染色证实miR-200b过表达还能诱导细胞核发生凋亡。荧光素酶报告基因实验表明miR-200b可与GATA43'非翻译区(3’-UTR)特异性结合。Western Blot实验进一步验证了miR-200b可以在蛋白水平上抑制GATA4的表达,表明GATA4确系：-niR-200b的靶基因。该研究证实了GATA4是miR-200b的靶基因,miR-200b通过转录后机制调控GATA4的蛋白表达,进而抑制细胞的增殖,致使细胞发生G0/G1期阻滞,并诱导细胞发生凋亡。这些结果表明miR-200b可能通过靶向GATA4在心脏发育、增殖与凋亡等方面发挥重要的调控作用。结论： 1、在PPARa激动剂fenofibrate刺激的心肌细胞中,转录因子GATA6招募核受体PPARa至葡萄糖转运蛋白Glut4启动子的GATA元件上,这种招募能够进一步激活Glut4的表达,并且伴随着线粒体功能的促进及葡萄糖利用的增加。2、PPARa激动剂fenofibrate对线粒体柠檬酸合酶和NADH氧化酶具有保护作用,可减轻抗癌药物Dox诱导的心、肾毒性。3、miR-200b作用于转录因子GATA4,抑制C2C12细胞和P19细胞的增殖,致使细胞发生G1/GO期阻滞并诱导细胞凋亡。本研究证实了GATA6是心肌能量代谢调控网络中的重要组成成分,并且作为PPARα的辅助激活因子协同PPARα调控心肌能量代谢。这为理解GATA6在心脏发育和心脏疾病的作用机制提供了新的分子基础。这一研究还揭示了miR-200b在细胞增殖与凋亡中的作用机制,为进一步理解GATA家族转录因子在心肌中的调控作用奠定了基础。
[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.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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