H19在心肌肥厚中的功能研究

发布时间：2019-06-22 09:09

【摘要】：心脏是人体在胚胎发育阶段最早形成的器官,也是人体最重要最保守的器官之一。心脏由心肌细胞和其他细胞(成纤维细胞,淋巴管,血管等)组成,心肌细胞数量占总数量的25%左右,体积却占心脏体积的75%左右。心衰是导致人类死亡的重要原因之一,而心衰往往由心肌肥厚发展而来,因此研究心肌肥厚的发病机制具有重大的现实意义。心肌肥厚是心脏在早期阶段为应对各种压力、维持心脏功能所表现出的适应性反应。然而持续的心肌肥厚和伴随着的心肌重塑往往导致心衰、心源性死亡的风险增加。虽然各种特殊的肽激素、生长因子和小RNA已经被证实参与心肌肥厚的调节,但心肌肥厚的机制依然没有被完全了解。长非编码RNA(long noncoding RNA,lncRNA)是一类长度大于200个核苷酸,不能编码蛋白质的RNA,其在生命过程中发挥重要而复杂的作用,参与诸如RNA加工,细胞命运决定,染色质修饰等过程。有研究证实lncRNA还可以作为竞争性内源RNA(competing endogenous RNA,ceRNA)发挥作用。最近几年有研究发现lncRNA参与调节心脏的发育与疾病过程。Braveheart lncRNA可以在小鼠胚胎干细胞分化期间刺激干细胞转变为心脏细胞。MIAT在第5外显子上的SNP变异增加了MIAT的转录,可能与心肌梗塞发生相关。LncRNA ANRIL定位于9p21,它的失调会引起心肌细胞增殖异常和冠脉疾病。对TAC手术导致心肌肥厚的小鼠心脏组织进行转录组测序研究发现众多lncRNA在心肌肥厚早期和心衰期表达水平发生明显改变,提示lncRNA在心肌肥厚和心衰中发挥了重要功能。因此lncRNA已逐渐成为心血管基础和临床研究的一个新的热点和重点。作为最早被发现的印记基因,lncRNA H19被证实在哺乳动物胚胎发育和肿瘤发生过程中具有重要作用。H19的第一外显子能够编码miR-675,已有研究证实H19在很多病理生理过程中发挥的功能都是通过miR-675介导的。近几年来的多项研究表明,在对病理性心肌肥厚小鼠模型进行差异表达的lncRNA筛选时,H19表达水平有明显的上调,提示其可能在心肌肥厚中发挥作用,但迄今为止关于H19在心脏组织中的具体功能及相关机制依然鲜见报导。本研究旨在探索H19在心肌肥厚过程中发挥的功能,并探索其相关的作用机制。我们首先在不同类型的心肌肥厚小鼠模型及人心衰标本中检测了H19及其编码的miR-675的表达,发现两者在病理性心肌肥厚模型和心衰样品中表达水平明显上调,而在运动诱导的生理性心肌肥厚小鼠模型中检测时发现两者的表达下调。为了研究H19在心肌细胞中的功能,我们构建和包装了用于过表达H19的腺病毒,并用其在体外感染分离的原代心肌细胞。通过对心肌细胞形态学分析和肥大标志基因表达水平的检测,发现过表达H19能够在体外抑制心肌细胞的肥大生长。另一方面,我们也在体外通过转染si-h19的方式来敲低原代心肌细胞中内源性h19的表达,发现h19的敲低会促进心肌细胞的肥大生长。为理解h19抑制心肌细胞肥大生长的机制,我们采取了以下策略探索h19在心肌细胞中的功能是否是由mir-675介导的。首先,我们在体外原代心肌细胞中过表达或敲低mir-675,发现mir-675也能够在体外抑制心肌细胞的肥大生长。进一步,我们在原代心肌细胞中过表达h19的同时敲低mir-675,心肌细胞形态学分析和肥大标志基因表达水平的检测显示,敲低mir-675能够有效地挽救由于h19过表达所引起的心肌细胞尺寸的减小。此外,我们还分别包装了包含缺失编码mir-675前体序列的h19截短体(h19-tru)和突变型mir-675前体序列的h19突变体(h19-mut)的腺病毒,并分别进行心肌细胞的感染。结果发现,两种形式的腺病毒都能有效地使h19在心肌细胞内过表达,但是对心肌细胞内mir-675的表达无明显影响。形态学分析以及肥大标志基因表达水平的检测显示h19-tru和h19-mut都失去了抑制心肌细胞肥大生长的能力。所有这些结果都表明mir-675能够介导h19抑制心肌细胞肥大生长的功能。我们对h19/mir-675的作用机制进行了进一步探索。利用targetscan预测了mir-675可能作用的靶分子,从中选择了具有促进心肌肥厚功能的钙/钙调蛋白依赖的蛋白激酶iiδ(camkiiδ)进行了系统分析。首先,我们构建了包含正常的及突变型的camkiiδ3’-utr的荧光素酶报告载体,并与mir-675共同转染293t细胞。结果显示mir-675能有效地抑制camkiiδ的3’-utr活性,而mir-675对突变型载体则丧失了这种抑制作用。进一步对camkiiδ表达水平的检测显示mir-675能明显抑制camkiiδ在mrna和蛋白水平的表达。这些结果表明camkiiδ是mir-675直接作用的靶标分子。为了验证心肌细胞内h19发挥的功能是否是由camkiiδ介导的,我们在体外原代心肌细胞中敲低h19的同时敲低camkiiδ,发现camkiiδ的敲低能部分缓解h19敲低所引起的心肌细胞尺寸的增加,表明camkiiδ能部分介导h19抑制心肌细胞肥大生长的功能。为了研究h19/mir-675在体内心脏稳态维持中的功能,我们考察了在体内抑制mir-675的表达对tac手术引起的心肌肥厚的影响。我们对2月龄的c57雄鼠进行了tac手术,在术后1周经确认发生心肌肥厚后,通过鼠尾静脉注射mir-675抑制剂(antagomir-675),并在注射3周后进行心脏取材和分析。我们发现在体内应用antagomir-675能有效地缓解tac手术引起的mir-675表达水平的升高,并且加重了压力性负荷引发的心肌肥厚,提示在体内抑制mir-675会促进心肌肥厚的发生。综上所述,我们的研究首次揭示了h19/mir-675可通过靶向camkiiδ抑制心肌肥厚的新功能,为了解心肌肥厚的发生机制提供了新的理论基础,为心脏疾病的治疗提供了新的靶标。
[Abstract]:The heart is one of the earliest organs of the human body in the development stage of the embryo, and is one of the most important organs of the human body. The heart is composed of cardiac muscle cells and other cells (fibroblasts, lymphatic vessels, blood vessels, etc.), and the quantity of the cardiac muscle cells is about 25% of the total quantity, but the volume accounts for about 75% of the volume of the heart. Heart failure is one of the important causes of human death, and heart failure is often derived from the development of cardiac hypertrophy, so it is of great practical significance to study the pathogenesis of cardiac hypertrophy. Myocardial hypertrophy is an adaptive response of the heart to various pressures and to maintain cardiac function at an early stage. However, persistent cardiac hypertrophy and concomitant myocardial remodeling often lead to heart failure, and the risk of cardiac death is increased. Although various specific peptide hormones, growth factors and small RNAs have been shown to be involved in the regulation of cardiac hypertrophy, the mechanism of cardiac hypertrophy is not fully understood. Long noncoding RNA (lncRNA) is a class of RNA which has a length of more than 200 nucleotides and cannot encode proteins, which plays an important and complex role in the process of life, and is involved in such processes as RNA processing, cell fate determination, chromatin modification, and the like. It has been found that lncRNA can also function as a competitive endogenous RNA (cRNA). In recent years, it has been found that lncRNA is involved in the regulation of the development and disease process of the heart. Braveheart lncRNA can stimulate the transformation of stem cells into cardiac cells during the differentiation of mouse embryonic stem cells. The mutation of the MIAT on exon 5 increases the transcription of MIAT and may be associated with the occurrence of myocardial infarction. LncRNA ANRIL is located at 9p21, and its offset can cause abnormal proliferation of myocardial cells and coronary artery disease. It was found that the expression of lncRNA in the early stage of cardiac hypertrophy and the stage of heart failure was significantly changed, suggesting that the lncRNA played an important role in cardiac hypertrophy and heart failure. Therefore, lncRNA has gradually become a new focus and focus of the cardiovascular and clinical research. As the earliest discovered imprinting gene, lncRNA H19 has been demonstrated to play an important role in the development and tumorigenesis of mammalian embryos. The first exon of H19 is capable of encoding miR-675, and it has been found that the function of H19 in many pathophysiological processes is mediated by miR-675. A number of studies in recent years have shown that the H19 expression level is up-regulated when screening for differentially expressed lncRNA in a pathological myocardial hypertrophy mouse model, suggesting that it may play a role in cardiac hypertrophy, However, the specific functions and related mechanisms of H19 in cardiac tissue have been reported to date. The purpose of this study was to explore the function of H19 in the course of myocardial hypertrophy and to explore its related mechanism. We first examined the expression of H19 and its coded miR-675 in different types of cardiac hypertrophy mouse model and human heart failure specimen, and found that the expression level of H19 and its coded miR-675 was up-regulated in pathological myocardial hypertrophy model and heart failure sample. While the expression of the two was down-regulated in the motion-induced physiological cardiac hypertrophy mouse model. In order to study the function of H19 in myocardial cells, we construct and package an adenovirus for overexpression of H19 and use it to infect isolated primary cardiomyocytes in vitro. It is found that the expression of H19 can inhibit the hypertrophy and growth of the cardiac muscle cells in vitro by the detection of the morphological analysis of the cardiac muscle and the expression level of the hypertrophy marker gene. On the other hand, we have also found the expression of endogenous h19 in the low primary cardiac muscle cells by transfecting si-h19 in vitro, and it is found that the knockdown of h19 can promote the hypertrophy and growth of the cardiac muscle cells. To understand the mechanism of h19 to inhibit the growth of cardiac myocyte hypertrophy, we have adopted the following strategy to explore whether the function of h19 in a cardiac muscle cell is mediated by mir-675. First, we overexpress or knock low mir-675 in an in vitro primary myocardial cell, and it is found that the mir-675 can also inhibit the growth of the hypertrophy of the cardiac muscle cells in vitro. Further, the detection of the simultaneous knockdown of mir-675, the morphological analysis of the cardiac myocyte and the level of the expression of the hypertrophy marker gene in primary myocardial cells shows that the knockdown of mir-675 can effectively save the decrease in the size of the myocardial cells due to the overexpression of h19. In addition, we also packaged the h19-tru and the h19-mut adenovirus containing the h19-truncated (h19-tru) and the mutant mir-675 precursor sequence of the mir-675 precursor sequence, respectively, and the infection of the cardiac muscle cells, respectively. It was found that both forms of adenovirus can effectively express h19 in the cardiac muscle cell, but it has no significant effect on the expression of mir-675 in the cardiac muscle cells. The morphological analysis and the detection of the expression level of the hypertrophy marker gene showed that both h19-tru and h19-mut both lost the ability to inhibit the growth of cardiac myocyte hypertrophy. All of these results show that mir-675 is capable of mediating the function of h19 to inhibit the growth of cardiac muscle cells. We have further explored the mechanism of the action of h19/ mir-675. The target molecules that are likely to act with the mir-675 are predicted using the targetscan, from which a system analysis of the calcium/ calmodulin-dependent protein kinase ii (camkii) with the function of promoting the cardiac hypertrophy is selected. First, we constructed a luciferase reporter vector containing normal and mutant camkii-3 '-utr and co-transfected 293T cells with mir-675. The results show that the mir-675 can effectively inhibit the 3 '-utr activity of camkiii, while the mir-675 has a loss of this inhibitory effect on the mutant vector. Further detection of the expression level of camkii showed that the mir-675 was able to significantly inhibit the expression of camkii in mrna and protein levels. These results show that camkii is a direct target molecule for mir-675. in order to verify whether the function of h19 in the cardiac muscle cell is mediated by camkii, we tap the low h19 in the in vitro primary myocardial cell and tap the camkii antigen, finding that the low-energy portion of the camkii antigen alleviates the increase in the size of the cardiac muscle resulting from the low h19 knockdown, It is shown that camkii can partially mediate the function of h19 to inhibit the growth of cardiac myocyte hypertrophy. In order to study the function of h19/ mir-675 in the steady-state maintenance of heart in vivo, we examined the effect of the expression of mir-675 in vivo on the cardiac hypertrophy caused by the tac operation. We performed a tac procedure on c57 males at 2 months of age, and after 1 week of post-operation, cardiac hypertrophy was confirmed, the mir-675 inhibitor (antagomir-675) was injected via the tail of the mouse, and the heart was taken and analyzed after 3 weeks of injection. We found that antagomir-675 was used in vivo to effectively relieve the increase in the level of mir-675 expression induced by the tac operation and to increase the cardiac hypertrophy induced by the stress load, suggesting that the inhibition of mir-675 in vivo would promote the occurrence of cardiac hypertrophy. To sum up, our study first revealed that h19/ mir-675 can provide a new theoretical basis for understanding the pathogenesis of cardiac hypertrophy by targeting camkii to inhibit cardiac hypertrophy, and provide a new target for the treatment of heart disease.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.2

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