H19在心肌肥厚中的功能研究
發(fā)布時間：2019-06-22 09:09
【摘要】：心臟是人體在胚胎發(fā)育階段最早形成的器官,也是人體最重要最保守的器官之一。心臟由心肌細胞和其他細胞(成纖維細胞,淋巴管,血管等)組成,心肌細胞數(shù)量占總數(shù)量的25%左右,體積卻占心臟體積的75%左右。心衰是導致人類死亡的重要原因之一,而心衰往往由心肌肥厚發(fā)展而來,因此研究心肌肥厚的發(fā)病機制具有重大的現(xiàn)實意義。心肌肥厚是心臟在早期階段為應對各種壓力、維持心臟功能所表現(xiàn)出的適應性反應。然而持續(xù)的心肌肥厚和伴隨著的心肌重塑往往導致心衰、心源性死亡的風險增加。雖然各種特殊的肽激素、生長因子和小RNA已經(jīng)被證實參與心肌肥厚的調(diào)節(jié),但心肌肥厚的機制依然沒有被完全了解。長非編碼RNA(long noncoding RNA,lncRNA)是一類長度大于200個核苷酸,不能編碼蛋白質(zhì)的RNA,其在生命過程中發(fā)揮重要而復雜的作用,參與諸如RNA加工,細胞命運決定,染色質(zhì)修飾等過程。有研究證實lncRNA還可以作為競爭性內(nèi)源RNA(competing endogenous RNA,ceRNA)發(fā)揮作用。最近幾年有研究發(fā)現(xiàn)lncRNA參與調(diào)節(jié)心臟的發(fā)育與疾病過程。Braveheart lncRNA可以在小鼠胚胎干細胞分化期間刺激干細胞轉(zhuǎn)變?yōu)樾呐K細胞。MIAT在第5外顯子上的SNP變異增加了MIAT的轉(zhuǎn)錄,可能與心肌梗塞發(fā)生相關。LncRNA ANRIL定位于9p21,它的失調(diào)會引起心肌細胞增殖異常和冠脈疾病。對TAC手術導致心肌肥厚的小鼠心臟組織進行轉(zhuǎn)錄組測序研究發(fā)現(xiàn)眾多l(xiāng)ncRNA在心肌肥厚早期和心衰期表達水平發(fā)生明顯改變,提示lncRNA在心肌肥厚和心衰中發(fā)揮了重要功能。因此lncRNA已逐漸成為心血管基礎和臨床研究的一個新的熱點和重點。作為最早被發(fā)現(xiàn)的印記基因,lncRNA H19被證實在哺乳動物胚胎發(fā)育和腫瘤發(fā)生過程中具有重要作用。H19的第一外顯子能夠編碼miR-675,已有研究證實H19在很多病理生理過程中發(fā)揮的功能都是通過miR-675介導的。近幾年來的多項研究表明,在對病理性心肌肥厚小鼠模型進行差異表達的lncRNA篩選時,H19表達水平有明顯的上調(diào),提示其可能在心肌肥厚中發(fā)揮作用,但迄今為止關于H19在心臟組織中的具體功能及相關機制依然鮮見報導。本研究旨在探索H19在心肌肥厚過程中發(fā)揮的功能,并探索其相關的作用機制。我們首先在不同類型的心肌肥厚小鼠模型及人心衰標本中檢測了H19及其編碼的miR-675的表達,發(fā)現(xiàn)兩者在病理性心肌肥厚模型和心衰樣品中表達水平明顯上調(diào),而在運動誘導的生理性心肌肥厚小鼠模型中檢測時發(fā)現(xiàn)兩者的表達下調(diào)。為了研究H19在心肌細胞中的功能,我們構建和包裝了用于過表達H19的腺病毒,并用其在體外感染分離的原代心肌細胞。通過對心肌細胞形態(tài)學分析和肥大標志基因表達水平的檢測,發(fā)現(xiàn)過表達H19能夠在體外抑制心肌細胞的肥大生長。另一方面,我們也在體外通過轉(zhuǎn)染si-h19的方式來敲低原代心肌細胞中內(nèi)源性h19的表達,發(fā)現(xiàn)h19的敲低會促進心肌細胞的肥大生長。為理解h19抑制心肌細胞肥大生長的機制,我們采取了以下策略探索h19在心肌細胞中的功能是否是由mir-675介導的。首先,我們在體外原代心肌細胞中過表達或敲低mir-675,發(fā)現(xiàn)mir-675也能夠在體外抑制心肌細胞的肥大生長。進一步,我們在原代心肌細胞中過表達h19的同時敲低mir-675,心肌細胞形態(tài)學分析和肥大標志基因表達水平的檢測顯示,敲低mir-675能夠有效地挽救由于h19過表達所引起的心肌細胞尺寸的減小。此外,我們還分別包裝了包含缺失編碼mir-675前體序列的h19截短體(h19-tru)和突變型mir-675前體序列的h19突變體(h19-mut)的腺病毒,并分別進行心肌細胞的感染。結果發(fā)現(xiàn),兩種形式的腺病毒都能有效地使h19在心肌細胞內(nèi)過表達,但是對心肌細胞內(nèi)mir-675的表達無明顯影響。形態(tài)學分析以及肥大標志基因表達水平的檢測顯示h19-tru和h19-mut都失去了抑制心肌細胞肥大生長的能力。所有這些結果都表明mir-675能夠介導h19抑制心肌細胞肥大生長的功能。我們對h19/mir-675的作用機制進行了進一步探索。利用targetscan預測了mir-675可能作用的靶分子,從中選擇了具有促進心肌肥厚功能的鈣/鈣調(diào)蛋白依賴的蛋白激酶iiδ(camkiiδ)進行了系統(tǒng)分析。首先,我們構建了包含正常的及突變型的camkiiδ3’-utr的熒光素酶報告載體,并與mir-675共同轉(zhuǎn)染293t細胞。結果顯示mir-675能有效地抑制camkiiδ的3’-utr活性,而mir-675對突變型載體則喪失了這種抑制作用。進一步對camkiiδ表達水平的檢測顯示mir-675能明顯抑制camkiiδ在mrna和蛋白水平的表達。這些結果表明camkiiδ是mir-675直接作用的靶標分子。為了驗證心肌細胞內(nèi)h19發(fā)揮的功能是否是由camkiiδ介導的,我們在體外原代心肌細胞中敲低h19的同時敲低camkiiδ,發(fā)現(xiàn)camkiiδ的敲低能部分緩解h19敲低所引起的心肌細胞尺寸的增加,表明camkiiδ能部分介導h19抑制心肌細胞肥大生長的功能。為了研究h19/mir-675在體內(nèi)心臟穩(wěn)態(tài)維持中的功能,我們考察了在體內(nèi)抑制mir-675的表達對tac手術引起的心肌肥厚的影響。我們對2月齡的c57雄鼠進行了tac手術,在術后1周經(jīng)確認發(fā)生心肌肥厚后,通過鼠尾靜脈注射mir-675抑制劑(antagomir-675),并在注射3周后進行心臟取材和分析。我們發(fā)現(xiàn)在體內(nèi)應用antagomir-675能有效地緩解tac手術引起的mir-675表達水平的升高,并且加重了壓力性負荷引發(fā)的心肌肥厚,提示在體內(nèi)抑制mir-675會促進心肌肥厚的發(fā)生。綜上所述,我們的研究首次揭示了h19/mir-675可通過靶向camkiiδ抑制心肌肥厚的新功能,為了解心肌肥厚的發(fā)生機制提供了新的理論基礎,為心臟疾病的治療提供了新的靶標。
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R542.2
本文編號：2504439
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R542.2
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