本文選題：致心律失常性右室心肌病 + microRNA��； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：研究背景致心律失常性右室心肌病(Arrhythmo genic right ventricular cardiomypathy ARVC)是原發(fā)性遺傳性心肌病,以右室心肌不同程度地被纖維脂肪組織代替為主要特征,往往伴有心室擴(kuò)大,心力衰竭,嚴(yán)重惡性心律失常甚至猝死發(fā)生,是青少年及運(yùn)動員猝死的主要病因。流行病學(xué)調(diào)查分析人群中發(fā)病率為1/2000-1/5000。一半以上的ARVC病例是由橋�；蛲蛔兯l(fā)的,最常見的橋粒基因突變?yōu)椋簆lako globin (PG), desmoplakin (DSP), plakophilin-2 (PKP2), desmoglein-2 (DSG2) and desmocollin-2 (DSC2)。非橋�；蛲蛔兊幕蛴修D(zhuǎn)錄因子β3 (transforming growth factor-β3TGF-β3)和連接蛋白(connexin43 Cx43)。Wnt/β-catenin 和 Hippo信號通路是與心臟發(fā)育相關(guān)的重要通路。Wnt/β-catenin信號通路調(diào)控心臟祖細(xì)胞向心肌細(xì)胞和脂肪細(xì)胞分化。研究發(fā)現(xiàn)通過沉默DSP基因表達(dá),會導(dǎo)致PG (γ-catenin)從細(xì)胞橋粒組合中脫落,從而和β-catenin共同競爭入核,進(jìn)而引起核內(nèi)脂肪轉(zhuǎn)化相關(guān)的轉(zhuǎn)錄因子PPARγ、C/EBP改變,從而促進(jìn)脂肪分化.Wnt/β-catenin 和 Hippo信號通路有交互作用,兩條通路中YAP, PG 及 β-catenin相互作用形成蛋白復(fù)合體。在橋�；騊KP2突變的情況下,激活NF2引起Hippo信號通路的重要因子MST1/2, LATS1/2 及 YAP發(fā)生激酶的級聯(lián)反應(yīng),通過一系列磷酸化,阻止YAP入核發(fā)揮作用,Hppo信號通路激活,發(fā)揮抑制Wnt通路作用,從而引起核內(nèi)轉(zhuǎn)錄因子改變促進(jìn)細(xì)胞脂肪化。因而,Hippo通路和Wnt通路可能共同參與了ARVC的發(fā)病。近年來研究發(fā)現(xiàn),micro RN A在心血管疾病的各種病理生理過程中發(fā)揮了重要的調(diào)控作用,研究表明microRNA調(diào)控心臟的發(fā)育和功能,在許多心血管疾病如急性心肌梗死、肥厚型心肌病、心力衰竭、動脈粥樣硬化等疾病中,microRNA都扮演了重要的角色。microRNA參與許多通路的調(diào)控而發(fā)揮作用,也參與了Wnt/β-catenin和Hippo信號通路調(diào)控疾病的病理生理過程。然而microRNA與ARVC的研究報(bào)道尚少,我們推測ARVC的發(fā)病過程,可能有microRNA的調(diào)控參與。研究目的探討microRNA 與 ARVC發(fā)病機(jī)制的關(guān)系,明確microRN 在 ARVC心肌病中的表達(dá)譜及microRNA可能通過Hippo信號通路調(diào)控ARVC發(fā)病機(jī)制,為ARVC致病機(jī)制研究及臨床診斷治療提供依據(jù)和方向。研究方法和結(jié)果本研究中應(yīng)用qRT-PCR檢測技術(shù)S-Poly(A)P lus對24例刂ARVC患者心臟移植后的心肌組織樣本microRNA進(jìn)行了檢測,共檢測1078個(gè)人類microRNA,同時(shí)以24例正常心肌組織樣本作為對照。通過檢測發(fā)現(xiàn)24個(gè)表達(dá)異常的microRNA,并進(jìn)一步在單獨(dú)樣品中進(jìn)行了驗(yàn)證,結(jié)果發(fā)現(xiàn)有12個(gè)microRNA表達(dá)上調(diào),11個(gè)microRNA表達(dá)下調(diào)。進(jìn)一步應(yīng)用ROC曲線分析,對每一個(gè)microRNA的敏感性和特異性進(jìn)行分析,排除兩個(gè)microRNA:miR-451a、miR-3647.應(yīng)用microRNA靶基因軟件進(jìn)行預(yù)測顯示：21個(gè)表達(dá)異常microRNA中,miR-21-5p與YAP基因的3'UTR具有潛在結(jié)合位點(diǎn)；miR-135b靶向MOB1b及LATS2兩個(gè)基因,分析了niR-21-5p、miR-135b在Wnt、Hippo信號通路中的靶基因,并繪制了microRNA-Gene網(wǎng)絡(luò)圖。通過對ARCV心肌組織microRNA表達(dá)譜研究,發(fā)現(xiàn)miR-21-5p.miR-135b可能調(diào)節(jié)Vnt及Hippo信號通路,調(diào)控細(xì)胞脂肪化形成。對miR-21-5p、miR-135b的靶基因和調(diào)控機(jī)制進(jìn)一步研究,在HL-1PKP2:shRNA細(xì)胞模型中檢測發(fā)現(xiàn)miR-21-5p表達(dá)上調(diào),而miR-135b表達(dá)下調(diào)。雙熒光素酶報(bào)告系統(tǒng)檢測結(jié)果證實(shí)miR-21-5p、miR-135b可以分別結(jié)合到靶基因mRNA的3'UTR端。在HL-1細(xì)胞中轉(zhuǎn)染miR-21-5p過表達(dá)使得YAP蛋白表達(dá)受到抑制；同樣過表達(dá)miR-135b時(shí)MoBlb及LATS2的蛋白表達(dá)量降低。細(xì)胞功能研究時(shí),HL-1PKP2:shRNA細(xì)胞過表達(dá)miR-21-5p會促進(jìn)細(xì)胞的脂肪分化,而miR-135b則會抑制心肌細(xì)胞脂肪分化,證實(shí)了miR-21-5p.miR-135b在Hippo信號通路中的調(diào)控功能。細(xì)胞學(xué)功能研究證實(shí)miR-21-5p促進(jìn)心肌細(xì)胞脂肪形成,而miR-135b起到抑制作用。進(jìn)一步研究證實(shí)YAP為miR-21-5p的靶基因,miR-135b則靶向MoBlb及LATS2.研究結(jié)論本實(shí)驗(yàn)應(yīng)用qRT-PCR檢測技術(shù)S-Poly(T)完成了ARVC患者的心肌組織microRNA表達(dá)譜研究,實(shí)驗(yàn)證實(shí)microRNA參與ARCV的發(fā)育和發(fā)病機(jī)制,構(gòu)建了miR-21-5p、miR-135b與Wnt及Hippo信號通路的調(diào)控基因網(wǎng)絡(luò)圖。本研究明確了ARVC患者心肌組織的microRNA表達(dá)譜；并通過細(xì)胞學(xué)實(shí)驗(yàn)探索了兩個(gè)microRNA miR-21-5p、miR-135b通過Hippo信號通路的調(diào)控基因調(diào)控基因表達(dá)而參與ARVC發(fā)病的分子機(jī)制。
[Abstract]:Background arrhythmogenic right ventricular cardiomyopathy (Arrhythmo genic right ventricular cardiomypathy ARVC) is a primary hereditary cardiomyopathy. The right ventricular myocardium is replaced by fibrous adipose tissue in different degrees, often accompanied by ventricular enlargement, heart failure, severe malignant arrhythmia and even sudden death. It is a teenager and The main cause of sudden death of athletes. Epidemiological investigation and analysis of the ARVC cases with more than half of the incidence of 1/2000-1/5000. in the population are caused by the mutation of the gene of the bridge granules. The most common mutation of the gene is: plako globin (PG), desmoplakin (DSP), plakophilin-2 (PKP2), desmoglein-2 (DSG2) and. Gene mutation genes are transcriptional factor beta 3 (transforming growth factor- beta 3TGF- beta 3) and connexin (connexin43 Cx43).Wnt/ beta -catenin and Hippo signaling pathway, an important pathway associated with cardiac development, the.Wnt/ beta -catenin signaling pathway regulates cardiac progenitor cells to differentiate into cardiomyocytes and adipocytes. The expression of DSP gene causes PG (gamma -catenin) to fall off from the cell bridge assemblage, thereby competing with beta -catenin to enter the nucleus, thereby causing the transcription factor related transcription factor PPAR gamma and C/EBP change in the nucleus, thus promoting the interaction of the fat differentiation.Wnt/ beta -catenin and Hippo signaling pathways, and the two pathways are YAP, PG, and beta -catenin. In the case of mutation of the gene PKP2, the activation of NF2 induces an important factor in the Hippo signaling pathway, MST1/2, the cascade of LATS1/2 and YAP, which prevents YAP into the nucleus through a series of phosphorylation, activation of the Hppo signaling pathway, and the inhibition of the Wnt pathway, resulting in the internal transcription factors. Hippo pathway and Wnt pathway may be involved in the pathogenesis of ARVC. In recent years, studies have shown that micro RN A plays an important role in the various pathophysiological processes of cardiovascular disease. The study shows that microRNA regulates the development and function of the heart, and in many cardiovascular diseases such as acute myocardial infarction. Death, hypertrophic cardiomyopathy, heart failure, atherosclerosis and other diseases, microRNA plays an important role in the regulation of many pathways and plays a role in the regulation of the pathophysiology of Wnt/ beta -catenin and Hippo signaling pathways. However, there are few reports on microRNA and ARVC, and we speculate on the hair of ARVC. The course of the disease may be involved in the regulation of microRNA. The purpose of this study is to explore the relationship between microRNA and the pathogenesis of ARVC, to clarify the expression of microRN in ARVC cardiomyopathy and to regulate the pathogenesis of ARVC through the Hippo signaling pathway, and to provide the basis and direction for the research and clinical diagnosis of ARVC pathogenesis and clinical diagnosis. In this study, the qRT-PCR detection technique S-Poly (A) P LUS was used to detect the myocardial tissue samples of 24 cases of ARVC patients after cardiac transplantation. A total of 1078 human microRNA were detected and 24 normal myocardial tissue samples were used as control. The results showed that 12 microRNA expressions were up-regulated and 11 microRNA expressions were downregulated. Further using ROC curve analysis, the sensitivity and specificity of each microRNA were analyzed, two microRNA:miR-451a were excluded, and miR-3647. application microRNA target gene software was used to predict 21 abnormal microRNA, miR-21-5p and YAP. The gene 3'UTR has a potential binding site; miR-135b targets MOB1b and LATS2 two genes to analyze the target genes of niR-21-5p and miR-135b in the Wnt, Hippo signaling pathway, and draw the microRNA-Gene network diagram. Cell adipose formation. The target gene and regulation mechanism of miR-21-5p, miR-135b were further studied. The expression of miR-21-5p was up-regulated in the HL-1PKP2:shRNA cell model and the expression of miR-135b was down regulated. The results of the dual luciferase reporter system detection confirmed that miR-21-5p, miR-135b could be combined to the 3'UTR end of the target gene mRNA. In HL-1 cells. The overexpression of miR-21-5p caused the expression of YAP protein to be inhibited, and the expression of MoBlb and LATS2 was reduced when the miR-135b was overexpressed. The over expression of miR-21-5p in HL-1PKP2:shRNA cells could promote the differentiation of fat in cells, while miR-135b inhibited the differentiation of myocardial cell fat, which confirmed miR-21-5p.miR-135b in Hip. The regulatory function in the Po signaling pathway. Cytological function studies confirm that miR-21-5p promotes the formation of adipose tissue in cardiac myocytes, and miR-135b plays an inhibitory role. Further studies have confirmed that YAP is the target gene for miR-21-5p, and miR-135b targets MoBlb and LATS2. in the conclusion of the experiment, the qRT-PCR detection technique S-Poly (T) was used to complete the myocardial tissue of the ARVC patients. The microRNA expression spectrum study shows that microRNA participates in the development and pathogenesis of ARCV, and constructs a network map of the regulatory gene of miR-21-5p, miR-135b and Wnt and Hippo signaling pathways. This study identified the microRNA expression profiles of cardiac tissue in ARVC patients. Two microRNA miR-21-5p were explored by cytological experiments. MiR-135b passed through the miR-135b. Signaling pathways regulate gene expression and participate in the molecular mechanism of ARVC pathogenesis.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R542.2

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