致心律失常性右室心肌病microRNA的表达谱及调控机制研究

发布时间：2018-05-02 04:54

本文选题：致心律失常性右室心肌病 + microRNA　；参考：《北京协和医学院》2016年博士论文

【摘要】：研究背景致心律失常性右室心肌病(Arrhythmo genic right ventricular cardiomypathy ARVC)是原发性遗传性心肌病,以右室心肌不同程度地被纤维脂肪组织代替为主要特征,往往伴有心室扩大,心力衰竭,严重恶性心律失常甚至猝死发生,是青少年及运动员猝死的主要病因。流行病学调查分析人群中发病率为1/2000-1/5000。一半以上的ARVC病例是由桥粒基因突变所引发的,最常见的桥粒基因突变为：plako globin (PG), desmoplakin (DSP), plakophilin-2 (PKP2), desmoglein-2 (DSG2) and desmocollin-2 (DSC2)。非桥粒基因突变的基因有转录因子β3 (transforming growth factor-β3TGF-β3)和连接蛋白(connexin43 Cx43)。Wnt/β-catenin 和 Hippo信号通路是与心脏发育相关的重要通路。Wnt/β-catenin信号通路调控心脏祖细胞向心肌细胞和脂肪细胞分化。研究发现通过沉默DSP基因表达,会导致PG (γ-catenin)从细胞桥粒组合中脱落,从而和β-catenin共同竞争入核,进而引起核内脂肪转化相关的转录因子PPARγ、C/EBP改变,从而促进脂肪分化.Wnt/β-catenin 和 Hippo信号通路有交互作用,两条通路中YAP, PG 及 β-catenin相互作用形成蛋白复合体。在桥粒基因PKP2突变的情况下,激活NF2引起Hippo信号通路的重要因子MST1/2, LATS1/2 及 YAP发生激酶的级联反应,通过一系列磷酸化,阻止YAP入核发挥作用,Hppo信号通路激活,发挥抑制Wnt通路作用,从而引起核内转录因子改变促进细胞脂肪化。因而,Hippo通路和Wnt通路可能共同参与了ARVC的发病。近年来研究发现,micro RN A在心血管疾病的各种病理生理过程中发挥了重要的调控作用,研究表明microRNA调控心脏的发育和功能,在许多心血管疾病如急性心肌梗死、肥厚型心肌病、心力衰竭、动脉粥样硬化等疾病中,microRNA都扮演了重要的角色。microRNA参与许多通路的调控而发挥作用,也参与了Wnt/β-catenin和Hippo信号通路调控疾病的病理生理过程。然而microRNA与ARVC的研究报道尚少,我们推测ARVC的发病过程,可能有microRNA的调控参与。研究目的探讨microRNA 与 ARVC发病机制的关系,明确microRN 在 ARVC心肌病中的表达谱及microRNA可能通过Hippo信号通路调控ARVC发病机制,为ARVC致病机制研究及临床诊断治疗提供依据和方向。研究方法和结果本研究中应用qRT-PCR检测技术S-Poly(A)P lus对24例刂ARVC患者心脏移植后的心肌组织样本microRNA进行了检测,共检测1078个人类microRNA,同时以24例正常心肌组织样本作为对照。通过检测发现24个表达异常的microRNA,并进一步在单独样品中进行了验证,结果发现有12个microRNA表达上调,11个microRNA表达下调。进一步应用ROC曲线分析,对每一个microRNA的敏感性和特异性进行分析,排除两个microRNA:miR-451a、miR-3647.应用microRNA靶基因软件进行预测显示：21个表达异常microRNA中,miR-21-5p与YAP基因的3'UTR具有潜在结合位点；miR-135b靶向MOB1b及LATS2两个基因,分析了niR-21-5p、miR-135b在Wnt、Hippo信号通路中的靶基因,并绘制了microRNA-Gene网络图。通过对ARCV心肌组织microRNA表达谱研究,发现miR-21-5p.miR-135b可能调节Vnt及Hippo信号通路,调控细胞脂肪化形成。对miR-21-5p、miR-135b的靶基因和调控机制进一步研究,在HL-1PKP2:shRNA细胞模型中检测发现miR-21-5p表达上调,而miR-135b表达下调。双荧光素酶报告系统检测结果证实miR-21-5p、miR-135b可以分别结合到靶基因mRNA的3'UTR端。在HL-1细胞中转染miR-21-5p过表达使得YAP蛋白表达受到抑制；同样过表达miR-135b时MoBlb及LATS2的蛋白表达量降低。细胞功能研究时,HL-1PKP2:shRNA细胞过表达miR-21-5p会促进细胞的脂肪分化,而miR-135b则会抑制心肌细胞脂肪分化,证实了miR-21-5p.miR-135b在Hippo信号通路中的调控功能。细胞学功能研究证实miR-21-5p促进心肌细胞脂肪形成,而miR-135b起到抑制作用。进一步研究证实YAP为miR-21-5p的靶基因,miR-135b则靶向MoBlb及LATS2.研究结论本实验应用qRT-PCR检测技术S-Poly(T)完成了ARVC患者的心肌组织microRNA表达谱研究,实验证实microRNA参与ARCV的发育和发病机制,构建了miR-21-5p、miR-135b与Wnt及Hippo信号通路的调控基因网络图。本研究明确了ARVC患者心肌组织的microRNA表达谱；并通过细胞学实验探索了两个microRNA miR-21-5p、miR-135b通过Hippo信号通路的调控基因调控基因表达而参与ARVC发病的分子机制。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.2

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