MicroRNA-184在致心律失常性心肌病中的病理作用机制研究

发布时间：2018-05-28 21:31

本文选题：Micro-RNA + DNA甲基化　；参考：《浙江大学》2016年博士论文

【摘要】：研究理由：致心律失常性心肌病特征性的心肌细胞脂肪化改变的病理机制尚不明确研究目的：研究Micro-RNA (miRNA)在致心律失常性心肌病(AC)发病机制中的作用研究方法与结果：对HL-1及Pkp2敲低HL-1心肌细胞(HL-1 Pkp2-shRNA)进行了含有750个miRNA的芯片筛查,发现了59个显著性差异表达的miRNA.其中miRNA-184 (miR-184)是在tHL_1Pkp2-shRNA中较对照组细胞表达下降最显著的一个miRNA.同时,miR-184在AC小鼠模型的心脏组织及心肌细胞中均较正常小鼠显著降低。miR-184在非成熟心肌细胞及心脏CD44+间充质祖细胞中表达最高,并且其表达量随心脏的发育逐渐降低。染色质免疫共沉淀(ChIP)实验表明,在AC模型中被抑制的E2F信号通路通过E2F1与miR-184 5’基因区域结合而调控miR-184的表达。通过对E2F1激动剂CCND1的过表达或对E2F1抑制剂RB1的抑制,均能增加miR-184的表达。另一方面,ChIP实验发现DNA甲基化转移酶1 (DNMT1)在HL-1 Pkp2-shRNA心肌细胞中与miR-184启动子区域大量结合,导致CpG位点过度甲基化,从而使miR-184表达受到沉默。去甲基化药物5-Aza-2'-Deoxycytidine (5-Aza-D)的使用以及转染针对Dnmtl的shRNA以抑制Dnmtl的表达均能增加miR-184的表达。在将miR-184的预测靶基因与HL-1和HL-1 Pkp2-ShRNA心肌细胞中差异表达的基因进行的配对研究显示,差异表达的miR-184靶基因在细胞生长,分化及死亡过程中起到主要作用。其中,Agpatl及Agpat3这两个脂肪代谢中的关键参与基因,被证明是miR-184的直接作用靶点。在HL-1及心脏CD44+细胞中进行的脂肪分化诱导实验中,miR-184敲低组与对照组相比均能更显著的诱导心肌细胞脂肪化。反之,miR-184过表达组与对照组相比则能够抑制心肌细胞脂肪化。结论与创新：本文第一次在AC模型中进行microRNA芯片筛查,发现miR-184表达量在AC细胞及小鼠模型中均显著下降,其下降机制包括E2F1通路的抑制及miR-184启动子区域的过度甲基化。同时,本文第一次发现miR-184的降低能够增加心肌细胞脂肪化从而参与了AC疾病的病理过程。
[Abstract]:Research reasons: the pathological mechanism of the characteristic fatty changes of cardiomyocytes in arrhythmia cardiomyopathy remains unclear objective: to study the role of Micro-RNA in the pathogenesis of arrhythmic cardiomyopathy Fruit: HL-1 and Pkp2 knockout HL-1 cardiomyocytes HL-1 Pkp2-shRNAs were screened with 750 miRNA microarrays. 59 differentially expressed miRNAs were found. The expression of miRNA in tHL_1Pkp2-shRNA was significantly decreased compared with that in control group. At the same time, the expression of miR-184 in heart tissue and cardiomyocytes of AC mouse model was significantly lower than that in normal mice. The expression of miR-184 was the highest in immature cardiomyocytes and cardiac CD44 mesenchymal progenitor cells, and its expression decreased gradually with the development of heart. Chromatin immunoprecipitation assay showed that the inhibited E2F signaling pathway regulated the expression of miR-184 through the binding of E2F1 to miR-184 5 'gene region in AC model. The expression of miR-184 was increased by overexpression of E2F1 agonist CCND1 or inhibition of E2F1 inhibitor RB1. On the other hand, we found that DNA methyltransferase 1 (DNMT1) binds to miR-184 promoter region in HL-1 Pkp2-shRNA cardiomyocytes, resulting in hypermethylation of CpG site and silencing of miR-184 expression. The use of demethylating drug 5-Aza-2'-Deoxycytidine 5-Aza-Dand the transfection of shRNA targeting Dnmtl to inhibit the expression of Dnmtl could increase the expression of miR-184. A pairing study of predicted target genes of miR-184 and differentially expressed genes in HL-1 and HL-1 Pkp2-ShRNA cardiomyocytes showed that differentially expressed miR-184 target genes played a major role in cell growth, differentiation and death. The two key genes involved in fat metabolism, such as Agpatl and Agpat3, have been proved to be the direct targets of miR-184. In the adipose differentiation induction experiment of HL-1 and cardiac CD44 cells, the myocytes in the low knockout group of miR-184 were significantly more likely to induce adipogenesis than those in the control group. On the contrary, the overexpression of miR-184 could inhibit the adipogenesis of cardiomyocytes compared with the control group. Conclusion and innovation: microRNA microarray screening was performed in AC model for the first time. It was found that the expression of miR-184 decreased significantly in both AC cells and mouse models. The mechanism of the decrease included the inhibition of E2F1 pathway and hypermethylation of miR-184 promoter region. At the same time, we found for the first time that the decrease of miR-184 could increase the adipogenesis of cardiac myocytes and participate in the pathological process of AC disease.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.7

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