自噬性心肌保护中基于Notch1信号相关miRNA和lncRNA的筛选

发布时间：2018-08-30 19:30

【摘要】：第1章引言急性心肌梗塞在心血管疾病中发病率和死亡率极高,缺血/再灌注损伤仍是急性心肌梗塞临床治疗中面临的难题。如何减轻缺血/再灌注损伤是心肌损伤与保护的研究热点。研究心肌损伤发生机制,将有助于我们为临床防治心肌缺血损伤提供理论和实践依据。因此,我们基于lncRNA-miRNA-Notch1调控假说,建立心肌细胞缺氧/复氧损伤及自噬保护模型,通过高通量测序技术及生物信息学比较分析,筛选出在自噬保护中具有调控Notch1蛋白作用的miRNA,以及具有调控该miRNA的lncRNA,为研究Notch1信号通路在自噬性心肌保护的分子机制提供科学依据。第2章Notch1信号通路增强自噬对抗心肌缺氧复氧损伤的保护作用目的:筛选有效调控自噬的3-MA及RAPA试剂浓度,以及探讨Notch1信号通路在自噬对抗心肌缺氧复氧损伤中的作用。方法:建立心肌细胞损伤模型,给予3-MA、RAPA干预,检测细胞活力,筛选3-MA及RAPA有效试剂浓度;利用Ad-N1ICD及Ad-N1ICD-shRNA干预Notch1信号通路,检测H/R损伤中心肌细胞活力情况。结果:本研究中3-MA和RAPA最佳试剂浓度分别为1μM和50nM;Notch1过表达可提高H/R的心肌细胞活力(p0.01),干扰Notch1可明显降低H/R心肌细胞活力(p0.05);3-MA加入Notch1过表达组中,H/R的心肌细胞活力再次降低(p0.01),RAPA加入Notch1干扰组中,H/R的心肌细胞活力恢复(p0.01)。结论:Notch1信号通路可增强自噬对抗心肌缺氧复氧损伤发挥保护作用。第3章高通量测序及生物信息学筛选自噬性心肌保护中Notch1信号通路相关miRNA及lncRNA目的:借助高通量测序及生物信息学比较分析筛选在自噬性心肌保护中具有调控Notch1信号通路的关键miRNA及lncRNA。方法:提取心肌缺氧/复氧损伤中的总RNA,借助高通量测序及生物信息学分析系统,筛选具有差异表达的miRNA及lncRNA。结果:筛选出了可能调控Notch1基因表达的miR-702-5p、miR-344g、miR-323-5p、miR-6334、miR-5132-5p、miR-15b-5p、miR-3562;生物信息学分析发现lncRNA-4321可与miR-702-5p直接结合并影响其活性;在RAPA和3-MA处理心肌细胞中,Realtime-PCR检测显示lncRNA-4321表达量与心肌自噬成正比,miR-702-5p表达量与心肌自噬成反比。结论:lncRNA-4321/miR-702-5p/Notch1可能在自噬性心肌保护中发挥重要作用。
[Abstract]:Chapter 1 introduces that the incidence and mortality of acute myocardial infarction in cardiovascular disease is very high. Ischemia / reperfusion injury is still a difficult problem in the clinical treatment of acute myocardial infarction. How to reduce ischemia / reperfusion injury is the focus of myocardial injury and protection. Studying the mechanism of myocardial injury will help us to provide theoretical and practical basis for clinical prevention and treatment of myocardial ischemia injury. Therefore, based on the lncRNA-miRNA-Notch1 regulation hypothesis, we established a cardiomyocyte model of hypoxia / reoxygenation injury and autophagy protection, and analyzed it by high-throughput sequencing and bioinformatics. The screening of miRNA, which has the function of regulating Notch1 protein in autophagy protection and lncRNA, which has the function of regulating miRNA provide scientific basis for studying the molecular mechanism of Notch1 signaling pathway in autophagy myocardial protection. Chapter 2 Notch1 signaling pathway enhances the protective effect of autophagy on myocardial hypoxic-reoxygenation injury objective: to screen the concentration of 3-MA and RAPA reagents that can effectively regulate autophagy and to explore the role of Notch1 signaling pathway in anti-hypoxia and reoxygenation injury of myocardium. Methods: myocardial injury model was established, 3-MARAPA was given to detect cell viability, 3-MA and RAPA effective reagent concentrations were screened, and Notch1 signaling pathway was interfered by Ad-N1ICD and Ad-N1ICD-shRNA to detect myocardial cell viability in H / R injury. Results: the best concentration of 3-MA and RAPA in this study was 1 渭 M and 50nM respectively, which increased the viability of H / R cardiomyocytes (p0.01). Interfering with Notch1 could significantly reduce the activity of H / R cardiomyocytes (p0.05) 3-MA added to Notch1 overexpression group. The secondary decrease (p0.01) and the activity recovery of H / R in the Notch1 interference group (p0.01). Conclusion the fraction Notch1 signaling pathway can enhance the protective effect of autophagy on myocardial hypoxia and reoxygenation injury. Chapter 3 High-throughput sequencing and Bioinformatics screening Notch1 signaling Path-Related miRNA and lncRNA in autophagy Myocardial Protection objective: to regulate Notch1 in autophagic myocardial protection by high-throughput sequencing and bioinformatics comparative analysis Key miRNA and lncRNA. of signal Pathway Methods: total RNA, from myocardial hypoxia / reoxygenation injury was extracted by high throughput sequencing and bioinformatics analysis system to screen miRNA and lncRNA. with differential expression. Results: the bioinformatics analysis of miR-702-5p,miR-344g,miR-323-5p,miR-6334,miR-5132-5p,miR-15b-5p,miR-3562;, which could regulate the expression of Notch1 gene, found that lncRNA-4321 could directly bind to miR-702-5p and affect its activity. In RAPA and 3-MA treated cardiomyocytes, the expression of lncRNA-4321 was directly proportional to myocardial autophagy and the expression of miR-702-5p was inversely proportional to myocardial autophagy. Conclusion: 1: lncRNA-4321% miR-702-5p / Notch1 may play an important role in the protection of autophagic myocardium.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54

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