长链非编码RNA (lncRNAs) 对衰老促心肌凋亡效应的调节作用及其机制的研究

Cardiovascular diseases are the leading causes of death in the elderly, as those older than 65 years account for greater than 80% of patients with ischemic heart disease. Additionally, the exponential increase in mortality rate related to cardiovascular diseases in the geriatric population implies that cardiac aging itself may be a major risk factor for cardiovascular pathology such as ischemic heart disease. In previous study, we determined aging-related myocardial apoptosis is the leading cause of ischemic heart disease. However, to date, the underlying mechanism still keep unclear. Currently, Long non-coding RNAs (lncRNAs) represent one of the most prominent but least understood genes in gene regulation. To address the role of lncRNAs in aging-related myocardial apoptosis, we carried out a genome-wide lncRNA microarray analysis, and the results revealed the level of some lncRNAs changed the age increasing, which could play the important roles in the apoptotic pathways. In order to clarify the function of the targeted lncRNAs, silencing and overexpressing of the above lncRNAs were carried out. The regulatory effects of the above lncRNAs on apoptosis were assessed. Additionally, the underlying mechanisms will be detected with Dual Luciferase Reporter, RNA pull down and RNA Binding Protein Immunoprecipitation (RIP). Furthermore, abandoned cardiac tissue from hypertrophic cardiomyopathy patients and blood samples from volunteers were detected with lncRNAs/mRNAs microarray and computational analysis. The targeted lncRNAs will be got with the following characters: 1. rich in the cardiac tissue of human being; 2. the strong aging-specific; 3. the significant regulatory effects on myocardial apoptosis. The targeted lncRNAs will be transfer in cardiac tissue of mouse. The regulatory effects and mechanisms of targeted lncRNAs on cardiac apoptosis will be investigated. The present study will provide the theoretical support for the future gene therapy to the aged heart failure patients.

缺血后心衰正成为亟待解决的临床问题。衰老诱发的心肌凋亡是导致缺血后心衰的首要原因。但是，衰老促心肌凋亡的机制尚不明了。由于具有重要的调控作用，长链非编码RNA(lncRNAs)日益成为学界聚焦的热点。我们采用基因芯片和生物信息方法，初步筛选出具有年龄特异性及心肌凋亡调节潜能的lncRNA。本项目旨在验证lncRNAs对衰老促凋亡效应的作用及其机制。我们使用基因干扰方法抑制心肌内目标lncRNAs表达(或上调)，检测其对凋亡的影响。进一步，拟使用双荧光素酶、RNA pull down等方法，探究目标lncRNAs对心肌凋亡调控的机制。在动物研究基础上，我们使用临床遗弃的心肌组织和志愿者的血液标本，检测出人类心肌富含、且具有年龄特异性和潜在凋亡调节作用的目标lncRNA, 拟将其转入小鼠体内，检测其对心肌凋亡的调节作用及机制。本研究将为发展针对老年心衰患者的基因治疗提供理论依据。

衰老诱发的心肌凋亡是导致缺血后心衰的首要原因。但是，衰老促心肌凋亡的机制尚不明了。本研究通过lncRNA/mRNA微阵列及生物信息学分析，发现小鼠lncRNA ENSMUST00000134285能够通过激活MAPK11及其信号通路，抑制年龄相关的心肌细胞凋亡的作用；此外，验证了miR-760能够调节MAPK11和 ENSMUST00000134285之间的相互作用。. 接着使用同样方法筛选在人体心肌和血液标本中富集的与衰老相关的lncRNA。其中lncRNA NONHSAT069381过表达后对缺氧/复氧的人心肌细胞AC16凋亡具有促进作用，hsa-miR-124-5p在NONHSAT069381和mCASP3之间起到桥接作用；而lncRNA NONHSAT140844过表达后对缺氧/复氧的AC16凋亡具有抑制作用，hsa-miR-6507-5p在NONHSAT140841和mXIAP间起到桥接作用。. 我们通过蛋白组学和蛋白磷酸化组学分析了青年和老年小鼠心肌组织，在对心肌内表达的196个凋亡调节蛋白比对后，仅有7个凋亡调节因子因衰老发生总量的变化；但是在许多重要的凋亡调节蛋白上都出现了磷酸化水平的改变，其中细胞色素C中T50（CytC T50）这一位点磷酸化水平在老年组与青年组之间改变差异倍数较大，而且发生在线粒体凋亡的重要凋亡通路上，未被注释；因此我们进一步证实了CytC T50的磷酸化具有抗凋亡作用。而CytC T50的磷酸化状态对于Bax和Bcl-2蛋白则无明显影响。. 本研究进一步探究衰老的促凋亡机制，不仅仅存在巨大学术价值，而且可能为未来发展心衰治疗的新技术、新靶点提供有力的理论支持。