Role of small RNAs in ischemic tissue repair

项目总结 心血管疾病(CVD)仍然是主要的死亡原因，迫切需要确定 潜在的机制，并开发新的治疗方法。卡哈尔小体特异性RNA(ScaRNAs) 是进化上保守的非编码RNA，指导特定核苷酸的生化修饰，如 假尿嘧啶(Ψ)和2‘-O-核糖甲基化(2’-OME)。这些修改在后一阶段是必不可少的- 剪接体小核糖核酸的转录变化和功能。最近的研究强调了一个 Scarna在保持剪接体保真度和心脏发育中的关键作用；然而，scaRNAs的作用 在急性心脏损伤中，其修复尚未建立。 这 该提案旨在确定斯卡纳的角色 在小鼠心肌梗死(MI)模型的心血管过程中，可能开发新的治疗工具 密西西比。我们的中心假设是，心肌梗死后心脏中scaRNA18的过度表达促进了正性心脏 通过U4SnRNA的2‘-端组诱导治疗性血管生成和调节Wilms肿瘤1(WT1)的重塑 表达，从而赋予心脏保护和改善心脏功能。我们的初步数据 证明1)使用无偏小RNA可降低心肌梗死后小鼠心脏中scaRNA18的表达 测序，2)scaRNA18在体外培养的心肌内皮细胞中表达下调 心肌梗死后小鼠心脏，3)scaRNA18基因敲除(KD)损伤小鼠心脏成管并诱导细胞凋亡 未诱导应激的心脏内皮细胞，4)scaRNA18过表达促进内皮细胞血管生成并抑制血管生成 应激诱导的体外内皮细胞凋亡；5)scaRNA18过表达的心肌内皮细胞外囊泡保护作用 IPSC-通过血管生成蛋白导致的心肌细胞死亡，6)心肌梗死心脏2‘-O-甲基化水平降低 而在scaRNA18KD小鼠的心脏内皮细胞中，7)突变的scaRNA18缺乏结合位点来引导U4上的2‘-OME SnRNA在没有诱导应力的情况下诱导EC细胞凋亡，无偏质谱结果显示 ScaRNA18可降低心脏内皮细胞WT1的表达水平。 ScaRNA18过表达使scaRNA18介导的EC存活效应无效，以及 VE-钙粘附素驱动的AAV9介导的scaRNA18减少体外心肌缺血/再灌注损伤后的心肌梗死范围 并改善心肌梗死后小鼠(体内)的心功能。假设scaRNA18是 心脏功能和修复将在以下三个具体目标中进行测试：在目标1中，我们将定义 ScaRNA18在心脏EC功能和心肌细胞存活中的作用；在目标2中，我们将确定 ScaRNA18调节心脏内皮细胞功能和心肌细胞存活的机制，以及在目标3中 将使用心肌梗塞后的小鼠来证明scaRNA18在心肌损伤修复中的关键作用。建议数 研究可以建立scaRNAs作为治疗心肌梗死的新靶点。

PROJECT SUMMARY Cardiovascular disease (CVD) remains the leading cause of death, and there is a critical need to determine underlying mechanisms and develop novel therapies for treatment. Small Cajal body-specific RNAs (scaRNAs) are evolutionarily conserved non-coding RNAs that guide biochemical modification on specific nucleotides such as pseudouridylation (Ψ) and 2′-O-ribose methylation (2′-OMe). These modifications are essential in the post- transcriptional changes and functions of spliceosomal small nuclear RNAs (snRNA). Recent studies highlight a crucial role of scaRNA in preserving spliceosome fidelity and cardiac development; however, the role of scaRNAs in acute cardiac injury and its repair has not been established. This proposal aims to determine scaRNA’s role in cardiovascular processes in a mouse myocardial infarction (MI) model that could develop new tools for treating MI. Our central hypothesis is that overexpression of scaRNA18 in the post-MI heart promotes positive cardiac remodeling by inducing therapeutic angiogenesis via 2′-OMe of U4 snRNA and regulating Wilms tumor1 (WT1) expression, thereby conferring cardioprotection and improving cardiac function. Our preliminary data demonstrates 1) decreased scaRNA18 expression in post-MI mouse hearts using unbiased small RNA sequencing, 2) scaRNA18 expression exclusively downregulated in isolated cardiac endothelial cells (EC) in post-MI mouse hearts, 3) scaRNA18 knockdown (KD) impairs tube formation and induces apoptosis in mouse cardiac ECs without induced stress, 4) scaRNA18 overexpression enhances EC angiogenesis and inhibits stress-induced EC apoptosis in vitro, 5) scaRNA18-overexpressing cardiac EC extracellular vesicles protect iPSC-cardiomyocyte cell death via angiogenic proteins, 6) 2’-O-methylation levels were reduced in MI hearts and in cardiac ECs from scaRNA18 KD mice, 7) mutant scaRNA18 lacking binding sites to guide 2′-OMe on U4 snRNA induces EC cell apoptosis without induced stress, 8) unbiased mass spectrometry results revealed that scaRNA18 reduces WT1 expression levels in scaRNA18 KD cardiac ECs, 9) WT-1 KD in the presence of scaRNA18 overexpression nullified scaRNA18 mediated EC survival effects, and 10) overexpression of scaRNA18 by VE-cadherin-driven AAV9 reduces infarct size after ex vivo cardiac ischemia/reperfusion injury and improves cardiac function post-MI mice (in vivo). The hypothesis that scaRNA18 is a critical component in cardiac function and repair will be tested in the following three specific aims: in Aim 1, we will define the role of scaRNA18 in cardiac EC function and cardiomyocyte survival; in Aim 2, we will determine the molecular mechanisms by which scaRNA18 regulates cardiac EC function and cardiomyocyte survival, and in Aim 3 we will use post-MI mice to demonstrate the critical role of scaRNA18 in myocardial injury repair. The proposed studies could establish scaRNAs as novel therapeutic targets for MI.