tRNA-derived non-coding RNAs in ASM function and in asthma

项目概要和摘要：我们提出的研究的目标是阐明表达谱， 短链非编码RNA（ncRNA）在哮喘发病机制中的分子功能。哮喘是一种慢性 炎症性疾病，其特征在于炎症、粘液产生、气道重塑和过度- 响应能力。在哮喘中，过敏性炎症介质，如细胞因子，作用于常驻气道细胞 [气道平滑肌（ASM）细胞，上皮（AE）细胞和成纤维细胞（LF）]导致其结构和 功能变化。然而，在我们对这些机制的理解方面仍然存在知识差距， 炎症介质调节细胞表型。虽然基因表达的转录调控在 已经广泛研究了常驻气道细胞，转录后步骤的调节机制仍然存在， 难以捉摸。在这种背景下，短ncRNA已经进化为基因转录后的关键调控因子之一， 表情以前用于短ncRNA分析的转录组分析主要依赖于标准RNA-seq 不能检测许多RNA种类的方法。含有环状磷酸盐的RNA（cP-RNA）， 环磷酸（cP）在其3 ′-末端，是一种这样的RNA物种不被RNA-seq捕获，因为cP阻止 3 ′-接头连接。它们在RNA-seq数据中的缺失使得cP-RNA成为一种不可见的，隐藏的成分， 转录组重要的是，cP-RNA被表达为功能分子。例如，血管生成素- 产生的5 ′-tRNA半体含有cP，因此属于cP-RNA，在胁迫中具有功能意义 反应，翻译调节和细胞增殖，并与神经退行性疾病有关 和癌症。我们认为5 ′-tRNA和其他cP-RNA在哮喘病理学中起重要作用。 在初步研究中，我们发现小鼠肺表达特异的5 ′-tRNA半体和cP-RNA，其水平 在屋尘螨（HDM）的吸入攻击引起的过敏性炎症期间上调。 此外，在人ASM细胞中，5 ′-tRNA半体的功能是调节细胞的粘着斑、迁移和增殖。 增殖这些结果使我们能够假设炎症介质上调5 ′- tRNA halves/cP-RNAs，其在分子发病机制中促进气道细胞声型改变 哮喘通过使用我们开发的cP-RNA-seq，我们建议完全阐明表达的调节 人肺和血浆样本中以及人肺和血浆样本中哮喘条件介导的5 ′-tRNA半体/cP-RNA ASM细胞、AE细胞和LF（Aim 1）。我们进一步建议评估5 ′-tRNA半体/cP-1的功能效应。 RNA对ASM细胞的细胞粘着斑、迁移、增殖和形态的影响（目的2），并研究 这些RNA的功能效应的分子机制（目标3）。拟议的研究将 揭示了哮喘发病机制中一种新的ncRNA参与途径，并将支持生物标志物的探索 哮喘病

Project Summary and Abstract: The goal of our proposed studies is to elucidate the expression profiles and molecular functions of short non-coding RNAs (ncRNAs) in the pathogenesis of asthma. Asthma is a chronic inflammatory disease characterized by inflammation, mucus production, airway re-modeling, and hyper- responsiveness. In asthma, allergic inflammatory mediators, such as cytokines, act on resident airway cells [airway smooth muscle (ASM) cells, epithelial (AE) cells and fibroblasts (LF)] causing their structural and functional changes. However, knowledge gaps remain in our understanding of the mechanisms by which inflammatory mediators modulate cellular phenotypes. Although transcriptional regulation of gene expression in resident airway cells has been extensively studied, regulatory mechanisms at post-transcriptional steps remain elusive. In this context, short ncRNAs have evolved as one of the key post-transcriptional regulators of gene expression. Previous transcriptome profiling for short ncRNA analyses relied mainly on standard RNA-seq methods which fail to detect many RNA species. Cyclic phosphate-containing RNAs (cP-RNAs), that harbor a cyclic phosphate (cP) at their 3′-end, are one such RNA species not captured by RNA-seq, because cP prevents 3′-adapter ligation. Their absence in RNA-seq data makes cP-RNAs an invisible, hidden component of transcriptomes. Importantly, cP-RNAs are expressed as functional molecules. For example, angiogenin- generated 5′-tRNA halves, containing a cP and thus belonging to cP-RNAs, have functional significance in stress response, translational regulation, and cell proliferation, and are associated with neurodegenerative diseases and cancers. We propose that 5′-tRNA halves and other cP-RNAs play important roles in asthma pathobiology. In preliminary studies, we found that mouse lung expresses specific 5′-tRNA halves and cP-RNAs whose levels are upregulated during allergic inflammation caused by inhaled challenge of house dust mite (HDM). Furthermore, in human ASM cells, 5′-tRNA halves function to regulate cellular focal adhesion, migration, and proliferation. These results allowed us to hypothesize that inflammatory mediators upregulate the levels of 5′- tRNA halves/cP-RNAs, which contributes to airway cellular phonotypic changes in the molecular pathogenesis of asthma. By using our developed cP-RNA-seq, we propose to fully elucidate the regulation of the expression of 5′-tRNA halves/cP-RNAs mediated by asthmatic conditions in human lung and plasma samples and in human ASM cells, AE cells, and LFs (Aim 1). We further propose to assess the functional effects of 5′-tRNA halves/cP- RNAs on cellular focal adhesion, migration, proliferation, and morphology of ASM cells (Aim 2) and to investigate the molecular mechanisms underlying the functional effects of those RNAs (Aim 3). The proposed studies will reveal a novel ncRNA-engaged pathway in asthma pathogenesis and will support the exploration of biomarkers in asthma.