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

项目摘要和摘要：我们建议的研究的目标是阐明表达谱和 短非编码RNA(NcRNAs)在哮喘发病机制中的分子功能哮喘是一种慢性疾病 炎症性疾病的特征是炎症、粘液产生、呼吸道重建和过度兴奋。 响应性。在哮喘中，过敏性炎症介质，如细胞因子，作用于驻留的呼吸道细胞。 [气道平滑肌(ASM)细胞、上皮(AE)细胞和成纤维细胞(LF)]引起它们的结构和 功能变化。然而，知识差距仍然存在于我们对机制的理解中 炎症介质调节细胞表型。尽管对基因表达的转录调控 驻留的呼吸道细胞已经被广泛研究，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ncRNAs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分析的转录组图谱主要依赖于标准的rna-seq 无法检测到许多RNA物种的方法。环状含磷RNA(CP-RNAs)，含有一种 环磷酸盐(CP)在其3‘端，是一种这样的RNA物种，不被RNA-seq捕获，因为CP阻止 3‘-接头结扎术。它们在RNA-seq数据中的缺失使得CP-RNA成为不可见的、隐藏的 抄本。重要的是，CP-RNA以功能分子的形式表达。例如，血管生成素- 生成的含有CP的5‘-tRNA半片段属于CP-RNAs，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相关 和癌症。我们认为5‘-tRNA半部分和其他CP-RNAs在哮喘的病理生物学中起重要作用。 在初步研究中，我们发现小鼠肺表达特异性的5‘-tRNA片段和CP-RNA，它们的水平 在吸入屋尘螨(HDM)引起的过敏性炎症过程中上调。 此外，在人ASM细胞中，5‘-tRNA调节细胞局部黏附、迁移和转移的功能减半。 扩散。这些结果允许我们假设炎症介质上调5‘- TRNA减半/CP-RNAs，在分子发病机制中促进呼吸道细胞音型变化 哮喘的症状。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 哮喘条件对人肺、血浆和人体5‘-tRNA半组分/CP-RNAs的影响 ASM细胞、AE细胞和LFS(目标1)。我们进一步建议评估5‘-tRNA Halves/CP-的功能效应。 RNA对ASM细胞灶性黏附、迁移、增殖和形态的影响(目标2)并探讨 这些RNA功能效应的分子机制(目标3)。拟议的研究将 揭示ncRNA参与哮喘发病机制的新途径，并将支持生物标志物的探索 得了哮喘。

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.