Functional characterization of hsa-mir-939 as a novel regulator of pain

hsa-mir-939 作为新型疼痛调节剂的功能表征

• 批准号: 8626458
• 负责人: Seena Ajit
• 金额: $ 19.12万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8626458
• 关键词: 3' Untranslated Regions; Arginine; Attention; Base Sequence; Binding; Binding Sites; Bioinformatics; Biological Assay; Biological Markers; Biological Models; Blood; Blood Circulation; Blood specimen; Cell Culture Techniques; Cells; Chronic; Collecting Cell; Communication; Complex Regional Pain Syndromes; Culture Media; Data; Down-Regulation; Electron Microscopy; Enzyme-Linked Immunosorbent Assay; Foundations; Functional disorder; Future; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Immune; In Vitro; Inflammation; Inflammatory; Injury; Interleukin-1; Investigation; Light; Lipids; Lipopolysaccharides; Liquid substance; Luc Gene; Measures; Mediating; Membrane; Messenger RNA; MicroRNAs; Molecular; Monitor; Morphology; Motor; NOS2A gene; Nervous system structure; Neurogenic Inflammation; Neuropathy; Pain; Pain Disorder; Patients; Pharmaceutical Preparations; Plasmids; Play; Process; Proteins; Regulator Genes; Reporter; Reporting; Role; Sampling; Seeds; Sensory; Signal Transduction; Stimulus; Symptoms; System; Therapeutic Intervention; Transforming Growth Factors; Translational Repression; Tumor Necrosis Factor-alpha; Untranslated Regions; Up-Regulation; Validation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vesicle; Western Blotting; Whole Blood; clinical Diagnosis; functional disability; human NOS2A protein; human disease; in vitro Model; insight; interest; lipid mediator; novel; novel therapeutics; overexpression; painful neuropathy; public health relevance; response; tool

DESCRIPTION (provided by applicant): In this proposal we will investigate the mechanistic significance of downregulation of microRNA (miRNA) hsa-miR-939 in complex regional pain syndrome (CRPS) and the functional implication of the presence of this miRNA in blood. Small noncoding miRNAs play an important role in the regulation of gene expression and they function by binding to the 3' untranslated region (3'-UTR) of target mRNAs. Recent identification of stable miRNAs in bodily fluids paved the way for their use as novel biomarkers amenable to clinical diagnosis. CRPS is a debilitating pain disorder with a broad spectrum of symptoms that are poorly managed by most available drugs. We investigated miRNA changes in whole blood from 41 patients with CRPS and 20 controls and identified differential expression of 18 miRNAs. We specifically chose to focus on hsa-miR-939 because it is the top candidate in our list of 18 miRNAs altered in CRPS patients. Neurogenic inflammation is common in CRPS and hence we will investigate if modulating a miRNA capable of targeting several genes known to have critical role in inflammation and pain, can amplify the pain signal transduction cascade. Thus we will focus our studies on three putative hsa-miR-939 target mRNAs including vascular endothelial growth factor A (VEGFA), inducible nitric oxide synthase (NOS2A), and tumor necrosis factor alpha (TNFalpha) to assess if downregulation of hsa-miR- 939 can result in the simultaneous upregulation of these proinflammatory genes. To demonstrate a direct role for hsa-miR-939 in regulating the expression of VEGFA, TNFalpha and NOS2A, we will overexpress and knockdown hsa-miR-939 and monitor changes in mRNA and protein levels of these genes. To investigate the functional implication of the presence of circulating miRNAs, we will characterize the exosomes collected from cell culture media and blood. Exosomes carry mRNAs, miRNAs, proteins and lipid mediators to recipient cells with functional gene regulatory consequences via blood indicating a novel mechanism of cellular communication. We will validate the exosomal markers and morphology using western blot analysis and electron microscopy respectively. Using exosomes secreted by THP-1 cells stimulated with lipopolysaccharides (LPS) and exosomes purified from patient blood samples, we will measure the alterations in miRNAs including hsa-miR-939 and mRNAs (VEGFA, TNFalpha and NOS2A) that occur with inflammation. Our studies will shed light on exosome-mediated information transfer in the context of inflammation and pain. In addition to providing insight into the molecular underpinnings of the multifactorial pathophysiological mechanisms in CRPS, these studies can help determine if miRNAs or the genes they modulate can be direct targets for future therapeutic interventions.

描述（由申请人提供）：在本提案中，我们将研究microRNA（miRNA）hsa-miR-939在复杂区域疼痛综合征（CRPS）中下调的机制意义以及血液中存在该miRNA的功能意义。小的非编码miRNAs在基因表达调控中起重要作用，它们通过与靶mRNA的3'非翻译区（3'-UTR）结合来发挥功能。最近在体液中鉴定出稳定的miRNA，为它们作为适合临床诊断的新型生物标志物的用途铺平了道路。CRPS是一种使人衰弱的疼痛疾病，具有广泛的症状，大多数可用药物都无法有效控制。我们研究了41名CRPS患者和20名对照者全血中miRNA的变化，并确定了18种miRNA的差异表达。我们特别选择关注hsa-miR-939，因为它是我们在CRPS患者中改变的18种miRNA列表中的首选。神经源性炎症在CRPS中很常见，因此我们将研究调节能够靶向已知在炎症和疼痛中起关键作用的几个基因的miRNA是否可以放大疼痛信号转导级联。因此，我们将集中我们的研究在三个假定的hsa-miR-939靶mRNA，包括血管内皮生长因子A（VEGFA），诱导型一氧化氮合酶（NOS 2A），和肿瘤坏死因子α（TNF α），以评估是否下调hsa-miR- 939可以导致这些促炎基因的同时上调。为了证明hsa-miR-939在调节VEGFA、TNF α和NOS 2A表达中的直接作用，我们将过表达和敲低hsa-miR-939，并监测这些基因的mRNA和蛋白水平的变化。为了研究循环miRNA存在的功能意义，我们将表征从细胞培养基和血液中收集的外泌体。外泌体通过血液将mRNA、miRNA、蛋白质和脂质介质携带至受体细胞，具有功能性基因调控结果，表明细胞通讯的新机制。我们将分别使用蛋白质印迹分析和电子显微镜验证外泌体标记和形态学。使用由脂多糖（LPS）刺激的THP-1细胞分泌的外泌体和从患者血液样品纯化的外泌体，我们将测量伴随炎症发生的包括hsa-miR-939和mRNA（VEGFA、TNF α和NOS 2A）在内的miRNA的改变。我们的研究将阐明炎症和疼痛背景下外泌体介导的信息传递。除了深入了解CRPS中多因素病理生理机制的分子基础外，这些研究还可以帮助确定miRNAs或它们调节的基因是否可以成为未来治疗干预的直接靶点。