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

描述(由申请人提供)：在这项建议中，我们将研究microRNA(MiRNA)hsa-miR-939下调在复杂区域疼痛综合征(CRPS)中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着重要作用，它们通过与靶mRNAs的3‘非翻译区(3’-UTR)结合而发挥作用。最近在体液中发现了稳定的miRNAs，为将其用作符合临床诊断的新生物标志物铺平了道路。CRPS是一种衰弱的疼痛障碍，具有广泛的症状，大多数可用的药物对这些症状处理不善。我们研究了41例CRPS患者和20例对照组全血中miRNA的变化，发现了18个miRNAs的差异表达。我们特别选择关注hsa-miR-939，因为它是我们列出的18个CRPS患者改变的miRNAs中的首选候选基因。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几个已知在炎症和疼痛中起关键作用的基因的miRNA是否可以放大疼痛信号转导级联。因此，我们将集中研究三个可能的hsa-miR-939靶基因，包括血管内皮生长因子A(VEGFA)、诱导型一氧化氮合酶(NOS2A)和肿瘤坏死因子α(TNFpha)，以评估hSA-miR-939的下调是否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、TNFpha和NOS2A表达中的直接作用，我们将过表达和敲除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miRNAs的存在的功能意义，我们将对从细胞培养液和血液中收集的外切体进行鉴定。Exosome通过血液携带mRNAs、miRNAs、蛋白质和脂质介体到受体细胞，具有功能基因调节作用，表明了一种新的细胞通讯机制。我们将分别使用蛋白质印迹分析和电子显微镜对外体标记和形态进行验证。利用内毒素刺激的THP-1细胞分泌的外切体和从患者血液样本中纯化的外切体，我们将测量炎症发生时miRNAs包括hsa-miR-939和mRNAs(VEGFA、TNFpha和NOS2A)的变化。我们的研究将阐明外切体在炎症和疼痛背景下的信息传递。除了深入了解CRPS多因素病理生理机制的分子基础外，这些研究还有助于确定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。

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.