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

描述（由申请人提供）：在本提案中，我们将研究microRNA (miRNA) hsa-miR-939在复杂区域性疼痛综合征（CRPS）中下调的机制意义以及该miRNA在血液中存在的功能意义。小的非编码mirna在基因表达调控中发挥重要作用，它们通过结合靶mrna的3‘非翻译区（3’-UTR）发挥作用。最近在体液中稳定的mirna的鉴定为它们作为适合临床诊断的新型生物标志物的使用铺平了道路。CRPS是一种使人衰弱的疼痛障碍，具有广泛的症状，大多数现有药物治疗效果都很差。我们研究了41例CRPS患者和20例对照组的全血miRNA变化，并鉴定了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中的首选候选。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几种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，我们将重点研究血管内皮生长因子A （VEGFA）、诱导型一氧化氮合酶（NOS2A）和肿瘤坏死因子α (TNFalpha)这三种可能的hsa-miR-939靶mrna，以评估下调hsa-miR-939是否会导致这些促炎基因同时上调。为了证明hsa-miR-939在调节VEGFA、TNFalpha和NOS2A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，我们将对从细胞培养基和血液中收集的外泌体进行表征。外泌体携带mrna、mirna、蛋白质和脂质介质通过血液到达受体细胞，具有功能基因调控作用，这表明了一种新的细胞通讯机制。我们将分别使用western blot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本中纯化的外泌体，我们将测量炎症发生时mirna的变化，包括hsa-miR-939和mrna （VEGFA， TNFalpha和NOS2A）。我们的研究将揭示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多因子病理生理机制的分子基础外，这些研究还可以帮助确定mirna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.