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.