Abstract
Circulating microRNAs are beneficial biomarkers because of their stability and dysregulation in diseases. Here we sought to determine the role of miR-939, a miRNA downregulated in patients with complex regional pain syndrome (CRPS). Hsa-miR-939 is predicted to target several proinflammatory genes, including IL-6, VEGFA, TNFα, NFκB2, and nitric oxide synthase 2 (NOS2A). Binding of miR-939 to the 3′ untranslated region of these genes was confirmed by reporter assay. Overexpression of miR-939 in vitro resulted in reduction of IL-6, NOS2A and NFκB2 mRNAs, IL-6, VEGFA, and NOS2 proteins and NFκB activation. We observed a significant decrease in the NOS substrate l-arginine in plasma from CRPS patients, suggesting reduced miR-939 levels may contribute to an increase in endogenous NOS2A levels and NO, and thereby to pain and inflammation. Pathway analysis showed that miR-939 represents a critical regulatory node in a network of inflammatory mediators. Collectively, our data suggest that miR-939 may regulate multiple proinflammatory genes and that downregulation of miR-939 in CRPS patients may increase expression of these genes, resulting in amplification of the inflammatory pain signal transduction cascade. Circulating miRNAs may function as crucial signaling nodes, and small changes in miRNA levels may influence target gene expression and thus disease.
Highlights
Distinct expression patterns of circulating microRNAs have been associated with a wide range of diseases[1]
Since several of the predicted miR-939 target genes play a central role in regulation of the immune system[11,12], we hypothesized that the downregulation of miR-939 may result in the upregulation of several mRNAs harboring miR-939 binding sites, known to regulate the inflammatory response in patients
HEK293 cells were transiently transfected with plasmids encoding the reporter 3′ untranslated region (3′UTR) constructs and either precursor miR-939 or a scrambled precursor miRNA control
Summary
Distinct expression patterns of circulating microRNAs (miRNAs) have been associated with a wide range of diseases[1]. Bioinformatic predictions showed that miR-939 can potentially target several mRNAs encoding various proinflammatory mediators, including interleukin-6 (IL-6), vascular endothelial growth factor (VEGFA), tumor necrosis factor α (TNFα), nitric oxide synthase 2 (NOS2A or iNOS), and nuclear factor-κB2 (NFκB2)[9,10] Among these putative target genes, plasma levels of IL-6 and VEGF protein were significantly negatively correlated with miR-939 expression in patients with CRPS when compared to control[8]. This suggests that a reduction in miR-939 may contribute to an increase in the translation of these target mRNAs. The classic inflammatory response occurring after injury includes secretion of proinflammatory cytokines. Results from our in vitro studies and analyses of total RNA from whole blood and plasma from CRPS patients and controls suggest that downregulation of miR-939 in CRPS patients may increase the translation of proinflammatory target mRNAs
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