RESUMEN

Complex regional pain syndrome (CRPS) is a chronic pain condition characterized by inflammation and debilitating pain. CRPS patients with pain refractory to more conventional analgesics can be treated with subanesthetic doses of ketamine. Our previous studies found that poor responders to ketamine had a 22-fold downregulation of the miRNA hsa-miR-605 in blood prior to ketamine treatment. Hence, we sought to investigate the functional significance of miR-605 downregulation and its impact on target gene expression, as investigating target mRNAs of differentially expressed miRNAs can provide important insights on aberrant gene expression that may contribute to disease etiology. Using a bioinformatics prediction, we identified that miR-605 can target the proinflammatory chemokine CXCL5, which plays a role in leukocyte recruitment and activation. We hypothesized that downregulation of miR-605 in poor responders to ketamine could increase CXCL5 expression and thereby contribute to inflammation in these patients. We confirmed that miR-605 regulates CXCL5 by using a miRNA mimic and inhibitor in human primary endothelial cells. Inhibition of miR-605 increased CXCL5 secretion and migration of human monocytic cells, thereby demonstrating a functional impact of miR-605 on chemotaxis. Additionally, CXCL5 mRNA was upregulated in whole blood from poor responders to ketamine, and CXCL5 protein was increased in plasma from CRPS patients. Thus, our studies suggest that miR-605 regulation of CXCL5 can regulate inflammation.

Asunto(s)

Quimiocina CXCL5/inmunología , Síndromes de Dolor Regional Complejo/inmunología , MicroARNs/inmunología , Analgésicos/uso terapéutico , Movimiento Celular , Quimiocina CXCL5/sangre , Quimiocina CXCL5/genética , Síndromes de Dolor Regional Complejo/sangre , Síndromes de Dolor Regional Complejo/tratamiento farmacológico , Síndromes de Dolor Regional Complejo/genética , Regulación hacia Abajo , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Ketamina/uso terapéutico , MicroARNs/metabolismo , Monocitos/inmunología , Monocitos/fisiología , Células THP-1 , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Biological sex influences inflammatory response, as there is a greater incidence of acute inflammation in men and chronic inflammation in women. Here, we report that acute inflammation is attenuated by X-inactive specific transcript (Xist), a female cell-specific nuclear long noncoding RNA crucial for X-chromosome inactivation. Lipopolysaccharide-mediated acute inflammation increased Xist levels in the cytoplasm of female mouse J774A.1 macrophages and human AML193 monocytes. In both cell types, cytoplasmic Xist colocalizes with the p65 subunit of NF-κB. This interaction was associated with reduced NF-κB nuclear migration, suggesting a novel mechanism to suppress acute inflammation. Further supporting this hypothesis, expression of 5' XIST in male cells significantly reduced IL-6 and NF-κB activity. Adoptive transfer of male splenocytes expressing Xist reduced acute paw swelling in male mice indicating that Xist can have a protective anti-inflammatory effect. These findings show that XIST has functions beyond X chromosome inactivation and suggest that XIST can contribute to sex-specific differences underlying inflammatory response by attenuating acute inflammation in women.

Asunto(s)

Inflamación/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Células Cultivadas , Citoplasma/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Inflamación/patología , Inflamación/prevención & control , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Monocitos/metabolismo , FN-kappa B/metabolismo , Interferencia de ARN , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismo , Factores Sexuales , Factor de Transcripción ReIA/metabolismo

RESUMEN

Extracellular RNA in circulation mediates intercellular communication in normal and pathological processes. One mode of circulating miRNA transport in bodily fluids is within 30-150 nm small extracellular vesicles (sEVs) or exosomes. Uptake of sEVs can regulate gene expression in recipient cells enabling circulating miRNAs to exert paracrine and systemic effects. Complex regional pain syndrome (CRPS) is a debilitating pain disorder characterized by chronic inflammation. Our previous investigations identified a significant decrease of hsa-miR-939 in whole blood from CRPS patients compared to control; we also observed that overexpression of miR-939 can negatively regulate several proinflammatory genes in vitro. Though downregulated in whole blood, miR-939 was significantly upregulated in sEVs isolated from patient serum. Here we investigated miR-939 packaging into sEVs in vitro under inflammation induced by monocyte chemoattractant protein-1 (MCP-1), a chemokine that is upregulated in CRPS patients. Stimulation of THP-1 monocytes by MCP-1 led to elevated levels of miR-939 in sEVs, which was abrogated using inhibitors of exosome secretion. miRNAs loaded into exosomes largely contain short miRNA sequence motifs called EXOmotifs. Mutation analysis of miR-939 showed that EXOmotif is one of the possible cellular mechanisms responsible for packaging miR-939 into sEVs. We confirmed gene expression changes in recipient cells following the uptake of sEVs enriched in miR-939 using RNA sequencing. Additionally, our data from primary immune cell-derived sEVs of CRPS patients and controls demonstrate that while the relative expression of miR-939 is higher in sEVs derived from B cells, T cells and NK cells relative to monocyte-derived sEVs in controls, only the B cell-derived sEVs showed a significantly higher level of miR-939 in CRPS patients. Differential miRNA sorting into exosomes and its functional impact on recipient cells may contribute to the underlying pathophysiology of CRPS.

RESUMEN

BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employed to treat immunological disorders. Exosomes, nanosized vesicles of endosomal origin, mediate intercellular communication by transferring cargo proteins and nucleic acids and regulate many pathophysiological processes. Exosomal miRNAs are potential biomarkers due to their stability and dysregulation in diseases including complex regional pain syndrome (CRPS), a chronic pain disorder with persistent inflammation. A previous study showed that a subset of CRPS patients responded to PE. METHODS: As a proof-of-concept, we investigated the PE-induced exosomal miRNA changes in six CRPS patients. Plasma cytokine levels were measured by HPLC and correlated with miRNA expression. Luciferase assay following co-transfection of HEK293 cells with target 3'UTR constructs and miRNA mimics was used to evaluate miRNA mediated gene regulation of target mRNA. Transient transfection of THP-1 cells with miRNA mimics followed by estimation of target gene and protein expression was used to validate the findings. RESULTS: Comparison of miRNAs in exosomes from the serum of three responders and three poor-responders showed that 17 miRNAs differed significantly before and after therapy. Of these, poor responders had lower exosomal hsa-miR-338-5p. We show that miR-338-5p can bind to the interleukin 6 (IL-6) 3' untranslated region and can regulate IL-6 mRNA and protein levels in vitro. PE resulted in a significant reduction of IL-6 in CRPS patients. CONCLUSIONS: We propose that lower pretreatment levels of miR-338-5p in poor responders are linked to IL-6 levels and inflammation in CRPS. Our data suggests the feasibility of exploring exosomal miRNAs as a strategy in patient stratification for maximizing therapeutic outcome of PE.

Asunto(s)

Síndromes de Dolor Regional Complejo/sangre , Síndromes de Dolor Regional Complejo/genética , Exosomas/genética , MicroARNs/genética , Intercambio Plasmático , Regiones no Traducidas 3'/genética , Adulto , Secuencia de Bases , Exosomas/ultraestructura , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Interleucina-6/sangre , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células THP-1 , Factor de Necrosis Tumoral alfa/sangre

RESUMEN

Pharmacogenomic approaches used to investigate how genes affect drug responses are critical for designing personalized therapies aimed at maximizing efficacy and minimizing adverse effects. Drug efficacy is often dependent on the sequence and expression levels of drug target genes or those involved in the metabolism and transport of the therapeutic agent. Expression of these genes, in turn, is negatively regulated by small noncoding miRNAs. The levels of miRNAs in bodily fluids have been studied extensively as potential diagnostic and prognostic biomarkers. Studies have shown that miRNAs regulate multiple genes and sequence homology is used to predict which genes are subject to regulation by a particular miRNA. Once a gene is identified as a potential target for an miRNA of interest, experiments are undertaken to confirm that the miRNA interacts with the target gene and can alter its level of expression and/or its activity. For example, the differential expression of miRNAs in whole blood obtained from good and poor responders to ketamine has been reported both prior to, and following treatment for complex regional pain syndrome. In this case, hsa-miR-548d-5p was significantly lower in poor responders relative to good responders. This miRNA was predicted to target UDP-glucuronyl transferase 1A1 (UGT1A1), a key drug metabolizing enzyme. Described in this unit are protocols used to confirm miR-548d-5p-mediated UGT1A1 regulation. The approaches described can be employed broadly for the validation of miRNA-mediated negative regulation of any gene. Determining miRNA-mediated regulation of enzymes and transporters affecting drug metabolism is a critical step in designing personalized therapy and for understanding the mechanisms responsible for variations in the responses to therapeutic agents. © 2017 by John Wiley & Sons, Inc.

Asunto(s)

Glucuronosiltransferasa/genética , MicroARNs/genética , Regulación de la Expresión Génica , Glucuronosiltransferasa/metabolismo , Células HEK293 , Células Hep G2 , Humanos , MicroARNs/metabolismo , Preparaciones Farmacéuticas/metabolismo , ARN Mensajero/genética

RESUMEN

MicroRNA(miRNA)-mediated gene regulation underlies cellular processes, playing an important role in homeostasis and diseases. The expression and function of miRNAs are altered by various pharmacological agents, with differences in the endogenous levels of miRNAs influencing drug efficacy and toxicity. Thus, miRNA levels could be a biomarker for predicting treatment response, efficacy, and safety. In addition, elucidating the mechanistic significance of miRNA alterations can aid in the identification of therapeutic targets and patient selection, and guide personalized therapy. Discussed in this overview are the properties of miRNA, their modulation, and the ways to measure them. The effects of different classes of analgesics, including opioid and non-opioid, are described as examples of drug-induced modifications of miRNA, with a discussion on how measurement of miRNA levels in patients receiving analgesic therapy can assist in maximizing effectiveness while minimizing the untoward responses to this drug class. © 2017 by John Wiley & Sons, Inc.

Asunto(s)

Analgésicos/farmacología , MicroARNs/metabolismo , Dolor/metabolismo , Analgésicos/uso terapéutico , Animales , Humanos , Dolor/tratamiento farmacológico , Dolor/genética

RESUMEN

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.

Asunto(s)

MicroARN Circulante/metabolismo , Síndromes de Dolor Regional Complejo/patología , Regulación de la Expresión Génica , Factores Inmunológicos/biosíntesis , Inflamación/patología , MicroARNs/metabolismo , Células Cultivadas , Perfilación de la Expresión Génica , Humanos

RESUMEN

Biomarkers are measurable characteristics reflective of the physiological or diseased state and a crucial feature in rendering personalized medicine more precise. Dysregulated expression of circulating microRNAs (miRNAs) in bodily fluids is being explored as noninvasive clinical biomarker for a variety of disorders including chronic pain. High-precision qPCR-based signal amplification of these miRNAs enables the detection of small changes making them ideal biomarker candidates. Presence of circulating miRNAs in exosomes, small vesicles that mediate intercellular communication, opens up novel avenues for target intervention and biomarker discovery. miRNA signatures specific to different pain conditions, and their reversal on treatment in patients and animal models can be beneficial in patient stratification, prognosis, and in bridging preclinical and clinical results. Identification of multiple miRNAs as opposed to reliance on one specific molecule as a biomarker could improve treatment efficacies in an extremely heterogeneous pain patient population. Additionally, owing to the stability of miRNAs, retrospective studies could be performed using banked samples from completed clinical trials. Irrespective of the phase and outcome, these studies can provide insights on molecular underpinnings influencing treatment outcome, or specific therapeutic intervention. Identification of miRNAs altered in chronic pain states will have a significant impact on the identification of right leads, targets, doses, and patients. Effective implementation of miRNA-based biomarkers would provide treatment guidance for clinicians, better clinical trial designs for pharmaceutical companies, all leading to individualized care and better treatment outcome for chronic pain patients.

Asunto(s)

MicroARNs , Dolor/genética , Animales , Biomarcadores , Humanos , MicroARNs/biosíntesis , Dolor/tratamiento farmacológico