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MiR-146a ameliorates liver ischemia/reperfusion injury by suppressing IRAK1 and TRAF6.

ABSTRACT: A critical role of the Toll-like receptor(TLR) and its downstream molecules, including IL-1 receptor-associated kinase 1(IRAK1) and tumor necrosis factor receptor- associated factor 6(TRAF6), in the pathogenesis of liver ischemia/reperfusion (I/R) injury has been documented. Recently a microRNA, miR-146a, was identified as a potent negative regulator of the TLR signaling pathway. In this study, we investigated the role of miR-146a to attenuate TLR signaling and liver I/R injury in vivo and in vitro. miR-146a was decreased in mice Kupffer cells following hepatic I/R, whereas IRAK1 and TRAF6 increased. Overexpression of miR-146a directly decreased IRAK1 and TRAF6 expression and attenuated the release of proinflammatory cytokines through the inactivation of NF-?B P65 in hypoxia/reoxygenation (H/R)-induced macrophages, RAW264.7 cells. Knockdown experiments demonstrated that IRAK1 and TRAF6 are two potential targets for reducing the release of proinflammatory cytokines. Moreover, co-culture assays indicated that miR-146a decreases the apoptosis of hepatocytes after H/R. In vivo administration of Ago-miR-146a, a stable version of miR-146a in vivo, protected against liver injury in mice after I/R via inactivation of the TLR signaling pathway. We conclude that miR-146a ameliorates liver ischemia/reperfusion injury in vivo and hypoxia/reoxygenation injury in vitro by directly suppressing IRAK1 and TRAF6.

SUBMITTER: Jiang W

PROVIDER: S-EPMC4079695 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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miR-146a ameliorates liver ischemia/reperfusion injury by suppressing IRAK1 and TRAF6.

Jiang Weiwei W Kong Liangliang L Ni Qingfeng Q Lu Yeting Y Ding Wenzhou W Liu Guoqing G Pu Liyong L Tang Weibing W Kong Lianbao L

PloS one 20140702 7

A critical role of the Toll-like receptor(TLR) and its downstream molecules, including IL-1 receptor-associated kinase 1(IRAK1) and tumor necrosis factor receptor- associated factor 6(TRAF6), in the pathogenesis of liver ischemia/reperfusion (I/R) injury has been documented. Recently a microRNA, miR-146a, was identified as a potent negative regulator of the TLR signaling pathway. In this study, we investigated the role of miR-146a to attenuate TLR signaling and liver I/R injury in vivo and in vi ...[more]

PMID: 24987958

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Project description:Rationale: Ischemia/reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) that is associated with high morbidity and mortality, and for which specific treatments are lacking. In this study, we investigated the protective effect of human urine-derived stem cells (USCs) and their exosomes against IRI-induced AKI to explore the potential of these cells as a new therapeutic strategy. Methods: USCs were derived from fresh human urine. Cell surface marker expression was analyzed by flow cytometry to determine the characteristics of the stem cells. Adult male Sprague-Dawley rats were used to generate a lethal renal IRI model. One dose of USCs (2×106 cells/ml) or exosomes (20 µg/1 ml) in the experimental groups or saline (1 ml) in the control group was administered intravenously immediately after blood reperfusion. Blood was drawn every other day for measurement of serum creatinine (sCr) and blood urea nitrogen (BUN) levels. The kidneys were harvested for RNA and protein extraction to examine the levels of apoptosis and tubule injury. In vitro, the hypoxia-reoxygenation (H/R) model in human kidney cortex/proximal tubule cells (HK2) was used to analyze the protective ability of USC-derived exosomes (USC-Exo). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), western blotting, superoxide dismutase activity, and malonaldehyde content analyses were used to evaluate oxidative stress in HK2 cells treated with USC-Exo after H/R. Exosomal microRNA sequencing techniques and bioinformatics analysis were used to search for enriched miRNAs in the exosomes and interacting genes. The interaction between miRNAs and the 3' untranslated region of the target gene was detected using a dual luciferase reporting system. The miRNA mimic and inhibitor were used to regulate the miRNA level in HK2 cells. Results: Treatment with USCs led to reductions in the levels of sCr, BUN, and renal tubular cell apoptosis; inhibited the infiltration of inflammatory cells; and protected renal function in the rat IRI model. Additionally, USC-derived exosomes protected against IRI-induced renal damage. miR-146a-5p was the most abundant miRNA in exosomes obtained from the conditioned medium (CM) of USCs. miR-146a-5p targeted and degraded the 3'UTR of interleukin-1 receptor-associated kinase 1 (IRAK1) mRNA, subsequently inhibited the activation of nuclear factor (NF)-κB signaling, and protected HK2 cells from H/R injury. USC transplantation also upregulated miR-146a-5p expression, downregulated IRAK1 expression and inhibited nuclear translocation of NF-κB p65 in the kidney of the rat IRI model. Conclusions: According to our experimental results, USCs could protect against renal IRI via exosomal miR-146a-5p, which could target the 3'UTR of IRAK1 and subsequently inhibit the activation of NF-κB signaling and infiltration of inflammatory cells to protect renal function. As a novel cell source, USCs represent a promising non-invasive approach for the treatment of IRI.

Project description:BACKGROUND:microRNA-146a-5p (miRNA-146a-5p) is a key molecule in the negative regulation pathway of TLRs and IL-1 receptor (TIR) signaling. Our recent study demonstrated that MyD88-dependent signaling pathway of TIR in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) plays a role in peripheral nerve injury-induced neuropathic pain. However, it was not clear whether and how miRNA-146a-5p regulates the TIR pathway of DRG and SDH in the development of neuropathic pain. METHODS:The sciatic nerve chronic constriction injury (CCI) model of rat was used to induce chronic neuropathic pain. The levels and cellular distribution of miRNA-146a-5p were detected with quantitative real-time PCR (qPCR) and fluorescent in situ hybridization (FISH). The RNA level, protein level, and cellular distribution of IRAK1 and TRAF6 that is targeted by miRNA-146a-5p were detected with qPCR, western blot, and immunofluorescent. The pain-related behavioral effect of miRNA-146a-5p was accessed after intrathecal administration. Mechanical stimuli and radiant heat were used to evaluate mechanical allodynia and thermal hyperalgesia. RESULTS:We found that the level of miRNA-146a-5p significantly increased in L4-L6 DRGs and SDH after CCI surgery; meanwhile, the protein level of IRAK1 and TRAF6 in DRGs was significantly increased after CCI. Intrathecal injection of miR146a-5p agomir or miRNA-146a-5p antagomir regulates miRNA-146a-5p level of L4-L6 DRGs and SDH. We found that intrathecal injection of miR146a-5p agomir can alleviate mechanical and thermal hyperalgesia in CCI rats and reverse the upregulation of IRAK1 and TRAF6 of L4-L6 DRGs and SDH induced by CCI. We furthermore found that intrathecal injection of miRNA-146a-5p antagomir can exacerbate the mechanical and thermal pain-related behavior of CCI rats and meanwhile increase IRAK1 and TRAF6 of L4-L6 DRGs and SDH expression even further. CONCLUSIONS:miRNA-146a-5p of DRG and SDH can modulate the development of CCI-induced neuropathic pain through inhibition of IRAK1 and TRAF6 in the TIR signaling pathway. Hence, miRNA-146a-5p may serve as a potential therapeutic target for neuropathic pain.

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MiR-146a ameliorates liver ischemia/reperfusion injury by suppressing IRAK1 and TRAF6.

Publications

miR-146a ameliorates liver ischemia/reperfusion injury by suppressing IRAK1 and TRAF6.

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