miRNA-23a/CXCR4 regulates neuropathic pain via directly targeting TXNIP/NLRP3 inflammasome axis

Pan et al. Journal of Neuroinflammation (2018) 15:29 DOI 10.1186/s12974-018-1073-0 RESEARCH Open Access miRNA-23a/CXCR4 regulates neuropathic pain via directly targeting TXNIP/NLRP3 inflammasome axis Zhiqiang Pan1,2,5*†, Qun Shan3,2†, Pan Gu2†, Xiao Min Wang2, Lydia Wai Tai2, Menglan Sun1, Xin Luo2, Liting Sun2 and Chi Wai Cheung2,4,5* Abstract Background: Chemokine CXC receptor 4 (CXCR4) in spinal glial cells has been implicated in neuropathic pain. However, the regulatory cascades of CXCR4 in neuropathic pain remain elusive. Here, we investigated the functional regulatory role of miRNAs in the pain process and its interplay with CXCR4 and its downstream signaling. Methods: miRNAs and CXCR4 and its downstream signaling molecules were measured in the spinal cords of mice with sciatic nerve injury via partial sciatic nerve ligation (pSNL). Immunoblotting, immunofluorescence, immunoprecipitation, and mammal two-hybrid and behavioral tests were used to explore the downstream CXCR4dependent signaling pathway. Results: CXCR4 expression increased in spinal glial cells of mice with pSNL-induced neuropathic pain. Blocking CXCR4 alleviated the pain behavior; contrarily, overexpressing CXCR4 induced pain hypersensitivity. MicroRNA-23a3p (miR-23a) directly bounds to 3′ UTR of CXCR4 mRNA. pSNL-induced neuropathic pain significantly reduced mRNA expression of miR-23a. Overexpression of miR-23a by intrathecal injection of miR-23a mimics or lentivirus reduced spinal CXCR4 and prevented pSNL-induced neuropathic pain. In contrast, knockdown of miR-23a by intrathecal injection of miR-23a inhibitor or lentivirus induced pain-like behavior, which was reduced by CXCR4 inhibition. Additionally, miR-23a knockdown or CXCR4 overexpression in naïve mice could increase the thioredoxininteracting protein (TXNIP), which was associated with induction of NOD-like receptor protein 3 (NLRP3) inflammasome. Indeed, CXCR4 and TXNIP were co-expressed. The mammal two-hybrid assay revealed the direct interaction between CXCR4 and TXNIP, which was increased in the spinal cord of pSNL mice. In particular, inhibition of TXNIP reversed pain behavior elicited by pSNL, miR-23a knockdown, or CXCR4 overexpression. Moreover, miR-23a overexpression or CXCR4 knockdown inhibited the increase of TXNIP and NLRP3 inflammasome in pSNL mice. Conclusions: miR-23a, by directly targeting CXCR4, regulates neuropathic pain via TXNIP/NLRP3 inflammasome axis in spinal glial cells. Epigenetic interventions against miR-23a, CXCR4, or TXNIP may potentially serve as novel therapeutic avenues in treating peripheral nerve injury-induced nociceptive hypersensitivity. Keywords: miRNA-23a, CXCR4, TXNIP, NLRP3 inflammasome, Sciatic nerve injury, Spinal glia cell * Correspondence: ; † Equal contributors 1 Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou 221002, China 2 Laboratory and Clinical Research Institute for Pain, Department of Anesthesiology, The University of Hong Kong, Hong Kong SAR, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pan et al. Journal of Neuroinflammation (2018) 15:29 Background Chemokine CXC receptor 4 (CXCR4) belongs to the family of G protein-coupled receptors. CXCR4 has been confirmed to have glia-modulatory and neuromodulatory properties in the central nervous system (CNS) [1]. Mounting evidence has shown that CXCR4 is involved in the process of different nociceptive responses such as neuropathic pain or cancer pain in glial cells of the dorsal root ganglion (DRG) or in the spinal cord [2–4]. Recently, it has been found that both chemokine C-X-C motif ligand 12 (CXCL12) and its receptor CXCR4 were upregulated in spinal glial cells of mice with partial sciatic nerve ligation (pSNL)-induced neuropathic pain or chronic post-ischemia-induced inflammatory pain [5]. Inhibition of CXCR4 attenuated pain induced by CXCL12, suggesting that the crosstalk between astrocytic CXCL12 and microglial CXCR4 contributes to the development of neuropathic pain [5]. However, the functional regulatory mechanisms of spinal CXCR4 in neuropathic pain remain unclear. Recent reports have strongly linked miRNA to nociceptive processing. MicroRNA-23a-3p (miR-23a) is highly conserved across species, and it modulates various disease processes, such as cancer [6], inflammation [7], Harada Miuji syndrome [8], and cognitive impairment [9]. Moreover, miR-23a was found not only decreased in the blood of patients with multiple sclerosis [10] or acute ischemic stroke [11], but also rapidly downregulated in the injured cortex following traumatic brain injury [12], suggesting a potential modulatory function of miR-23a in CNS diseases. Here, miR-23a was predicted to bind to CXCR4 mRNA; however, it is still unknown whether miR-23a regulated neuropathic pain via directly targeting CXCR4. Thioredoxin-interacting protein (TXNIP) is ubiquitously expressed in a variety of cells and acts as an endogenous suppressor of reactive oxygen species scavenging protein thioredoxin, as well as a crucial molecular nutrient sensor to oxidative stress and inflammation in the regulation of energy metabolism [13, 14]. TXNIP is associated with stroke, depression, Alzheimer’s disease, and spinal or brain injury [15–18]. Knockdown of hippocampal TXNIP significantly improves brain injury [15], cognitive impairment, and neuroinflammation [16], suggesting that TXNIP is a potential target for the treatment of these CNS disorders. In particular, overexpression of TXNIP attenuates CXCL12-induced bladder carcinogenesis, while knockout of TXNIP enhances CXCR4 expression in bladder carcinogenesis in urothelial cells [19], indicating an interaction may exist between TXNIP and CXCR4. Therefore, the present study aimed to determine the functional and regulatory role of miR-23a in pain processing in the CNS and its interplay with CXCR4 and TXNIP at spinal level, which Page 2 of 19 may provide potential therapeutic targets for peripheral injury-induced neuropathic pain. Methods Animals Pathogen-free adult male C57BL/6J wild-type mice (25– 30 g, n = 3 or 5 per group for each experiment) were housed at 23 ± 3 °C, with humidity ranges between 25 and 45% under a 12-h light/12-h dark cycle (lights on at 07:00). Mice were allowed for free access to water and standard lab diet (1.0% calcium, 0.5% phosphorus, and 3.3 IU/g of (...truncated)