SYNAPSE 70:317–324 (2016)
Changes in the Expression of IL-6Mediated MicroRNAs in the Dorsal Root Ganglion Under Neuropathic Pain in Mice NAOSUKE HORI,1,2 MICHIKO NARITA,1 AKIRA YAMASHITA,1 HIROSHI HORIUCHI,1 YUSUKE HAMADA,1 TAKASHIGE KONDO,1 MOE WATANABE,1 KATSUHIDE IGARASHI,2 MIHO KAWATA,1 MASAHIRO SHIBASAKI,1 MITSUAKI YAMAZAKI,3 NAOKO KUZUMAKI,1 EIICHI INADA,2 TAKAHIRO OCHIYA,1,4 MASAKO ISEKI,2 TOMOHISA MORI,1 AND MINORU NARITA1,2,3,5* 1 Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Shinagawa-ku, Tokyo 142-8501, Japan 2 Department of Anesthesiology and Pain Medicine, Juntendo University School of Medicine, Tokyo 113-8421, Japan 3 Department of Anesthesiology, Graduate School of Medical and Pharmaceutical Sciences for Education, Toyama University, Sugitani 930-0194, Japan 4 Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan 5 Life Science Tokyo Advanced Research Center (L-StaR), Hoshi University School of Pharmacy and Pharmaceutical Sciences, Shinagawa-ku, Tokyo 142-8501, Japan
KEY WORDS
microRNA; neuropathic pain; exosome; DRG
ABSTRACT A multiplex analysis for profiling the expression of candidate microRNAs (miRNAs), which are small noncoding RNAs that function as key posttranscriptional regulators, may lead to a better understanding of the complex machinery of neuropathic pain. In the present study, we performed a miRNA array analysis using tissues of the dorsal root ganglion (DRG), a primary site for pain processing, obtained from mice with partial sciatic nerve ligation. Among 1135 total miRNAs, 26 miRNAs showed up-regulation (more than 2-fold change) and only 4 miRNAs showed downregulation (less than 0.5-fold change) in the DRG of nerve-ligated mice. In a RT-qPCR assay, the levels of miR-21, miR-431, and miR-511-3p were significantly increased on the ipsilateral side of the DRG from 3 to 7 days after sciatic nerve ligation. These elevations were almost absent in IL-6 knockout mice. Furthermore, the expression level of miR-21, but not those of miR-431 or miR511-3p, was significantly increased in exosomes extracted from blood of nerve-ligated mice. These findings suggest that the increased expression of IL-6-regulated miR-21, miR-431, and miR-511-3p in the DRG and increased exosomal miR-21 extracted from blood after sciatic nerve ligation may play at least a partial role in neuropathic pain. Synapse 70:317–324, 2016. VC 2016 Wiley Periodicals, Inc. INTRODUCTION Neuropathic pain is defined as chronic pain that is caused by lesions of the peripheral or central nervous system. Chronic pain stimuli are now considered to promote a large number of persistent adaptations at the cellular and molecular levels and elicit epigenetically abnormal transcription/translation and posttranslational modification in related cells depending on the degree or kind of injury or associated conditions (Descalzi et al., 2015). The elucidation of such epigenetic modification may lead to a better understanding of chronic or neuropathic pain. Major epigenetic modifications include DNA methylation and histone modification, and these changes have longlasting effects on gene functions. Epigenetic abnormality is an important mechanism for cancers, and abnormal DNA methylation and histone modification Ó 2016 WILEY PERIODICALS, INC.
have been detected in various cancers. However, such epigenetic abnormalities are also present in noncancer tissues. For example, studies have shown that epigenetic modifications may occur under stress(Papadopoulos et al., 2011), in the metabolic syndrome (Barres et al., 2009), and in association with a mental disorder (Murgatroyd et al., 2009; Roth et al., 2009). We recently demonstrated that epigenetic transcriptional activation of monocyte chemotactic protein-3 Contract grant sponsor: MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2014–2018; Contract grant number: S1411019. *Correspondence to: Minoru Narita, Ph.D. Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. E-mail:
[email protected] Received 27 January 2016; Revised 2 March 2016; Accepted 7 March 2016 DOI: 10.1002/syn.21902 Published online 7 April 2016 in Wiley Online Library (wileyonlinelibrary. com).
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(MCP-3) in the spinal cord contributes to long-lasting neuropathic pain (Imai et al., 2013). MicroRNA (miRNA) is small noncoding RNA that represses transcription and translation and serve as the key modulators of epigenetics (Chuang and Jones, 2007). In 2009, Aldrich et al. showed for the first time the down-regulation of miR-96, miR-182, and miR-183 in the dorsal root ganglion (DRG) of rats with neuropathic pain (Aldrich et al., 2009). Since then, a significant decrease in miR-103 expression was found in the ipsilateral dorsal horn of neuropathic pain models (Favereaux et al., 2011), whereas a significant increase in miR-21 expression in the DRG was observed after various types of peripheral nerve injury (Sakai and Suzuki, 2013; Strickland et al., 2011; Wu et al., 2011; Yu et al., 2011). Based on these findings, it is highly likely that miRNA plays some key roles in the formation of pain and intractable pain. Exosomes are 40–100 nm nano-sized vesicles that can be released from many cell types into various body fluids (Simons and Raposo, 2009). Recently, mRNAs and miRNAs have been identified in exosomes (Valadi et al., 2007; Zhang et al., 2015). A growing body of evidence suggests that exosomal miRNAs may play an important role in disease progression (Candelario and Steindler, 2014). In this study, to further understand the relationship between neuropathic pain and epigenetic modulation related to miRNAs, we performed a miRNA array analysis in the DRG of mice with sciatic nerve ligation and investigated the possible changes in DRG-associated miRNAs in exosomes derived from blood serum in mice. MATERIALS AND METHODS Animals In this study, we used male C57BL/6J mice (aged 6–8 weeks, males; Tokyo Laboratory Animals Science) and male IL-6 knockout mice (Jackson Laboratory). All animals were housed with ad libitum access to food and water and maintained on a 12 h light/dark cycle. This study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, Hoshi University, as adopted by the Committee on Animal Research of Hoshi University, which is accredited by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Neuropathic pain model mice We produced a partial sciatic nerve ligation model by tying a tight ligature with an 8–0 silk suture around approximately one-third to one-half the diameter the sciatic nerve on the right side (ipsilateral side) of mice under a light microscope (SD30, Olympus, Tokyo, Japan) and under anesthesia with 3% isoflurane. In sham-operated mice, the nerve was exposed without ligation. To assess the sensitivity to Synapse
thermal stimulation, each of the hind paws of mice was tested individually using a thermal stimulus apparatus (UGO-BASILE, Biological Research Apparatus, VA, Italy). Briefly, the intensity of the thermal stimulus was adjusted to achieve an average baseline paw-withdrawal latency of 8–10 s in naive mice. Only quick hind paw movements (with or without licking of the hind paws) away from the stimulus were considered to be a withdrawal response. Paw movements associated with locomotion or weightshifting were not counted as a response. The paws were measured alternating between the left and right with an interval of >3 min between measurements. The latency of paw withdrawal after the thermal stimulus was determined as the average of three measurements per paw. Von Frey filament test Mechanical allodynia was measured by the von Frey filament test. The rats were placed in individual stainless steel chambers (25 3 20 3 22 cm3) with a wire mesh floor, and allowed to explore the chamber. Seven von Frey filaments of from 1.4 to 15 g (TouchTest Sensory Evaluators, Remington Medical Equipment, ON, Canada) were applied to the mid-plantar surface of both hind paws (3 trials for each paw, 6 trials for both paws per filament). Stimulation with the filaments was performed for 3 s every 5 s. Withdrawal of the hind paw was considered to be a positive response, and neuropathy was evaluated by the average of the three values for both hind paws. Each von Frey filament test was performed before the injection of oxaliplatin or paclitaxel under single blind fashion. TaqMan miRNA arrays Total RNA including miRNAs was extracted from L3-L5 DRGs, pooled from 6 mice at 7 days after sciatic nerve ligation, using the mirVana miRNA Isolation Kit (Applied Biosystems). For miRNA cDNA synthesis, RNA was reverse-transcribed using the TaqMan miRNA Reverse Transcription (RT) kit (Applied Biosystems Inc.) in combination with the stemloop Megaplex primer pool (Applied Biosystems). Cycling conditions were as follows: 40 cycles of 168C for 2 min, 428C for 1 min, and 508C for 1 s, followed by 858C for 5 min. miRNA expression profiles were acquired using a TaqMan rodent miRNA array v2.0 A (Applied Biosystems). Quantitative RT-PCR was performed at Life Technologies Japan. Using an Applied Biosystems 7900HT Fast Real-Time PCR system. Cycling threshold (Ct) values were calculated using SDS2.2.2 with an automatic baseline and a threshold of 0.2. Normalized expression (NE) was calculated as NE 2Ct, where Ct is the threshold cycle for detecting fluorescence. The data were normalized to snoRNA202.
CHANGES IN MICRORNAS UNDER NEUROPATHIC PAIN
Extraction of exosomes from blood serum Blood was collected from abdominal aorta of mice by 26 gauge needle (BD Vacutainer, 0.7 3 25 mm2) and transferred to a test tube (Terumo Venoject II) under anesthesia with 3% isoflurane at 7 days after sciatic nerve ligation. The test tubes were left to stand for >30 min after blood collection. To separate serum fraction, the blood sample was centrifuged at 2000 rcf for 15 min and upper layer was gently collected. For exosome preparation, PBS was added to the serum fraction. Then, they were ultracentrifuged at 100,000 rcf for 70 min twice at 48C. The pellets were resuspended in PBS for elution exosomes. Extracted exosomes were identified using NanoSight analysis according to previous article (Harp et al., 2016). Briefly, Nanoparticle tracking analysis (NTA) measurements were performed by using a NanoSight NS500 instrument (NanoSight NTA 2.3 Nanoparticle Tracking and Analysis Release Version Build 0025). The size distribution and quantification of exosome preparations were analyzed by measuring the rate of Brownian motion with a NanoSight LM10 system (NanoSight, Wiltshire, United Kingdom) equipped with fast video capture and particle-tracking software. Quantification of miRNAs with a real-time PCR Mice were sacrificed by decapitation at 7 days after sciatic nerve ligation. Individual TaqMan assays (Applied Biosystems) were used to analyze the expression of the mature mouse miRNAs. Total RNA (10 ng) extracted from the DRG or blood exosomes of mice was used in the RT reaction and the transcribed cDNA was then used for subsequent PCR amplification using TaqMan 23 Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems). Real-time PCR was conducted at 958C for 10 min, followed by 45 cycles of 958C for 15 s and 608C for 1 min. The threshold cycle (Ct) method was used to determine the relative quantity of each miRNA. Statistical data analysis Data are expressed as the mean with SEM. Twoway ANOVAs with independent and repeated measures, as well as planed comparisons or Student’s t tests, were used as appropriate for the experimental design. A value of P < 0.05 was considered statistically significant. All statistical analyses were performed with Prism version 5.0a (GraphPad Software). RESULTS miRNA array using the DRG of mice with nerve injury First, we confirmed that partial sciatic nerve ligation produced a significant thermal hyperalgesia and tactile allodynia from 4 to 7 days after surgery in mice (data not shown). To evaluate the changes in the
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expression of miRNAs on the ipsilateral side of the DRG of sham-operated or sciatic nerve-ligated mice, miRNA array analysis was performed at 7 days after sciatic nerve ligation. The expression levels of the 1135 miRNAs spotted on microarrays from sham-operated and nerve-ligated mice were analyzed with correlation plots (r 5 0.994). This analysis revealed 26 miRNAs that were up-regulated (>2-fold change) after nerve ligation and only 4 miRNAs that were down-regulated ( 2). C: List of down-regulated miRNAs (Ligation/Sham ratio
