Biomolecules & Therapeutics 2019; 27(4): 414-422  https://doi.org/10.4062/biomolther.2018.073
Inhibition of MicroRNA-15a/16 Expression Alleviates Neuropathic Pain Development through Upregulation of G Protein-Coupled Receptor Kinase 2
Tao Li1, Yingchun Wan2, Lijuan Sun2, Shoujun Tao3, Peng Chen1, Caihua Liu4, Ke Wang5, Changyu Zhou6, and Guoqing Zhao1,*
1Department of Anesthesiology, China-Japan Union Hospital, Jilin University, Jilin 130033, China, 2Department of Endocrinology, China-Japan Union Hospital, Jilin University, Jilin 130033, China, 3Department of Anesthesiology, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Zhejiang 310006, China, 4Department of Anaesthesiology, The Central Hospital of Wuhan Affiliated with Tongji Medical College of Huazhong University of Science and Technology, Hubei 430014, China, 5Department of Gynaecology and Obstetrics, China-Japan Union Hospital, Jilin University, Jilin 130033, China, 6Department of Gastroenterology, China-Japan Union Hospital, Jilin University, Jilin 130033, China
*E-mail: zhaoguoqing_cjuh@163.com, Tel: +86-0431-84995999, Fax: +86-0431-84995999
Received: April 30, 2018; Revised: July 24, 2018; Accepted: August 14, 2018; Published online: June 13, 2019.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

There is accumulating evidence that microRNAs are emerging as pivotal regulators in the development and progression of neuropathic pain. MicroRNA-15a/16 (miR-15a/16) have been reported to play an important role in various diseases and inflammation response processes. However, whether miR-15a/16 participates in the regulation of neuroinflammation and neuropathic pain development remains unknown. In this study, we established a mouse model of neuropathic pain by chronic constriction injury (CCI) of the sciatic nerves. Our results showed that both miR-15a and miR-16 expression was significantly upregulated in the spinal cord of CCI rats. Downregulation of the expression of miR-15a and miR-16 by intrathecal injection of a specific inhibitor significantly attenuated the mechanical allodynia and thermal hyperalgesia of CCI rats. Furthermore, inhibition of miR-15a and miR-16 downregulated the expression of interleukin-1β and tumor-necrosis factor-α in the spinal cord of CCI rats. Bioinformatic analysis predicted that G protein-coupled receptor kinase 2 (GRK2), an important regulator in neuropathic pain and inflammation, was a potential target gene of miR-15a and miR-16. Inhibition of miR-15a and miR-16 markedly increased the expression of GRK2 while downregulating the activation of p38 mitogen-activated protein kinase and NF-κB in CCI rats. Notably, the silencing of GRK2 significantly reversed the inhibitory effects of miR-15a/16 inhibition in neuropathic pain. In conclusion, our results suggest that inhibition of miR-15a/16 expression alleviates neuropathic pain development by targeting GRK2. These findings provide novel insights into the molecular pathogenesis of neuropathic pain and suggest potential therapeutic targets for preventing neuropathic pain development.

Keywords: GRK2, miR-15a/16, Neuropathic pain, p38 MAPK
INTRODUCTION

Neuropathic pain is a neurological disease characterized by hyperalgesia and allodynia of the somatosensory system (Haanpaa et al., 2011). Neuropathic pain has emerged as a global problem affecting approximately 6.9–10% of the population worldwide, with enormous socioeconomic costs (Denk and McMahon, 2012; van Hecke et al., 2014). Despite advances in drug development for neuropathic pain, treatment efficacy is still limited (O’Connor and Dworkin, 2009). Neuropathic pain is a complicated disease and the molecular mechanisms underlying its pathogenesis remain poorly understood. Therefore, a better understanding of the molecular pathogenesis of neuropathic pain, and the search for novel and effective targets for treatment, are essential.

MicroRNAs (miRNAs), non-coding RNAs, are emerging as novel and important mediators for gene expression (Ambros, 2004). Mature miRNAs consist of 19–25 nucleotides which can bind to the 3′-untranslated region (UTR) of target mRNA, with complementary base-pair sequences, leading to mRNA degradation and translational repression (Bartel, 2004). miRNAs are highly involved in the development and progression of various diseases via regulation of various biological activities and may be potential and critical targets for treatment (Krol et al., 2010). There is accumulating evidence that miRNAs play a pivotal role in the pathogenesis of neuropathic pain and are novel targets for prevention and treatment (Andersen et al., 2014; Sakai and Suzuki, 2014; Jiangpan et al., 2016). Therefore, a better understanding of the regulatory network of miRNAs involved in the pathogenesis of neuropathic pain will aid development of novel therapeutic strategies.

G protein-coupled receptor kinase 2 (GRK2), also known as beta-adrenergic receptor kinase 1, is an important in regulating the responsiveness of G protein-coupled receptors (GPCRs) (Lombardi et al., 2002). GRK2 can prevent agonist-induced overstimulation via desensitization of a series of GPCRs, while a deficiency of GRK2 prolongs the signaling in response to activation of GPCRs (Vroon et al., 2006). Moreover, GRK2 is involved in regulating various intracellular signaling pathways, such as mitogen activated protein kinases (MAPK) and PI3K/Akt (Peregrin et al., 2006; Zhang et al., 2017). Accumulating evidence has suggested that GRK2 plays an important role in various diseases associated with inflammation (Penela et al., 2008; Lucas et al., 2015; Woodall et al., 2016). Notably, GRK2 is also reported to regulate the development and progression of neuropathic pain. In a rodent model of neuropathic pain, GRK2 expression is decreased in the lumbar spinal cord (Kleibeuker et al., 2007). Additionally, neuropathic pain is prolonged in GRK2-deficient mice (Willemen et al., 2010; Wang et al., 2011). Therefore, GRK2 may serve as a promising target for treatment of neuropathic pain (Kavelaars et al., 2011).

The miR-15a/16 cluster located on chromosome 13q14 is a highly conserved miRNA group that plays an important role in various diseases, such as cancer, cardiovascular disease, and neurological disease (Calin et al., 2008; Spinetti et al., 2013; Yang et al., 2017b). However, whether miR-15a/16 is involved in regulating neuropathic pain remains unclear. In this study, we aimed to investigate the potential role of miR-15a/16 in the development and progression of neuropathic pain.

MATERIALS AND METHODS

Animals

Adult male Sprague-Dawley (SD) rats (weighing 200–250 g, aged 7–8 weeks) were purchased from Experimental Animal Center of Jilin University. The rats were housed in separate cages (20 ± 1°C; 12-h light/dark cycle) with ad libitum access to water and food. The animal procedures were performed in accordance with the guidelines of the International Association for the Study of Pain and the National Institute of Health Guide for the Care and Use of Laboratory Animals. This study was authorized and approved by the Institutional Animal Care and Use Committee of China-Japan Union Hospital.

Neuropathic pain model

Neuropathic pain in rats was induced by CCI based on procedures described previously (Bennett and Xie, 1988). The rats were allowed to acclimate to their environment for 2 days prior to the experiments. The rats were anesthetized by an intraperitoneal injection of phenobarbital sodium (40 mg/kg). The sciatic nerves on both sides were revealed by blunt dissection and isolated from surrounding tissues. The sciatic nerves were loosely ligated using a 4-0 catgut thread with about 1 mm between ligatures. A sham surgery was performed with the sciatic nerve revealed but not ligated. Rats with sham surgery were used as controls. After the surgery, the muscle and skin layers were sutured with thread and the area of surgery was sterilized with iodine.

Intrathecal injection procedure

The rats were anesthetized and a Hamilton syringe with a 30-gauge needle was inserted into the subarachnoid space of the spinal cord between the L4 and L5 lumbar. Proper location of the intrathecal implantation was systemically confirmed by injection of 2% lidocaine to induce bilateral hind limb paralysis. Intrathecal delivery of miR-15a/16 antagomir (anti-miR-15a, anti-miR-16, or a mixture of miR-15a and miR-16) was performed using a microinjection syringe linked to the intrathecal catheter. After the experiments, the L4–L5 lumbar spinal cords were dissected for biological detection.

Real-time quantitative polymerase chain reaction (RT-qPCR) analysis

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s instructions. The reverse transcription of the total RNA was conducted using M-MLV reverse transcriptase (Takara, Dalian, China) for mRNA detection and the TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) for miRNA detection. The amplification of cDNA was performed using SYBR Green PCR Master Mix (Applied Biosystems) following the thermal cycling conditions: initial denaturation (94°C, 5 min), 40 cycles at 94°C (20 s), 55°C (30 s) and 72°C (30 s), and a final cycle at 72°C (5 min). GAPDH and U6 were used as reference genes for normalization of mRNA and miRNA expression, respectively. The relative expression was calculated using the 2−ΔΔCt method.

Measurement of pain threshold

Thermal hyperalgesia, indicated by paw withdrawal latencies (PWL) in response to radiant heat stimulation, was determined using a pain threshold detector. Mechanical allodynia, indicated by paw withdrawal threshold (PWT) in response to the mechanical stimulus, was detected using Von Frey hair (IITC, Woodland Hills, CA, USA). The experimental detections were performed according to standard procedures described previously (Hargreaves et al., 1988; Chaplan et al., 1994).

Enzyme-linked immunosorbent assay (ELISA)

The protein concentrations of IL-1β and TNF-α in lumbar spinal cords were determined using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) as per the protocol recommended by the manufacturer.

Luciferase reporter assay

293T cells purchased from Stem Cell Bank of Chinese Academy of Sciences (Shanghai, China) were used to perform the luciferase reporter assay. The 293T cells were cultured in DMEM (Gibco, Rockville, MD, USA), containing 10% fetal bovine serum and a 1% penicillin/streptomycin mix, and grown in a humidified atmosphere with 95% air and 5% CO2 at 37°C. The fragments of GRK2 3′-UTRs harboring the predicted wild-type seed-matched (WT) or mutant (MT) binding sites were inserted into pmirGLO dual-luciferase reporter plasmids (Promega, Madison, WI, USA). The WT or MT constructs were cotransfected with miR-15a/16 mimics (RiboBio, Guangzhou, China) into 293T cells using Lipofectamine 2000 (Invitrogen) as per the protocols of the manufacturer. After incubation for 48 h, the luciferase reporter activity was determined using the dual-luciferases reporter system (Promega).

Western blot analysis

Tissues were homogenized in cold phosphate-buffered saline for protein extraction. Protein concentrations were measured using a BCA protein assay kit (Beyotime Biotechnology, Haimen, China). Equal amounts of protein (40 μg) from each sample were separated by electrophoresis with 10% sodium dodecyl sulfate polyacrylamide gel. The separated proteins were transferred into a polyvinylidene fluoride (PVDF) membrane by the electro-blotting method. Thereafter, the membrane was blocked using 5% skimmed milk for 1 h at 37°C, followed by incubation with primary antibodies (4°C, overnight). The primary antibodies, including anti-GRK2, anti-p38, anti-p-p38, and anti-GAPDH, were purchased from Cell Signaling Technology (Danvers, MA, USA). After washing with TBST (Tris-buffered saline with Tween 20), the membrane was probed with horseradish peroxidase (HRP)-labeled secondary antibodies for 1 h at room temperature. Afterwards, target proteins in the membrane were visualized using a chemiluminescent agent. Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Bethesda, MD, USA) was used to calculate the grey value of target bands.

Data analysis

All data were presented as the mean ± standard deviation (SD). All statistical comparisons were performed using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA) by student’s t-test and one-way analysis of variance. Differences were regarded as statistically significant at p<0.05.

RESULTS

miR-15a/16 expression is upregulated in the spinal cord of rats after CCI

To explore the role of miR-15a/16 in neuropathic pain, we first determined the expression of miR-15a/16 in a rat model of neuropathic pain induced by CCI. RT-qPCR detection showed that both miR-15a and miR-16 expression were significantly upregulated in the spinal cord of rats after CCI compared with the sham group (Fig. 1A, 1B). The data indicate that dysregulation of miR-15a/16 may be associated with the pathogenesis of neuropathic pain.

Inhibition of miR-15a/16 alleviates thermal hyperalgesia and mechanical allodynia in CCI rats

To investigate whether restored dysregulation of miR-15a/16 expression affects neuropathic pain development, we inhibited miR-15a/16 expression by intrathecal injection of miR-15a/16 antagomir (anti-miR-15a/16) into CCI rats. We showed that administration of miR-15a/16 antagomir markedly alleviated thermal hyperalgesia as indicated by PWL (Fig. 2A) and mechanical allodynia as indicated by PWT (Fig. 2B). These results suggest that inhibition of miR-15a/16 alleviates neuropathic pain development in CCI rats.

Inhibition of miR-15a/16 decreases the expression of IL-1β and TNF-α in CCI rats

We next investigated the effect of miR-15a/16 inhibition on the expression of inflammatory cytokines, IL-1β and TNF-α, in the spinal cords of CCI rats. Our results showed that CCI rats had high levels of expression of IL-1β and TNF-α in their spinal cords (Fig. 3A–3D). However, inhibition of miR-15a/16 significantly suppressed the mRNA expression levels of IL-1β and TNF-α in the spinal cords of CCI rats (Fig. 3A, 3B). Consistently, protein expression of IL-1β and TNF-α in the spinal cords of CCI rats was also reduced by miR-15a/16 inhibition (Fig. 3C, 3D). Moreover, miR-15a/16 inhibition promoted the protein expression of IL-2 and decreased the protein expression of IL-6 (Supplementary Fig. 1). These data suggest that inhibition of miR-15a/16 impedes the inflammation in CCI rats.

miR-15a/16 binds to the 3′-UTR of GRK2

To determine the underlying mechanism by which miR-15a/16 regulates neuropathic pain development, we performed bioinformatics analysis to predict the target genes of miR-15a/16 using TargetScan-Prediction of microRNA targets. Interestingly, we found that GRK2, an important regulator of neuropathic pain (Willemen et al., 2010; Wang et al., 2011), was a potential target gene of miR-15a/16 (Fig. 4A). We found that the GRK2 3′-UTR has an identical seed region for miR-15a and miR-16 (Fig. 4A, 4B). To confirm whether miR-15a and miR-16 can directly bind to the GRK2 3′-UTR, we performed a dual-luciferase reporter assay. The results showed that overexpression of miR-15a or miR-16 markedly decreased the luciferase activity of the reporter plasmid containing the predicted wild-type seed region for miR-15a and miR-16 (Fig. 4C). However, these effects were not observed in reporter plasmids containing mutant binding sites in the seed region of the GRK2 3′-UTR (Fig. 4C). Collectively, these data suggest that miR-15a/16 directly binds to the 3′-UTR of GRK2.

Inhibition of miR-15a/16 upregulates the expression of GRK2 in CCI rats

Considering the interaction between miR-15a/16 and the GRK2 3′-UTR, we investigated the effect of miR-15a/16 inhibition on GRK2 expression in CCI rats. The results showed that CCI surgery induced a significant decrease in GRK2 mRNA and protein expression in rat spinal cords (Fig. 5A, 5B). Interestingly, inhibition of miR-15a/16 significantly promoted the expression of GRK2 in CCI rats (Fig. 5A, 5B). Overall, these results suggest that miR-15a/16 inhibition can promote the expression of GRK2 in CCI rats.

Inhibition of miR-15a/16 suppresses the activation of p38 MAPK

GRK2 has been reported to regulate neuropathic pain and the inflammatory response associated with regulation of p38 MAPK (Peregrin et al., 2006). We investigated whether inhibition of miR-15a/16 has a regulatory effect on p38 MAPK. We found that the phosphorylation of p38 MAPK was significantly increased in CCI rats (Fig. 6A–6D). Inhibition of miR-15a/16 showed no obvious effect on total protein expression of p38 MAPK (Fig. 6A, 6B). However, inhibition of miR-15a/16 significantly inhibited phosphorylation of p38 MAPK in CCI rats (Fig. 6A, 6C, 6D). In addition, we further found that inhibition of miR-15a/16 significantly decreased the phosphorylation of NF-κB p65 in CCI rats (Fig. 6E–6H). These data suggest that miR-15a/16 inhibition alleviates neuropathic pain that may be associated with regulation of GRK2/p38 MAPK/NF-κB signaling.

Knockdown of GRK2 eliminates the protective effects of miR-15a/16 inhibition in neuropathic pain rats

To validate whether inhibition of miR-15a/16 alleviates neuropathic pain through promoting GRK2 expression, we investigated the effect of GRK2 silencing on miR-15a/16 inhibition-mediated effects. LV-GRK2 shRNA was used to silence the expression of GRK2. We found that knockdown of GRK2 significantly blocked the promotive effect of miR-15a/16 inhibition on GRK2 expression in CCI rats (Fig. 7A). Furthermore, our results showed that knockdown of GRK2 eliminated the inhibitory effect of miR-15a/16 inhibition on neuroinflammation (Fig. 7B, 7C) and neuropathic pain (Fig. 7D, 7E) in CCI rats. Collectively, these results suggest that inhibition of miR-15a/16 alleviates neuropathic pain through upregulation of GRK2.

Knockdown of GRK2 reverses the inhibitory effect of miR-15a/16 inhibition on p38 MAPK activation

To investigate whether inhibition of miR-15a/16 suppresses the activation of p38 MAPK through regulation of GRK2, we evaluated the effect of GRK2 knockdown on the miR-15a/16 inhibition-mediated effect on the phosphorylation of p38 MAPK. As expected, we found that knockdown of GRK2 markedly reversed the inhibitory effect of miR-15a/16 inhibition on the phosphorylation of p38 MAPK (Fig. 8A–8D), indicating that GRK2 contributes to miR-15a/16 inhibition-mediated p38 MAPK phosphorylation.

DISCUSSION

There is a growing body of evidence that miRNAs are critical regulators in neuropathic pain development, and are potential therapeutic targets for neuropathic pain prevention in rodent models (Su et al., 2017; Yang et al., 2017a; Ji et al., 2018). However, the precise role of miRNAs in neuropathic pain remains largely unknown. In this study, we newly identified miR-15a/16 as a regulator in neuropathic pain. We found that both miR-15a and miR-16 expression was upregulated in CCI rats, and inhibition of miR-15a/16 had a protective effect on CCI-induced neuropathic pain. The underlying molecular mechanism is associated with the regulatory effect of miR-15a/16 on GRK2 expression. Our study will allow further development of therapeutic strategies for neuropathic pain.

Increasing evidence suggests that miR-15a/16 plays an important role in various diseases (Yue and Tigyi, 2010), especially those involving inflammatory responses. One study showed that miR-15a/16 regulates phagocytosis and Toll-like receptor 4-mediated pro-inflammatory cytokine/chemokine release in macrophages, suggesting an important role of miR-15a/16 in sepsis (Moon et al., 2014). Moreover, miR-15a/16 regulates high glucose-induced pro-inflammatory signaling in human retinal endothelial cells (Ye et al., 2016). Interestingly, inhibition of miR-15a/16 has been shown to ameliorate ischemic brain injury in experimental stroke associated with inhibition of pro-inflammatory cytokines (Yang et al., 2017b). However, little is known about the role of miR-15a/16 in neuropathic pain. In this study, we showed that miR-15a/16 expression was significantly increased in CCI rats and the inhibition of miR-15a/16 alleviated the progression and development of neuropathic pain. Moreover, we showed that inhibition of miR-15a/16 also inhibited the expression of IL-1β and TNF-α, suggesting that miR-15a/16 is involved in regulating inflammation in neuropathic pain. Conversely, a recent study reported that miR-16 inhibits inflammatory pain by targeting Ras-related protein 23 (Chen et al., 2016). Therefore, the exact role of miR-15a/16 in neuropathic pain and neuroinflammation requires further investigation. Nevertheless, our study suggests that inhibition of miR-15a/16 attenuates neuropathic pain in a rat model.

Studies have shown that GRK2 plays an important role in the neuroinflammatory processes of the nervous system. GRK2 expression is decreased in hypoxic-ischemic brain damage, which precedes the loss of neurons (Lombardi et al., 2004). Beta-amyloid peptide induces a decrease in GRK2 expression in the temporal cortex, which is associated with activation of microglia-mediated neuroinflammation, indicating an important role of GRK2 in the pathogenesis of Alzheimer’s disease (Suo et al., 2004). Moreover, reduced expression of GRK2 is associated with the pathogenesis of Parkinson disease (Ahmed et al., 2008). These findings suggest that GRK2 exerts a neuroprotective effect in neurological diseases. The function of GRK2 in neuropathic pain has also been widely studied. Kleibeuker et al. (2007) first reported that GRK2 expression was decreased in the lumbar spinal cord of neuropathic pain rats induced by CCI, and decreased expression of GRK2 could potentiate inflammation-induced mechanical allodynia. In GRK2-deficient mice, microglia/macrophage GRK2 expression was reduced in the lumbar spinal cord during neuropathic pain, alongside increased microglial activation and pro-inflammation signaling in the spinal cord (Eijkelkamp et al., 2010). Willemen et al. (2012) reported that GRK2-deficient mice were associated with increased levels of pro-inflammatory M1 activation markers in spinal cord microglia/macrophages, which contribute to the persistent microglia activation during neuropathic pain. These findings suggest that GRK2 is a novel and potential target for treatment of neuropathic pain. The decreased expression of GRK2 is related to pathogenesis of neuropathic pain. However, the upstream regulation of GRK2 remains unknown. In this study, we found that GRK2 was a target gene of miR-15a/16. We found that miR-15a/16 expression was significantly upregulated in CCI rats, which may contribute to the downregulation of GRK2 during neuropathic pain. As expected, we showed that inhibition of miR-15a/16 significantly upregulated the expression of GRK2 in the spinal cord of CCI rats. Moreover, knockdown of GRK2 eliminated the miR-15a/16 inhibition-mediated protective effect. Our study may provide novel insights into understanding the dysregulation of GRK2 in the pathogenesis of neuropathic pain.

GRK2 has been reported to inhibit pro-inflammation by inhibiting the activation of p38 MAPK (Peregrin et al., 2006). In GRK2-deficient microglia, LPS promotes TNF-α expression through a p38 MAPK-dependent pathway (Nijboer et al., 2010). The increased phosphorylation and activation of p38 MAPK in the spinal cord contributes to the development and progression of neuropathic pain (Tsuda et al., 2004; Svensson et al., 2005). In GRK2-deficient mice, chronic hyperalgesia is associated with increased phosphorylation of p38 MAPK and TNF-α in the spinal cord (Eijkelkamp et al., 2010). The p38 MAPK inhibitor can inhibit IL-1β-induced hyperalgesia in GRK2-deficient mice (Willemen et al., 2010). In line with these findings, our results showed that miR-15a/16 inhibition attenuated neuropathic pain and neuroinflammation through promotion of GRK2 expression, which impeded the activation of p38 MAPK in CCI rats.

As well-known, one miRNA can target numerous target genes and one target gene can be targeted by various miRNAs. In this study, we showed that inhibition of GRK2 partially reversed the miR-15a/16 inhibition-mediated suppressive effect on neuropathic pain and neuroinflammation, indicating that other target gene of miR-15a/16 may be involved in this process. Therefore, the precise regulatory mechanism of miR-15a/16 in neuropathic pain remains to be determined.

Taken together, our results show that epigenetic regulation of GRK2 by miR-15a/16 contributes to the dysregulation of GRK2 during the development and progression of neuropathic pain. Inhibition of miR-15a/16 attenuates neuropathic pain and neuroinflammation through promotion of GRK2 expression and inhibition of the activation of p38 MAPK. These findings provide novel insights into the molecular pathogenesis of neuropathic pain and will allow further development of promising and effective therapeutic strategies for the treatment and prevention of neuropathic pain.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

SUPPLEMENTARY
Figures
Fig. 1. Expression profile of miR-15a/16 in CCI rats. Relative expression of miR-15a (A) and miR-16 (B) in spinal cords from sham or CCI rats were detected by RT-qPCR. At postoperative days 1, 3, 7, and 14, the L4–L5 lumbar spinal cords were removed for investigation. *p<0.05 and **p<0.01 versus sham.
Fig. 2. Inhibition of miR-15a/16 alleviates neuropathic pain development in CCI rats. (A) Thermal hyperalgesia was determined by the PWL in response to radiant heat stimulation. (B) Mechanical allodynia was determined by the PWT in response to Von Frey hair stimulation. Anti-miR-15a, anti-miR-16, or the negative control (NC) were intrathecally injected into CCI rats. *p<0.05 versus sham, &p<0.05 versus CCI+NC.
Fig. 3. Inhibition of miR-15a/16 suppresses neuroinflammation in CCI rats. Relative mRNA expression of IL-1β (A) and TNF-α (B) in the spinal cord was assessed by RT-qPCR analysis on postoperative day 7. Protein concentration of IL-1β (C) and TNF-α (D) in the spinal cord was determined by the ELISA method on postoperative day 7. *p<0.05 versus sham, &p<0.05 versus CCI+NC.
Fig. 4. GRK2 is a potential target gene of miR-15a/16. (A) Bioinformatic analysis showing sequence alignment between miR-15a/16 and GRK2 3′-UTR. (B) Construction of WT and MT GRK2 3′-UTR reporter vectors. (C) Dual-luciferase reporter assay of miR-15a/16 and the GRK2 3′-UTR. The WT or MT GRK2 3′-UTR constructs were contransfected with miR-15a or miR-16 mimics into 293T cells and incubated for 48 h before detection. *p<0.05 versus NC.
Fig. 5. Inhibition of miR-15a/16 upregulates the expression of GRK2 in CCI rats. (A) Relative mRNA expression of GRK2 in the spinal cords was measured by RT-qPCR at postoperative day 7. (B) Relative protein expression of GRK2 in the spinal cords was determined by Western blot at postoperative day 7. *p<0.05 versus sham, &p<0.05 versus CCI+NC.
Fig. 6. Inhibition of miR-15a/16 suppresses the phosphorylation of p38 MAPK. (A) Western blot analysis of total p38 and phosphorylated p38 (p-p38) MAPK expression in the spinal cords from sham and CCI rats at postoperative day 7. Quantitative analysis of protein expression of total p38/GAPDH (B), p-p38 MAPK/GAPDH (C) and p-p38 MAPK/total p38 MAPK (D). (E) Western blot analysis of total NF-κB p65 and phosphorylated NF-κB p65 (p-NF-κB p65) expression in the spinal cords from sham and CCI rats at postoperative day 7. Quantitative analysis of protein expression of total NF-κB p65/GAPDH (F), p-NF-κB p65/GAPDH (G) and p-NF-κB p65/total NF-κB p65 (H). *p<0.05 versus sham, &p<0.05 versus CCI+NC.
Fig. 7. Inhibition of miR-15a/16 alleviates neuropathic pain through upregulation of GRK2. CCI rats were treated with anti-miR-15a/16 combined with LV-GRK2 shRNA by intrathecal injection. (A) Relative protein expression of GRK2 was detected by Western blot at postoperative day 7. Protein expression of IL-1β (B) and TNF-α (C) was measured by the ELISA method. (D) Thermal hyperalgesia in rats was determined by the PWL in response to radiant heat stimulation. (E) Mechanical allodynia in rats was determined by the PWT in response to Von Frey hair stimulation. *p<0.05.
Fig. 8. Knockdown of GRK2 reverses the inhibitory effect of miR-15a/16 inhibition on p38 MAPK activation. CCI rats were treated with anti-miR-15a/16 in combination with LV-GRK2 shRNA by intrathecal injection. (A) Relative protein expression of total p38 and p-p38 MAPK were observed by Western blot at postoperative day 7. Quantitative analysis of protein expression of total p38 MAPK/GAPDH (B), p-p38 MAPK/GAPDH (C) and p-p38 MAPK/total p38 MAPK (D). *p<0.05.
References
  1. Ahmed, MR, Bychkov, E, Gurevich, VV, Benovic, JL, and Gurevich, EV (2008). Altered expression and subcellular distribution of GRK subtypes in the dopamine-depleted rat basal ganglia is not normalized by l-DOPA treatment. J Neurochem. 104, 1622-1636.
    Pubmed KoreaMed CrossRef
  2. Ambros, V (2004). The functions of animal microRNAs. Nature. 431, 350-355.
    Pubmed CrossRef
  3. Andersen, HH, Duroux, M, and Gazerani, P (2014). MicroRNAs as modulators and biomarkers of inflammatory and neuropathic pain conditions. Neurobiol Dis. 71, 159-168.
    Pubmed CrossRef
  4. Bartel, DP (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 116, 281-297.
    Pubmed CrossRef
  5. Bennett, GJ, and Xie, YK (1988). A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 33, 87-107.
    Pubmed CrossRef
  6. Calin, GA, Cimmino, A, Fabbri, M, Ferracin, M, Wojcik, SE, Shimizu, M, Taccioli, C, Zanesi, N, Garzon, R, Aqeilan, RI, Alder, H, Volinia, S, Rassenti, L, Liu, X, Liu, CG, Kipps, TJ, Negrini, M, and Croce, CM (2008). MiR-15a and miR-16-1 cluster functions in human leukemia. Proc Natl Acad Sci USA. 105, 5166-5171.
    Pubmed KoreaMed CrossRef
  7. Chaplan, SR, Bach, FW, Pogrel, JW, Chung, JM, and Yaksh, TL (1994). Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 53, 55-63.
    Pubmed CrossRef
  8. Chen, W, Guo, S, and Wang, S (2016). MicroRNA-16 alleviates inflammatory pain by targeting Ras-related protein 23 (RAB23) and inhibiting p38 MAPK activation. Med Sci Monit. 22, 3894-3901.
    Pubmed KoreaMed CrossRef
  9. Denk, F, and McMahon, SB (2012). Chronic pain: emerging evidence for the involvement of epigenetics. Neuron. 73, 435-444.
    Pubmed KoreaMed CrossRef
  10. Eijkelkamp, N, Heijnen, CJ, Willemen, HL, Deumens, R, Joosten, EA, Kleibeuker, W, den Hartog, IJ, van Velthoven, CT, Nijboer, C, Nassar, MA, Dorn, GW, Wood, JN, and Kavelaars, A (2010). GRK2: a novel cell-specific regulator of severity and duration of inflammatory pain. J Neurosci. 30, 2138-2149.
    Pubmed KoreaMed CrossRef
  11. Haanpaa, M, Attal, N, Backonja, M, Baron, R, Bennett, M, Bouhassira, D, Cruccu, G, Hansson, P, Haythornthwaite, JA, Iannetti, GD, Jensen, TS, Kauppila, T, Nurmikko, TJ, Rice, AS, Rowbotham, M, Serra, J, Sommer, C, Smith, BH, and Treede, RD (2011). NeuPSIG guidelines on neuropathic pain assessment. Pain. 152, 14-27.
    Pubmed CrossRef
  12. Hargreaves, K, Dubner, R, Brown, F, Flores, C, and Joris, J (1988). A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 32, 77-88.
    Pubmed CrossRef
  13. Ji, LJ, Shi, J, Lu, JM, and Huang, QM (2018). MiR-150 alleviates neuropathic pain via inhibiting toll-like receptor 5. J Cell Biochem. 119, 1017-1026.
    Pubmed CrossRef
  14. Jiangpan, P, Qingsheng, M, Zhiwen, Y, and Tao, Z (2016). Emerging role of microRNA in neuropathic pain. Curr Drug Metab. 17, 336-344.
    CrossRef
  15. Kavelaars, A, Eijkelkamp, N, Willemen, HL, Wang, H, Carbajal, AG, and Heijnen, CJ (2011). Microglial GRK2: a novel regulator of transition from acute to chronic pain. Brain Behav Immun. 25, 1055-1060.
    Pubmed CrossRef
  16. Kleibeuker, W, Ledeboer, A, Eijkelkamp, N, Watkins, LR, Maier, SF, Zijlstra, J, Heijnen, CJ, and Kavelaars, A (2007). A role for G protein-coupled receptor kinase 2 in mechanical allodynia. Eur J Neurosci. 25, 1696-1704.
    Pubmed CrossRef
  17. Krol, J, Loedige, I, and Filipowicz, W (2010). The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet. 11, 597-610.
    Pubmed CrossRef
  18. Lombardi, MS, Kavelaars, A, and Heijnen, CJ (2002). Role and modulation of G protein-coupled receptor signaling in inflammatory processes. Crit Rev Immunol. 22, 141-163.
    Pubmed CrossRef
  19. Lombardi, MS, van den Tweel, E, Kavelaars, A, Groenendaal, F, van Bel, F, and Heijnen, CJ (2004). Hypoxia/ischemia modulates G protein-coupled receptor kinase 2 and beta-arrestin-1 levels in the neonatal rat brain. Stroke. 35, 981-986.
    Pubmed CrossRef
  20. Lucas, E, Cruces-Sande, M, Briones, AM, Salaices, M, Mayor, F, Murga, C, and Vila-Bedmar, R (2015). Molecular physiopathology of obesity-related diseases: multi-organ integration by GRK2. Arch Physiol Biochem. 121, 163-177.
    Pubmed CrossRef
  21. Moon, HG, Yang, J, Zheng, Y, and Jin, Y (2014). miR-15a/16 regulates macrophage phagocytosis after bacterial infection. J Immunol. 193, 4558-4567.
    Pubmed KoreaMed CrossRef
  22. Nijboer, CH, Heijnen, CJ, Willemen, HL, Groenendaal, F, Dorn, GW, van Bel, F, and Kavelaars, A (2010). Cell-specific roles of GRK2 in onset and severity of hypoxic-ischemic brain damage in neonatal mice. Brain Behav Immun. 24, 420-426.
    Pubmed KoreaMed CrossRef
  23. O’Connor, AB, and Dworkin, RH (2009). Treatment of neuropathic pain: an overview of recent guidelines. Am J Med. 122, S22-32.
    Pubmed CrossRef
  24. Penela, P, Murga, C, Ribas, C, Salcedo, A, Jurado-Pueyo, M, Rivas, V, Aymerich, I, and Mayor, F (2008). G protein-coupled receptor kinase 2 (GRK2) in migration and inflammation. Arch Physiol Biochem. 114, 195-200.
    Pubmed CrossRef
  25. Peregrin, S, Jurado-Pueyo, M, Campos, PM, Sanz-Moreno, V, Ruiz-Gomez, A, Crespo, P, Mayor, F, and Murga, C (2006). Phosphorylation of p38 by GRK2 at the docking groove unveils a novel mechanism for inactivating p38MAPK. Curr Biol. 16, 2042-2047.
    Pubmed CrossRef
  26. Sakai, A, and Suzuki, H (2014). Emerging roles of microRNAs in chronic pain. Neurochem Int. 77, 58-67.
    Pubmed CrossRef
  27. Spinetti, G, Fortunato, O, Caporali, A, Shantikumar, S, Marchetti, M, Meloni, M, Descamps, B, Floris, I, Sangalli, E, Vono, R, Faglia, E, Specchia, C, Pintus, G, Madeddu, P, and Emanueli, C (2013). MicroRNA-15a and microRNA-16 impair human circulating proangiogenic cell functions and are increased in the proangiogenic cells and serum of patients with critical limb ischemia. Circ Res. 112, 335-346.
    Pubmed KoreaMed CrossRef
  28. Su, S, Shao, J, Zhao, Q, Ren, X, Cai, W, Li, L, Bai, Q, Chen, X, Xu, B, Wang, J, Cao, J, and Zang, W (2017). MiR-30b attenuates neuropathic pain by regulating voltage-gated sodium channel Nav1.3 in rats. Front Mol Neurosci. 10, 126.
    Pubmed KoreaMed CrossRef
  29. Suo, Z, Wu, M, Citron, BA, Wong, GT, and Festoff, BW (2004). Abnormality of G-protein-coupled receptor kinases at prodromal and early stages of Alzheimer’s disease: an association with early beta-amyloid accumulation. J Neurosci. 24, 3444-3452.
    Pubmed CrossRef
  30. Svensson, CI, Schafers, M, Jones, TL, Powell, H, and Sorkin, LS (2005). Spinal blockade of TNF blocks spinal nerve ligation-induced increases in spinal P-p38. Neurosci Lett. 379, 209-213.
    Pubmed CrossRef
  31. Tsuda, M, Mizokoshi, A, Shigemoto-Mogami, Y, Koizumi, S, and Inoue, K (2004). Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury. Glia. 45, 89-95.
    Pubmed CrossRef
  32. van Hecke, O, Austin, SK, Khan, RA, Smith, BH, and Torrance, N (2014). Neuropathic pain in the general population: a systematic review of epidemiological studies. Pain. 155, 654-662.
    Pubmed CrossRef
  33. Vroon, A, Heijnen, CJ, and Kavelaars, A (2006). GRKs and arrestins: regulators of migration and inflammation. J Leukoc Biol. 80, 1214-1221.
    Pubmed CrossRef
  34. Wang, H, Heijnen, CJ, Eijkelkamp, N, Garza Carbajal, A, Schedlowski, M, Kelley, KW, Dantzer, R, and Kavelaars, A (2011). GRK2 in sensory neurons regulates epinephrine-induced signalling and duration of mechanical hyperalgesia. Pain. 152, 1649-1658.
    Pubmed CrossRef
  35. Willemen, HL, Eijkelkamp, N, Wang, H, Dantzer, R, Dorn, GW, Kelley, KW, Heijnen, CJ, and Kavelaars, A (2010). Microglial/macrophage GRK2 determines duration of peripheral IL-1beta-induced hyperalgesia: contribution of spinal cord CX3CR1 , p38 and IL-1 signaling. Pain. 150, 550-560.
    Pubmed KoreaMed CrossRef
  36. Willemen, HL, Huo, XJ, Mao-Ying, QL, Zijlstra, J, Heijnen, CJ, and Kavelaars, A (2012). MicroRNA-124 as a novel treatment for persistent hyperalgesia.. J Neuroinflammation. 9, 143.
    Pubmed KoreaMed CrossRef
  37. Woodall, MC, Woodall, BP, Gao, E, Yuan, A, and Koch, WJ (2016). Cardiac fibroblast GRK2 deletion enhances contractility and remodeling following ischemia/reperfusion injury. Circ Res. 119, 1116-1127.
    Pubmed KoreaMed CrossRef
  38. Yang, D, Yang, Q, Wei, X, Liu, Y, Ma, D, Li, J, Wan, Y, and Luo, Y (2017a). The role of miR-190a-5p contributes to diabetic neuropathic pain via targeting SLC17A6. J Pain Res. 10, 2395-2403.
    Pubmed KoreaMed CrossRef
  39. Yang, X, Tang, X, Sun, P, Shi, Y, Liu, K, Hassan, SH, Stetler, RA, Chen, J, and Yin, KJ (2017b). MicroRNA-15a/16-1 antagomir ameliorates ischemic brain injury in experimental stroke. Stroke. 48, 1941-1947.
    Pubmed KoreaMed CrossRef
  40. Ye, EA, Liu, L, Jiang, Y, Jan, J, Gaddipati, S, Suvas, S, and Steinle, JJ (2016). miR-15a/16 reduces retinal leukostasis through decreased pro-inflammatory signaling.. J Neuroinflammation. 13, 305.
    Pubmed KoreaMed CrossRef
  41. Yue, J, and Tigyi, G (2010). Conservation of miR-15a/16-1 and miR-15b/16-2 clusters.. Mamm Genome. 21, 88-94.
    Pubmed KoreaMed CrossRef
  42. Zhang, F, Xiang, S, Cao, Y, Li, M, Ma, Q, Liang, H, Li, H, Ye, Y, Zhang, Y, Jiang, L, Hu, Y, Zhou, J, Wang, X, Nie, L, Liang, X, Gong, W, and Liu, Y (2017). EIF3D promotes gallbladder cancer development by stabilizing GRK2 kinase and activating PI3K-AKT signaling pathway. Cell Death Dis. 8, e2868.
    Pubmed KoreaMed CrossRef


This Article

e-submission

Archives