MiR‐101 promotes pain hypersensitivity in rats with chronic constriction injury via the MKP‐1 mediated MAPK pathway

Abstract This study was performed to characterize the effect of microRNA‐101 (miR‐101) on the pain hypersensitivity in CCI rat models with the involvement of mitogen‐activated protein kinase phosphatase 1 (MKP‐1) in spinal cord microglial cells. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in the developed CCI models were determined to assess the hypersensitivity of rats to mechanical stimulation and thermal pain. To assess inflammation, the levels of interleukin (IL)‐1β, IL‐6 and tumour necrosis factor‐α (TNF‐α) in the spinal dorsal horns of CCI rats and lipopolysaccharide (LPS)‐activated microglial cells were examined. miR‐101 and MKP‐1 gain‐ and loss‐of‐function experiments were conducted in in vivo and in vitro settings to examine the roles of miR‐101 and MKP‐1 in CCI hypersensitivity and inflammation. The results showed that miR‐101 was highly expressed in the spinal dorsal horn and microglial cells of CCI rat models. Furthermore, overexpression of miR‐101 promoted the pain hypersensitivity in CCI rat models by reducing MWT and TWL. The overexpression of miR‐101 also promoted inflammation in LPS‐exposed microglial cells, as indicated by increased levels of IL‐1β, IL‐6 and TNF‐α. MiR‐101 was shown to target MKP‐1, inhibiting its expression. Moreover, miR‐101 promoted pain hypersensitivity in CCI rat models by inhibiting MKP‐1 expression and activating the mitogen‐activated protein kinase (MAPK) signalling pathway. Taken together, miR‐101 could potentially promote hypersensitivity and inflammatory response of microglial cells and aggravate neuropathic pain in CCI rat models by inhibiting MKP‐1 in the MAPK signalling pathway.


| INTRODUC TI ON
Chronic pain pre-disposes approximately 20% of the population across the world to physical and emotional incapacity and is regarded as one of the leading causes of disability. 1 Notably, approximately, 20% of the cases of chronic pain comprise neuropathic pain arising from impairment of the nervous system, which may be attributed to direct damage to the spinal cord, the brain or the peripheral nerves, or dysfunction due to degenerative or chronic inflammation. 2 In particular, the activation of spinal cord microglial cells is responsible for persistent neuropathic pain. 3 The treatment strategies for nerve injury and subsequent neuropathic pain are usually aimed at reducing inflammation, a primary cause underlying neuropathic pain. 4 However, neuropathic pain is a frequently occurred condition, which often has poor treatment outcome due to resistance to therapies. 5 The genesis and development of neuropathic pain are still poorly understood, and thus current therapies only focused on symptomatic management instead of targeting the underlying aetiology. 6 Therefore, the search on more effective and safer treatment options for neuropathic pain warrants investigations into its relevant molecular mechanisms.
MicroRNAs (miRNAs) are a group of small non-coding RNAs and known to mediate gene expression through either translational inhibition or mRNA degradation. 7 Interestingly, multiple miRNAs have been reported as molecular players in the generation and development of chronic neuropathic pain 8 owing to their roles as key modulators of gene expression and neuronal network plasticity within the nervous system. 9 The aberrant expression of miRNAs has been observed in the spinal cord dorsal horn in rat models of chronic constriction injury (CCI) and plays important roles in the induction and persistence of neuropathic pain. 10,11 Interestingly, a previous study has indicated that miR-101a could induce the differentiation of bone marrow cells into microglial cells, whose activation has been associated with neuropathic pain. 12 Moreover, miR-101 has been demonstrated to attenuate neuropathic pain in CCI rat models. 13 The interaction of miRNAs and mRNAs has been characterized in inflammatory pain in the rat spinal cord and is also implicated in pain regulating pathways. 14 miR-101 has been reported to negatively regulate mitogen-activated protein kinase phosphatase 1 (MKP-1) expression. 15 MKP-1 is considered a key factor in the mitogen-activated protein kinase (MAPK) signalling pathway. 16 As a mediator of phosphorylated p38, MKP-1 exerts regulatory effects on pro-inflammatory factor in the spinal cord following peripheral nerve injury, thereby regulating chronic mechanical hypersensitivity. 17 Inactivation of the MAPK signalling pathway has been documented to attenuate neuropathic pain in CCI rats. 18 Therefore, we hypothesized that miR-101 might participate in the induction of pain hypersensitivity following CCI through MKP-1-mediated MAPK signalling pathway.

| Ethics statement
Animal experiments were performed with the approval of the Ethics Committee of Linyi People's Hospital and in accordance with the Guide for the Care and Use of Laboratory animals published by the US National Institutes of Health. All efforts were made to minimize the suffering of the animals included in the study.

| Rat CCI model establishment
Adult female Sprague Dawley (SD) rats (weighing 180-210 g) were purchased from Shanghai, Lab. Animal Research Center. All rats were housed in cages with a constant temperature of 25°C. The CCI rat model was developed according to previously described methods. 19 Briefly, the rats were anaesthetized by intraperitoneal injections of 40 mg/kg pentobarbital sodium. The sciatic nerves on both sides of the rats were exposed and isolated from the surrounding tissues. The posterior medial sciatic nerve of the left femur was exposed, set free for 5-6 mm before bifurcating and ligated 4 times within l mm. After the surgery, the incision was sutured layer by layer. Rats in the sham group were subjected to exposure and isolation of the sciatic nerve without ligation. There were 18 rats in the sham group and CCI group (without infection with lentivirus), respectively. The rats developed with CCI were then infected with lentiviruses expressing miR-101, miR-101-inhibitor, MKP-1, negative control (NC), namely, LV-miR-101, LV-miR-101-inhibitor, LV-MKP-1 and LV-NC, with 9 rats in each group. The remaining rats developed with CCI were treated with MKP-1 inhibitor, RO318220 or infected with LV-miR-101 and LV-MKP-1 or LV-NC in combination, with 9 rats in each group. Three rats were randomly selected and euthanized at each time when the spinal cord tissue was harvested, and six randomly selected rats were used for behavioural tests. Next, L4-L6 spinal cord segment was harvested on days 0, 3, 7, 14 and 21 after the CCI induction surgery.

| Intrathecal catheterization and injection
The rats were intraperitoneally anaesthetized with 40 mg/kg pentobarbital sodium. Thereafter, the occipital muscles were separated, and the PE-10 polyethylene catheter was placed in the cisterna magna of the cerebellum. Next, 80 000 units of penicillin sodium were injected into the right upper extremity to prevent infection. On the next day, after the rats were awake, 20 μL of 2% lidocaine was injected through a microcatheter, and both lower extremities were paralysed within 30 seconds. The rats that recovered within 30 minutes were considered to have a successful catheterization. After catheterization for 2 days, the rats with movement and sensory disturbances such as monoplegia, paraplegia and hemiplegia were excluded. Three days prior to CCI surgery, 10 μL of the corresponding recombinant lentivirus was injected through the intrathecal catheter using a micro-injector.

| Behavioural test
Von Frey's method was used to measure the mechanical withdrawal threshold (MWT) to evaluate the mechanical abnormal pain. The rats were kept in a clear plastic box with a metal mesh bottom. Pressure was applied to the rat hind paw using Calibrated Electronic von Frey filaments (Electronic von Frey 2393; IITC) and the time for paw withdrawal was recorded. Thermal withdrawal latency (TWL) was measured using a 336 analgesic model (IITC Life Science Instruments) to assess thermal hyperalgesia. The rats were kept in a plexiglass box, and in order to avoid tissue damage caused by long-term thermal stimulation, the heat intensity was set to 10 seconds, and the cut-off time was 20 seconds (the power was stopped automatically 30 seconds later). The duration between stimulation and withdrawal was recorded. Each test was repeated 5 times at an interval of 5 minutes for each claw, with the mean value obtained. At the end of the behavioural test, the rats were euthanized, and bilateral L4-L6 spinal segments were harvested in a chronological order.

| Lentiviral vector construction and transfection
Full-length miR-101 and its inhibitor, MKP-1 oligonucleotide or NC oligonucleotide were subcloned into GV280 lentiviral vectors

| Cell separation and culture
The isolation of rat spinal cord microglial cells was performed as described in previous study. 20  10100147, Gibco BRL/Invitrogen) and 100 U/mL penicillin/streptomycin (15140122, Gibco BRL/Invitrogen), followed by culture at 37°C with 5% CO 2 .

| Dual-luciferase reporter assay
Possible interaction between miR-101 and MKP-1 was first determined in silico using a web-based biological prediction website (https://cm.jeffe rson.edu/rna22/ Inter activ e/) and the binding site was predicted. A dual-luciferase reporter assay was then used to verify the predicted interaction between miR-101 and MKP-1. Artificially synthesized MKP-1 mRNA 3′ untranslated region (UTR) fragment was inserted into pmirGLO luciferase vector (E1330, Promega) according to the predicted binding site, which was designated as MKP-1-wild-type (wt). The complementary sequence mutation site of the seed sequence was designed on the basis of MKP-1-wt, which was inserted to the reporter plasmid, and the recombinant plasmid MKP-1-mutant (mut) was obtained. The correctly sequenced luciferase reporter plasmid was co-transfected into HEK-293T cells with miR-101 and miR-101-NC, respectively. Luciferase activity was measured using a dual-luciferase assay kit (E1910, Promega) and luminance was detected using a Promega's GLoma × 20/20 Luminometer (E5311, Shaanxi Zhongmei Biotechnology Co., Ltd.). All experiments were repeated three times independently.

| RNA isolation and quantitation
Total RNA was extracted from the spinal cord or cells of each group after 36 hours of transfection using TRIzol reagent (10296010, Invitrogen) according to the manufacturer's protocol. The concentration, purity and integrity of the extracted RNA were determined by Nano-Drop ND-1000 spectrophotometry and 1% agarose gel electrophoresis. All primers were synthesized by Beijing Genomics Institute, Co., Ltd. (Table S1). The miRNA-specific complementary DNA was synthesized using a TaqMan™ MicroRNA Reverse Transcription Kit  An antagonistic miR-101 probe was set up as a NC and all following procedures were performed according to manufacturer's instructions. Fluorescence images were obtained using a Zeiss LSM880 laser confocal scanning microscope (Leica Microsystems). The experiment was repeated three times independently.

| Enzyme-linked immunosorbent assay (ELISA)
The levels of inflammatory factors interleukin (IL)-6, tumour necrosis factor-α (TNF-α), IL-1β and COX-2 in rat spinal cord and microglia samples were measured following the instructions of the Simple Step ELISA ® kits (ab100712, ab208348, ab100768, ab52237) purchased from Abcam. An EON spectrophotometer (BioTek Instruments) was employed to quantify the levels of the microplate at 450 nm.

| RNA pull-down assay
The binding of miR-101 to MKP-1 mRNA was examined using the Magnetic RNA-Protein Pull-Down kit (20164, Pierce). After the microglial cells were routinely detached, the pellet was collected after centrifugation and RIP Lysis Buffer was added to lyse the cells, followed by incubation on ice for 30 minutes. After centrifugation at 12 000 g for 10 minutes at 4°C, the supernatant of the lysate was equally divided into several aliquots and stored at −80°C, and one aliquot was used as the input for the pull-down experiment. According to the kit instructions, the biotinylated miR-101, miR-101-NC, MKP-1-wt, MKP-1-mut were enriched with streptavidin-labelled magnetic beads, which were then incubated overnight with lysis buffer at 4°C. Finally, RNA was extracted by conventional TRIzol method for purification, followed by measurement of MKP-1 mRNA expression by RT-qPCR.

| Immunohistochemistry
On the 7th day post-operation, rats were deeply anaesthetized with sodium pentobarbital (50 mg/kg, ip) and intracardially perfused with saline. Next, 4.0% paraformaldehydein 0.1 mol/L PBS (pH = 7.4, Sigma) was used for perfusion. Spinal cord segments L4-L6 were extracted and fixed at 4°C for 12 hours in the same fixative, and then transferred to PBS containing sucrose (15%-20%). On the next day, the segments were sliced continuously at a thickness of 30 μm.

| Statistical analysis
Statistical analysis was performed using SPSS 21.0 statistical software (IBM Corp). The measurement data were summarized as mean ± standard deviation. Normality and homogeneity of variance were tested, and the data obeying normal distribution and homogeneity of variance between two groups were compared using unpaired t test. The comparison among multiple groups was performed using one-way analysis of variance (ANOVA) or repeated measures ANOVA, followed by Tukey's post hoc test for pairwise comparison.
The rank sum test was performed for data with unequal variances or skewed distribution. P < .01 indicated the difference was statistically significant.

F I G U R E 1
MiR-101 is highly expressed in CCI rat models. A, MWT indicating the hypersensitivity to mechanical stimulation on the 0, 3rd, 7th, 14th and 21st days after operation in the sham-operated rats and CCI rat models. n = 6. B, TWL indicating the hypersensitivity to thermal hyperalgesia at the 0, 3rd, 7th, 14th and 21st days after operation in the sham-operated rats and CCI rat models. n = 6. C, levels of IL-1β, IL-6 and TNF-α in the L4-L6 segments of spinal dorsal horn in sham-operated rats and CCI rat models on the 7th day after operation detected by ELISA. n = 3. D, immunohistochemical detection of the expression of microglial cell marker CD11b in the L4-L6 spinal cord in the sham-operated rats and CCI rat models on the 7th day after operation. n = 3. E, the relative expression of miR-101 in L4-L6 spinal cord dorsal horn of sham-operated rats and CCI rat models at the 0, 3rd, 7th, 14th and 21st days after operation detected by RT-qPCR. n = 3. F, miR-101 expression in L4-L6 spinal dorsal horn of sham-operated rats and CCI rat models on the 7th day after operation identified using FISH assay. n = 3. G, microscopic observation from morphological characteristics of microglia isolated from the spinal cord. H, immunofluorescence assay of the isolated microglia marker CD11b expression to identify the purity of isolation. I, the expression of miR-101 in microglial cells isolated from the L4-L6 spinal cord of the sham-operated rats and CCI rat models at the 0, 3rd, 7th, 14th and 21st days after operation determined by RT-qPCR. J, the localization of miR-101 in microglial cells isolated from the L4-L6 spinal cord on the 7th day post-CCI model construction detected by FISH assay. Measurement data were expressed as mean ± standard deviation. Comparison between time-based measurements within each group was performed with repeated measures ANOVA (panels A, B, E, I), followed by Tukey's post hoc test. Comparison between two groups was performed by independent sample t test (panel C). * P < .05 vs the sham group. All cell experiments were repeated three times

| MiR-101 is highly expressed in CCI rat models
Foot licking, biting or shaking after the ligation of bilateral sciatic nerves indicated the occurrence of spontaneous pain. Besides, MWT was shown to be significantly lower in the CCI group than that in the sham group, suggesting mechanical allodynia (P < .05) ( Figure 1A).
Moreover, TWL in the CCI group was significantly lower than that in the sham group, suggesting thermal hyperalgesia (P < .05) ( Figure 1B).

| MiR-101 loss-of-function reduces hypersensitivity to pain in CCI rat models
As depicted in Figure 2A MWT and TWL in rats in the CCI-LV-miR-101 group were significantly increased, indicating a significantly higher sensitivity to the pain caused by stimulation. However, the MWT and TWL in rats in the CCI-LV-miR-101-inhibitor group were significantly decreased, indicating that these rats were less sensitive to pain (P < .05) ( Figure 2B,C). miR-101 overexpression was found to increase the levels of IL-1β, IL-6 and TNF-α in the spinal dorsal horn of rats with CCI, and all of which were significantly decreased by miR-101 knockdown (P < .05) ( Figure 2D). Taken together, the results demonstrated that up-regulation of miR-101 promoted pain hypersensitivity in CCI rat models, whereas the down-regulation of miR-101 attenuated this sensitization.

| MiR-101 loss-of-function impedes lipopolysaccharide (LPS)-induced activation of spinal cord microglia and reduces inflammation
As shown in Figure 3A were activated by LPS. The LPS-exposed cells showed a morphological change from amoebiform and long shape with asymmetrical branches to an amoeba-like shape, with enrichment of the markers CD11b and Iba1 ( Figure 3B). In the LPS-exposed microglial cells, the secreted levels of IL-1β, IL-6 and TNF-α were increased upon miR-101 overexpression ( Figure 3C). On the contrary, the secretion of those pro-inflammatory factors IL-1β, IL-6 and TNF-α in the LPS-exposed microglial cells was inhibited by miR-101 loss-offunction inhibited the secretion of these pro-inflammatory factors, accompanied by decreased expression of CD11b and Iba1 (P < .05) ( Figure 3B,C). These results suggested that inhibition of miR-101 hindered LPS-induced activation of spinal cord microglia and reduced inflammation.

| MiR-101 binds to the 3′UTR of MKP-1 mRNA
The miRNA target prediction websites (miRanda and TargetScan) predicted the complementary sequence between miR-101 and MKP-1 3′UTR both in the human genome and the rat genome ( Figure 4A). As miR-101 was highly expressed in CCI model rats ( Figure 1E,F

| MiR-101 enhances inflammation in spinal cord microglia by inhibiting MKP-1 expression
Next, we explored whether miR-101 can affect the MAPK signalling pathway by inhibiting MKP-1. Western blot analysis ( Figure 5A) showed that the extent of p38, JNK and c-Jun phosphorylation in LPS-activated microglial cells was significantly higher than that in control cells without LPS stimulation, indicating that the p38 MAPK/ JNK signalling pathway was activated ( Figure 5B). This activation  Figure 5D). Together, these findings indicated that miR-101 inhibited MKP-1 to activate the MAPK signalling pathway in spinal cord microglial cells, thereby increasing inflammation.

| MiR-101 aggravates pathological pain in CCI rat models through suppression of MKP-1 in the MAPK signalling pathway
In CCI rat models, the overexpression of MKP-1 diminished the allodynia response reflected by significant increases in MWT and TWL ( Figure 6A 3,23 A previous study demonstrated that the exacerbated inflammatory response is linked with activated microglial cells following traumatic brain injury. 24 In neuropathic pain, microglia plays an important role in the process of pain sensitization and thus comprises a promising target for neuropathic and post-operative pain, and morphine tolerance. 25 The secreted inflammatory mediators act to stimulate induction and cascade expansion during pain transmission between microglial cells and neurons. 26 Our results highlighted that miR-101 was highly expressed in the spinal cord dorsal horn in CCI rat models and the microglial cells isolated from these rats. These results are consistent with recent evidence indicating that miRNAs have critical roles in the induction and progression of chronic pain. 9 More specifically, evidence has demonstrated that miRNAs are involved in the pathophysiologic processes of spinal cord injury and can be considered potential therapeutic targets. 27 In addition, reports indicate that miR-101 expression is clearly associated with inflammation and it mediates the secretion of inflammatory factors. 28,29 Our study also demonstrated that overexpression of miR-101 enhanced inflammation in LPS-exposed spinal cord microglial cells and in CCI rat models, as reflected by the of IL-6 and TNF-α is shown to promote the expression of miR-101, which subsequently augments intracellular cholesterol retention under inflammation conditions. 31 This highlights another possible mechanism associated with the neuroinflammation, which warrants further verification. Furthermore, we verified MKP-1 to be a target gene of miR-101. It is well established that MKP-1 is a member of the dual-specificity phosphatase family that deactivates MAPKs. 32 Our results showed that miR-101 targets the 3'UTR of MKP-1 and inhibits its expression. miR-101 similarly modulates immune responses of macrophages to LPS by targeting MKP-1. 33  Based on the aforementioned evidence, the pain hypersensitivity and inflammation in rats with CCI could be accelerated by miR-101-mediated MKP-1 inhibition in microglial cells (Figure 7). miR-101 and MKP-1 may therefore have the potential to serve as therapeutic targets for chronic neuropathic pain management. More generally, the miRNA-based clinical trials may provide potential therapeutic tools for chronic neuropathic pain, which enable improved categorization of risk, prognosis and possibly translate to personalized therapy. However, future researches should confirm the findings with larger patient cohorts and identify the best strategy to limit the side effects.

ACK N OWLED G EM ENTS
We would like express sincere appreciation to the reviewers for their critical comments on this article.

CO N FLI C T S O F I NTE R E S T S
None.

CO N S E NT FO R PU B LI C ATI O N
Consent for publication was obtained from the participants.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated/analysed during the current study are available.