MiR-106b-5p represses neuropathic pain by regulating P2X4 receptor in the spinal cord in mice


 Background: P2X4 receptor (P2X4R)-mediated spinal microglial activation makes a critical contribution to pathologically enhanced pain processing in the dorsal horn. It can be upregulated under conditions of neuropathic pain. However, the specific mechanism of pathogenesis and potential molecular targets has not yet been made explicit. MicroRNAs (miRNAs) are commonly recognized as indicators in neuropathic pain pathophysiology.Methods: We established the pain model of spared nerve injury (SNI), and the 50% paw withdrawal thresholds (PWMTs) were used to assess behavior of mouse. MiRNA expression profiling was performed to detect differential expressed miRNA. The western-bolt and quantitative real time PCR to examine P2X4R and miRNA expression in the mouse. Dual-luciferase reporter assays confirmed the correlation between P2X4R and miRNA. Fluorescence in situ hybridization was used to show location between P2X4R and miRNA. Results: In the present study, we found that P2X4R was up-regulated in the spinal dorsal horn of mice following spared nerve injury (SNI), and we identified 69 miRNAs (46 up-regulated and 23 down-regulated miRNAs) were differently expressed (fold change > 2, P < 0.05). P2X4R was a major target of miR-106b-5p (one of down-regulated miRNAs in SNI) with bioinformatics technology and quantitative real time PCR analysis validated the expressed change of miR-106b-5p, and dual-luciferase reporter assays confirmed the correlation between them. Fluorescence in situ hybridization showed that miR-106b-5p was co-localized with P2X4R in the spinal cord. Transfection with miR-106b-5p mimic on BV2 cells reversed the up-regulation of P2X4R induced by LPS. Moreover, miR-106b-5p overexpression significantly attenuated neuropathic pain induced by SNI, with decreased expression of P2X4R mRNA and protein in the spinal cord.Conclusion: Taken together, our results suggest that miR-106b-5p can serve as an important regulator of neuropathic pain development by targeting P2X4R.


Introduction
Pain is de ned as an unpleasant sensory and emotional experience associated with actual or potential tissue damage by the International Association for the Study of pain [1], and neuropathic pain is de ned as "pain caused by a lesion or disease of the somatosensory nervous system" [2]. Neuropathic pain treatment is a great Page 4/23 high glucose medium was added. 2 ml LPS (100 ng/mL, Sigma) was added to each 2 mL well 6 h before the the cells were collected 48 h later for qPCR and western-blot examinations.

Cell Proliferation Assay
Cell proliferation was evaluated using a cell counting kit-8 (CCK-8) assay (Beyotime, China). BV2 cells (5 × 10 3 ) were plated onto 96-well plates, then the LPS or miRNA mimic or miRNA inhibitor were added in the BV2 cells.
Each well contained 100 µL of medium supplemented with 10 µL of CCK8 solution. The absorbance at 450 nm was measured to assess the degree of cell proliferation after incubating the cells at 37 °C for 1 hr.

Luciferase Assay
A dual luciferase reporter assay was performed as outlined for a previous procedure [23]. The pmirGLO dualluciferase vector (pmirGLO vector), which contained both the re y luciferase gene and the renilla luciferase gene, was purchased from Promega (Madison, WI, USA). P2X 4 R 3'UTR, including the predicted binding sites of miR-106b-5p, was inserted into the 3'UTR region downstream of the re y luciferase gene of the pmirGLO vector (pmir-GLO-UTR). HEK293 cells were cultivated in high glucose medium (Gibco) with 10% FBS (JRScienti c, Woodland, CA, USA), 100 mg/mL streptomycin, and 100 units/mL penicillin (Quality Biological, Gaithersburg, MD, USA). The cells were incubated in a humidi ed incubator with 5% CO2 at 37℃. When the HEK293T cells had a con uency of 70-80%, A P2X 4 R-luciferase-reporter construct (pmiRGLO-P2X 4 R 3′-UTR vector), containing the miR-106b-5p binding motif, was co-transfected with miR-106b-5p mimic at different doses of 10, 20, and 50 nM (20 umol/L) or NC mimic into HEK293 cells by using RNAiMAX (Invitrogen). After another 48 h of culture, we used 1 * passive lysis buffer to lyse the transfected cells, and 20 ul supernatant was achieved for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega). The ratio of re y activity to renilla activity was recognized as relative reporter activity. Experiments were performed in triplicate and repeated three times.

Surgical Procedures And Drug Infusion
SNI surgeries were performed to mice according to our previous work [24]. In brief, mice were deeply anesthetized after the behavioral tests. The skin of the lateral thigh was incised and the biceps femoris muscle was dissected bluntly to expose the left sciatic nerve and its three terminal branches (the sural, common peroneal and tibial nerves). The common peroneal and the tibial nerves were tightly ligated with 5 − 0 silk. Then, the nerve was transected distal to the ligation, and 2-4 mm length of nerve ber was removed. Great care was taken to avoid any contact with or stretching of the intact sural nerve. The wound was closed in two layers. The sham group was given all procedures except ligation and transection.
The method of intrathecal injections was described previously [25]. In brief, mice were restrained with the left hand and the injection was performed with the right hand. The vertebral landmarks for L5 and L6 vertebrate were identi ed by palpation. An injection into the subarachnoid space between the L5 and the L6 vertebrae was done via a 27-gage needle. Entry of the needle was con rmed with the presence of a tail ick. The injection volume of all other compounds was 5 µl. In on part, the mice were divided into three groups: sham + scramble (saline), SNI + scramble (saline) and SNI + miR-106b-5p agomir (a selective mimic of miR-106b-5p). In another part, the mice were divided into two groups, naïve + scramble and naïve + miR-106b-5p antagomir. Beginning 1 day after naïve or 7 days after SNI surgery, continuous intrathecal infusion was delivered once a day for 3 days, from day 1 to 3 for naïve mice and from day 7 to 9 for SNI mice. Mice with neurological de cits were excluded from the experiment.
They were measured on days 0, 1, 2, and 3 during continuous miR-106b-5p antagomir injection in naïve mice, always between 8 and 10 AM in the morning. PWMT was assessed with method described previously [26]. The lament force evoking paw withdrawals for more than 3 times in a round of testing was de ned as the mechanical threshold. The cutoff force was 4 g. The observation of a positive response (paw lifting, shaking, or licking) within 5 s.

Mirna Microarray Analysis
The method was described as our previous study [13]. MiRNA expression pro ling was performed using the RiboArray platform (RiboBio, Guangzhou, China). In brief, 1 lg of total RNA was labeled with a Cy3 using a ULS™ microRNA Labeling Kit (Kreatech, Amsterdam, Netherlands) and hybridized on the microarray. Based on Sanger miRBase version 19.0 database, RiboBio designed 1263 speci c oligos for 1281 mouse miRNA, where 1263 are non-redundant sequence. In addition, 54 RiboArray™ internal controls were used. We also put some probes for location identify function. T-test P-value of < 0.05 and fold-change (> 2) were applied to determine two differentially expressed (DE) sets of genes of six experimental samples.

Rna Extraction And Quantitative Real Time Pcr (qpcr)
Total RNA was extracted using the Trizol method [24,27]. The quality and quantity of RNA were measured using a Nanodrop Spectrophotometer (Thermo Scienti c) and samples with an absorbance ratio at 260/280 between 1.8-2.2 were considered acceptable. RNA degradation was not assessed. RNA dilutions were made up in nuclease-free water. For analysis of mRNA, the method was described previously [24,27]. Reverse transcription was generated using the SuperScriptTM III Reverse Transcriptase (Invitrogen) with a Gene Amp PCR System 9700 (Applied Biosystems). For miRNA analysis, 1 ug RNA was used for reverse transcription by miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, #MR101) according to the instructions. Quantitative Real-time PCR (qPCR) was carried out on a real-time detection instrument ViiA 7 Real-time PCR System (Applied Biosystems) using 2X PCR master mix (Arraystar). All experiments were replicated three times.
The relative expression of genes was calculated based on the 2 −△△Ct method using the mouse housekeeping GAPDH/U6 gene as an endogenous control. Expression ratios were subjected to a log2 transform to produce fold change data.

Western Blot
To ensure a su cient amount of protein, BV2 cells and a section of ipsilateral Lumbar enlargement was prepared. Based on established protocol [28], tissues were homogenized in a RIPA buffer (Sangon, Shanghai, China). After centrifugation at 12 000 × g for 15 min at 4 °C, the supernatant was collected to analyze cytosolic proteins, and protein concentrations were determined with a BCA Kit (Thermo Fisher Scienti c, Rockford, IL, USA). The contents of the proteins in the samples were measured using the Bio-Rad protein assay (Bio-Rad) and were then heated at 99 °C for 5 min. Samples of 20 mg total protein were separated by 10% SDSpolyacrylamide gel electrophoresis and electrophoretically transferred onto a polyvinylidene di uoride membrane. After the membranes were blocked with 5% Milk in Tris-buffered PBS containing 0.1% Tween-20 for 1 h, then rabbit anti-P2X 4 R (1:1000, Alomone Labs), rabbit anti-tubulin (1:10000, Sigma), and rabbit anti-GAPDH (1:5000, Cell Signaling Technology) primary antibodies would be used. The proteins were detected using horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:1000, Jackson) and visualized using Western peroxide reagent and luminol/enhancer reagent (Clarity Western ECL Substrate, Bio-Rad). The intensity of the blots was quanti ed via densitometry using Image J software. All cytosol protein bands were normalized to tubulin or GAPDH.

Fluorescence In Situ Hybridization
The uorescence in situ hybridization technology was performed by Servicebio (wuhan, China). The spinal cord of animals was sectioned at 3 mm, and sections were dehydrated in series of ethanol washes (70%, 85%, and 100%) and air-dried. After incubated in hybridization solution at 37 °C for 1 h, the sections were incubated overnight in hybridization solution with 6 ng/µL of DIG (488) labeled probes for miR-106b-5p (5'-DIG-ATCTGCACTGTCAGCACTTTA-DIG-3') at 37 °C. Following hybridization, the sections were washed twice at 37 °C with 2 × SSC for 10 min, then twice at 37 °C with 1 × SSC for 5 min and in 0.5 × SSC at room temperature for 10 min. After they were blocked for 30 min at room temperature, we incubated them with anti-DIG-HRP at 37 °C for 50 min. Finally, FITC-TSA was added at dark for 5 min. To identify the cell types expressing miR-106b-5p, the above sections were incubated overnight at 4 °C with primary antibodies against ATP-gated cation channel protein (P2X 4 R, rabbit, 1:100, ), the ionized calcium-binding adapter molecule (IBA-1, rabbit, 1:300;), glial brillary acidic protein (GFAP, mouse, 1:1000;) or the neuronal-speci c nuclear protein (NeuN, mouse, 1:50;) for double staining as described previously.

Statistical analysis
The data are presented as means ± SEM. For comparisons between two groups, the P value was evaluated and calculated using a two-tailed unpaired t-test. When there were multiple groups involved, a one-way analysis of variance (ANOVA) was used; multiple factors were compared using two-way analysis of variance (ANOVA).
Values of P < 0.05 were considered statistically signi cant.

Results
Up-regulation of P2X 4 R in the spinal cord after SNI 0.001, Fig. 1A). In contrast, no changes in the PWMTs were observed in the sham group during the observation period. To determine the expression of P2X 4 R in the spinal cord of SNI mice, qPCR and western-blot analysis were performed. Compared to the sham group, the SNI group showed an obvious up-regulation of P2X 4 R mRNA expression in the spinal cord (P < 0.05, Fig. 1B). The western-blot results showed that the P2X 4 R protein strongly increased after nerve injury (P < 0.01, Fig. 1C), which was consistent with the data from the behavioral test. As a consequence, the increased expression of P2X 4 R mRNA and protein in the spinal cord of SNI mice con rmed the potential ability of P2X 4 R to aggravate SNI-induced neuropathic pain.

The Differentially Expressed Mirnas In Neuropathic Pain Mice
To investigate the expression of miRNAs in neuropathic pain, the expression levels of miRNAs in the spinal cord of neuropathic pain mice with microarray were analyzed. Results showed 69 miRNAs were differently expressed (fold change > 2.0, P < 0.05), including 46 up-regulated miRNAs ( Fig. 2A) and 23 down-regulated miRNAs (Fig. 2B).
Focus on the downregulated miRNAs, we discovered that P2X 4 R was the target of miR-106b-5p using TargetScan software. The matched seed sequences between miR-106b-5p and P2X 4 R 3'UTR were highly matched between human and mice (Fig. 2C). The TargetScan software demonstrated that the seed sequence for the miR-106b-5p position (1-7) was paired with P2X 4 R 3'UTR from 363 bps to 388 bps in humans and from 1165 bps to 1190 bps in the mice. Then, the expression of miR-106b-5p in SNI mice was veri ed by the qPCR method, and the results were consistent with those of the microarray. MiR-106b-5p showed a down-regulation trend in the spinal dorsal horn of the SNI mice (P < 0.01, Fig. 2D). To verify whether miR-106b-5p targets P2X 4 R 3'UTR, a dual-luciferase reporter vector containing the sequence of P2X 4 R 3'UTR was designed (pmirGLO P2X 4 R 3'UTR). When transfecting the P2X 4 R 3'UTR vector with three different doses of miR-106b-5p mimic (10, 20 and 50 nM) into HEK293 cells with RNAiMax, the miR-106b-5p mimic reduced relative luciferase activity in a dosedependent manner (P < 0.05, Fig. 2E).
MiR-106b-5p mimic transfection inhibits the expression of P2X 4 R mRNA in BV2 cells The result was consistent with previous study [22], BV2 cells were activated after stimulating by LPS (Fig. 3A-B). Further, the cell proliferation was veri ed by using CCK-8 assay. The results showed that BV2 cells viability increased signi cantly in a dose-dependent relationship with LPS (P < 0.05, Fig. 3C). Besides, miR-106b-5p mimic or inhibitor did not affect BV2 cells viability (P > 0.05, Fig. 3D), which indicated miR-106b-5p mimic or inhibitor has no toxic effect on BV2 cells viability.
P2X 4 R are co-localized with miR-106b-5p in spinal cord of SNI mice In order to de ne the localization of miR-106b-5p, the immuno uorescence in situ hybridization was performed in the spinal cord. As shown in Fig. 5, miR-106b-5p with P2X 4 R, IBA1 (a marker for microglia cells), GFAP (a marker for glial cells), and NeuN (a marker for neurons) were stained. Results showed that miR-106b-5p was mainly co-localized with P2X 4 R, IBA1 and NeuN, but not with GFAP.
Intrathecal miR-106b-5p antagomir induces pain behaviors and increases the expression of P2X 4 R in the spinal cord of naïve mice intrathecal injection with miR-106b-5p antagomir (P < 0.05, Fig. 7B). MiR-106b-5p antagomir was applied to naïve mice for 3 days and the sensitivity to the mechanical thresholds were observed. We found that the 50% paw withdrawal threshold was signi cantly lower in miR-106b-5p antagomir treated mice than that of scrambled miRNAs treated mice (P < 0.01, Fig. 7A), demonstrating that miR-106b-5p antagomir produced pain behaviors in naïve mice. Furthermore, the spinal cord tissue was harvested at day 3 to assess the expression of P2X 4 R at the mRNA and protein levels. As shown in Fig. 7, intrathecal miR-106b-5p antagomir increases the expression of P2X 4 R mRNA (P < 0.01, Fig. 7C), as well as P2X 4 R protein (P < 0.05, Fig. 7D) in naïve mice.

Discussion
Nervous system damage results in neuropathic pain, and miRNAs are reported to regulate neuropathic pain. In our study, P2X 4 R mRNA and protein were increased in the SNI mouse model. Then, 69 DE miRNAs were identi ed in SNI mice; among them, we also found that miR-106b-5p was decreased and that it could repress neuropathic pain development by targeting P2X 4 R. Our ndings indicated that miR-106b-5p could be used as a novel therapeutic target of neuropathic pain through regulating P2X 4 R.
As is already known, purinergic receptor-mediated spinal microglial functions contribute signi cantly to pathologically enhanced pain management. [8,9]. Consistent with previous study, the expression of spinal P2X 4 R was up-regulated in SNI rats and DNP rats [29][30][31]. Additionally, P2X 4 R was mostly expressed in microglial cell [32,33]. Some researchers found that duloxetine, a drug that inhibits neurotransmitters and microglial P2X 4 R function and alleviates neuropathic pain after peripheral nerve injury [10]. Our results together with these ndings, show that P2X 4 R is upregulated in neuropathic pain model, which strongly suggests that P2X 4 R plays a crucial role in neuropathic pain. Despite recent advances, understanding the transcriptional or translational regulatory mechanisms underlying changes in the expression and function of P2X 4 R under neuropathic pain conditions remains a major challenge.
MiRNAs are commonly recognized as mediators of gene expression and can serve as indicators in neuropathic pain pathophysiology [18][19][20]. There has been a focus on studies associated miRNAs with chronic neuropathic pain states. For example, miR-98 might depress neuropathic pain development through modulating HMGA2 [21]. MiRNAs are highly deregulated in neuropathic pain. [13,14] and might be critical molecules for treatment.
These ndings provide us with information that the mechanism of P2X 4 R in neuropathic pain can be investigated from the perspective of miRNA.
We identi ed 69 miRNAs showing differential expression in the spinal cord after SNI by microarray analysis (fold change > 2, P < 0.05, Fig. 2A and B). Based on common mechanisms, miRNA plays a regulatory role mainly by silencing its target genes [34,35], thus we focused on the down-regulated miRNAs in our present study. Among the down-regulated miRNAs, miR-106b-5p was found to target P2X 4 R through TargetScan software (Fig. 2C). Consistent with the results of the microarray, miR-106b-5p was decreased in SNI mice by the qPCR method (Fig. 2D). Interestingly, miR-106b-5p was also dysregulated in some psychiatric disorder diseases, such as Alzheimer's disease and attention-de cit hyperactivity disorder [36,37]. Further, the dualluciferase assay veri ed that miR-106b-5p negatively regulated P2X 4 R through combing with P2X 4 R 3'UTR ( Fig. 2E). Moreover, uorescence in situ hybridization showed that miR-106b-5p was co-localized with P2X 4 R, microglia and neurons in mice spinal cords (Fig. 4). A lot of evidence suggests that P2X 4 R are almost expressed in microglia to play a vital role in pain development [8,38], but also are also expressed in DRG neurons during chronic in ammatory pain [39]. Our results are consistent with these studies and provide evidence for the interaction between miR-106b-5p and P2X 4 R.
In our study, BV2 cells were activated after stimulating by LPS (Fig. 3A-B), which are consistent with classical theory [22]. And the viability of BV2 cells increased in a dose-dependent manner following LPS treatment (P < 0.05, Fig. 3C). BV2 cells are typically microglia cell lines, and P2X 4 R is almost exclusively expressed in microglia [10]. Further, we found that P2X 4 R is increased at both the mRNA and protein level under LPS-induced in ammatory conditions in BV2 cells ( Fig. 4B and C). What's more, the BV2 cells transfected with miR-106b-5p mimic reversed the up-regulation of P2X 4 R induced by LPS ( Fig. 4B and C). These data indicated that miR-106b-5p regulates P2X 4 R at the transcription level in BV2 cells. However, transfecting miR-106b-5p mimic did not alter the level of P2X 4 R in naïve BV2 cells ( Fig. 4B and C, P > 0.05).
Further, we demonstrated that miR-106b-5p alleviated pain by inhibiting P2X 4 R through the changes in molecular levels in SNI mice. Meanwhile, the increased expression of P2X 4 R was reversed ( Fig. 6C and D) with intrathecal administration of miR-106b-5p agomir in SNI mice. To further determine the role of miR-106b-5p in the SNI-induced neuropathic pain, we found that intrathecal administration of miR-106b-5p antagomir contributed to pain behaviors (Fig. 7A) and the up-regulation of P2X 4 R ( Fig. 7C and D) in naïve mice. These results revealed that miR-106b-5p may function as an effective therapeutic target for treating neuropathic pain through regulating P2X 4 R.
The activated P2X 4 R causes the phosphorylation of p38-MAPK, and the release of BDNF, all of which are essential to the persistence of pain hypersensitivity after nerve injury. Mounting evidence suggests that P2X 4 R may regulate the progress of neuropathic pain through the p38MAPK, ERK1/2, and PI3K/Akt pathways, which were core pathways in the development of neuropathic pain [11,[40][41][42]. Meanwhile, miR-106b could promote cervical cancer progression by modulating the expression of the GSK3B, VEGFA, and PTK2 genes. Importantly, these three genes play crucial roles in PI3K-Akt signaling, focal adhesion and cancer [43]. Further, the expression of miR-106b was inversely correlated with TGF-β type II receptor protein level; and miR-106b can directly inhibit the TGF-β type II receptor translation in vitro [44], while TGF-β receptor binding is an important molecular function in diabetic neuropathic pain from our previous study [27]. This accumulated evidence reveals that miR-106b-5p might regulate the expression of P2X 4 R in neuropathic pain through some important pathways. Therefore, further investigations illustrating the potential mechanism would provide a new direction and serviceable theoretical foundation for the clinical intervention of neuropathic pain.

Conclusion
The biological role of miR-106b-5p in neuropathic pain progression was the focus of our present research. We observed that miR-106b-5p alleviated neuropathic pain development by down-regulating P2X 4 R. P2X 4 R can enable neuropathic pain development, which can be reversed by miR-106b-5p in vitro. Our data revealed that miR-106b-5p may function as an effective therapeutic target for treating neuropathic pain through regulating

Availability of data and materials
The datasets generated and/or analyzed in present study will be available from the corresponding author upon reasonable request from any quali ed investigator.
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