For this study, healthy adult male pathogen-free grade Sprague-Dawley rats (200 ± 20 g) were selected as experimental animals, which were uniformly provided by the animal center of Shaanxi Provincial People’s hospital. The provided mice were acclimatized for 4 weeks to carry out the corresponding studies, and the mouse housing room conditions were set as: room temperature 20˚C - 25˚C, relative humidity 50% - 70%, and free access to food and water. All animal experiments were designed and operated in compliance with international guidelines for laboratory animals.

An animal model of neuropathic pain was established in rats by CCI [9]. Rats were anesthetized using an intraperitoneal injection of phenobarbital sodium (40 mg/kg) and subsequently placed prone on the experimental operating table. The skin was cut out using surgical scissors, the muscles were separated, and both sciatic nerves of the rats were exposed. A segment of the sciatic nerve was selected, and the two ends of the sciatic nerve were ligated using a 4 - 0 suture, cut at the middle portion of the ligated nerve, and the muscular and skin layers were sutured sequentially with sutures, sterilized, and wrapped. Sham group rats underwent blunt dissection to expose the sciatic nerve and branches after, but without ligation and transection.

After rats were anesthetized, a 30 gauge Hamilton syringe needle was inserted in the subarachnoid space of the spinal cord between L4 and L5. In order to induce bilateral hind limb paralysis, the intrathecal catheter implantation in the correct position was affirmed by injecting 2% lidocaine. MiR-362-3p mimic or LV-JMJD1A was delivered intrathecally using a microsyringe connected to an intrathecal catheter. At the end of the experiment, lumbar L4-L5 spinal cords were dissected for follow-up experiment.

Rat spinal cord tissue was first isolated and placed into cold Dulbecco′s Modified Eagle’s Medium (DMEM) medium, the tissue was sheared into small pieces of approximately 1 mm³ and digested with trypsin solution into a cell suspension, the supernatant was removed by centrifugation, and the cells were resuspended in complete medium containing DMEM. After that, they were plated in T75 flasks with a final volume of 25 ml and placed in an incubator at 37˚C with 5% CO₂ for two weeks. Finally, microglia was separated from other cells by shaking the flask with mixed glial cells.

On days 0, 3, 7, and 14 after CCI surgery, rats were measured for mechanical paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) according to previously described methods [10]. Rats were placed in cages before measurements and acclimatized for 30 min. PWT determination: rats were stimulated on the bottom of the left hind foot with a probe from the electronic von Frey pain meter at stimulus intensities ranging from 0 to 50 g in 2.5 g/s increments, which were stopped when the rat had a lifting and paw withdrawal response, and measured three times in consecutive intervals for 5 min each, and the mean value was taken as the PWT value (g) of the rat. PWL determination: when the left hind limb of rats was stimulated by heat radiation, the duration of paw retraction and heat radiation stimulation was recorded (s). The duration of paw retraction and heat radiation stimulation was measured continuously for 5 times with an interval of 20 min. The mean value was the PWL value of rats (s).

Treated rat spinal cord tissues were lysed with RIPA lysis buffer. According to the instructions of the respective ELISA kits, the concentrations of interleukin (IL)-6 and tumour necrosis factor-α (TNF-α) were measured.

After the rats were sacrificed, they were quickly perfused with 4% paraformaldehyde through the ascending aorta. Subsequently, spinal cord segments were collected, fixed, cryopreserved in 30% sucrose, embedded in cryostat (Sakura, 4583), and sectioned at 16 μm thick tissue sections. After sections were blocked with 4% normal Donkey Serum, primary antibody Iba1 were added for overnight incubation, followed by secondary antibody incubation. Subsequently, DAPI were added into prolong gold antifade reagent, which was then added dropwise to the sections and the images were acquired under a fluorescence microscope (Olympus).

Total protein was extracted from L4-L5 segments by utilized lysis buffer. The protein content was determined with a BCA kit. 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the protein suspension, and polyvinylidene difluoride (PVDF) membranes were used to transfer the protein band. Primary antibodies for JMJD1A, Iba1 and GAPDH were used to incubate the membranes after they had been blocked with 5% nonfat milk. After washing with Tris-buffered saline containing 0.1% tween-20 (TBST) buffer, HRP-conjugated goat anti-rabbit IgG was performed to incubate for 1 h. After washing with TBST, the protein bands were visualized with ECL detection reagent, the intensity of the protein bands was quantitatively analyzed using image software, and the relative expression of the protein of interest was calculated.

Total RNA was obtained from spinal cord tissue of rats through TRIzol reagent (Takara), extracted RNA was reverse transcribed to cDNA using primescript RT reagent kit. The single-strand cDNA was prepared employing Prime Script RT reagent Kit. Importantly, the software was then run in an ABI 7900HT RT-PCR system using the SYBR Premix Ex Taq II program. Ultimately, 2^−ΔΔCT method was used to calculate the expression of miR-362-3p.

The targeted binding sites between miR-362-3p and JMJD1A were predicted using Starbase bioinformatics tool. And miR-362-3p mimics or negative control were cotransfected with wild type JMJD1A (JMJD1A-wt) and mutant JMJD1A (JMJD1A-mut) luciferase reporter vectors into cells using Lipofectamine^TM 3000, followed by incubation 48 h and then detection luciferase activity.

Each experiment was independently repeated three times, and the results were expressed as mean ± standard deviation (SD) and analyzed using SPSS 21.0, with student’s t-test for differences between two groups and ANOVA for multiple groups. A value of P < 0.05 was considered statistically significant.

To examine the potential role of miR-362-3p in neuropathic pain, we established a rat model of CCI and further examined PWT and PWL at 0, 3, 7, and 14 days. The results demonstrated that CCI treatment caused a remarkable mechanical allodynia and thermal hyperalgesia (Figure 1(a) and Figure 1(b)). Moreover, RT-qPCR was performed to assess miR-362-3p expression in the spinal cord at segments L4-L5 and isolated microglias of CCI rats on day 0, 3, 7, and 14 following CCI. The results showed that downregulation of miR-362-3p was uncovered both in L4-L5 segments and isolated microglias (Figure 1(c) and Figure 1(d)).

Rats underwent CCI or sham surgery on day 3 after intrathecal injection of LV-miR-miR-362-3p or LV-NC. RT-qRCR demonstrated that injection of LV-miR-miR-362-3p lead to an effective increase on miR-miR-362-3p level (Figure 2(a)). The results of behavioral measurement of rats showed that miR-362-3p overexpression significantly reduced the PWT and PWL (Figure 2(b) and Figure 2(c)). Upregulation of miR-362-3 pdramatically increased IL-6 and TNF-α production in the L4-L5 segments of rats with CCI (Figure 2(d) and Figure 2(e)). Moreover, immunofluorescence revealed that overexpression of miR-362-3p markedly suppressed the expression of microglial marker Iba1 in the spinal L4-L5 segments of CCI rats (Figure 2(f)).

Through starbase databases retrieval, we found that there were highly conserved binding sites between miR-362-3p and JMJD1A, which suggested that JMJD1A might be a potential target of miR-362-3p (Figure 3(a)). Surprisingly, miR-362-3p mimics significantly reduced the luciferase activity of wild type JMJD1A, whereas the luciferase activity of mutant JMJD1A was not changed (Figure 3(b)). Moreover, miR-362-3p mimics downregulated JMJD1A protein expression, whereas miR-362-3p inhibitor upregulated JMJD1A protein expression (P < 0.01) (Figure 3(c)). All the results indicated that JMJD1A was the target gene of miR-362-3p.

To study whether JMJD1A plays an active part in miR-362-3p-mediated biological function in neuropathic pain development, we injected LV-miR-362-3p alone or together with LV-JMJD1A in CCI rats. We first found that JMJD1A expression was increased both in L4-L5 segments and isolated microglias following CCI (Figure 4(a) and Figure 4(b)). miR-362-3p elevation remarkably reduced JMJD1A level in CCI rats, which was counteracted by the JMJD1A overexpression (Figure 4(c)). JMJD1A overexpression substantially abolished miR-362-3p-mediated neuropathic pain-like behaviors in CCI rats (Figure 4(d) and Figure 4(e)).

ELISA and western blotting demonstrated miR-362-3p overexpression suppressed IL-6 and TNF-α secretion and Iba1 expression in CCI rats, whereas JMJD1A overexpression could inverted these changes (Figures 4(f)-(h)).