C57BL/6J mice (male, 25.0–30.0 g, 6–8 weeks old) were purchased from the Experimental Animal Centre of Chongqing Medical University (Chongqing, China) and randomly assigned to each experimental groups. Animal experimental protocols were approved by the Ethics Committee of Chongqing Medical University. Precautions were taken to minimise animal suffering and the number of animals used was kept to a minimum. All mice were housed under alternating 12-h light/dark cycles with constant temperature and humidity. Mice were given free access to food and water.
The experiments were conducted in 8-week-old male C57BL/6 mice weighing approximately 25.0–30.0 g at study initiation. The study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (approval number: 2020–127) and performed in accordance with the guidelines from the European Parliament and the International Association for the Study of Pain. The sciatic nerve injury model was constructed as described previously [12,13]. In brief, mice were anaesthetised with 1.5% isoflurane. The left common sciatic nerve was exposed by isolating it from the surrounding connective tissue and then crushed for 7 seconds using smooth forceps. Mice in the sham surgery group underwent the same procedure while the nerve remained intact.
Tactile allodynia evaluation
Paw mechanical withdrawal thresholds were evaluated using von Frey filaments in mice. The experimental mice were placed in a plexiglass cage with a grid at the bottom and adapted for 20 min until exploratory activities of the mice disappeared. The feet of mice were stimulated using a series of standardised von Frey filaments (0.4, 0.6, 1.4, 2.0, 4.0, 6.0, 8.0, and 15.0 g) according to the up-down method introduced by Chaplan et al. . The starting filament was set to 0.6 g and the mouse withdrawal or licking reaction was observed. Fibres were applied to the paw five times and the response ratio score was calculated as described previously . To avoid animal tolerance or hyperalgesia caused by frequent or prolonged stimulation, the stimulation time for each filament was limited to 1–2 seconds, and the interval between the two stimulations was 10 min.
Intrathecal (IT) injection of adeno-associated virus
All mice were weighed and randomly assigned to three groups: CircControl + control (n = 5); CircControl + chronic constriction injury (CCI)(n = 11); or circDym + CCI (n = 5). IT injections of 1×1012 vg /ml AAV9-shRNA-cZFP609 (adeno-associated virus-9 short-hairpin RNA; shcZFP609, HANBIO) or AAV9-shRNA-NC (shNC, HANBIO) were administered as previously described . For IT injections, animals were shaved, sterilised, and gripped at the pelvic girdle. A UMP3 micropump (80330, Sigma) with a 33 gauge 45° bevelled point needle (7803-05, Hamilton) was inserted at the marked mid-spinal line. After entering the lumbar cisterna, which was indicated by a sudden tail flick, 20 μl of AAV combined with 1% lidocaine hydrochloride were delivered to each mouse. Injections were conducted at a constant injection speed, with transient mouse limb weakness signifying a successful injection . The experimental group was further divided into two subgroups according to the time of injection. A one-time injection was performed in the low dose group with 20 μl AAV 21 days before surgery and a high dose group was injected two times at both 21 and 14 days before surgery.
Mouse spinal dorsal horn tissue isolation
Mice were deeply anaesthetised using isoflurane and rapidly decapitated. The lumbar enlargement segments of the spinal cords were transversely sectioned and dissected along the midline, and the dorsal part of the lumbar enlargement of sham surgery and CCI mice was collected and frozen at -80°C for subsequent processing.
Synaptosomes were prepared according to the detailed protocol of Dunkley et al. . In brief, spinal dorsal horns were homogenised in a modulated buffer by at least 10 strokes using a Teflon-glass grinder. The homogenate was centrifuged at 1000g for 10 min, the supernatant was isolated and applied onto a discontinuous Percoll gradient from 0% to 23%, and finally centrifuged again at 31,000g for 5 min to isolate the synaptosome fraction .
Cell culture and differentiation
Neuro-2a cells (N2a cells, mouse origin) obtained from American Type Culture Collection were cultured in dulbecco's modified eagle medium (Invitrogen Corporation) and 10% FBS (Wisent Canadian Laboratories) as previously described . During cell differentiation, the medium was switched to dulbecco's modified eagle medium with 0.5% FBS and 1 mM dbcAMP (Sigma) for 72 h . The cultures were then used for qRT-PCR and fluorescence in situ hybridization (FISH) analyses.
All procedures were approved by the ethics committee of Chongqing Medical University and written informed consent was obtained from all patients. The diagnosis of chronic NP was based on clinical symptoms, signs, and characteristic findings in the electrophysiological examinations of nerve conduction functions in nerves of all limbs. Patients with autoimmune, inflammatory, psychiatric, or neoplastic diseases were excluded. Nineteen patients with chronic NP symptoms were prospectively studied in the orthopaedics department at the First Affiliated Hospital of Chongqing Medical University, and the control group consisted of patients who lacked painful pathologies (n = 23). The Douleur Neuropathique 4 (DN4) questionnaire was used to assess the NP condition , which includes pain features and the occurrence of allodynia and hypoesthesia. During patient hospitalisation, 10 ml of peripheral venous blood was collected in an EDTA tube from the antecubital vein before treatment and the blood was centrifuged at 3000g for 5 min to separate the plasma. Plasma was recentrifuged for another 10 min at 1500g and stored at -80°C.
Total RNA extraction from cell lines, frozen neurological tissue, and synaptosomes was performed as previously described . Slight modifications were made according to the method described by Chen et al. . The concentration and purity of RNA were assessed using an SMA4000 microspectrophotometer (Merinton Instrument, Inc.) and a DYY-6C electrophoresis apparatus (Liuyi. Beijing).
Reverse transcription and qRT-PCR
RNA from cell lines and tissues was reverse-transcribed using a cDNA synthesis kit (RR047A, TaKaRa, China). CEP55, MTFR2, and PIMREG RNA levels were determined by quantitative PCR using a 2X SG Fast qPCR Master Mix (High Rox, B639273, BBI) and a fluorescence quantitative PCR instrument (ABI, Foster, CA, USA). GAPDH was used as an internal control gene and all primer sequences are listed below.
circDym (mouse) F: 5'-ctgatactccactcttttct-3'
circDym (mouse) R: 5'-GACCTTAGTTAGCGCAGCAA-3'
circDYM (human) F: 5'-TGGAAGAATTGCTGTGCTGTT-3'
circDYM (human) R: 5'-TGCACCAAGATTTCCTGTTCG-3'
Gapdh (mouse) F: 5'-TGACCTCAACTACATGGTCTACA-3'
Gapdh (mouse) R: 5'-CAAATCCGTTCACACCGACCT-3'
GAPDH (human) F: 5'-ACCATCTTCCAGGAGCGAGAT-3'
GAPDH (human) R: 5'-GGGCAGAGATGATGACCCTTT-3'
Nlgn2 (mouse) F: 5'-CTGCCCTACGTCTTTGGTGT-3'
Nlgn2 (mouse) R: 5'-AGCTCTGTGTGCAGGTTGTG-3'
Sequencing and bioinformatic analysis
The illumina Truseq RNA Sample Prep Kit (illumina, San Diego, USA) was used for library construction  and cDNA quantification was performed using TBS380 Picogreen (Invitrogen; Waltham, USA). Low Range Ultra Agarose (Bio-Rad Laboratories Hercules, USA), cBot TruseqPE Cluster Kit (llumina, San Diego, USA), and Hiseq 2000 Truseq SBS Kit (illumina, San Diego, USA) were used for library collection, amplification, and sequencing, respectively. The quality of raw data was assessed in an orderly manner using FastQC (version 0.11.4), Cutadapt (version 1.16), and HIST2 (version 2.1.0, with parameters set to -p 10 and RNA-strandness RF).
Differentially expressed circRNAs in the spinal dorsal horn (SDH) after injury were identified by Cao et al.  and synaptosome-specific expression of circRNAs were obtained from Agnieszka Rybak-Wolf et al. . circRNAs that are expressed at high levels in the mammalian brain were identified through GSE accession number GSE65926, while a synaptosome-specific expression set of circRNAs was downloaded from the Gene Expression Omnibus (GEO) database (GSE61991). The synaptosome-specific mRNA gene set was downloaded from the GEO database (GSE62573), while spatial single-cell transcriptome data were obtained from the Gene Expression Omnibus database (GSE103840).
StringTie (version 1.3.3b) and edgeR (version 3.24) were used to assess mRNA expression levels and differentially expressed gene (DEG) filtering. Next, DEGs were clustered and annotated with plot_cluster_exp (version 1.1.0) and GO_anot_exp (version 1.4.0).
The StarBase circinteractome was used as a screening target. The secondary and tertiary structures of circDym were formed by RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) and RNAComposer (http://rnacomposer.ibch.poznan.pl/). The crystal structure of Fmrp (binding site complex, PDB ID: 4OVA) was obtained from the Protein Data Bank and the nucleic acid-protein structures were predicted, scored, and visualised using NPDock (http://genesilico.pl/NPDock/help), which was performed using a hybrid docking algorithm by applying RNA-protein benchmarks. We also used catRAPID and PASSION to confirm our results (https://flagship.erc.monash.edu/PASSION/).
The Seurat package was used for integrated data analysis of single-cell seq data, principal components were filtered with P < 0.05, and cell subclusters were identified and visualised using t-distributed stochastic neighbour embedding with resolution = 0.50 . DEGs among cell subclusters were selected with a log2 fold change (FC) > 1.
Fluorescence in situ hybridization
The cells were incubated using the circDym FISH Probe (Red) and synthesised by Ribobio (Guangzhou, China). Hybridisation was performed at 55°C for 1.5 h using fluorescence-labelled probes in hybridisation buffer. After stringent washing with SSC buffer, the cell nuclei were counterstained with DAPI (Invitrogen) and images were acquired using a confocal laser-scanning microscope (Leica DM2500).
RNA immunoprecipitation (RIP)assay
The Magna RIP Kit (17–700, Millipore) was used to perform the RIP procedure. Tissues were lysed in RIP lysis buffer and immunoprecipitated with Fmrp antibody using protein magnetic beads. Next, complexes bound to the magnetic beads were immobilised by magnets, while the unbound materials were washed off. The remaining RNA was extracted.
Western blot analysis
The tissues were lysed with RIPA buffer containing protease inhibitors (Sigma-Aldrich). A BCA protein assay kit (Promega) was used for protein quantification. Western blot analysis was performed as described previously . The primary antibodies used in the western blot analysis were as follows: human/mouse anti-neuroligin 2 (PA5-18668, ThermoFisher Scientific, 1:500), mouse anti‐GABAARγ2 (MABN875, Millipore, 1:200), human/mouse anti-Fmrp (4317S, Cell Signaling Technology, 1:1000). Horseradish peroxidase-conjugated (HRP) goat anti-rabbit IgG (H+L) (4050–05, SouthernBiotech, 1:20000) was used as a secondary antibody. Protein levels were normalised to those of GAPDH.
Statistical analyses were performed using GraphPad Prism 8.0 and SPSS 21.0. Two groups were analysed using t-tests and differences among groups were compared by one-way analysis of variance. P < 0.05 was considered significant for all statistical comparisons.