Application of Nno gene in regulation and control of Kcnq2 expression and research method of Nno gene in regulation and control of Kcnq2 expression

Abstract

The invention discloses an application of a non-o gene in regulation and control of expression of Kcnq2. A chronic constriction injury rat model is combined with a multi-omics comprehensive analysis method, dynamic molecule and cell changes related to neuropathic pain are systematically analyzed, and it is found that overexpression of Non can recover expression of Kcnq2, pain sensitivity is relieved, and the neuropathic pain is relieved. The key effect of the Nono-Kcnq2 regulation axis in regulating neuronal excitability and neuropathic pain is emphasized, the application of the Nono-Kcnq2 regulation axis in preparing the medicine for treating the neuropathic pain is provided, and a new strategy is provided for studying treatment of the neuropathic pain.

Description

Technical Field

[0001] This invention belongs to the field of gene expression regulation technology, specifically involving the application and research method of Nono gene regulating Kcnq2 expression. Background Technology

[0002] Neuropathic pain is a debilitating condition characterized by spontaneous pain, hyperalgesia, and paresthesia, typically triggered by damage or disease of the somatosensory nervous system. It not only causes significant physical suffering but is also accompanied by a high incidence of mood disorders such as depression and anxiety, severely reducing patients' quality of life. Although current drug treatments can provide temporary relief by modulating nociceptive signals, their efficacy is often limited by side effects such as drug tolerance, psychological dependence, and neurotoxicity. Therefore, the effective management of neuropathic pain remains a significant challenge, primarily due to an insufficient understanding of its underlying pathophysiological mechanisms.

[0003] The pathogenesis of neuropathic pain involves complex peripheral and central processes. The spinal cord, as the primary pathway for transmitting nociceptive signals to the brain, involves intricate interactions between neurons, glial cells, and their supporting cells, driving the initiation and maintenance of pain. Following peripheral nerve injury, increased presynaptic glutamate release activates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and depolarization-dependent N-methyl-D-aspartate receptors (NMDARs) on the postsynaptic membrane. This triggers calcium-dependent signaling cascades and activation of extracellular signal-regulated kinases (ERKs), enhancing synaptic efficacy and long-term potentiation (LTP), a key process driving spinal cord sensitization. Microglia, resident immune cells in the spinal cord, undergo significant morphological, transcriptional, and functional changes upon activation, releasing pro-nociceptive and pro-inflammatory mediators that act on neurons, amplifying the activity of the spinal cord nociceptive circuit. This coordinated interaction between neurons and glial cells forms a maladaptive pain circuit that leads to the occurrence and persistence of neuropathic pain. It is worth noting that the above process involves a wide range of transcription factor (TF) changes, such as cAMP response element binding protein (CREB), activated transcription factor 3 (ATF3), and activated T cell nuclear factor type 1 (NFAT1), which affect the processing and transmission of pain signals by regulating the expression of target genes.

[0004] Despite significant progress in elucidating the molecular basis of neuropathic pain, the complex interactions between different cell types and their regulatory networks remain largely unexplored. This invention, using a rat model of chronic constrictive injury combined with multi-omics analysis, systematically elucidated the dynamic molecular and cellular changes associated with neuropathic pain. The study found that overexpression of Nono can restore Kcnq2 expression and reduce pain sensitivity, highlighting the crucial role of the Nono-Kcnq2 regulatory axis in regulating neuronal excitability and neuropathic pain, thus providing a new strategy for researching the treatment of neuropathic pain. Summary of the Invention

[0005] The purpose of this invention is to provide an application of Nono gene regulation of Kcnq2 expression.

[0006] Another objective of this invention is to provide a research method for regulating Kcnq2 expression by the Nono gene.

[0007] The application described in this invention is the use of the Nono-Kcnq2 regulatory axis in the preparation of drugs for treating neuropathic pain.

[0008] The Nono gene overexpression described in this invention increases the level of Nono protein and enhances the expression of Kcnq2 protein.

[0009] The research method described in this invention includes the following aspects: By integrating multi-omics approaches, the molecular characteristics of neuropathic pain in the CCI model were revealed; Proteomics analysis revealed the abnormal expression of glutamatergic synapse-related proteins in neuropathic pain; Phosphorylated proteomics analysis revealed the role of decreased potassium channel phosphorylation in glutamatergic synaptic activation during neuropathic pain; CatTFRE analysis revealed downregulation of the transcription factor Nono in neuropathic pain; To verify the role of Nono in Kcnq2 protein expression and the regulation of neuropathic pain.

[0010] Compared with the prior art, the present invention has the following beneficial effects: This invention uses a rat model of chronic constrictive injury combined with multi-omics comprehensive analysis to systematically analyze the dynamic molecular and cellular changes related to neuropathic pain. It found that overexpression of Nono can restore Kcnq2 expression and reduce pain sensitivity, highlighting the key role of the Nono-Kcnq2 regulatory axis in regulating neuronal excitability and neuropathic pain, and providing a new strategy for the study of neuropathic pain treatment. Attached Figure Description

[0011] Figure 1The workflow of pain-related behaviors and multi-omics approaches (where A represents the MWT values ​​(n=15) of the CCI group and sham-operated group at baseline and on days 3, 7, 11, 15, and 21 post-CCI, p<0.01 compared to the sham-operated group; B represents the TWL values ​​(p<0.01) of the CCI group and sham-operated group at baseline and on days 3, 7, 11, 15, and 21 post-CCI, p<0.01 compared to the sham-operated group; C represents the multi-omics analysis process, including scRNA-seq, spatial transcriptomics, catTFRE, proteomics, and phosphorylated proteomics; D is a bar chart showing the results at each...) E is a box plot showing the number of proteins identified in each sample (n=3); F is a protein abundance density distribution analysis (n=3); G is a bar plot showing the number of phosphorylation sites, phosphorylated peptides, and phosphorylated proteins identified in each sample (n=3); H is a box plot showing the relative abundance distribution of phosphorylated peptides identified in each sample (n=3); I is a phosphorylated peptide abundance density distribution analysis (n=3); J is a bar plot showing the number of transcription factors (TFs) and transcription regulatory factors (TCs) identified in each sample (n=3). Figure 2 Proteomics analysis revealed abnormal activation of glutamatergic neurons during the occurrence and progression of neuropathic pain (where A is a line graph of protein clusters showing significant differences in expression trends among the sham surgery 7-day group, CCI 7-day group, and CCI 21-day group; B is a heatmap of protein expression in different clusters among the sham surgery 7-day group, CCI 7-day group, and CCI 21-day group; CF is the pathway characteristics of each cluster of proteins shown by DAVID pathway enrichment analysis; G is a network diagram of the activation of glutamatergic synaptic pathways in the CCI7 and CCI21 groups). Figure 3Phosphoproteomics revealed a significant association between reduced potassium phosphorylation and excessive glutamatergic activation (where A is a bar chart showing the number of differentially expressed phosphoproteins and phosphorylation sites in the CCI7 and sham-operated groups; B is a bar chart showing the number of differentially expressed phosphoproteins and phosphorylation sites in the CCI21 and CCI7 groups; C is a Venn diagram showing the overlap between downregulated phosphorylation sites in the CCI7 and sham-operated groups and upregulated phosphorylation sites in the CCI21 and CCI7 groups; D is a bar chart showing the enrichment pathways of downregulated phosphorylation proteins in the CCI7 group; E presents the relationships between the three groups). Heatmap of expression patterns of phosphorylation sites related to potassium ion transport pathways; F is a network diagram of potassium ion channel interactions; G is a bar chart of potassium ion channel phosphorylation site expression levels (n=3); H is a volcano plot of the correlation between potassium ion channel phosphorylation sites and related molecules in the synaptic vesicle pathway; I is a scatter plot and regression curve of the correlation between Kcnq2_S352 and Stx2, and Kcnh2_S323 and Stx2; J is a flowchart illustrating the association mechanism between potassium ion channel phosphorylation downregulation and glutamatergic neuron overactivation in the CCI7 model. Figure 4 The role of downregulation of Nono activity in neuropathic pain based on TFRE analysis (wherein, A is a volcano plot showing the differentially expressed TFRE molecules between the CCI7 group and the 7-day sham surgery group; B is a volcano plot showing the differentially expressed TFRE molecules between the CCI21 group and the CCI7 group; C is a Venn diagram showing the specific upregulation and downregulation of TFRE molecules in the CCI7 group compared to the sham surgery group and the CCI21 group; DE are line graphs showing the specific upregulation and downregulation of TFRE molecules in the CCI7 group compared to the sham surgery group and the CCI21 group; F is a network diagram of differentially expressed TFRE molecules interactions; GH is a bar chart showing the differential expression of Nono and its phosphorylation sites, and interacting transcriptional cofactors in the three experimental groups (n=3); I is a kinase library prediction of regulatory kinases for phosphorylation sites Nono_T455 and Nono_T433; J is a scatter plot and regression analysis showing the correlation between Nono TFRE activity and potassium ion channel Kcnq2 expression; K is a flowchart illustrating the association mechanism between Nono phosphorylation downregulation and neuronal overactivation in neuropathic pain. Figure 5Nono overexpression relieves neuropathic pain by transcriptionally activating Kcnq2 in the spinal cord (where, A - B: After CCI surgery, mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) behavioral assessments were performed by intrathecal injection of an adenovirus expressing Nono (CCI + Ad - Nono - OE) or a control virus (CCI + Ad - NC) (n = 8); C: Representative Western blot results of Nono and Kcnq2 protein expression in L4 - L6 spinal cord tissues collected 21 days after surgery (n = 3); D: Quantitative Western blot results showed decreased levels of Nono and Kcnq2 in CCI rats, while Nono overexpression restored their expression (n = 3); E: ChIP experiments confirmed enhanced binding of Nono to the Kcnq2 promoter in the spinal cord (n = 3); F: Dual - luciferase reporter assays in HEK - 293T cells showed that Nono overexpression enhanced luciferase activity driven by the Kcnq2 promoter compared with the control group (n = 3); Data are presented as mean ± standard deviation; **p < 0.01 (Mann - Whitney U test) compared with the sham - operated group; #p < 0.05, ##p < 0.01 (Mann - Whitney U test) compared with the CCI + Ad - NC group (A, B); *p < 0.05, **p < 0.01 (one - way ANOVA) compared with the corresponding control group (C - F)). Detailed implementation manners

[0012] The technical solutions of the present invention will be further specifically described below through specific embodiments.

[0013] Example 1 Application of the Nono - Kcnq2 regulatory axis in the preparation of drugs for treating neuropathic pain: Overexpression of the Nono gene increases the level of Nono protein, enhances the expression of Kcnq2 protein, and thus relieves neuropathic pain.

[0014] Example 2 Research method for the Nono gene regulating Kcnq2 expression 1. Experimental method 1.1 Experimental materials Fifty - three Sprague - Dawley rats (7 - week - old, male, weighing 150 to 170 g) were purchased from Henan Skibes Biotechnology Co., Ltd. (certificate number: SCXK[Yu]2020 - 0005, Anyang, China) and housed in a specific - pathogen - free animal laboratory (temperature: 22 - 25°C, humidity: 50 - 60%, 12 - hour day - night cycle). The rats were randomly divided into a sham - operated group (n = 23) and a CCI group (n = 30). All animal experimental procedures were approved by the Experimental Animal Ethics Committee of Guizhou University of Traditional Chinese Medicine (approval number: 20240826003).

[0015] 1.2 CCI Model The method for preparing the CCI model was based on previous literature. In brief, rats were deeply anesthetized via intraperitoneal anesthesia with sodium pentobarbital and placed in a prone position. The left sciatic nerve was fully exposed, and after skin incision and muscle separation, it was ligated four times at 1 mm intervals with 4-0 silk sutures. The sham-operated group was identical to the experimental group, except that no ligation was performed.

[0016] 1.3 Construction and Intrathecal Injection of Nono Overexpressing Adenovirus in Rats The rat Nono gene was cloned into the GV314 vector, using BamHI and AgeI restriction enzyme sites during the cloning process. The recombinant plasmid was used for adenovirus packaging, and the virus was purified and titrated using a plaque formation assay, yielding a final titer of [missing value]. PFU / mL. Rats were randomly divided into four groups: sham-operated group, CCI group, CCI plus negative control adenovirus group (CCI + Ad-NC), and CCI plus Nono overexpressing adenovirus group (CCI + Ad-Nono-OE). The CCI model was established according to the previously described method. Intrathecal injection of adenovirus was performed on day 1 post-surgery. Rats in the Ad-NC group were injected with 10 µL of control virus (PFU / mL). PFU / mL), injected into each rat PFU; Rats in the Ad-Nono-OE group were injected with 67µL of Nono overexpression virus (PFU; Ad-Nono-OE group rats were injected with PFU; Ad-Nono-OE group rats ... (PFU / mL) to ensure the same dosage. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured before surgery and on days 0, 3, 7, 14, and 21 after surgery to assess nociceptive sensation.

[0017] 1.4 Behavioral Testing To assess mechanical and thermal hyperalgesia, mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured using von Frey tactile wires (RWD Life Science, Shenzhen, China) and a plantar thermal stimulator (Model XR1800, Shanghai Xinruan Information Technology Co., Ltd., Shanghai, China), respectively. Measurements were taken before CCI surgery and on days 3, 7, 11, 14, and 21 post-surgery. Before each test, rats were placed alone in a transparent plexiglass box for 30 minutes to acclimatize. Subsequently, a series of von Frey wires (target force 0.008–300 g) or thermal radiation sources were used to stimulate the mid-plantar surface of the ipsilateral hind paw until positive responses such as paw lifting or licking occurred. Each test was repeated three times, with a 5-minute interval, and the average value was taken as the MWT and TWL results.

[0018] 1.5 Sample Collection and Preparation On the 7th day after CCI surgery, 15 rats were randomly selected from each group and subjected to deep anesthesia to obtain ipsilateral L4-L6 segment spinal cord tissue; the remaining rats were sacrificed 21 days after surgery and spinal cord samples were collected.

[0019] 1.6 Proteomics Analysis Samples were rinsed three times with phosphate-buffered saline (PBS) and then finely minced. Tissue lysis was performed using a lysis buffer containing 8M urea, 100mM Tris-HCl, and a protease / phosphatase inhibitor. The lysate was sonicated (1 minute, 3-second on / 3-second off cycling, 25% amplitude), centrifuged at 14000g for 10 minutes to obtain the supernatant whole tissue protein extract, and protein concentration was determined using the Bradford method. 20 μL of the protein solution was mixed with 2× loading buffer and boiled for 5 minutes. Separation was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie Brilliant Blue R-250 staining and imaging. For each sample, 200 μg of protein was reduced with 10 mM dithiothreitol (DTT) at 56°C for 30 minutes, followed by alkylation with 10 mM iodoacetamide (IAA) at room temperature in the dark for 30 minutes. The protein was then transferred to an ultrafiltration device, and 200 μL of 8 M urea bicarbonate buffer was added. After centrifugation and washing, 50 mM NH4HCO3 buffer was added, and the protein was digested overnight at 37°C with 4 μg of trypsin (Promega). The filtrate was collected and the peptide samples were vacuum dried. Peptides were reconstituted in 0.1% formic acid aqueous solution (solvent A) and loaded onto a trap column using an EASY nLC 1200 system. Gradient elution was performed for 120 minutes at a flow rate of 600 nL / min on a 15 cm analytical column (solvent B: 0.1% formic acid - 80% acetonitrile). Detection was performed using quadrupole-orbitrap mass spectrometry (QE-Orbitrap) in data-dependent acquisition mode: primary scan resolution 120,000 (AGC 3e6), secondary scan resolution 7,500 (AGC 5e4, HCD collision energy 30%), dynamic exclusion for 40 seconds, and FAIMS dual-voltage (-45V, -65V) mode switching. Raw data were processed using the iProteome platform. Differential protein hierarchical clustering was visualized using the Pheatmap package. Gene Ontology (GO) and Kyoto Encyclopedia of Genetics and Genomes (KEGG) pathway enrichment analyses were performed using the DAVID tool. Protein-protein interaction networks were constructed using the STRING database.

[0020] 1.7 Phosphorylated Proteomics Protein extraction, SDS-PAGE electrophoresis, filter-assisted sample preparation (FASP), and mass spectrometry detection were performed according to the aforementioned proteomics methods. For trypsin-digested peptides, the High-Select Fe-NTA phosphorylated peptide enrichment kit (Thermo Fisher Scientific) was used for specific screening: peptides were resuspended in the kit's binding / washing buffer, mixed with pre-equilibrated resin, and incubated at room temperature for 30 minutes; they were then washed three times with binding / washing buffer and twice with deionized water; finally, the enriched peptides were collected with elution buffer, concentrated by vacuum centrifugation at 30°C (SpeedVac), and dried for analysis. Raw mass spectrometry data were processed using the iProteome platform, and bioinformatics analysis covered four modules: hierarchical clustering analysis of phosphorylated peptides; Gene Ontology (GO) functional annotation; Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation; and construction of a phosphorylated protein-protein interaction network (PPI) based on the STRING database.

[0021] 1.8 catTFRE analysis After being washed twice with pre-cooled PBS, tissue samples were resuspended in 800 μL of cytoplasmic extraction buffer I (NE-PER kit, Thermo Fisher) and homogenized. Nucleoprotein extracts were quantified using the Bradford method (Bio-Rad protein assay kit), and 20 μL of protein was verified by SDS-PAGE electrophoresis and Coomassie Brilliant Blue R-250 staining. 3 pmol of biotinylated catTFRE double-stranded DNA probes were immobilized on Dynabeads streptavidin magnetic beads (M-280, Thermo Fisher) and co-incubated with the nucleoprotein extracts. EDTA / EGTA chelating agent was added to the system, and the final NaCl concentration was adjusted to 200-250 mM. Incubation was carried out at 4°C for 2 hours. After discarding the supernatant, the magnetic beads were washed sequentially with NETN buffer (100 mM NaCl, 20 mM Tris-Cl, 0.5 mM EDTA, 0.5% NP-40) and PBS. The catTFRE capturing beads were co-incubated with 40 ng / μL trypsin / 50 mM NH4HCO3 solution and digested overnight at 37°C. The peptides were eluted with 50% acetonitrile solution containing 0.1% formic acid, concentrated by Concentrator Plus vacuum centrifugation (Eppendorf), and dried.

[0022] The dried peptide fragments were reconstituted with 0.1% formic acid aqueous solution (solvent A) and loaded into a self-packed trapping column (2cm×100μm, 3μm C18 packing material, Dr. Maisch GmbH) using an EASY nLC 1200 ultra-high performance liquid chromatography system. Linear gradient elution was then performed on an analytical column (30cm×150μm, 1.9μm C18 packing material) at a flow rate of 600 nL / min for 150 min (solvent B: 0.1% formic acid-80% acetonitrile). Eluted peptides were ionized by 2 kV electrospray ionization and analyzed by a Q Exactive HF-X quadrupole-orbit trap mass spectrometer in data-dependent acquisition mode: primary mass spectrometry scan range m / z 300-1400, resolution 120,000, AGC target value 3e6, ion implantation time 80 ms; high-order collisional dissociation (HCD) was performed using the top 60 precursor ions with a normalized collision energy of 27%; secondary fragmentation analysis resolution 7,500, AGC target value 5e4, ion implantation time 20 ms; dynamic exclusion time 25 seconds.

[0023] The raw data (.RAW files) were qualitatively and quantitatively analyzed using the iProteome one-stop cloud platform. Hierarchical clustering and visualization were performed using the PheatmapR package (v1.0.8). Gene ontology (GO) annotation mapping and R language visualization were completed using Blast2GO. Simultaneously, phosphorylated peptide clustering analysis, KEGG pathway annotation, and STRING protein interaction network construction were carried out.

[0024] 1.9 Immunofluorescence After dehydration with gradient ethanol and clearing with xylene, tissue blocks were embedded in paraffin in three stages at 60°C. Sections were mounted on glass slides and baked at 60°C for 3 hours. The dewaxing process involved xylene → gradient ethanol → distilled water. Antigen retrieval was performed by incubation at 95°C for 15 minutes with 0.01M EDTA buffer (pH 9.0), followed by blocking of endogenous peroxidase with 3% hydrogen peroxide for 15 minutes. Sections were blocked with normal goat serum, incubated with primary antibody overnight at 4°C, and then co-incubated with HRP-conjugated secondary antibody for 50 minutes. After reacting with tyramine signal-amplifying fluorescent dye (Carestream Biotech, catalog number RC0086Plus-45RM) for 5 minutes, antibody was eluted at 95°C for 25 minutes, and this process was repeated four times to complete multiple labeling. Finally, the sections were stained with DAPI, mounted with anti-quenching mounting medium, and imaged under a fluorescence microscope. The primary antibody information is as follows: neuronal marker RBFOX3 (Abcam ab177487), nuclear transcription factor Nono (Abcam ab70335), potassium channel protein Kcnq2 (Abcam ab22897), peripheral neurofilament protein Prph (Proteintech 17399-1-AP), and synaptic adhesion protein Nrxn3 (Novus Biologicals NBP1-88424).

[0025] 1.10 Western Blot Experiment Spinal cord tissue was homogenized and lysed with RIPA lysis buffer containing protease and phosphatase inhibitors. After incubation on ice for 30 minutes, the mixture was centrifuged at 12,000 rpm for 5 minutes at 4°C. The supernatant was collected and the protein concentration was determined using a BCA protein quantification kit (GBCBIO, China). 40 μg of protein sample was mixed with 5× loading buffer and boiled for 10 minutes to denature the protein. After separation by SDS-PAGE gel electrophoresis, the protein was transferred to a PVDF membrane using a semi-dry transfer system. The membrane was blocked with 5% skim milk powder / TBST solution at room temperature for 2 hours and incubated overnight at 4°C with primary antibody. The target proteins were Nono (Abcam ab70335) and Kcnq2 (Abcam ab22897), and the internal control was GAPDH (Proteintech 60004-1-Ig). After washing the membrane five times with TBST buffer, it was incubated with HRP-conjugated secondary antibody at room temperature for 2 hours. The protein bands were visualized using an enhanced chemiluminescence (ECL) system, and the optical density was quantified using Image-Pro Plus software with GAPDH as the internal control.

[0026] 1.11 ChIP Experiment ChIP analysis was performed on spinal cord tissues from sham-operated rats and CCI rats on day 21 post-surgery using the EpiQuik Chromatin Immunoprecipitation Kit (Epigentek, Farmingdale, NY, USA). Tissues were minced, homogenized with PBS, cross-linked with 1% formaldehyde at room temperature for 10 minutes, and the cross-linking was terminated with 1.25M glycine. The tissues were then centrifuged and washed with PBS. After nuclear lysis, chromatin was sheared by sonication. The supernatant was diluted with CP4 buffer, and a portion was retained for input samples. Immunoprecipitation was performed using CP2-pretreated strips, with 4 µg of anti-NONO antibody (Proteintech, ILLU, USA, catalog number 11058-1-AP) or normal mouse IgG (negative control) added. After incubation, DNA-protein complexes were eluted, and cross-linking was reversed at 65°C. The samples were then purified by column centrifugation. The final analysis was performed using AceQ qPCR SYBR. Green premix (Vazyme, Nanjing, China) and primers (forward 5'-AGGTAGGTGATGGTAAA-3' / reverse 5'-CTGGGTAAGGGAGGAGG-3') were used to quantify KCNQ2 promoter DNA enrichment by qPCR, and the input group, IgG and immunoprecipitation group of the two groups of samples were analyzed in parallel.

[0027] 1.12 Dual-luciferase reporter gene assay HEK-293T cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (37°C, 5%). Environment), inoculated into 12-well plates (per well) The following day, cells were transfected with Lipofectamine™ 2000 (Invitrogen, USA) according to the manufacturer's instructions: ① pcDNA3.1+pGL3-Basic+pRL-TK, ② pcDNA3.1-Nono+pGL3-Basic+pRL-TK, ③ pcDNA3.1+pGL3-Kcnq2 promoter+pRL-TK, ④ pcDNA3.1-Nono+pGL3-Kcnq2 promoter+pRL-TK. Cells were lysed 48 hours after transfection, and luciferase activity was measured using a dual-luciferase reporter gene assay kit (Beyond Biotech, Shanghai, China). The ratio of firefly luciferase activity to Renilla luciferase activity was used to assess the transcriptional activation effect of Nono protein on the Kcnq2 promoter.

[0028] 1.13 Quantitative and Statistical Analysis Data are expressed as mean ± standard deviation. Normality was tested using the Shapiro-Wilk method, and homogeneity of variance was verified using Levene's test. When data simultaneously met the conditions of normality (P > 0.05) and homogeneity of variance (P > 0.05), one-way ANOVA combined with the least significant difference (LSD) method was used for multiple comparisons. For non-normal data, the Kruskal-Wallis H test was used to assess overall differences between groups, and the Mann-Whitney U test was used for pairwise comparisons between groups. Statistical significance was defined as P < 0.05. All analyses were performed using IBM SPSS Statistics 26.0 software.

[0029] 2. Experimental Results 2.1 Integrating multi-omics approaches revealed the molecular characteristics of neuropathic pain in the CCI model. To assess mechanical and thermal hyperalgesia, the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured at baseline and on days 3, 7, 11, 15, and 21 post-CCI. From day 3 post-surgery, the MWT and TWL values ​​in the CCI model rats significantly decreased (p < 0.01) and remained at low levels, reflecting a marked state of hyperalgesia. Figure 1 A and 1B). To explore the molecular mechanisms of neuropathic pain, we performed multi-omics integrated analysis of spinal cord tissues from the sham-operated group, and the groups on day 7 (CCI7) and day 21 (CCI21) after CCI surgery, including catTFRE analysis, proteomics, and phosphoproteomics. Figure 1C). This integrated approach provides in-depth analysis of the characteristics of driving molecular changes in CCI-induced neuropathic pain. Proteomics and phosphoproteomics analyses identified a large number of proteins, phosphorylation sites, phosphorylated peptides, and phosphorylated proteins in all samples, with good consistency across biological replicates. Figure 1 D and 1G). Box plots show the distribution of relative protein abundance and phosphorylated peptide abundance in the sham-operated group, CCI7 group, and CCI21 group, with consistent patterns observed among biological replicates within each group ( Figure 1 E and 1H). Density distribution analysis further validated the trends in the abundance of proteins and phosphorylated peptides in each group, confirming the reliability and reproducibility of the proteomic and phosphomic data. Figure 1 F and 1I). catTFRE analysis detected a large number of transcription factors (TFs) and transcriptional cofactors (TCs) in spinal cord tissue. Figure 1 J).

[0030] 2.2 Proteomics analysis revealed abnormal expression of glutamatergic synapse-related proteins in neuropathic pain. We used proteomics to detect the dynamic changes in protein expression in the ipsilateral L4-L6 spinal cord tissue of a neuropathic pain model from day 7 to day 21 after CCI surgery. Analysis identified four protein clusters with different temporal expression trends in the sham7, CCI7, and CCI21 groups, and their expression patterns were visualized using line plots and Z-score heatmaps. Figure 2 A and 2B). Pathway enrichment analysis using the DAVID database revealed the specific biological processes and signaling pathways involved in each protein cluster. Figure 2 C-2F). Cluster 1 showed a sustained upregulation trend at both CCI7 and CCI21, enriched in glutamatergic synapses, Parkinson's disease, and intracellular protein transport pathways. Cluster 2 was significantly downregulated at CCI7 and maintained low expression at CCI21, mainly involved in ATP binding, phosphorylation, and peroxisome function. Cluster 3 reached its peak expression at CCI7 and then declined, enriched in pathways including glutamatergic synapses, synaptic structure organization, and neurotrophic factor signaling pathways. Cluster 4 was downregulated at CCI7 and gradually recovered at CCI21, closely related to neural development and synaptic plasticity pathways, such as long-term potentiation (LTP) and axonal guidance. The glutamatergic synapse-related signaling pathway map comprehensively summarizes its molecular activities involved in neuropathic pain. Figure 2G). At multiple time points, significant changes in the expression of proteins associated with neurotransmitter release (such as synaptic vesicle circulation), receptor activity (such as NMDA and AMPA receptors), and intracellular signal transduction pathways were observed, suggesting that abnormal expression of glutamatergic synaptic proteins may be associated with a potential mechanism for pain progression.

[0031] 2.3 Phosphorylated proteomics analysis reveals the role of decreased potassium channel phosphorylation in glutamatergic synaptic activation during neuropathic pain. like Figure 3 As shown in Figure A, compared with the sham surgery group, the levels of phosphorylated proteins and their phosphorylation sites (serine, threonine, and tyrosine) were significantly decreased on day 7 after CCI surgery. However, on day 21 after CCI surgery, a significant upregulation of numerous phosphorylated proteins and phosphorylation sites was observed compared to day 7, particularly at the serine and threonine sites. Figure 3 B). The Venn diagram revealed a total of 169 phosphorylation sites downregulated in CCI7 vs. sham7 and upregulated in CCI21 vs. CCI7, involving 147 co-phosphorylated proteins, which may serve as regulators of early pathological damage or recovery processes in neuropathic pain. Figure 3 C). These phosphorylated proteins are mainly enriched in biological processes related to neurons and synapses, including pathways such as glutamatergic synapses, neuronal cell bodies, neuronal projections, chemosynaptic transmission, and potassium ion transport. Figure 3 D). Among these pathways, aberrant changes in potassium channel phosphorylation levels have received particular attention as an important potential mechanism of neuropathic pain. The heatmap shows the expression dynamics of potassium channel-related phosphorylated proteins in the sham7, CCI7, and CCI21 groups: phosphorylation levels were generally downregulated in the CCI7 group, while partial recovery was observed in the CCI21 group. Figure 3 E). The network diagram illustrates the interactions between potassium channel-related proteins, including Kcnq2, Kcnh2, Slc12a5, Kcna6, Kcnab2, and Kcnc2, etc. Figure 3 F); the box plot details the trend of significantly reduced phosphorylation levels of the aforementioned key proteins in CCI7, with partial recovery in CCI21. Figure 3 G). Notably, potassium channel-related phosphorylation sites—particularly Kcnq2_S352 and Kcnh2_S323—are significantly enriched in the synaptic vesicle pathway in the WikiPathways database. Figure 3H). Correlation analysis showed that Kcnq2_S352 expression was significantly negatively correlated with Stx2 expression (R = -0.75, p = 0.021), and Kcnh2_S323 expression was also negatively correlated with Stx2 (R = -0.70, p = 0.035), suggesting that Kcnq2 and Kcnh2 may be involved in the regulation of synaptic vesicles. Figure 3 I). In summary, the decrease in phosphorylation levels at key potassium channel sites is closely related to increased pain sensitivity, consistent with the classic mechanism by which decreased potassium permeability leads to neuronal overexcitation (I). Figure 3 J).

[0032] 2.4 catTFRE analysis showed that the activity of transcription factor Nono was downregulated in neuropathic pain. catTFRE analysis was used to identify changes in the activity of transcription factors (TFs) in the spinal cord during neuropathic pain, highlighting their role in regulating glutamatergic excitability. On day 7 after CCI surgery, compared with the sham-operated group, significant changes were observed in TFs and transcriptional cofactors (TCs) in the spinal cord, with 37 upregulated genes (e.g., Nedd8, Trim25, Tcf25) and 27 downregulated genes (e.g., Nono, Spen) detected. Figure 4 A). On day 21 post-CCI surgery, compared to day 7, 9 genes were upregulated (e.g., Arid2, Hmnp, Nsd2) and 21 genes were downregulated (e.g., Dhx9, Mixip); Figure 4 B). Six distinct gene expression clusters revealed different temporal expression dynamics among sham7, CCI7, and CCI21, with trends displayed using line plots and expression levels highlighted by Z-score heatmaps (Figure S1A). Venn plot analysis identified 10 differentially expressed TFs and TCs common between CCI7 vs. sham7 and CCI21 vs. CCI7. Figure 4 C). These shared factors exhibited different expression dynamics at three time points: factors downregulated in CCI7 (such as Hnrnpu, Psip1, Rbm15) showed a "V"-shaped recovery trend, while upregulated factors (such as Dhx9, E2f2, Ilf2, Lrprc, Paf1, Raly, Sox9) showed an inverted "V"-shaped change, reflecting their dynamic regulatory role in the progression of neuropathic pain. Figure 4 D and 4E). The interaction network diagram of differentially expressed TFs and TCs depicts the changes in the regulatory relationship between CCI7 vs. sham7 and CCI21 vs. CCI7. Figure 4 F).

[0033] To further elucidate the regulatory dynamics of the aforementioned TFs, we analyzed their TFRE activities in detail. The transcription factor activity of Nono significantly decreased on day 7 after CCI surgery and remained at a low level on day 21 (p < 0.05). Its interacting TCs (such as Spen, Taf15, Hnrnpu, and Smarca5) also showed a similar downregulation trend. Figure 4 G). Meanwhile, phosphorylation levels of Nono at T433 and T455 sites were significantly reduced on days 7 and 21 after CCI surgery (p < 0.05), suggesting that Nono may play a key regulatory role in the development of neuropathic pain. Figure 4 H). Kinase library analysis results indicate that VRK2 is the most likely candidate kinase to simultaneously phosphorylate both T455 and T433 sites of Nono, suggesting it may serve as a core regulator of Nono phosphorylation. Figure 4 I). Scatter plots and regression analysis showed a significant positive correlation between Nono transcriptional activity and Kcnq2 protein abundance (R = 0.72, p = 0.028). Figure 4 J). Multiple potential Nono binding sites were identified in a 2 kb region upstream of the Kcnq2 gene transcription start site. These sites were labeled with scores, strand orientation, and sequence patterns, supporting the possibility that Nono may regulate Kcnq2 transcriptional expression by binding to this region (Figures S1B and S1C). In summary, CCI leads to a decrease in Nono phosphorylation at T433 / T455, which in turn causes a decrease in its transcription factor activity, resulting in reduced Kcnq2 protein expression and ultimately promoting neuronal overactivation. Figure 4 K).

[0034] 2.5 Nono overexpression alleviates neuropathic pain by transcribedly activating spinal cord Kcnq2. To determine whether Nono plays a causal role in the regulation of neuropathic pain, we constructed a recombinant adenovirus carrying Nono and injected it intrathecally into rats after CCI surgery. Compared with CCI rats receiving the control virus, Nono overexpression significantly increased the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in rats from day 3 to day 21 post-surgery. Figure 5 (A and 5B) indicates a continued decrease in mechanical and thermal hypersensitivity responses. To further verify the molecular effects of Nono overexpression in vivo, we collected spinal cord tissue on day 21 post-surgery for protein level detection. Western blot results showed that, compared with the sham-operated group, the expression levels of Nono and Kcnq2 proteins in the spinal cord of CCI rats were significantly downregulated ( Figure 5 C and 5D).

[0035] Notably, the level of Nono protein in the spinal cord of Nono-overexpressing rats was significantly restored, accompanied by a significant increase in Kcnq2 protein expression. Figure 5 The results (C and 5D) suggest that Nono can positively regulate Kcnq2 expression in damaged spinal cord. To investigate its mechanism of action, we performed chromatin immunoprecipitation (ChIP) experiments on spinal cord tissue. The results showed that Nono can directly bind to the conserved region of the Kcnq2 promoter, indicating that it may function as a transcriptional regulator of Kcnq2. Figure 5 E). Consistent with this, dual-luciferase reporter assays showed that Nono co-transfection significantly enhanced luciferase activity driven by the Kcnq2 promoter. Figure 5 F), further supporting its transcriptional activation function. In summary, downregulation of Nono caused by nerve injury can inhibit the transcriptional activation of Kcnq2, thereby reducing the expression of Kcnq2 channels in the spinal cord and promoting pain sensitization in neuropathic pain. The restoration of Nono expression, however, reverses this process by enhancing Kcnq2 transcription, thus alleviating the pain hypersensitivity state.

[0036] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Use of a Nono gene to regulate expression of Kcnq2, characterized in that, The application of a Nono-Kcnq2 regulatory axis in the preparation of a drug for treating neuropathic pain.

2. Use according to claim 1, characterized in that, The overexpression of the Nono gene increases the level of Nono protein and enhances the expression of Kcnq2 protein.

3. A research method for use as claimed in any of claims 1-2, characterized in that, The method The application relates to the following aspects: Molecular characteristics of neuropathic pain in a CCI model are revealed by integrating multi-omics methods; Abnormal expression of glutamatergic synapse-related proteins in neuropathic pain is revealed by proteomic analysis; The role of reduced potassium channel phosphorylation level in glutamatergic synapse activation in neuropathic pain is revealed by phosphoproteomic analysis; The down-regulation of the activity of transcription factor Nono in neuropathic pain is found by catTFRE analysis; The role of Nono in the expression of Kcnq2 protein and the regulation of neuropathic pain is verified.

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