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Spinal CircKcnk9 Regulates Chronic Visceral Hypersensitivity of Irritable Bowel Syndrome

  • Zhong Chen
    Affiliations
    Pain Research Institute, Fujian Provincial Key Laboratory of Brain Aging and Neurodegenerative Diseases, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China.
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  • Yuan Liu
    Affiliations
    Pain Research Institute, Fujian Provincial Key Laboratory of Brain Aging and Neurodegenerative Diseases, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China.
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  • Xianhe Wu
    Affiliations
    Pain Research Institute, Fujian Provincial Key Laboratory of Brain Aging and Neurodegenerative Diseases, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China.
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  • Wei Lin
    Affiliations
    Department of Pediatrics, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China.
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  • Zihan Liu
    Affiliations
    Pain Research Institute, Fujian Provincial Key Laboratory of Brain Aging and Neurodegenerative Diseases, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China.
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  • Yang Huang
    Affiliations
    Pain Research Institute, Fujian Provincial Key Laboratory of Brain Aging and Neurodegenerative Diseases, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China.
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  • Yu Chen
    Affiliations
    Pain Research Institute, Fujian Provincial Key Laboratory of Brain Aging and Neurodegenerative Diseases, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China.
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  • Ying Tang
    Affiliations
    Pain Research Institute, Fujian Provincial Key Laboratory of Brain Aging and Neurodegenerative Diseases, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China.
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  • Aiqin Chen
    Affiliations
    Pain Research Institute, Fujian Provincial Key Laboratory of Brain Aging and Neurodegenerative Diseases, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China.
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  • Chun Lin
    Correspondence
    Address reprint requests to Chun Lin, MD, PhD, Department of Pediatrics, The First Affiliated Hospital of Fujian Medical University, No. 20, Chazhong Road, Taijiang District, Fuzhou 350005, Fujian, China.
    Affiliations
    Pain Research Institute, Fujian Provincial Key Laboratory of Brain Aging and Neurodegenerative Diseases, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, China.

    Department of Pediatrics, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China.
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Open AccessPublished:October 14, 2022DOI:https://doi.org/10.1016/j.jpain.2022.10.007

      Highlights

      • Spinal circKcnk9 was involved in visceral hypersensitivity of IBS-like rats.
      • Spinal high circKcnk9 facilitated STAT3 expression by inhibiting miR-124-3p silencing effect in Irritable bowel syndrome (IBS).
      • The study reveals a novel epigenetic mechanism of visceral hypersensitivity in IBS.
      • ShcircKcnk9 could be used as a candidate for the treatment of chronic functional visceral pain.

      Abstract

      Dysregulation of circular RNAs (circRNAs) has been reported to be functionally associated with chronic pain, but it is unknown whether and how circRNAs participate in visceral hypersensitivity. The expression of circKcnk9 was increased in spinal neurons of irritable bowel syndrome (IBS)-like rats. ShcircKcnk9 attenuated visceral hypersensitivity and inhibited c-Fos expression in IBS-like rats, whereas overexpression of spinal circKcnk9 induced visceral hypersensitivity and increased c-Fos expression in control rats. Furthermore, circKcnk9 was found to act as a miR-124-3p sponge. MiR-124-3p antagomir restored pain responses downregulated by shcircKcnk9 in IBS-like rats. Finally, the signal transducer and activator of transcription 3 (STAT3), validated as a target of miR-124-3p, could play a critical role in visceral hypersensitivity by regulating NSF/GluR2.
      Perspective. Spinal circKcnk9 functions as a miR-124-3p sponge to promote visceral hypersensitivity by regulating the STAT3/NSF/GluR2 pathway. This pathway might provide a novel epigenetic mechanism of visceral hypersensitivity and a potential circRNA therapeutic target for IBS.

      Graphical abstract

      Keywords

      Irritable bowel syndrome (IBS) is a chronic functional intestinal disease characterized by chronic abdominal pain or discomfort that affects approximately 11.2% of the worldwide population.
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      However, it remains unknown whether circRNA is involved in visceral hypersensitivity.
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      CircAnks1a in the spinal cord regulates hypersensitivity in a rodent model of neuropathic pain.
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      Our circRNA sequencing indicated that the expression of circKcnk9 was increased in the spinal dorsal horn of IBS-like rats. However, the role of circKcnk9 in visceral hypersensitivity remains unknown.
      CircRNA coexpression with miRNA in the cytoplasm serves as a ceRNA mechanism that regulates the expression of target genes.
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      Natural RNA circles function as efficient microRNA sponges.
      MiRNAs also belong to noncoding RNAs characteristic of short chains that participate in multiple physiological and pathological processes. MiR-124 is highly expressed in the nervous system.
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      An updated role of microRNA-124 in central nervous system disorders: A review.
      Spinal miR-124 is vital to regulate the roles of neurons and microglia in neuropathic pain.
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      MicroRNA-124-3p attenuates the development of nerve injury-induced neuropathic pain by targeting early growth response 1 in the dorsal root ganglia and spinal dorsal horn.
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      miR-124-3p attenuates neuropathic pain induced by chronic sciatic nerve injury in rats via targeting EZH2.
      Study has shown that miRNA could regulate the expression of signal transducer and activator of transcription 3 (STAT3) by targeting the 3′-UTR, including miR-124-3p.
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      miR-17 family of microRNAs controls FGF10-mediated embryonic lung epithelial branching morphogenesis through MAPK14 and STAT3 regulation of E-Cadherin distribution.
      MiR-124-3p negatively regulated STAT3 to inhibit breast cancer development.
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      We found that circKcnk9 could bind to the sequence of miR-124-3p by bioinformatics analysis, but it is unknown whether circKcnk9 regulates visceral hypersensitivity by the miR-124-3p/STAT3 axis in IBS-like rats.
      In this study, we first determined the role of spinal circKcnk9 in the visceral hypersensitivity of IBS-like rats by molecular biological and behavioral methods. Then, bioinformatic tools and dual luciferase assay were used to reveal the miRNA molecule interacted with circKcnk9. Finally, we explored their downstream gene contributed to visceral hypersensitivity. The study might provide a novel epigenetic mechanism for visceral hypersensitivity of IBS. In this study, the role of spinal circKcnk9 was determined by molecular biological and behavioral methods in the visceral hypersensitivity of IBS-like rats. Then, bioinformatic tools and dual luciferase assay were used to reveal the miRNA molecule interacted with circKcnk9. Finally, the downstream gene was explored in visceral hypersensitivity. The study might provide a novel epigenetic mechanism for visceral hypersensitivity of IBS.

      Methods

      Animals

      Male Sprague–Dawley rats (< 5 days old) were obtained from the Experimental Animal Center of Fujian Medical University (SCXK(Fujian Province)2016–0006), Fuzhou, China). The animals were randomly allocated to each group. All animals were kept in a greenhouse with a controlled humidity of 65 to 77%. They were maintained on a 12 hours light/dark cycle. All procedures were approved by the Animal Care and Use Committee. All animal experiments were double-blind.

      IBS Models and Behavioral Tests

      The IBS models were established as previously described.
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      • Chen A-q
      • Luo X-q
      • Guo L-x
      • Tang Y
      • Bao C-j
      • Lin L
      • Lin C.
      Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain.
      Briefly, newborn rats were exposed to colorectal distention stimulation at a pressure of 60 mm Hg for 1 minute from 8 to 14 days after birth. Avascular reconstruction balloon of 20.0 mm in length and 2.5 mm in diameter was inserted into the descending colon. Control rats were subjected to the same procedure, except for colorectal distention (CRD). After stimulation, the rats were housed until they were at least 6 weeks old (about 200 g).
      Visceral hypersensitivity in IBS was evaluated by recording changes in the electromyographic (EMG) response to CRD (40 mm Hg, 60 mm Hg), as described in our previous study.
      • Fan F
      • Tang Y
      • Dai H
      • Cao Y
      • Sun P
      • Chen Y
      • Chen A
      • Lin C.
      Blockade of BDNF signalling attenuates chronic visceral hypersensitivity in an IBS-like rat model.
      Under isoflurane anesthesia, balloons were placed for descending colon distention. Silver bipolar electrodes were inserted into the muscle to record spikes in the abdominal muscle. Colon distention was induced by rapidly inflating the balloon for 10 seconds with 4 minutes resting period. EMG signals were collected using an RM6240BD (Chengdu, China). The change in EMG was expressed as a relative percentage change, EMG = (EMGc-EMGb) * 100%/EMGb, where EMGc is the response to CRD and EMGb is the baseline average for 20 seconds.

      Spinal Tissue Collection

      Rats were anesthetized with isoflurane, and the dorsal half of the spinal thoracic segment (T13–L2) tissues were collected and stored in a freezer at −80 °C.

      CircRNA Sequence and Bioinformatics Analysis

      After the IBS rat model was established, transcriptome sequencing of the spinal dorsal horn tissues was performed. Total RNA extraction, RNA sequencing, and analysis were performed using OE Biotech Co., Ltd. (Shanghai, China). An FDR q-value threshold of .05 was used to determine significance. The target miRNA of circRNA was predicted using the miRanda software. MiRNAs target genes were predicted using 3 bioinformatics databases (miRanda, TargetScan, and microstarbase).

      Spinal Neuron Culture

      Primary spinal neurons were collected from the rats after birth for 24 hours. Spinal sections were dissociated using .05% trypsin (Thermo Fisher, USA) at 37 °C for 10 minutes in Hank's balanced salt solution. The suspensions were filtered successively through a 70 μm and 40 μm (Corning, USA) cell strainer. Cells were seeded in 24-well plates coated with poly d-lysine (Sigma, USA) and cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA). After 4 hours, neurobasal culture medium supplemented with B27 was replaced with 10% FBS plating medium at 37 °C in a humidified incubator with 5% CO2/95% air.

      Fluorescence In situ Hybridization (FISH) and Immunofluorescence (IF)

      The ascending aorta of anesthetized rats was filled with 4% paraformaldehyde. The spinal cord was dehydrated with 30% sucrose and cut into 20 μm frozen sections for staining. The sections were then hybridized for 12 hours at 37 °C with the circkcnk9 probe 5’-TCCAGGTGCAGCATGTCCATATCAT-3’ (1:100, EXIQON) and the 3’ and 5’-FAM-labeled miR-124-3p probe 5’-CATTCACCGCGTGCCTT-3’ (1:100, EXIQON)). The sections were incubated with primary antibodies against STAT3 (Abcam; ab109085; 1:100), GFAP (CST; 3670; 1:300), Iba1 (Wako;019-19741; 1:10k), or NeuN (Millipore; MAB377; 1:500) overnight at 4 °C. c-Fos immunocytochemistry was performed using c-Fos (Abcam; ab208942; 1:1000) primary antibodies. Alexa 488 (abcam; ab150077; 1:500) or Alexa 594 (abcam; ab150116; 1:500) was then used to conjugate the secondary antibody at 37 °C for 2 hours. Sections were photographed using a Leica TCS SP5 confocal microscope.

      Intrathecal Injection

      Neuronal hSyn promoter was used to drive gene expression in neuron.
      • Haery L
      • Deverman BE
      • Matho KS
      • Cetin A
      • Woodard K
      • Cepko C
      • Guerin KI
      • Rego MA
      • Ersing I
      • Bachle SM
      • Kamens J
      • Fan M.
      Adeno-associated virus technologies and methods for targeted neuronal manipulation.
      LV-hSyn-mCherry-5’miR-30a-shRNA (circKcnk9)-3’miR-30a-wpres (shcircKcnk9 and LV-scramble), rAVV (2/9)-hSyn-circKcnk9-nEF1α-EGFP-Pa (AAV-circKcnk9 and AAV-NC), and LV-hSyn-mCherry-5’miR-30a-shRNA (STAT3)-3’miR-30a-wpres (shSTAT3 and sh-NC) were designed and synthesized by BrainVTA (Wuhan, China). Kcnk9 siRNA (si-mKcnk9), miR-324-3p agomir, and antagomir were obtained from GenePharma Biotech (Shanghai, China). S3I-201 (MCE, HY-15146, USA) and actinomycin D (MCE, HY-17559, USA) were purchased from MCE. A sterile polyethylene-10 catheter (Scientific Commodities Inc., BB31695-PE/1, USA) was implanted between the L4 and L5 vertebrae. Animals with numbness or paralysis in their hindlimbs were removed from the trial. Agents were administered 5 days after the surgery. ShRNA was intrathecally injected (10 μL), mCherry was observed after 1 week. AAV was intrathecally injected (10 μL), and GFP was observed after 21 days. MiR-324-3p agomir, miR-124-3p antagomir and si-mKcnk9 (20 μM, 10 μL/d) were intrathecally injected for 3 days. S3I-201 (10 mM, 10 μL/d) was intrathecally injected for 5 days. Actinomycin D (100 μM/10 μL) was intrathecally injected. Intrathecal administration was at a rate of 20 μL/min. A dose of 10 μL drug was injected into the catheter, followed by 10 μL of saline.

      RNA Extraction and Quantitative PCR (qPCR)

      The PARIS Kit (Invitrogen, #AM1921) was used to isolate nuclear and cytoplasmic RNA from cultured spinal neurons, following the manufacturer's instructions. Total RNA was collected from cells and tissues using TRIzol reagent. For RNase R experiment, RNA was treated with RNase R (Epicenter, USA) at 37 °C for 10 minutes. CircRNA and mRNA cDNA were synthesized using the Evo M-MLV RT Kit (Accurate Biology, AG11705, China). MiRNA cDNA was synthesized using the Mir-X miRNA First-Strand Synthesis Kit (Takara, Japan). SYBR (Accurate Biology, China) was used to detect mRNA expression. The 2−∆∆CT method was used to evaluate the relative expression. CircRNA primers were designed across the back-splice junction. The Sanger sequencing of the amplification products were used to verify the circularization site. The primers used were in Table S2.

      RNA Immunoprecipitation (RIP) Assay

      The RIP assay was performed using the RIP Kit (BersinBio, China) and Argonaute-2 (Ago2) antibody (Abcam, ab186733, 5 μg) according to the recommended protocol.
      • Zhang SB
      • Lin SY
      • Liu M
      • Liu CC
      • Ding HH
      • Sun Y
      • Ma C
      • Guo RX
      • Lv YY
      • Wu SL
      • Xu T
      • Xin WJ.
      CircAnks1a in the spinal cord regulates hypersensitivity in a rodent model of neuropathic pain.

      Cell Culture and Transfection

      PC12 cells were obtained from Institute of Neuroscience (Soochow University, China). 293T cells were obtained from the Provincial Key Laboratory of Neuroglia and Diseases (Fujian Medical University, China). Cells were cultured in DMEM supplemented with 10% FBS (Gibco, USA) at 37 °C and 5% CO2. CircKcnk9 plasmids (CircKcnk9-OE), circKcnk9-OE control plasmids (control), shcircKcnk9 plasmids (shcircKcnk9), and scramble plasmids (scramble) were constructed by BrainVTA Biotech (Wuhan, China). MiR-124-3p mimics, inhibitor, and the corresponding controls were obtained from GenePharma Biotech (Shanghai, China). Lipofectamine 3000 (Invitrogen, USA) was used for transfection, as described in the manufacturer's instructions.

      Western Blotting (WB)

      Spinal tissues and PC12 cells were lysed in RIPA buffer (Beyotime, P0013B, China). The proteins were separated by electrophoresis and transferred onto a PVDF membrane. The following antibodies were used: STAT3 (CST; 9139; 1:2000), NSF (CST; 3924; 1:1000), GluR2 (Millipore; MAB397; 1:2000), and GAPDH (Abclonal; A19056; 1:4000). Protein bands were detected using an enhanced chemiluminescence reaction. GAPDH were used as internal controls.

      Luciferase Reporter Assays

      The pmirGLO Dual-Luciferase vector containing wild-type circKcnk9 (circKcnk9-wt), wild-type STAT3 (STAT3-wt), circKcnk9-mutant (circKcnk9-mut), and STAT3-mutant (STAT3-mut) were synthesized by GenePharma Biotech (Shanghai, China). The 293T cells were seeded in 96-well plates and cotransfected with circKcnk9-wt plasmids, circKcnk9-mut plasmids, STAT3-wt, STAT3-mut, miR-124-3p mimics, or mimic NC using Lipofectamine 3000. Cell lysates were collected after transfection for 48 hours, and the Dual-Luciferase Reporter Assay System (Promega) was used to detect firefly and Renilla luciferase activities.

      Chromatin Immunoprecipitation Assay (ChIP)

      Spinal tissues were homogenized in PBS. The ChIP assay was performed using the SimpleChIP Enzymatic Chromatin IP Kit following the manufacturer's instructions. An antibody against STAT3 (CST, 9139) was used to immunoprecipitate the NSF-DNA complex. qPCR was used to assess the enrichment of NSF. The primers used were as follows: NSF, forward: 5′-ACTTCAGCAGCAGGTATTTGT-3′, reverse: 5′-TCGATCATTTGTCCACCTT-3′.

      Statistical Analysis

      All data are expressed as (mean±S.E.M). SPSS 26 was used for the statistical analyses. The Student's t-test (2 groups) or One-way ANOVA (3 groups) was used for the immunoblot, qPCR, and c-Fos immunolabeling data. For RNase R treatment, actinomycin D, and behavior experiments, comparisons between groups were carried out using 2-way ANOVA. 2 × 3 ANOVA was applied to analyze the expression of circKcnk9 for each time point. Correlation coefficients between circKcnk9 and miR-124-3p were calculated using the Pearson product–moment correlation coefficient. Statistical significance was set at P<.05. GraphPad Prism 8.0 was used for plotting. ImageJ2X was used to analyze immunostaining images and protein band intensity.

      Results

      Sequencing of Spinal circRNAs in Rats

      To estimate visceral sensitivity, we recorded the EMG response to CRD in control and IBS-like rats. Compared with control rats, the magnitude of EMG increased markedly under 40 and 60 mm Hg CRD in IBS-like rats (n = 6, 2-way ANOVA, F = 63.92, P<.0001) (Supplementary Fig S1A, B and Table S1). The HE staining results also showed no obvious edema or ulceration in the colon of IBS-like rats compared with those of the controls (Supplementary Fig S1C). These results suggested that IBS-like rats were successfully established. To explore the role of spinal circRNAs in IBS-like rats, circRNA expression profiling was performed by RNA-Seq analysis of the spinal dorsal horn (T13-L2). CIRI software analysis indicated that 95% of the circRNAs were derived from exons (Fig 1A), and most of circRNAs include 1 to 4 exons (Fig 1B). DESeq software showed that 37 circRNAs were upregulated, and 40 circRNAs were downregulated in IBS-like rats (Fig 1C). To confirm the RNA-Seq results, 11 candidate circRNAs were selected for qPCR analysis according to Foldchange (FC ≥ 2 or FC ≤ 0.4). Among them, spinal circRNA_7685 increased most significantly in IBS-like rats compared with controls (n = 9, 2-tailed Student t-test, P<.05) (Fig 1D). Sanger sequencing analysis showed that circRNA_7685 originated from exon 2 of the Kcnk9 gene (Fig 1E). Therefore, circRNA_7685 was named as circKcnk9.
      Figure 1
      Figure 1Profiling of circRNAs and identification of circKcnk9. (A) Genomic origin of circRNAs. (B) The number of exons in each circRNA. (C) Volcano plot of DEcircRNAs from control and IBS-like rats (n = 3). (D) qPCR showed the expression of 11 different circRNAs. CircRNA_7685 significantly upregulated in the spinal cord of IBS-like rats. n = 9 per group. *P<.05 versus controls (2-tailed Student t-test). (E) Schematic representation of circKcnk9 production. CircKcnk9 was formed by back splicing from exon 2 of the Kcnk9 gene and validated by Sanger sequencing.

      Expression Characteristics of CircKcnk9 in IBS-like Rats

      To confirm the circular property of circKcnk9, RNase R digestion assay was performed. qPCR showed that circKcnk9 was resistant to RNase R, while linear Kcnk9 mRNA was degraded (n = 4, 2-way ANOVA, F = 196.2, P<.0001) (Fig 2A). Random hexamers or oligo (dT) 18 primers were used for reverse transcription assays. Compared with random primers, the expression of circKcnk9 was decreased using oligo (dT) 18 primers, implying that circKcnk9  lacks a poly (A) tail (n = 4, 2-tailed Student t-test, t = 24.04, P<.0001) (Fig 2B). Next, actinomycin D was intrathecally injected to inhibit transcription and then determine the expression of circKcnk9 and Kcnk9 in IBS-like rats. Treatment with actinomycin D decreased the mRNA level of Kcnk9 but not of circKcnk9 (n = 3, 2-way ANOVA, F = 7.223, P=.0161) (Fig 2C). To determine whether circKcnk9 was temporal-specific, circKcnk9 expressions in spinal tissues of different ages were tested. The qPCR results showed that the expressions of circKcnk9 increased significantly from 4 to 10 weeks of age (n = 3, 2 × 3 ANOVA, F = 24.522, P = .014) (Fig 2D). To assess the cell specificity of circKcnk9, costaining of circKcnk9 FISH with a neuronal marker (NeuN), astrocytic marker (GFAP), and microglial marker (Iba-1) was performed. CircKcnk9 was expressed in neurons of T13 spinal cord (Fig 2E), suggesting that circKcnk9 is a neuron-specific circRNA in the spinal dorsal horn of IBS-like rats. In addition, localization of circKcnk9 in primary spinal cord neurons was examined by cytoplasm qPCR, nucleus qPCR, and FISH. The results showed that circKcnk9 was mainly localized in the cytoplasm of neurons (Fig. 2F and 2G). These results indicate that circKcnk9 is a circRNA abundantly and stably localized in the spinal cord. Furthermore, the sequence of the full-length circKcnk9 was blasted against the human circRNAs in circBase. The result showed that circKcnk9 shared homology with 82 human circRNAs (Table S3).
      Figure 2
      Figure 2CircKcnk9 distribution and expression in the spinal cord. (A) The RNase R digestion assay showed circKcnk9 was resistant to RNase R. n = 4, 2-way ANOVA, F = 196.2, ***P<.0001 versus the corresponding groups. (B) qPCR showed circKcnk9 significantly decreased using oligo dT primer. n = 4, 2-tailed Student t-test, t = 24.04, ***P<.0001 versus random primers group. (C) qPCR showed actinomycin D treatment lowered mRNA level of mKcnk9 but not circKcnk9. n = 3, 2-way ANOVA, F = 7.223, *P = .0161 versus the corresponding groups. (D) qPCR showed that circKcnk9 expression in different periods of IBS-like rats. n = 3, 2 × 3 ANOVA, F = 24.522, *P = .014 versus controls. (E) FISH and IF showed that circKcnk9 (red) was mainly expressed in neurons (green), but not in astrocytes (green) and microglias (green) of T13 spinal cord. And nuclei were fluorescently labeled with DAPI (blue). Scale bar, 100 μm (n = 3). FISH (F) and qPCR (G) showed circKcnk9 expressed mainly in cytoplasm of cultured spinal neuron (n = 3).

      CircKcnk9 was Involved in Visceral Hypersensitivity and Neuronal Activity in IBS-like Rats

      To determine whether circKcnk9 participated in visceral hypersensitivity, a lentiviral vector (shcircKcnk9) that carried the mCherry reporter gene under the control of hSyn (neuron-specific synapsin promoter) was used to suppress the expression of endogenous circKcnk9 in neurons (Fig 3A and Supplementary Fig S2A). qPCR analysis showed that the expression of circKcnk9 decreased in the spinal dorsal horn of IBS-like rats after intrathecal administration of shcircKcnk9 (n = 6, 1-way ANOVA, F = 11.5, P = .0009) (Fig 3B). Visceral hypersensitivity was alleviated 1 week after intrathecal injection of shcircKcnk9 compared to scramble in IBS-like rats (n = 6, 2-way ANOVA, F = 23.38, P = .0001) (Fig 3C). However, Kcnk9 siRNA had no effect on EMG (n = 5, 2-way ANOVA, F = 0.1233, P = .7301) (Supplementary Fig S3A). qPCR result showed linear transcript of Kcnk9 (mkcnk9) but not circKcnk9 expression was decreased after Kcnk9 siRNA was intrathecally injected for 3 days (n = 5, 2-tailed Student t-test, t = 4.497, P = .0108) (Supplementary Fig S3B). After behavioral experiment, spinal c-Fos expression was detected to assess neuronal activity. Confocal analysis showed that knockdown of circKcnk9 suppressed the expression of c-Fos in IBS-like rats (n = 3, 2-tailed Student t-test, t = 8.81, P = .0009) (Fig. 3D and 3E). To further clarify the role of spinal circKcnk9 in visceral hypersensitivity modulation, overexpression of circKcnk9 in spinal neurons was performed by intrathecal injection of AAV-circKcnk9 in control rats (Fig 3A and Supplementary Fig S2B). Compared with the AAV-NC group, circKcnk9 expression increased significantly in the AAV-circKcnk9 group (n = 6, 1-way ANOVA, F = 7.67, P = .0051) (Fig 3F). Moreover, intrathecal injection of AAV-circKcnk9 induced pain behavior compared to injection of AAV-NC (n = 6, 2-way ANOVA, F = 5.532, P = .0303) (Fig 3G). The c-Fos expression was enhanced after injection of AAV-circKcnk9 (n = 3, 2-tailed Student t-test, t = 4.339, P = .0123) (Fig. 3H and 3I). These results demonstrate that spinal circKcnk9 plays a significant role in visceral hypersensitivity. This may serve as a new molecular target for pain treatment.
      Figure 3
      Figure 3Upregulation of spinal circKcnk9 facilitated visceral hypersensitivity of IBS-like rats. (A) Time schedule of experimental design. (B) CircKcnk9 expression was significantly inhibited after intraspinal injection of shcircKcnk9. n = 6, 1-way ANOVA, F = 11.5, ***P = .0009 versuss scramble group. (C) Intrathecal injection of shcircKcnk9 attenuated the visceral hypersensitivity. n = 6, 2-way ANOVA, F = 23.38, ***P = .0001 versus scramble group. (D) and (E) Intrathecal injection of shcircKcnk9 suppressed c-Fos expression. n = 3, 2-tailed Student t-test, t = 8.81, ***P = .0009 versus scramble group. Nuclei were fluorescently labeled with DAPI (blue). (F) CircKcnk9 expression was elevated after intraspinal AAV-circKcnk9. n = 6, 1-way ANOVA, F = 7.67, **P = .0051 versus AAV-NC group. (G) Intraspinal injection of recombinant AAV-circKcnk9 induced pain behavior. n = 6, 2-way ANOVA, F = 5.532, *P = .0303 versus AAV-NC group. (H) and (I) Intraspinal injection of recombinant AAV-circKcnk9 increased c-Fos expression. n = 3, 2-tailed Student t-test, t = 4.339, *P = .0123 versus AAV-NC group. And nuclei were fluorescently labeled with DAPI (blue).

      CircKcnk9 Acted as a Sponge for miR-124-3p

      Generally, the subcellular localization of circRNA determines its mechanism of action. CircKcnk9 was mainly localized in the cytoplasm of spinal neurons. RIP assay was performed to determine whether cytoplasmic circRNAs can function as miRNA sponges. The results showed that endogenous circKcnk9 was significantly enriched in Ago2 immunoprecipitants compared with control IgG (n = 3, 2-tailed Student t-test, t = 6.163, P = .0035) (Fig 4A), indicating that circKcnk9 can bind to miRNA. MiRanda database analysis showed that circKcnk9 had potential binding sites with 104 miRNAs (Fig 4B and Table S4). Furthermore, 11 miRNAs that showed at least 2 potential binding sites in circKcnk9 from IBS-like rats were analyzed by qPCR. The result showed that miR-124-3p, let-7b-5p, miR-352, and miR-3583-5p were strongly downregulated in the spinal dorsal horn (n = 6, 2-tailed Student t-test, P<.05) (Fig 4C). Bioinformatics analysis showed that miR-124-3p was the only 1 with 3 potential binding sites (218, 413, 528) in circKcnk9 (Supplementary Fig S4). Therefore, we focused on the role of miR-124-3p in chronic visceral hypersensitivity. A negative correlation between circKcnk9 and miR-124-3p expression in IBS-like rats was found (n = 6, Pearson's correlation coefficient, r = -.870, P =.024) (Fig. 4D). To test miR-124-3p as a target of circKcnk9, luciferase reporter plasmids containing miR-124-3p binding site in the 3-UTRs (528) of circKcnk9 were constructed in 293T cells. Relative luciferase reporter activity of 293T cells co-transfected circKcnk9-wild type (wt) with miR-124-3p mimics was significantly lower than that cotransfected circKcnk9-mutant (mut) with miR-124-3p mimics (n = 3, 2-tailed Student t-test, t = 12.36, P = .0002) (Fig 4E). Furthermore, FISH assay showed that coexistence of circKcnk9 and miR-124-3p in the spinal dorsal horn of IBS-like rats (Fig 4F). Overall, these results suggest that circKcnk9 acts as a sponge for miR-124-3p.
      Figure 4
      Figure 4CircKcnk9 functioned as a sponge for miR-124-3p in the spinal dorsal horn of IBS-like rats. (A) RIP assay showed the interaction between circKcnk9 and Ago2. n = 3, 2-tailed Student t-test, t = 6.163, **P = .0035 versus IgG group. (B) The circKcnk9/miRNA network analysis and qPCR (C) showed miR-124-3p, let-7b-5p, miR-352 and miR-3583-5p interacted with circKcnk9 and decreased in the spinal dorsal horn of IBS-like rats. n = 6 per group. *P<.05 versus control group (2-tailed Student t-test). (D) Inverse correlation between circKcnk9 and miR-124-3p expressions in IBS-like rats. n = 6, Pearson's correlation coefficient, r = -.870, P = .024. (E) Schematic representation of predicted circKcnk9 binding sites and mut sites (upper). Results from the luciferase assay of miR-124-3p binding to circKcnk9 in the 293T cell (below). n = 3, 2-tailed Student t-test, t = 12.36, ***P = .0002 versus NC group. (F) FISH showed the colocalization of circKcnk9 and miR-124-3p. Scale bar, 25 μm (n = 3).

      CircKcnk9 Enhanced Visceral Hypersensitivity Through miR-124-3p

      To investigate whether circKcnk9 enhances visceral hypersensitivity through a miRNA-mediated pathway, the pain responses were evaluated after miR-124-3p antagomir was intrathecal injected for 3 days in IBS-like rats with spinal shcircKcnk9. MiR-124-3p antagomir significantly restored the pain responses relieved by shcircKcnk9 (n = 4, 2-way ANOVA, F = 36.82, P < .0001) (Fig 5A). Next, miR-124-3p was costained with NeuN, GFAP, and Iba-1 by FISH. The results showed that miR-124-3p was mainly expressed in neurons (Fig 5B), with only a few in microglia, but not in astrocytes of the spinal dorsal horn (Supplementary Fig S5). To explain the potential role of miR-124-3p in chronic visceral pain, miR-124-3p agomir and antagomir were injected intrathecally. First, the expression of miR-124-3p after miR-124-3p agomir administration for 3 days was determined. The results showed that miR-124-3p agomir enhanced miR-124-3p expression in the spinal dorsal horn (C, n = 6, 1-way ANOVA, F = 6.479, P = .0094, D, n = 3, 2-tailed Student t-test, t = 5.655, P =.0048) (Fig. 5C and 5D) and decreased visceral hypersensitivity in IBS-like rats (n = 6, 2-way ANOVA F = 18.99, P = .0003) (Fig 5E). Whether the inhibition of spinal miR-124-3p could induce visceral hypersensitivity in control rats was evaluated. The results showed that miR-124-3p antagomir decreased the expression of miR-124-3p (F, n = 4, 1-way ANOVA, F = 5.553, P = .0269, G, n = 3, 2-tailed Student t-test, t = 3.315, P = .0295) (Fig. 5F and 5G) and induced pain behavior in control rats (n = 6, 2-way ANOVA, F = 15.98, P = .0007) (Fig 5H). These findings demonstrate that spinal miR-124-3p could involve in spinal central sensitization. Overall, these findings imply that spinal circKcnk9 enhances visceral hypersensitivity by targeting miR-124-3p.
      Figure 5
      Figure 5CircKcnk9 enhanced visceral hypersensitivity through miR-124-3p. (A) EMG showed miR-124-3p antagomir restored antinociception of shcircKcnk9 silencing after coadministration of shcircKcnk9/miR-124-3p antagomir. n = 4, 2-way ANOVA, F = 36.82, ***P<.0001 versus the corresponding groups. (B) FISH and IF showed that miR-124-3p (green) was expressed in neurons (red) of spinal cord. And nuclei were fluorescently labeled with DAPI (blue). Scale bar, 100 μm (n = 3). (C) and (D) qPCR and FISH showed that intrathecal injection of miR-124-3p agomir increased miR-124-3p expression (C, n = 6, 1-way ANOVA, F = 6.479, **P = .0094, D, n = 3, 2-tailed Student t-test, t = 5.655, **P = .0048). And nuclei were fluorescently labeled with DAPI (blue). Scale bar, 100 μm. (E) Intrathecal injection of miR-124-3p agomir reduced the visceral hypersensitivity following IBS. n = 6, 2-way ANOVA, F = 18.99, ***P = .0003 versus agoNC group. (F) and (G) qPCR and FISH showed that intrathecal injection of miR-124-3p-antagomir inhibited the expression of miR-124-3p (F, n = 4, 1-way ANOVA, F = 5.553, *P = .0269, G, n = 3, 2-tailed Student t-test, t = 3.315, *P = .0295). And nuclei were fluorescently labeled with DAPI (blue). Scale bar, 100 μm. (H) Intrathecal injection of miR-124-3p-antagomir induced pain behavior in the controls. n = 6, 2-way ANOVA, F = 15.98, ***P = .0007 versus antaNC group.

      MiR-124-3p Inhibited STAT3 Expression In vivo and In vitro

      To explore the mechanisms underlying the effects of miR-124-3p on spinal central sensitization, 3 databases (TargetScan, miRanda, and microstarbase) were used to predict miR-124-3p target genes. There were 4 genes (ITGB1, STAT3, NR3C1, and LAMC1) at the intersection of the 3 databases (Fig 6A and Supplementary Fig S6). The expression of them were investigated by qPCR. Only STAT3 expression was significantly upregulated in the spinal dorsal horn of IBS-like rats (n = 4, 2-tailed Student t-test, P<.05) (Fig 6B). STAT3 has previously been reported to be an important regulator of synaptic plasticity.
      • Han JK
      • Kwon SH
      • Kim YG
      • Choi J
      • Kim JI
      • Lee YS
      • Ye SK
      • Kim SJ.
      Ablation of STAT3 in Purkinje cells reorganizes cerebellar synaptic plasticity in long-term fear memory network.
      Therefore, STAT3 was selected as the miR-124-3p target candidate. To verify this hypothesis, a luciferase reporter plasmid containing the binding site of WT or Mut 3-UTR STAT3 was transfected together with miR-124-3p mimics in 293T cells. The results showed that relative luciferase reporter activity of 293T cells cotransfected STAT3-wild type (wt) with miR-124-3p mimics was significantly lower than that cotransfected STAT3-mutant (mut) with miR-124-3p mimics (n = 3, 2-tailed Student t-test, t = 9.469, P = .0007) (Fig 6C). To further confirm that STAT3 is regulated by miR-124-3p, STAT3 expression of rats was determined after the administration of miR-124-3p agomir, ago NC, miR-124-3p antagomir, or antaNC for 3 days. Overexpression of miR-124-3p decreased the mRNA and protein expression of STAT3 (D, n = 4, 1-way ANOVA, F = 10.67, P = .0042, E, n = 3, 1-way ANOVA, F = 5.346, P = .0465) (Fig. 6D and 6E), while suppression of miR-124-3p increased STAT3 expression in vivo (F, n = 4, 1-way ANOVA, F = 7.568, P = .0118, G, n = 3, 1-way ANOVA, F = 23.57, P = .0014) (Fig. 6F and 6G). Similarly, PC12 cells were transfected with miR-124-3p mimics, mimic control, miR-124-3p inhibitor, or inhibitor NC plasmid. The qPCR and WB results showed that the expression level of miR-124-3p was increased, while the expression levels of STAT3 mRNA and protein were reduced in PC12 cells after transfection with miR-124-3p mimics (n = 3, 2-tailed Student t-test, P<.05) (Fig. 6H and 6I). In contrast, the expression level of miR-124-3p was inhibited, but STAT3 expression at mRNA and protein levels was enhanced in PC12 cells after transfection with the miR-124-3p inhibitor (n = 3, 2-tailed Student t-test, P<.05) (Fig. 6H and 6I). These results indicated that miR-124-3p could downregulate STAT3 expression. FISH assay further verified the coexistence of miR-124-3p and STAT3 in PC12 cells (Fig 6J) and spinal dorsal horn (Fig 6K). These results show that miR-124-3p negatively regulates STAT3 expression in vivo and in vitro.
      Figure 6
      Figure 6MiR-124-3p regulated STAT3 expression. (A) Predicted miRNA-target by 3 kinds of software. (B) The mRNA level of STAT3 was increased in the spinal cord of IBS-like rats. n = 4 per group. *P<.05 versus controls (2-tailed Student t-test). (C) Overexpression of miR-124-3p significantly decreased STAT3 transcription in the 293T cells, as assessed by the luciferase reporter assay. n = 3, 2-tailed Student t-test, t = 9.469, ***P = .0007 versus mimicsNC group. (D) and (E) The overexpression of miR-124-3p suppressed the STAT3 mRNA (D, n = 4, F = 10.67, **P = .0042) and protein (E, n = 3, F = 5.346, P = .0465) expression levels in vivo versus the agoNC group (1-way ANOVA). (F) and (G) The downregulation of miR-124-3p increased the STAT3 mRNA (F, n = 4, F = 7.568, *P = .0118) and protein (G, n = 3, F = 23.57, **P = .0014) expression levels in vivo versus antaNC group (1-way ANOVA). (H) qPCR showed miR-124-3p expression level was increased and STAT3 mRNA expression was suppressed after transfected with miR-124-3p mimics, miR-124-3p expression level was inhibited and STAT3 mRNA expression was increased after transfected with miR-124-3p inhibitor in the PC12 cells. n = 3 per group. *P<.05 versus the corresponding groups (2-tailed Student t-test). (I) MiR-124-3p mimics inhibited STAT3 protein expression, and miR-124-3p inhibitor increased STAT3 protein expression in the PC12 cells. n = 3 per group. *P<.05 versus the corresponding groups (2-tailed Student t-test). (J) and (K) MiR-324-3p colocalized with STAT3 in the PC12 cells (J) and spinal dorsal horn (K). Scale bar, 75μm (n = 3). SDH: spinal dorsal horn.

      CircKcnk9 Upregulated STAT3 Expression Through miR-124-3p

      In vivo experiments showed that intrathecal administration of AAV-circKcnk9 enhanced the expression of STAT3 mRNA (n = 6, 1-way ANOVA, F = 19.68, P<.0001) (Fig 7A) and protein (n = 3, 1-way ANOVA, F = 7.259, P = .025) (Fig 7B) in control rats. Furthermore, intrathecal administration of shcircKcnk9 attenuated STAT3 mRNA (n = 6, 1-way ANOVA, F = 20.84, P<.0001) (Fig 7C) and protein (n = 3, 1-way ANOVA, F = 762.6, P<.0001) (Fig 7D) levels in IBS-like rats. To demonstrate that circKcnk9 regulates STAT3 expression by targeting miR-124-3p, STAT3 expression was examined after miR-124-3p agomir or antagomir was intrathecal injected for 3 days in IBS-like rats with spinal shcircKcnk9. The miR-124-3p inhibitor reversed STAT3 expression by silencing circKcnk9 (n = 3, 1-way ANOVA, F = 10.29, P = .0014) (Fig 7E). Then, circKcnk9 shRNA vector (shcircKcnk9), scramble, AAV-circKcnk9 vector (circKcnk9-OE), or control was transfected into PC12 cells. The results showed that the expression of circKcnk9 increased in PC12 cells after transfection with circKcnk9-OE, while the expression level of circKcnk9 was inhibited after transfection with shcircKcnk9, (n = 3, 2-tailed Student t-test, P<.05) (Fig 7F). Overexpression of circKcnk9 increased the expression of STAT3 mRNA and protein, while suppression of circKcnk9 inhibited STAT3 expression (n = 3, 2-tailed Student t-test, P<.05) (Fig. 7G and 7H). CircKcnk9 was colocalized with STAT3 in the PC12 cells (Fig 7I) and spinal dorsal horn (Fig 7J). These results indicate that circKcnk9 enhances STAT3 expression by absorbing miR-124-3p in IBS-like rats.
      Figure 7
      Figure 7CircKcnk9 upregulated STAT3 expression via absorbing miR-124-3p. (A) and (B) The overexpression of circKcnk9 enhanced STAT3 mRNA (A, n = 6, F = 19.68, ***P<.0001) and protein (B, n = 3, F = 7.259, *P = .025) expression levels in vivo versus AAV-NC group (1-way ANOVA). (C) and (D) The downregulation of circKcnk9 inhibited STAT3 mRNA (C, n = 6, F = 20.84, ***P<.0001) and protein expression (D, n = 3, F = 762.6, ***P<.0001) levels in vivo versus scramble group (1-way ANOVA). (E) MiR-124-3p antagomir restored STAT3 protein expression inhibited by shcircKcnk9. n = 3, 1-way ANOVA, F = 10.29, **P = .0014. (F) and (G) qPCR showed circKcnk9 (F) and STAT3 (G) expression levels were increased after transfected with circKcnk9-OE plasmid, the expression levels of circKcnk9 and STAT3 were decreased after transfected with shcircKcnk9 plasmid in the PC12 cells. n = 3 per group. *P<.05 versus the corresponding groups (2-tailed Student t-test). (H) CircKcnk9 overexpression increased STAT3 protein expression, while circKcnk9 downregulation inhibited STAT3 protein expression in the PC12 cells. n = 3 per group. *P<.05 versus the corresponding groups (2-tailed Student t-test). (I) and (J) CircKcnk9 colocalized with STAT3 in the PC12 cells (I) and spinal dorsal horn (J). Scale bar, 50 μm (n = 3). SDH, spinal dorsal horn.

      STAT3 Contributed to Visceral Hypersensitivity

      To determine the role of STAT3 in IBS-like rats, the protein expression of STAT3 was examined in the spinal dorsal horn. The result showed that STAT3 protein level was increased in IBS-like rats (n = 4, 2-tailed Student t-test, t = 2.886, P = .0162) (Fig 8A). Immunofluorescence showed STAT3 immunoreactivity in neurons of spinal dorsal horn (Fig 8B). Lentivirus-shRNA of STAT3 (shSTAT3) was intrathecally injected to block STAT3 in IBS-like rats. The results of qPCR and western blot showed that the expression of STAT3 was decreased after shSTAT3 treatment (C, n = 5, 2-tailed Student t-test, t = 9.039, P<.0001, D, n = 3, 2-tailed Student t-test, t = 4.417, P = .0115) (Fig. 8C and 8D). Behavioral result showed that STAT3 inhibition relieved visceral hypersensitivity in IBS-like rats (n = 9, 2-way ANOVA, F = 17.71, P = .0003) (Fig 8E). Interesting, western blot results showed that the synaptosomal - associated protein levels of GluR2 and NSF were increased in the IBS-like rats (n = 3, 2-tailed Student t-test, P<.05) (Fig 8F). To investigate whether STAT3 regulated visceral hypersensitivity through GluR2 and NSF, the expression of GluR2 and NSF were tested after shSTAT3, the expressions of GluR2 and NSF were inhibited by shSTAT3 (n = 3, 2-tailed Student t-test, P<.05) (Fig 8G). S3I-201 (an inhibitor of STAT3 to block STAT3 DNA-binding and transcriptional activity) was also used for 5 days by intrathecal injection. The western blot result showed that the expression of GluR2 and NSF were repressed by S3I-201 (n = 3, 2-tailed Student t-test, P<.05) (Fig. 8H). The EMG result showed that S3I-201 alleviated visceral hypersensitivity in IBS-like rats (n = 4, 2-way ANOVA, F = 19.97, P = .0008) (Fig 8I). Since our results indicate NSF/GluR2 could be involved in STAT3 signaling in IBS-like rats, we used the Cistrome data browser and JASPAR database to predict the existence of binding sites between STAT3 and NSF promoter region. The promoter region of NSF was predicted to be the binding site of STAT3. ChIP assays showed direct binding of STAT3 to the promoter region of the endogenous NSF gene (n = 3, 2-tailed Student t-test, t = 8.689, P = .001) (Fig 8J). These results suggest that STAT3 activation is necessary for visceral hypersensitivity in IBS-like rats.
      Figure 8
      Figure 8STAT3 was involved in visceral hypersensitivity of IBS-like rats. (A) Quantified result of protein expression level of STAT3 in the spinal dorsal horn of IBS-like rats. n = 4, 2-tailed Student t-test, t = 2.886, P = .0162 versus controls. (B) Immunofluorescence showed STAT3 expression in neurons of spinal cord. Scale bar, 75 μm (n = 3). (C) and (D) Intrathecal injection of shSTAT3 inhibited STAT3 mRNA (C, n = 5, t = 9.039, ***P<.0001) and protein expression (D, n = 3, t = 4.417, *P = .0115) versus sh-NC group (2-tailed Student t-test). (E) Intrathecal injection of shSTAT3 attenuated visceral hypersensitivity. n = 9, 2-way ANOVA, F = 17.71, ***P = .0003 versus sh-NC group. (F) WB assays showed the protein expression of NSF and GluR2 were increased in IBS-like rats. n = 3 per group. *P<.05 versus the control group (2-tailed Student t-test). (G) WB assays showed the protein expression of NSF and GluR2 were decreased after intrathecal injection of shSTAT3. n = 3 per group. *P<.05 versus the sh-Nc group (2-tailed Student t-test). (H) Intraspinal injection of S3I-201 reduced NSF and GluR2 protein expressions. n = 3 per group. *P<.05 versus DMSO group (2-tailed Student t-test). (I) Intraspinal injection of S3I-201 (STAT3 inhibitor) alleviated the visceral hypersensitivity. n = 4, 2-way ANOVA, F = 19.97, ***P = .0008 versus DMSO group. (J) Chromatin immunoprecipitation was performed with anti-STAT3 antibody. n = 3, 2-tailed Student t-test, t = 8.689, ***P = .001 versus IgG group.

      Discussion

      In this study, different circRNAs expressions in the spinal dorsal horn of IBS-like rats were screened using circRNA sequencing analysis. Eleven circRNAs were selected for qPCR, and circKcnk9 was the most highly expressed among them. Inhibition of circKcnk9 suppressed spinal c-Fos expression and visceral hypersensitivity in IBS-like rats, while overexpression of circKcnk9 induced c-Fos expression in spinal neurons and pain behavior in control rats. CircKcnk9 was found to serve as a miR-124-3p sponge. Furthermore, miR-124-3p antagomir significantly restored the pain responses downregulated by shcircKcnk9 in IBS-like rats. STAT3 was validated as a target of miR-124-3p to play a critical role in visceral hypersensitivity by regulating NSF/GluR2.
      CircKcnk9 is an exonic circRNA. It was mainly distributed in the cytoplasm of spinal dorsal horn neurons. Suppression of spinal circKcnk9 blocked visceral hypersensitivity and inhibited c-Fos expression in IBS-like rats, whereas upregulation of circKcnk9 expression induced pain behavior and enhanced the activity of neurons in controls. Studies have shown that abnormal expression of circRNA is related to multiple diseases of the nervous system, which are widely expressed in the central and peripheral tissues, such as the hippocampus,
      • Gasparini S
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      • Rinaldi A
      • Presutti C
      • Mannironi C
      Differential expression of hippocampal circular RNAs in the BTBR mouse model for autism spectrum disorder.
      PFC,
      • Zimmerman AJ
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      • Mellios N.
      A psychiatric disease-related circular RNA controls synaptic gene expression and cognition.
      and DRG.
      • Mao S
      • Huang T
      • Chen Y
      • Shen L
      • Zhou S
      • Zhang S
      • Yu B.
      Circ-Spidr enhances axon regeneration after peripheral nerve injury.
      In the peripheral region, circ-Spidr inhibits axon reconstruction after nerve damage by modulating the PI3K-Akt pathway.
      • Mao S
      • Huang T
      • Chen Y
      • Shen L
      • Zhou S
      • Zhang S
      • Yu B.
      Circ-Spidr enhances axon regeneration after peripheral nerve injury.
      In the central region, the hippocampal circUBQLN1 accelerates neuronal proliferation and inhibits neuronal apoptosis.
      • Zhu Z
      • Wang S
      • Cao Q
      • Li G.
      CircUBQLN1 promotes proliferation but inhibits apoptosis and oxidative stress of hippocampal neurons in epilepsy via the miR-155-mediated SOX7 upregulation.
      Pain-related studies have shown that circSlc7a11 induces bone cancer pain by regulating LLC-WRC 256 cell proliferation and apoptosis.
      • Chen HW
      • Zhang XX
      • Peng ZD
      • Xing ZM
      • Zhang YW
      • Li YL.
      The circular RNA circSlc7a11 promotes bone cancer pain pathogenesis in rats by modulating LLC-WRC 256 cell proliferation and apoptosis.
      CircRNA ZRANB1 targets miR-24-3p/LPAR3 to modulate neuropathic pain via the wnt5a/β-catenin pathway following CCI.
      • Wei M
      • Li L
      • Zhang Y
      • Zhang M
      • Su Z.
      Downregulated circular RNA zRANB1 mediates Wnt5a/beta-Catenin signaling to promote neuropathic pain via miR-24-3p/LPAR3 axis in CCI rat models.
      This study indicates that the high expression of spinal circKcnk9 induces pain behavior and neuronal activity, whereas knockdown of circKcnk9 alleviates visceral hypersensitivity and neuronal excitability. CircRNA may be specific to different pains. Our data pinpoint circKcnk9, a novel circRNA that regulates chronic visceral pain.
      Recent study indicated that circRNAs could be involved in the regulation of biological processes by the circRNA/miRNA interaction network.
      • Hansen TB
      • Jensen TI
      • Clausen BH
      • Bramsen JB
      • Finsen B
      • Damgaard CK
      • Kjems J.
      Natural RNA circles function as efficient microRNA sponges.
      Our study showed that circKcnk9 could interact with miR-124-3p in spinal dorsal horn neurons. Importantly, rescue experiments showed that the miR-124-3p inhibitor restored the analgesic effects of shcircKcnk9. In previous studies, miRNA modulation is reflected in peripheral tissues of IBS, such as miR-16, miR-125b,
      • Martinez C
      • Rodino-Janeiro BK
      • Lobo B
      • Stanifer ML
      • Klaus B
      • Granzow M
      • Gonzalez-Castro AM
      • Salvo-Romero E
      • Alonso-Cotoner C
      • Pigrau M
      • Roeth R
      • Rappold G
      • Huber W
      • Gonzalez-Silos R
      • Lorenzo J
      • de Torres I
      • Azpiroz F
      • Boulant S
      • Vicario M
      • Niesler B
      • Santos J
      miR-16 and miR-125b are involved in barrier function dysregulation through the modulation of claudin-2 and cingulin expression in the jejunum in IBS with diarrhoea.
      miR-199,
      • Zhou Q
      • Yang L
      • Larson S
      • Basra S
      • Merwat S
      • Tan A
      • Croce C
      • Verne GN.
      Decreased miR-199 augments visceral pain in patients with IBS through translational upregulation of TRPV1.
      miR-485
      • Xu X
      • Li YC
      • Wu YY
      • Xu YC
      • Weng RX
      • Wang CL
      • Zhang PA
      • Zhang Y
      • Xu GY.
      Upregulation of spinal ASIC1 by miR-485 mediates enterodynia in adult offspring rats with prenatal maternal stress.
      etc. These miRNAs played important roles in visceral pain response or intestinal permeability by regulating the expression of genes. However, the roles of spinal miRNAs in IBS-like rats have not been reported. In this study, spinal miR-124-3p was found to be involved in visceral hypersensitivity in IBS-like rats. Different circRNAs have different miRNA binding sites. For example, circ-Ankib1 could promote Schwann cell proliferation by adsorbing miR-423-5p, miR-485-5p, and miR-666-3p in peripheral nerve injury.
      • Mao S
      • Zhang S
      • Zhou S
      • Huang T
      • Feng W
      • Gu X
      • Yu B.
      A Schwann cell-enriched circular RNA circ-Ankib1 regulates Schwann cell proliferation following peripheral nerve injury.
      Circ-Serpine2 promoted tumor progression by interacting with miR-124-3p and increasing KIF20A expression in glioma.
      • Li G
      • Lan Q.
      Bioinformatics analysis reveals a stem cell-expressed circ-Serpine2-mediated miRNA-mRNA regulatory subnetwork in the malignant progression of glioma.
      In this study, we first verified the association of circKcnk9 with miR-124-3p in rats and the site 528 of circKcnk9 could combine with miR-124-3p, which is consistent to the prediction by miRanda software.
      MiRNA functions as silencing gene expression by inhibiting the transcription of target genes.
      • Agarwal V
      • Bell GW
      • Nam JW
      • Bartel DP
      Predicting effective microRNA target sites in mammalian mRNAs.
      We used 3 databases to predict and found 4 genes (ITGB1, STAT3, NR3C1, and LAMC1) at the intersection. Furthermore, only STAT3 expression was significantly upregulated in IBS-like rats. It was reported that STAT3 could be an important regulator of synaptic plasticity.
      • Han JK
      • Kwon SH
      • Kim YG
      • Choi J
      • Kim JI
      • Lee YS
      • Ye SK
      • Kim SJ.
      Ablation of STAT3 in Purkinje cells reorganizes cerebellar synaptic plasticity in long-term fear memory network.
      Therefore, we demonstrated that the STAT3 gene is the target of miR-124-3p by a Dual-Luciferase reporter assay. Increased miR-124-3p expression inhibited STAT3 mRNA and protein expression levels, while blocking miR-124-3p upregulated STAT3 expression in rats. Similarly, miR-124-3p negatively regulates STAT3 expression in PC12 cells. Studies have reported that the miR-124-3p/STAT3 axis could play roles in traumatic brain injury and colorectal cancer.
      • Roshani Asl E
      • Rasmi Y
      • Baradaran B
      MicroRNA-124-3p suppresses PD-L1 expression and inhibits tumorigenesis of colorectal cancer cells via modulating STAT3 signaling.
      ,
      • Vuokila N
      • Lukasiuk K
      • Bot AM
      • van Vliet EA
      • Aronica E
      • Pitkanen A
      • Puhakka N.
      miR-124-3p is a chronic regulator of gene expression after brain injury.
      In this study, we demonstrated that the miR-124-3p/STAT3 axis is involved in visceral hypersensitivity. CircRNA can remove miRNA-mediated inhibitory effects on their target genes by binding to specific miRNAs.
      • Hansen TB
      • Jensen TI
      • Clausen BH
      • Bramsen JB
      • Finsen B
      • Damgaard CK
      • Kjems J.
      Natural RNA circles function as efficient microRNA sponges.
      The modulatory effect of circKcnk9 on STAT3 expression was further investigated. Overexpression of circKcnk9 increased STAT3 expression, whereas downregulation of circKcnk9 decreased the expression of STAT3 in vivo and in vitro. The miR-124-3p inhibitor restored STAT3 expression inhibited by shcircKcnk9. Furthermore, circKcnk9 colocalized with STAT3 in PC12 cells and the spinal dorsal horn. In other study, circHIPK3 facilitated the progression of tumor by sponging miR-124-3p to upregulate STAT3 expression in glioma.
      • Hu D
      • Zhang Y.
      Circular RNA HIPK3 promotes glioma progression by binding to miR-124-3p.
      In this study, we demonstrated that circKcnk9 could induce visceral hypersensitivity as a sponge of miR-124-3p to increase STAT3 expression in IBS-like rats. CircKcnk9/ miR-124-3p/ STAT3 may be a novel ceRNA mechanism in IBS-like rats.
      Our previous study found that GluR2 is involved in central sensitivity in IBS-like rats.
      • Chen A
      • Chen Y
      • Tang Y
      • Bao C
      • Cui Z
      • Xiao M
      • Lin C.
      Hippocampal AMPARs involve the central sensitization of rats with irritable bowel syndrome.
      Studies reported that NSF could interact with GluR2 to mediate the transport of AMPA receptors to the postsynaptic membrane.
      • Nishimune A
      • Isaac JT
      • Molnar E
      • Noel J
      • Nash SR
      • Tagaya M
      • Collingridge GL
      • Nakanishi S
      • Henley JM.
      NSF binding to GluR2 regulates synaptic transmission.
      ,
      • Xiong H
      • Cassé F
      • Zhou M
      • Xiong Z-Q
      • Joels M
      • Martin S
      • Krugers HJ.
      Interactions betweenN-Ethylmaleimide-sensitive factor and GluA2 contribute to effects of glucocorticoid hormones on AMPA receptor function in the rodent hippocampus.
      In our experiment, the expression of STAT3, NSF and GluR2 were increased in the spinal dorsal horn of IBS-like rats. Inhibition of STAT3 alleviated visceral hypersensitivity and decreased the expression of STAT3, NSF, and GluR2 after intrathecal administration of shRNA or STAT3 inhibitor (S3I-201). ChIP-qPCR showed that STAT3 could bind to the NSF promoter. Herein, we demonstrated that spinal STAT3 could regulate visceral hypersensitivity by enhancing the NSF transport of GluR2 in IBS-like rats. Electrophysiological study showed that STAT3 could participate in the regulation of AMPA receptors and synaptic plasticity in Purkinje cells of the cerebellum.
      • Han JK
      • Kwon SH
      • Kim YG
      • Choi J
      • Kim JI
      • Lee YS
      • Ye SK
      • Kim SJ.
      Ablation of STAT3 in Purkinje cells reorganizes cerebellar synaptic plasticity in long-term fear memory network.
      Studies have reported that inhibition of STAT3 relieved mechanical pain caused by cancer drugs and pathological pain caused by nerve damage.
      • Chen K
      • Fan J
      • Luo ZF
      • Yang Y
      • Xin WJ
      • Liu CC.
      Reduction of SIRT1 epigenetically upregulates NALP1 expression and contributes to neuropathic pain induced by chemotherapeutic drug bortezomib.
      ,
      • Ono T
      • Kohro Y
      • Kohno K
      • Tozaki-Saitoh H
      • Nakashima Y
      • Tsuda M.
      Mechanical pain of the lower extremity after compression of the upper spinal cord involves signal transducer and activator of transcription 3-dependent reactive astrocytes and interleukin-6.
      STAT3 may be widely involved in chronic pain. In our study, STAT3 acts as a downstream molecule of circKcnk9/miR-124-3p to promote visceral hypersensitivity of IBS.

      Conclusions

      The mechanism of IBS has not been fully elucidated. In the current study, we found spinal circKcnk9 was involved in visceral hypersensitivity of IBS and demonstrated that spinal circKcnk9 served as a miR-124-3p sponge to promote visceral hypersensitivity by regulating the STAT3/NSF/GluR2 pathway. These findings suggest a novel spinal mechanism and a potential circRNA therapeutic target for IBS.

      Author contributions

      Zhong Chen: draft preparation, experiment design, in vivo experiments and data analysis. Yuan Liu and Xianhe Wu: in vitro experiments and data analysis. Zihan Liu: IBS model construct. Wei Lin,Yang Huang, Ying Tang and Yu Chen:data analysis. Aiqin Chen and Chun Lin: experiment design, supervision, editing and writing the manuscript.

      Acknowledgments

      We thank the Public Technology Service Center of Fujian Medical University (China) for providing technical support and experimental platforms.

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