miR-132-3p targets and modulates Ptch1 to reduce neuropathic pain in mice with chronic constriction injury

doi:10.12307/2022.934

Abstract

Abstract: BACKGROUND: The pathogenesis of neuropathic pain involving many pathological processes is complex. miR-132, which is abnormally expressed in neuropathic pain conduction pathway, affects the occurrence and development of pain.
OBJECTIVE: To investigate the role and mechanism of miR-132-3p in neuropathic pain in mice with chronic constriction injury.
METHODS: Mouse microglia BV2 cells were stimulated with 100 ng/mL lipopolysaccharide to establish an activation model. The expression of ionized calcium binding adapter molecule 1 (IBA1), a marker for microglia activation, was detected by western blot. The expression of miR-132-3p was detected by RT-qPCR. After transfecting miR-132-3p mimic or inhibitor into activated BV2 cells, the expression of IBA1 and P2X4 receptor (P2X4R) was detected by western blot. miR-132-3p targeted and regulated Ptch1, which was predicted and verified. pcDNA-Ptch1 or si-Ptch1 was transfected into BV2 cells for 24 hours and then stimulated with lipopolysaccharide for 24 hours. The expression of IBA1, P2X4R and Sonic Hedgehog (Shh) signaling pathway marker proteins was detected by Western blot. BV2 cells were co-transfected with miR-132-3p-mimic and pcDNA-Ptch1 for 24 hours and then stimulated by lipopolysaccharide for 24 hours. The expression of the above proteins was further detected by western blot. Thirty C57BL/6 mice were randomly divided into control group (n=6), sham group (n=6), model group (n=6), NC-mimic group (n=6) and miR-132-3p-mimic group (n=6). The latter two groups were injected intrathecally with NC-mimic and miR-132-3p-mimic, respectively. The mechanical withdrawal threshold and thermal withdrawal latency were monitored. The expression of miR-132-3p, Ptch1, P2X4R and Shh pathway marker proteins in dorsal root ganglion was detected by RT-qPCR and western blot to clarify the role and mechanism of miR-132-3p in chronic constriction injury-induced neuropathic pain.
RESULTS AND CONCLUSION: Compared with the control group, lipopolysaccharide stimulation promoted the activation of BV2 cells (P < 0.05) and down-regulated the expression of miR-132-3p (P < 0.05). miR-132-3p inhibited the activation of Shh signaling pathway through targeted regulation of Ptch1, and further inhibited the activation of BV2 cells and the expression of P2X4R. Overexpression of Ptch1 could reverse the effect of miR-132-3p-mimic (P < 0.05). The chronic constriction injury model was successfully established. Compared with the NC-mimic injection group, intrathecal injection of miR-132-3p-mimic could down-regulate the expression of Ptch1 (P < 0.05), inhibit the activation of the Shh signaling pathway (P < 0.05), reduce the expression of P2X4R (P < 0.05), and significantly increase the mechanical withdrawal threshold and thermal withdrawal latency of mice with chronic constriction injury (P < 0.01). Overall, miR-132-3p can reduce neuropathic pain in mice with chronic constriction injury by targeting Ptch1 and inhibiting the activation of Shh signaling pathway.

Key words: miR-132-3p, Ptch1, the Sonic Hedgehog (Shh) signaling pathway, chronic constriction injury, neuropathic pain, microglia

CLC Number:

Zhao Feng, Fan Shaoqing, Cheng Xiaoyan, Li Xiaona, Li Changsheng, Ma Haojie. miR-132-3p targets and modulates Ptch1 to reduce neuropathic pain in mice with chronic constriction injury[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(2): 230-236.

Figures/Tables 7

References

[1] BICKER S, LACKINGER M, WEIß K, et al. MicroRNA-132, -134, and -138: a microRNA troika rules in neuronal dendrites. Cell Mol Life Sci. 2014;71(20):3987-4005.
[2] CHEN D, HU S, WU Z, et al. The Role of MiR-132 in Regulating Neural Stem Cell Proliferation, Differentiation and Neuronal Maturation. Cell Physiol Biochem. 2018;47(6):2319-2330.
[3] ATEN S, PAGE CE, KALIDINDI A, et al. miR-132/212 is induced by stress and its dysregulation triggers anxiety-related behavior. Neuropharmacology. 2019;144:256-270.
[4] LIU G, YIN F, ZHANG C, et al. Effects of regulating miR-132 mediated GSK-3β on learning and memory function in mice. Exp Ther Med. 2020;20(2):1191-1197.
[5] ARAI M, GENDA Y, ISHIKAWA M, et al. The miRNA and mRNA changes in rat hippocampi after chronic constriction injury. Pain Med. 2013;14(5): 720-729.
[6] 施燕渲,朱涛.微RNA-132在神经病理性疼痛中的研究进展[J].医学综述,2017,23(16):3170-3173.
[7] 施燕渲,朱涛.miR-132在神经病理性疼痛传导通路中的表达[J].实用医学杂志,2017,33(19):3189-3192.
[8] ZHANG R, HUANG M, CAO Z, et al. MeCP2 plays an analgesic role in pain transmission through regulating CREB / miR-132 pathway. Mol Pain. 2015;11:19.
[9] WU B, LI J, WANG H, et al. MiR-132-3p serves as a tumor suppressor in mantle cell lymphoma via directly targeting SOX11. Naunyn Schmiedebergs Arch Pharmacol. 2020;393(11):2197-2208.
[10] GE Y, SONG X, LIU J, et al. The Combined Therapy of Berberine Treatment with lncRNA BACE1-AS Depletion Attenuates Aβ25-35 Induced Neuronal Injury Through Regulating the Expression of miR-132-3p in Neuronal Cells. Neurochem Res. 2020;45(4):741-751.
[11] 杨秀方,褚凯丽,李媛,等.miR-132调节非小细胞肺癌hedgehog信号通路受体基因PTCH1及细胞增殖、迁移和侵袭[J].复旦学报(医学版),2015,42(3):291-299,306.
[12] YANG Z, GAO L, JIA H, et al. The Expression of Shh, Ptch1, and Gli1 in the Developing Caudal Spinal Cord of Fetal Rats With Anorectal Malformations. J Surg Res. 2019;233:173-182.
[13] TUKACHINSKY H, PETROV K, WATANABE M, et al. Mechanism of inhibition of the tumor suppressor Patched by Sonic Hedgehog. Proc Natl Acad Sci U S A. 2016;113(40):E5866-E5875.
[14] MOREAU N, MAUBORGNE A, BOURGOIN S, et al. Early alterations of Hedgehog signaling pathway in vascular endothelial cells after peripheral nerve injury elicit blood-nerve barrier disruption, nerve inflammation, and neuropathic pain development. Pain. 2016;157(4):827-839.
[15] MOREAU N, BOUCHER Y. Hedging against Neuropathic Pain: Role of Hedgehog Signaling in Pathological Nerve Healing. Int J Mol Sci. 2020; 21(23):9115.
[16] FURUBE E, KAWAI S, INAGAKI H, et al. Brain Region-dependent Heterogeneity and Dose-dependent Difference in Transient Microglia Population Increase during Lipopolysaccharide-induced Inflammation. Sci Rep. 2018;8(1):2203.
[17] LV R, DU L, ZHANG L, et al. Polydatin attenuates spinal cord injury in rats by inhibiting oxidative stress and microglia apoptosis via Nrf2/HO-1 pathway. Life Sci. 2019;217:119-127.
[18] 李永丰,任维,刘一辉.CB1受体通过钾离子通道介导外周镇痛作用[J].中国病理生理杂志,2019,35(4):92-98.
[19] 刘宇临.Sonic hedgehog信号通路在神经病理性疼痛模型大鼠中的表达及其意义的研究[D].重庆:重庆医科大学,2017.
[20] COLLOCA L, LUDMAN T, BOUHASSIRA D, et al. Neuropathic pain. Nat Rev Dis Primers. 2017;3:17002.
[21] BARON R, MAIER C, ATTAL N, et al. Peripheral neuropathic pain: a mechanism-related organizing principle based on sensory profiles. Pain. 2017;158(2):261-272.
[22] NI HD, YAO M, HUANG B, et al. Glial activation in the periaqueductal gray promotes descending facilitation of neuropathic pain through the p38 MAPK signaling pathway. J Neurosci Res. 2016;94(1):50-61.
[23] KUAN YH, SHYU BC. Nociceptive transmission and modulation via P2X receptors in central pain syndrome. Mol Brain. 2016;9(1):58.
[24] CHEN L, LIU YW, YUE K, et al. Differential expression of ATP-gated P2X receptors in DRG between chronic neuropathic pain and visceralgia rat models. Purinergic Signal. 2016;12(1):79-87.
[25] ZHANG WJ, LUO HL, ZHU ZM. The role of P2X4 receptors in chronic pain: A potential pharmacological target. Biomed Pharmacother. 2020; 129:110447.
[26] INOUE K, TSUDA M. Nociceptive signaling mediated by P2X3, P2X4 and P2X7 receptors. Biochem Pharmacol. 2021;187:114309.
[27] 陈桂玲,张加强.右美托咪定对神经病理性痛大鼠脊髓P2X3和P2X4受体表达的影响[J].中国现代医学杂志,2017,27(6):27-31.
[28] TRANG M, SCHMALZING G, MÜLLER CE, et al. Dissection of P2X4 and P2X7 Receptor Current Components in BV-2 Microglia. Int J Mol Sci. 2020;21(22):8489.
[29] INOUE K. Role of the P2X4 receptor in neuropathic pain. Curr Opin Pharmacol. 2019;47:33-39.
[30] ZHANG WJ, ZHU ZM, LIU ZX. The role of P2X4 receptor in neuropathic pain and its pharmacological properties. Pharmacol Res. 2020;158:104875.
[31] HALIEVSKI K, GHAZISAEIDI S, SALTER MW. Sex-Dependent Mechanisms of Chronic Pain: A Focus on Microglia and P2X4R. J Pharmacol Exp Ther. 2020;375(1):202-209.
[32] XU R, YANG F, LI L, et al. CB2/miR-124 signaling down-regulate the expression of purinergic P2X4 and P2X7 receptor in dorsal spinal cord of CCI rats. 2020.
[33] GONG X, HUANG MY, CHEN L. Mechanism of miR-132-3p Promoting Neuroinflammation and Dopaminergic Neurodegeneration in Parkinson’s Disease. eNeuro. 2022;9(1):ENEURO.0393-21.2021.
[34] WANG X, WANG H, ZHANG T, et al. Inhibition of MicroRNA-195 Alleviates Neuropathic Pain by Targeting Patched1 and Inhibiting SHH Signaling Pathway Activation. Neurochem Res. 2019;44(7):1690-1702.
[35] COHEN M, KICHEVA A, RIBEIRO A, et al. Ptch1 and Gli regulate Shh signalling dynamics via multiple mechanisms. Nat Commun. 2015;6: 6709.
[36] 孙艳.2,5-己二酮致人卵巢颗粒细胞凋亡及Sonichedgehog信号通路调控机制的研究[D].福州:福建医科大学,2012.
[37] CHOUDHRY Z, RIKANI AA, CHOUDHRY AM, et al. Sonic hedgehog signalling pathway: a complex network. Ann Neurosci. 2014;21(1):28-31.
[38] 牛小莉.丁苯酞对血管性痴呆大鼠认知功能及Shh/Ptch1信号通路和内质网应激相关蛋白表达的影响[D].石家庄:河北医科大学, 2019.
[39] BELGACEM YH, HAMILTON AM, SHIM S, et al. The Many Hats of Sonic Hedgehog Signaling in Nervous System Development and Disease. J Dev Biol. 2016;4(4):35.
[40] HE W, CUI L, ZHANG C, et al. Sonic Hedgehog Promotes Neurite Outgrowth of Primary Cortical Neurons Through Up-Regulating BDNF Expression. Neurochem Res. 2016;41(4):687-695.
[41] LI X, LI Y, LI S, et al. The role of Shh signalling pathway in central nervous system development and related diseases. Cell Biochem Funct. 2021;39(2):180-189.
[42] 刘宇临,刘丹彦.Sonic hedgehog信号通路在神经病理性疼痛模型大鼠中的表达及其意义[J].重庆医科大学学报,2016,41(1):61-65.
[43] 冯晓雪,蔡孟,杨晓秋,等.大鼠神经病理性痛时脊髓Sonic hedgehog信号通路与炎症反应的关系[J].中华麻醉学杂志,2018, 38(11):1347-1350.
[44] 冯晓雪,杨晓秋,刘丹彦. 环巴明抑制SHH信号通路缓解大鼠神经病理性疼痛[J].基础医学与临床,2016,36(5):661-665.