MicroRNA 374 as an Epigenetic Regulator of Chronic Pain

MicroRNA 374 作为慢性疼痛的表观遗传调节剂

• 批准号: 10747237
• 负责人: Nathaniel Hernandez
• 金额: $ 4.28万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10747237
• 关键词: Adipocytes; Adipose tissue; Affect; Agonist; American; Analgesics; Androgens; Animal Model; Aromatase Inhibitors; Binding; Biological Assay; CRK gene; Career Mobility; Case/Control Studies; Catechol O-Methyltransferase; Catecholamines; Cell Line; Cells; Chronic; Clinical; Data; Development; Dose; Down-Regulation; Environmental Risk Factor; Enzymes; Epigenetic Process; Estrogens; Etiology; Exhibits; Exposure to; Female; Fibromyalgia; Fostering; Functional disorder; Gene Expression; Genes; Genetic; Genotype; Gonadal Steroid Hormones; HIF1A gene; Healthcare; Immune; Immune response; Immune signaling; In Vitro; Incubated; Individual; Inflammation; Link; Luciferases; Measures; Mediating; Mediator; Messenger RNA; Metabolism; Methodology; Methods; MicroRNAs; Modeling; Molecular; Mus; Nerve; Nervous System Physiology; Neuroglia; Neurons; Norepinephrine; Pain; Pathology; Patients; Peripheral; Persistent pain; Plasma; Play; Production; Proteins; Quality of life; RNA; Receptor Activation; Receptors, Adrenergic, beta-3; Regulation; Reporter; Reporting; Risk; Role; Sex Differences; Signal Transduction; Site; Spinal Cord; Stress; Stressful Event; Syndrome; System; TGFBR2 gene; Techniques; Temporomandibular Joint Disorders; Testing; Tissues; Training; Universities; Untranslated RNA; Variant; Vehicle crash; Visualization; Work; antagonist; cell type; chronic pain; clinically relevant; collaborative environment; cytokine; enhancing factor; genetic variant; immune function; improved; in vivo; mRNA Expression; male; mouse model; neuronal excitability; novel; overexpression; pain behavior; pharmacologic; pre-clinical; prevent; sex; supportive environment; transcriptome sequencing

Abstract Chronic primary pain conditions (CPPCs) such as fibromyalgia and temporomandibular disorder (TMD) constitute a significant healthcare problem that affects over 100 million, predominately female, Americans. The origin of CPPCs is linked to genetic and environmental factors that enhance catecholamine tone. An estimated 2 in 3 patients with CPPCs have variants in the gene encoding catechol-O-methyltransferase (COMT; an enzyme that metabolizes catecholamines) that result in low COMT activity and increased catecholamine levels. Pain in these individuals is enhanced by stressful events (eg, motor vehicle collision) resulting in increased release of catecholamines from sympathetic nerves. Our lab has shown that catecholamines drive pain via activation of peripheral beta-adrenergic receptor 3 (Adrb3) and downstream mediators that regulate neuronal excitability and immune responses. Catecholamine signaling has also been shown to alter the expression of microRNAs (miRNAs), which are small non-coding RNAs that negatively regulate mRNA targets. However, the role of miRNA dysregulation in CPPC pathophysiology remains understudied and unclear. Preliminary data from our case- control study reveal that patients with TMD have decreased levels of miR-374. We replicated this finding in an animal model of CPPCs where mice with low COMT activity exposed to stress exhibited pain and decreased levels of miR-374. In the same mice, 5 miR-374 mRNA targets that were dysregulated in patients with TMD (ATXN7, CRK, HIF1A, NUMB, and TGFBR2) were also dysregulated in adipose and spinal cord tissues, where they are predicted to influence immune signaling and pain. HIF1A, NUMB, and TGFBR2 were upregulated in adipose from female mice, while ATXN7 and TGFBR2 were downregulated in spinal cord from male mice. These findings point to new RNA targets that may play an important role in pain related to heightened catecholamine tone, yet mechanistic studies are needed to determine their causal role. Thus, the objective of this proposal is to directly test the relationship between miR-374, its mRNA targets, pain, and inflammation. My central hypothesis is that catecholamine activation of Adrb3 reduces levels of miR-374, leading to dysregulation of mRNAs that promote inflammation and chronic pain in a sex hormone-dependent manner. I will use primary adipocytes and neurons to measure 1) miR-374 binding to mRNA targets using a luciferase reporter system and 2) the effects of Adrb3 activation and sex hormones on miR-374 and mRNA target expression using qPCR. In our CPPC mouse model, I will also measure 3) the effects of synthetic miR-374 overexpression and antagonism on pain and cytokine production, and 4) miR-374 and mRNA target expression in distinct cell types using combined RNAscope and immunohistochemical methods. These results will elucidate our understanding of epigenetic mechanisms contributing to CPPCs and identify novel targets for improved treatment options for those with these conditions. The proposed training plan will promote development of new in vitro and in vivo techniques and foster career advancement in a highly supportive and collaborative environment at Duke University.

摘要 慢性原发性疼痛疾病（CPPC），如纤维肌痛和颞下颌关节紊乱病（TMD） 这是一个严重的健康问题，影响了1亿多美国人，其中主要是女性。的 CPPC的起源与增强儿茶酚胺张力的遗传和环境因素有关。估计 3例CPPC患者中有2例在编码儿茶酚-O-甲基转移酶（COMT;一种酶）的基因中存在变异， 代谢儿茶酚胺），导致COMT活性降低和儿茶酚胺水平升高。疼痛 这些人被压力事件（例如，机动车碰撞）增强，导致增加释放 交感神经分泌的儿茶酚胺我们的实验室已经表明，儿茶酚胺通过激活 外周β-肾上腺素能受体3（Adrb 3）和调节神经元兴奋性的下游介质， 免疫反应。儿茶酚胺信号也被证明可以改变microRNA的表达， miRNA是一种小的非编码RNA，负调控mRNA靶点。然而，miRNA的作用 CPPC病理生理学中的调节失调仍未得到充分研究且尚不清楚。我们的初步数据- 对照研究显示，TMD患者的miR-374水平降低。我们在一个 在CPPC动物模型中，暴露于应激的低COMT活性小鼠表现出疼痛和降低 miR-374的水平。在相同的小鼠中，TMD患者中5个失调的miR-374 mRNA靶点， （ATXN 7，CRK，HIF 1A，NUMB和TGFBR 2）也在脂肪和脊髓组织中失调， 它们被预测会影响免疫信号和疼痛。HIF 1A、NUMB和TGFBR 2在正常对照组中表达上调。 ATXN 7和TGFBR 2在雌性小鼠的脊髓中表达下调。这些 研究结果指出，新的RNA靶点可能在与升高的儿茶酚胺相关的疼痛中发挥重要作用， 然而，需要进行机械研究来确定它们的因果作用。因此，本提案的目的是 直接测试miR-374及其mRNA靶点、疼痛和炎症之间的关系。我的中枢 假设Adrb 3的儿茶酚胺激活降低了miR-374的水平，导致了 以性别依赖方式促进炎症和慢性疼痛的mRNA。我将使用primary 1）使用荧光素酶报告系统测量miR-374与mRNA靶标的结合， 2)Adrb 3激活和性激素对miR-374和mRNA靶表达的影响，使用qPCR。在 我们的CPPC小鼠模型，我还将测量3）合成的miR-374过表达和拮抗作用的影响 对疼痛和细胞因子产生的影响，以及4）使用 RNA显微镜和免疫组织化学相结合的方法。这些结果将阐明我们对 表观遗传机制有助于CPPC，并确定新的目标，以改善这些治疗方案 在这些条件下。拟议的培训计划将促进新的体外和体内技术的发展 并在杜克大学高度支持和协作的环境中促进职业发展。