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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的慢性原发性疼痛条件（CPPC），例如纤维肌痛和颞下颌疾病（TMD）构成一个重大的医疗保健问题，影响了超过1亿，主要是女性的美国人。这 CPPC的起源与增强儿茶酚胺张力的遗传和环境因素有关。估计在编码儿茶醇-O-甲基转移酶的基因中，有3例CPPC患者具有变异性（COMT；酶这代表了儿茶酚胺），这会导致COMT活性低和儿茶酚胺水平升高。疼痛这些人通过压力事件（例如，机动车碰撞）增强了这些人，从而增加了同情神经的儿茶酚胺。我们的实验室表明，儿茶酚胺通过激活驱动疼痛外围β-肾上腺素受体3（ADRB3）和下游介质调节神经元兴奋性和免疫反应。儿茶酚胺信号传导也已被证明改变了microRNA的表达（miRNA），它是对mRNA靶标负调控的小非编码RNA。但是，mirna的作用 CPPC病理生理学的失调仍在研究和尚不清楚。我们案件的初步数据 - 对照研究表明，TMD患者的miR-374水平降低。我们在一个中复制了这一发现 CPPC的动物模型，其中低COMT活性的小鼠表现出疼痛，减轻 miR-374的水平。在同一只小鼠中，TMD患者的5个miR-374 mRNA靶标的失调（ATXN7，CRK，HIF1A，NUMB和TGFBR2）在脂肪和脊髓组织中也失调预计它们会影响免疫信号传导和疼痛。 HIF1A，Numb和TGFBR2在雌性小鼠的脂肪，而ATXN7和TGFBR2在雄性小鼠的脊髓中下调。这些调查结果指出了可能在与增强的儿茶酚胺有关的疼痛中起重要作用的新RNA靶标语气，需要机械研究来确定其因果关系。因此，该提议的目的是直接测试miR-374，其mRNA靶标，疼痛和炎症之间的关系。我的中央假设是ADRB3的儿茶酚胺激活降低了miR-374的水平，导致失调的失调以性激素依赖性方式促进炎症和慢性疼痛的mRNA。我将使用主要脂肪细胞和神经元测量1）miR-374使用荧光素酶报告器系统与mRNA靶标结合，并 2）使用qPCR，ADRB3激活和性激素对miR-374和mRNA靶标表达的影响。在我们的CPPC小鼠模型，我还将测量3）合成miR-374过表达和拮抗作用的影响关于疼痛和细胞因子的产生，以及4）miR-374和mRNA靶标在不同的细胞类型中使用合并的rnascope和免疫组织化学方法。这些结果将阐明我们对有助于CPPC的表观遗传机制，并确定了改进治疗选择的新目标在这些条件下。拟议的培训计划将促进新的体外和体内技术的开发并在杜克大学的高度支持和协作环境中促进职业发展。