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 (CPPC)，例如纤维肌痛和颞下颌关节紊乱病 (TMD) 构成了影响超过 1 亿美国人（主要是女性）的重大医疗保健问题。这 CPPC 的起源与增强儿茶酚胺张力的遗传和环境因素有关。估计三分之二的 CPPC 患者的儿茶酚-O-甲基转移酶编码基因（COMT；一种酶）存在变异代谢儿茶酚胺），导致 COMT 活性低和儿茶酚胺水平升高。疼痛在这些人因压力事件（例如机动车碰撞）而增强，导致释放更多的来自交感神经的儿茶酚胺。我们的实验室表明，儿茶酚胺通过激活外周 β 肾上腺素能受体 3 (Adrb3) 和调节神经元兴奋性的下游介质免疫反应。儿茶酚胺信号传导也被证明可以改变 microRNA 的表达 (miRNA)，它们是负向调节 mRNA 靶标的小非编码 RNA。然而，miRNA 的作用 CPPC 病理生理学的失调仍然没有得到充分研究和不清楚。我们案例的初步数据- 对照研究表明，TMD 患者的 miR-374 水平降低。我们在一项研究中重复了这一发现 CPPC 动物模型，其中 COMT 活性低的小鼠暴露于压力时表现出疼痛并减少 miR-374 的水平。在同一只小鼠中，TMD 患者的 5 个 miR-374 mRNA 靶点失调（ATXN7、CRK、HIF1A、NUMB 和 TGFBR2）在脂肪和脊髓组织中也失调，其中预计它们会影响免疫信号和疼痛。 HIF1A、NUMB 和 TGFBR2 在雌性小鼠的脂肪中，ATXN7 和 TGFBR2 在雄性小鼠的脊髓中下调。这些研究结果指出新的 RNA 靶标可能在儿茶酚胺升高相关的疼痛中发挥重要作用语气，但需要进行机制研究来确定其因果作用。因此，本提案的目标目的是直接测试 miR-374、其 mRNA 靶标、疼痛和炎症之间的关系。我的中央假设儿茶酚胺激活 Adrb3 会降低 miR-374 的水平，从而导致以性激素依赖性方式促进炎症和慢性疼痛的 mRNA。我将使用初级脂肪细胞和神经元使用荧光素酶报告系统测量 1) miR-374 与 mRNA 靶标的结合，以及 2) 使用 qPCR 观察 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疼痛和细胞因子产生，4) miR-374 和 mRNA 在不同细胞类型中的靶表达结合RNAscope和免疫组织化学方法。这些结果将阐明我们的理解有助于 CPPC 的表观遗传机制并确定改善治疗方案的新靶标具备这些条件。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高度支持和协作的环境中促进职业发展。