MicroRNA 374 as an Epigenetic Regulator of Chronic Pain

摘要 慢性原发性疼痛状况(CPPC)，如纤维肌痛和颞下颌关节紊乱病(TMD) 构成一个重大的医疗保健问题，影响着1亿多美国人，其中主要是女性。这个 CPPC的起源与基因和环境因素有关，这些因素增强了儿茶酚胺的张力。据估计 3例CPPC患者中有2例存在编码儿茶酚-O-甲基转移酶(COMT；一种酶)的基因变异 这会代谢儿茶酚胺)，导致COMT活性降低，儿茶酚胺水平升高。疼痛在 这些人会因压力事件(如汽车碰撞)而得到加强，导致释放更多的 来自交感神经的儿茶酚胺。我们的实验室已经证明，儿茶酚胺通过激活 外周β-肾上腺素能受体3(ADRB3)及其下游调节神经元兴奋性和 免疫反应。儿茶酚胺信号也被证明改变了microRNAs的表达 (MiRNAs)，这是一种小的非编码RNA，负向调节mRNA靶标。然而，miRNA的作用 CPPC病理生理学中的失调仍未得到充分研究，也不清楚。我们案件的初步数据- 对照研究显示，TMD患者miR-374水平降低。我们将这一发现复制到了一个 CPPC动物模型，COMT活性低的小鼠暴露在应激状态下表现出疼痛和减少 MiR-374水平。在同一只小鼠中，5个miR-374mRNA靶点在TMD患者中表达失调 (ATXN7、CRK、HIF1a、Numb和TGFBR2)在脂肪和脊髓组织中也表达失调， 据预测，它们会影响免疫信号和疼痛。HIF1a、Numb和TGFBR2在 雌性小鼠脊髓中ATXN7和TGFBR2表达下调，而雄性小鼠脊髓中ATXN7和TGFBR2表达下调。这些 研究结果指出，新的rna靶点可能在与儿茶酚胺升高相关的疼痛中发挥重要作用。 然而，需要机械论的研究来确定它们的因果作用。因此，这项提案的目标是 直接测试miR-374及其信使核糖核酸靶标与疼痛、炎症的关系。我的中央 假设ADRB3的儿茶酚胺激活降低了miR-374的水平，导致了 以性激素依赖的方式促进炎症和慢性疼痛的mRNA。我将使用主要的 使用荧光素酶报告系统测量1)miR-374与mRNA靶标结合的脂肪细胞和神经元 2)定量聚合酶链式反应检测ADRB3激活和性激素对miR-374和mRNA靶基因表达的影响。在……里面 我们的CPPC小鼠模型，我还将测量3)合成miR-374的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)miR-374和mRNA在不同细胞类型中的靶向表达 结合RNAScope和免疫组织化学方法。这些结果将澄清我们对 促进CPPC的表观遗传机制并确定新的靶点，以改进这些患者的治疗方案 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。

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.