The miR-183/96/182 Cluster in Pseudomonas aeruginosa-induced Keratitis

铜绿假单胞菌(PA)角膜炎是近年来发展最迅速、破坏性最大的疾病之一。 眼角膜是导致视力受损和失明的全球性原因。抗生素耐药菌株的出现构成 有效的疾病管理面临更多挑战。开发替代疗法是当务之急。 在这方面，microRNAs(MiRNAs)是一种小的、非编码的RNA，是基因表达的重要调节因子。 MiRNAs在人类疾病中发挥关键作用，是可行的治疗靶点。然而，miRNAs的作用 在PA中，角膜炎在很大程度上仍未被研究。我们的长期目标是揭开分子机制 眼部感染性疾病中miRNAs的研究，寻找新的基于miRNA的治疗靶点并开发 这些疾病的替代疗法。拟议的研究将直接解决这一知识差距。它是 基于我们最近公布的强有力的初步数据，这些数据表明应用抗MIR靶向 MiR-183/96/182簇(以下简称miR-183C)和miR-183C在小鼠中的敲除 减少角膜神经密度和神经肽的产生，同时减轻PA角膜炎的严重程度；miR- 183C靶向调控角膜感觉神经支配的关键基因，如Nrp1，细菌诱导的感觉神经元 激活和神经肽的产生，如Toll样受体(TLR)4、甲酰蛋白受体(FPR)1和 P物质(SP)前体基因Tac1。这些数据让我们得出了一个重要的假设，除了 先天免疫，miR-183C通过调节角膜感觉神经功能和调节PA角膜炎 靶向感觉神经关键基因的神经免疫相互作用--PA诱导的感觉神经元 激活和促炎神经肽的产生。在此应用程序中提出了三个目标来进行测试 这个假说。在目标1中，荧光素酰亚胺(FAM)标记的抗miR-183C将应用于 野生型(WT)小鼠，以验证抗miR-183C治疗上调Tac1，TLR4，FPR1和 Nrp1在角膜感觉神经中的表达，导致促炎神经肽(如SP)的增加和早期的 免疫/炎症反应(感染后24小时)和感觉神经加速减少和 疾病后期促炎神经肽和免疫/炎症反应降低 决议。Aim 2将使用感觉神经元特异性miR-183C条件基因敲除来检验这一假设 老鼠模型。Aim 3将验证以下假设：miR-183C在体外/体外都被敲除或敲除 人类和小鼠的感觉神经元与体内的数据相似，因为它将上调Tac1、TLR4和FPR1以 促进PA诱导的感觉神经元分泌促炎神经肽，并增加Nrp1 表达抑制轴突生长。人和人miR-183C靶基因的全基因组鉴定 小鼠三叉神经节感觉神经元也将由RNA序列进行传导。 这项研究将为miR-183C调节PA角膜炎的分子基础提供新的生物学见解 以及其他眼部感染性疾病。它将揭示机制，并揭示抗miR治疗的新靶点。

Pseudomonas aeruginosa (PA) keratitis is one of the most rapidly developing and destructive diseases of the cornea and a global cause of visual impairment and blindness. Emergence of antibiotic-resistant strains poses additional challenges for effective disease management. Development of alternative treatment is urgent. In this regard, microRNAs (miRNAs) are small, non-coding RNAs and important regulators of gene expression. miRNAs play critical roles in human diseases and are viable therapeutic targets. However, the roles of miRNAs in PA keratitis remain largely unexplored. Our long-term objectives are to uncover the molecular mechanisms of miRNAs in ocular infectious diseases, identify novel miRNA-based therapeutic targets and develop alternative treatment of these diseases. The proposed research will directly address this knowledge gap. It is built upon our recently published and strong preliminary data showing that application of anti-miRs targeting the miR-183/96/182 cluster (referred to as miR-183C from here on) and knockout of miR-183C in mice decreases corneal nerve density and neuropeptide production, while reducing the severity of PA keratitis; miR- 183C targets key genes regulating corneal sensory innervation, e.g., Nrp1, bacterium-induced sensory-neuron activation and neuropeptide production, e.g., Toll-Like Receptor (TLR)4, Formyl Protein Receptor (Fpr)1 and substance P (sP) precursor gene Tac1. These data lead us to the overarching hypothesis that, in addition to innate immunity, miR-183C modulates PA keratitis through its regulation of corneal sensory nerve function and neuroimmune interaction by targeting key genes involved in sensory innervation, PA-induced sensory-neuron activation and pro-inflammatory neuropeptide production. Three Aims are proposed in this application to test this hypothesis. In Aim 1, fluorescein amidites (FAM)-labeled anti-miR-183C will be applied to the cornea of wild-type (WT) mice to test the hypothesis that anti-miR-183C treatment upregulates Tac1, TLR4, Fpr1 and Nrp1 in corneal sensory nerves, resulting in increased pro-inflammatory neuropeptides (e.g., sP) and an early immune/inflammatory response (<24 hours post-infection) and an accelerated sensory-nerve reduction and decreased pro-inflammatory neuropeptides and immune/inflammatory response in a later stage during disease resolution. Aim 2 will test the hypothesis using a sensory neuron-specific miR-183C conditional knockout mouse model. Aim 3 will test the hypothesis that knockdown or knockout of miR-183C in vitro/ex vivo in both human and mouse sensory neurons parallels the in vivo data in that it will upregulate Tac1, TLR4 and Fpr1 to enhance PA-induced secretion of pro-inflammatory neuropeptides by sensory neurons, and increase Nrp1 expression to inhibit neurite growth. A genome-wide identification of miR-183C target genes in both human and mouse trigeminal ganglion sensory neurons will also be conducted by RNA seq. This study will provide novel biological insights into molecular bases of miR-183C's regulation of PA keratitis and other ocular infectious diseases. It will uncover mechanisms and reveal new targets for anti-miR therapy.