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

铜绿假单胞菌（PA）角膜炎是发展最迅速和破坏性的疾病之一， 角膜和视力障碍和失明的全球性原因。抗药性菌株的出现 有效的疾病管理面临更多挑战。开发替代疗法迫在眉睫。 在这方面，微小RNA（miRNAs）是小的非编码RNA，是基因表达的重要调节因子。 miRNAs在人类疾病中起关键作用，是可行的治疗靶点。然而，miRNAs的作用 在PA角膜炎中仍有很大一部分未被探索。我们的长期目标是揭示 在眼部感染性疾病中的miRNAs，识别新的基于miRNAs的治疗靶点，并开发 这些疾病的替代治疗。拟议的研究将直接解决这一知识差距。是 基于我们最近发表的强有力的初步数据，显示抗miR靶向的应用 小鼠中miR-183/96/182聚簇（此后称为miR-183 C）和miR-183 C敲除 减少角膜神经密度和神经肽产生，同时降低PA角膜炎的严重程度; miR- 183 C靶向调节角膜感觉神经支配的关键基因，例如，细菌诱导的感觉神经元 活化和神经肽产生，例如，Toll样受体（TLR）4、甲酰蛋白受体（Fpr）1和 P物质（sP）前体基因Tac 1。这些数据使我们得出一个总体假设，即除了 在先天免疫中，miR-183 C通过调节角膜感觉神经功能调节PA角膜炎， 通过靶向参与感觉神经支配的关键基因的神经免疫相互作用，PA诱导的感觉神经元 活化和促炎性神经肽产生。本申请提出了三个目标， 这个假设。在目标1中，将荧光素酰胺（FAM）标记的抗miR-183 C应用于受试者的角膜。 野生型（WT）小鼠，以检验抗miR-183 C处理上调Tac 1、TLR 4、Fpr 1和 角膜感觉神经中的nrp 1，导致促炎神经肽增加（例如，sP）和早期 免疫/炎症反应（感染后<24小时）和加速的感觉神经减少， 在疾病的后期阶段，促炎性神经肽和免疫/炎症反应减少 分辨率目的2将使用感觉神经元特异性miR-183 C条件性敲除来检验该假设 小鼠模型目的3将检验miR-183 C的体外/离体敲低或敲除在两种细胞中均是有效的假设。 人类和小鼠感觉神经元与体内数据相似，因为它将上调Tac 1、TLR 4和Fpr 1， 增强PA诱导的感觉神经元分泌促炎神经肽，并增加Nrp 1 表达以抑制神经突生长。人类和哺乳动物中miR-183 C靶基因的全基因组鉴定 小鼠三叉神经节感觉神经元也将通过RNA测序进行。 本研究将为miR-183 C调控PA角膜炎的分子基础提供新的生物学见解 和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.