Dominant microRNAs as biomarkers in innate immunity and periodontitis

摘要 牙周病仅在美国就影响了数百万人，并已被证实为前驱疾病 其他使人衰弱的系统性疾病，包括心血管疾病、阿尔茨海默病、类风湿 关节炎和不良妊娠结局。我们的实验室已经表明，某些microRNA的表达 (MiRNAs)在小鼠多菌性牙周炎中升高。目前的范例是miRNAs是 一般参与微调基因表达。然而，我们的体外研究表明，miR- 146a是一种主要的miRNA，在内毒素刺激下可以上调30到200倍， 更重要的是，这种增长持续了几天。我们已经证明miR-146a是一个关键 利用单核/巨噬细胞系统调节内毒素诱导的耐受和交叉耐受。 类似地，我们已经证明miR-132是肽聚糖刺激的单核细胞中的主要miRNA。 并可诱导交叉耐受。在目前的提案中，这些主要的miRNA将使用 体外和体内系统，以确定它们在我们建立的小鼠牙周炎模型中的作用 牙周主要病原菌(牙龈卟啉单胞菌、齿密螺旋体、坦纳氏菌 连翘属、核梭杆菌)和戈登链球菌为早期定殖者。总体假设是 这些miRNAs是调控Toll样受体(TLR)/IL-1信号通路的主要miRNAs。四 提出了具体的目标。具体目标1将定义这些miRNAs的机制作用，包括 原代人类口腔上皮细胞中靶mRNAs的定位，参照人类单核细胞。特定的 目标2将确定单一或时序多菌体中主要miRNA的表达动力学 小鼠感染性牙周炎模型的建立及TLR2、TLR4基因敲除的相关性研究 老鼠。特殊目标3将评估这些主要miRNAs在牙周炎模型中的相对贡献。 使用特定的miRNA基因敲除小鼠。特定目标4将调查这些主要miRNAs之间的关联 慢性牙周炎患者龈沟液和牙周组织中的生物标志物及其相关性 治疗效果。长期目标是确定这些占主导地位的miRNA能提供多大范围的服务。 作为功能生物标记物，它们调节导致牙周炎的先天免疫反应途径。在……里面 在未来的研究中，这种小鼠牙周炎模型将成为帮助发展对这些miRNA的操纵的关键 功能和/或TLR途径进入牙周炎的新疗法。因为先天免疫反应是 已知在许多其他疾病中起关键作用，我们的发现可能适用于其他慢性疾病 炎症性和自身免疫性疾病。

Abstract Periodontal disease affects millions of individuals in the US alone and has been substantiated as a precursor to other debilitating systemic diseases, including cardiovascular disease, Alzheimer’s disease, rheumatoid arthritis, and adverse pregnancy outcomes. Our laboratories have shown that expression of certain microRNAs (miRNAs) are elevated in murine polymicrobial periodontitis. The current paradigm is that miRNAs are generally involved in fine-tuning gene expression. However, our in vitro studies have demonstrated that miR- 146a is a dominant miRNA that can be up-regulated 30 to 200+ fold during lipopolysaccharide stimulation and, more importantly, that this increase is sustained for days. We have demonstrated that miR-146a is a key regulator in endotoxin-induced tolerance and cross-tolerance using a monocyte/macrophage-based system. Similarly, we have demonstrated that miR-132 is a dominant miRNA in peptidoglycan-stimulated monocytes and can induce cross-tolerance. In the current proposal, these dominant miRNAs will be examined using in vitro and in vivo systems to critically determine their role in our established murine model of periodontitis with 4 major well-characterized periodontal pathogens (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Fusobacterium nucleatum) and Streptococcus gordonii as early colonizer. The overall hypothesis is that these miRNAs are the dominant miRNAs regulating toll-like receptor (TLR)/IL-1-signaling pathways. Four Specific Aims are proposed. Specific Aim 1 will define the mechanistic role of these miRNAs, including mapping of target mRNAs, in primary human oral epithelial cells in reference to human monocytes. Specific Aim 2 will determine the expression kinetics of the dominant miRNA in mono- or time-sequential polymicrobial infection-induced periodontitis in mice and examine the relative effects of TLR2 and TLR4 using gene knockout mice. Specific Aim 3 will evaluate the relative contribution of these dominant miRNAs in this periodontitis model using specific miRNA knockout mice. Specific Aim 4 will investigate the association of these dominant miRNAs as biomarkers in gingival crevicular fluid and gingival tissues in chronic periodontitis and correlation with therapeutic outcomes. The long-term goal is to determine how extensively these dominant miRNAs can serve as functional biomarkers and they regulate innate immune response pathways contributing to periodontitis. In future studies, this mouse periodontitis model will become critical to help develop manipulation of these miRNA functions and/or the TLR pathway into novel therapeutics for periodontitis. Since innate immune response is known to play critical roles in many other diseases, our findings will likely be applicable to other chronic inflammatory and autoimmune diseases.