Dominant microRNAs as biomarkers in innate immunity and periodontitis

摘要 仅在美国就有数百万人患上牙周病， 其他使人衰弱的全身性疾病，包括心血管疾病、阿尔茨海默病、类风湿性关节炎、 关节炎和不良妊娠结局。我们的实验室已经证明某些microRNA的表达 在鼠多微生物牙周炎中，微RNA（miRNAs）升高。目前的范式是， 通常参与基因表达的微调。然而，我们的体外研究表明，miR- 146 a是一种显性的miRNA，在脂多糖刺激期间可以上调30至200+倍， 更重要的是，这种增长持续了好几天。我们已经证明了miR-146 a是一个关键， 使用基于单核细胞/巨噬细胞的系统，研究内毒素诱导的耐受性和交叉耐受性的调节剂。 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 并能诱导交叉耐受。在目前的建议中，这些占主导地位的miRNAs将使用 体外和体内系统，以严格确定它们在我们建立的小鼠牙周炎模型中的作用， 主要的特征性牙周病原体（牙龈卟啉单胞菌、齿垢密螺旋体、坦纳氏菌 革兰氏阴性杆菌、具核梭杆菌）和戈登链球菌作为早期定殖者。总的假设是 这些miRNAs是调节Toll样受体（TLR）/IL-1信号通路的主要miRNAs。四 提出了具体目标。具体目标1将定义这些miRNA的机制作用，包括 在原代人口腔上皮细胞中相对于人单核细胞的靶mRNA的定位。具体 目的2将确定单一或时间顺序的多微生物中优势miRNA的表达动力学， 感染诱导的小鼠牙周炎，并使用基因敲除检测TLR 2和TLR 4的相对作用 小鼠具体目标3将评估这些显性miRNA在该牙周炎模型中的相对贡献 使用特定的miRNA敲除小鼠。Specific Aim 4将研究这些显性miRNAs之间的关联， 作为生物标志物在慢性牙周炎龈沟液和牙龈组织中的表达及其与 治疗结果。长期目标是确定这些占主导地位的miRNAs在多大程度上可以发挥作用， 作为功能性生物标志物，它们调节导致牙周炎的先天免疫应答途径。在 在未来的研究中，这种小鼠牙周炎模型将成为帮助开发这些miRNA操作的关键 功能和/或TLR途径转化为牙周炎的新疗法。由于先天免疫反应是 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.