Suppression of MMP-13 Expression in Arthritis by Pomegranate

描述（由申请人提供）：骨关节炎（OA）是最常见的肌肉骨骼疾病，其特征是软骨退化和关节炎症。目前唯一有效的治疗方法是手术关节置换术。MicroRNAs （miRNA）是一类通过序列特异性抑制靶mRNA翻译来调节基因表达的非编码rna。特异性mirna在RA滑膜和其他风湿性疾病中表现出改变的表达模式，但它们在OA发病机制中的作用尚未明确。在我们的初步研究中，我们分析了受IL-12刺激的人软骨细胞中miRNA的全球表达，发现miRNA mir-27b （mir-27b）的功能未知，下调了数倍。计算机分析确定了一些可能的靶mRNA，包括基质金属蛋白酶-13 (MMP-13) mRNA。我们之前已经证明，标准化石榴提取物（PE）通过抑制体外人软骨外植体中MMP-13的表达和体内小鼠模型中炎症性关节炎的发展，发挥软骨和软骨保护作用。与本应用中提出的研究相关的是，我们发现IL-12对mir-27b的下调被PE阻断。我们的基本假设是“PE通过抑制人软骨细胞中mir-27b和其他靶向MMP-13 mRNA的microrna的下调来抑制il -12诱导的OA软骨分解代谢作用”。这一假设的一个推论是，“PE的生物活性成分通过调节体内负调节MMP-13表达的特定mirna的表达来发挥软骨/软骨保护作用”。我们提出了以下具体目标：具体目标-1：我们将确定人类软骨细胞中靶向MMP-13 mRNA并受IL-12差异调节的其他mirna。通过更大量的患者样本，我们将证实IL-12对上述mir-27b和其他mirna表达的影响，并确定PE是否阻止IL-12在体外对人OA软骨细胞的调节。特异性Aim-2：确定mir-27b和上述其他mirna表达改变对体外IL-12刺激的人OA软骨细胞中MMP-13表达的影响。特异性Aim-3：在OA小鼠模型中，我们将在疾病诱导和进展过程中开发关节中的miRNA表达谱。我们还将研究PE消耗对小鼠OA关节中miR-27b和其他靶向MMP-13的mirna表达的影响，并将其表达谱与疾病进展相关联。从这些研究中获得的知识可能为开发治疗/预防OA的新颖和经济有效的治疗方法提供见解。

DESCRIPTION (provided by applicant): Osteoarthritis (OA) is the most common musculoskeletal disorder characterized by cartilage degradation and joint inflammation. Currently the only effective treatment is surgical joint replacement. MicroRNAs (miRNA) are a class of non-coding RNAs regulating gene expression by sequence specific inhibition of target mRNA translation. Specific miRNAs has been shown to exhibit altered pattern of expression in RA synovium and in other rheumatic diseases but their role in OA pathogenesis is yet to be defined. In our preliminary studies we analyzed the global expression of miRNAs in human chondrocytes stimulated with IL-12 and discovered that miRNA mir-27b (mir-27b), with no known function, was downregulated several fold. In silico analysis identified a number of possible target mRNAs including the matrix metalloproteinase-13 (MMP-13) mRNA. We have previously shown that a standardized pomegranate extract (PE) exert cartilage and chondroprotective effects by inhibiting the expression of MMP-13 in human cartilage explants in vitro and the development of inflammatory arthritis in a mouse model in vivo. Of relevance to the studies proposed in this application is our finding that downregulation of mir-27b by IL-12 was blocked by PE. Our basic hypothesis is that "PE suppresses the IL-12-induced cartilage catabolic effects in OA by inhibiting the downregulation of mir-27b and other selected microRNAs that target MMP-13 mRNA in human chondrocytes". A corollary of this hypothesis is that "bioactive constituents of PE exert their cartilage/ chondroprotective effects by modulating the expression of specific miRNAs that negatively regulate the expression of MMP-13 in vivo". We propose the following specific aims: Specific Aim- 1: We will identify other miRNAs that target MMP-13 mRNA in human chondrocytes and are differentially modulated by IL-12. Using a larger number of patient samples we will confirm the effect of IL-12 on the expression of mir-27b and other miRNAs identified above and determine whether PE prevents their modulation by IL-12 in human OA chondrocytes in vitro. Specific Aim-2: To determine the impact of altered expression of mir-27b and of other miRNAs identified above on the expression MMP-13 in human OA chondrocytes stimulated with IL-12 in vitro. Specific Aim-3: In a mouse model of OA we will develop the miRNA expression profile in the joints during disease induction and progression. We will also examine the effect of PE consumption on the expression of miR-27b and other miRNAs that target MMP-13 in mouse OA joints and correlate their expression profile with the disease progression. Knowledge gained from these studies may provide insight into developing novel and cost effective therapeutic approaches for the treatment/ prevention of OA. PUBLIC HEALTH RELEVANCE: Osteoarthritis (OA) is characterized by high levels of IL-12, excessive production of ROS and articular cartilage degeneration in the affected joints. However, its etiology and precise pathogenetic mechanisms remain unclear. MicroRNAs (miRNA) are a class of non-coding RNAs regulating gene expression by sequence-specific inhibition of target mRNA translation. Specific miRNAs have been shown to exhibit an altered pattern of expression in RA synovium and in other diseases. Currently there is limited information available on the expression of miRNAs in human chondrocytes but their role in OA is yet to be defined. In addition there is no information on the effects of dietary polyphenols on miRNA expression in any cell type. By studying the regulation of miRNA expression by bioavailable dietary constituents, our findings are likely to open the door for the development of novel therapeutic strategies for the treatment of OA and other diseases.