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，其特征为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。MicroRNA（miRNA）是一类通过序列特异性抑制靶mRNA翻译来调控基因表达的非编码RNA。在RA滑膜和其他风湿性疾病中，特定的miRNA表现出改变的表达模式，但其在OA发病机制中的作用尚未确定。在我们的初步研究中，我们分析了IL-12刺激的人软骨细胞中miRNA的整体表达，发现功能未知的miRNA mir-27 b（mir-27 b）下调了数倍。计算机模拟分析鉴定了许多可能的靶mRNA，包括基质金属蛋白酶-13（MMP-13）mRNA。我们以前已经表明，标准化的石榴提取物（PE）发挥软骨和软骨保护作用，通过抑制MMP-13在体外人软骨外植体的表达和体内小鼠模型中炎症性关节炎的发展。与本申请中提出的研究相关的是，我们发现IL-12对mir-27 b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靶向MMP-13 mRNA的mir-27 b和其他选定microRNA的下调来抑制OA中IL-12诱导的软骨分解代谢作用”。该假设的推论是“PE的生物活性成分通过调节体内负调节MMP-13表达的特异性miRNA的表达来发挥其软骨/软骨保护作用”。我们提出了以下具体目标：具体目标-1：我们将确定其他miRNA的目标MMP-13的mRNA在人类软骨细胞和差异调节IL-12。使用大量的患者样本，我们将证实IL-12对mir-27 b和上文鉴定的其他miRNA表达的影响，并确定PE是否在体外阻止人OA软骨细胞中IL-12对其的调节。具体目标2：为了确定mir-27 b和上文鉴定的其他miRNA的表达改变对体外用IL-12刺激的人OA软骨细胞中MMP-13表达的影响。具体目标-3：在OA小鼠模型中，我们将开发疾病诱导和进展期间关节中的miRNA表达谱。我们还将研究PE消耗对小鼠OA关节中靶向MMP-13的miR-27 b和其他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的新型和成本有效的治疗方法提供见解。 公共卫生相关性：骨关节炎（OA）的特征在于受影响关节中高水平的IL-12、过量产生的ROS和关节软骨退变。然而，其病因和确切的发病机制仍不清楚。MicroRNA（miRNA）是一类通过序列特异性抑制靶mRNA翻译来调控基因表达的非编码RNA。特定的miRNA已被证明在RA滑膜和其他疾病中表现出改变的表达模式。目前，关于miRNAs在人软骨细胞中的表达的信息有限，但它们在OA中的作用尚未确定。此外，没有关于膳食多酚对任何细胞类型中的miRNA表达的影响的信息。通过研究生物可利用的饮食成分对miRNA表达的调节，我们的发现可能为开发治疗OA和其他疾病的新治疗策略打开大门。

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.