Micro-RNAs as biomarkers and targets of herbal CAM in autoimmune arthritis.

项目摘要 类风湿性关节炎(RA)是一种多因素疾病，既涉及遗传因素，也涉及环境因素 组件。美国约有0.5%-1%的人口患有类风湿关节炎。目前的治疗方法， 包括生物制剂，如抗肿瘤坏死因子-α，是有效的，但只对大约50%-60%的RA患者有效。 此外，它们的使用与严重的不良反应有关，例如导致RA的免疫抑制 患者更容易受到感染。为了开发更好的治疗方法，更全面的 了解类风湿关节炎的发病机制是必要的。我们的实验室之前已经证明，有一个动态 Lewis大鼠佐剂性关节炎不同时相基因表达的变化。此外，a 中药南蛇藤及其活性成分雷公藤红素具有抑制再生障碍性贫血的作用。然而，他们的 行动机制尚未得到充分界定。MicroRNAs(MiRNAs)是短的(~19-22个核苷酸长)， 抑制特定基因活性的非编码RNA序列。这些短序列要么抑制 翻译或导致靶信使RNA(MRNA)的降解。我们假设miRNAs扮演着一个 在自身免疫性关节炎的发生和发展中起着至关重要的作用，而且特定的miRNAs也可以 作为这种疾病的生物标志物。此外，我们认为南蛇舌草/雷公藤红素通过部分途径调节其作用。 调节特定miRNAs的水平。目的1.确定早期关节炎Lewis大鼠的miRNA图谱 以及AA的后期阶段，并识别显著改变的相关miRNAs及其潜在靶点 参与关节炎发病机制的基因。目的2.确定其表达的特定miRNAs 用南蛇藤或其活性成分雷公藤红素治疗关节炎大鼠后，显著改变，以及 为了确定这些miRNAs在调节这些草药的抗关节炎活性中的作用，补充和 替代医学(CAM)产品。使用从引流的淋巴结细胞中纯化的总RNA 致关节炎攻击后Lewis大鼠的滑膜浸润性细胞，有无治疗 我们计划使用miRNA微阵列来鉴定感兴趣的miRNAs。为了进一步验证 选择miRNA，我们将用miRNA模拟物/拮抗剂分别转染一株细胞，使其过表达/低表达 感兴趣的miRNA。然后我们将使用qRT-PCR、Western blotting和免疫化学方法来确定 它们对关节炎发病机制相关靶基因的影响及其中药CAM的调控 心理治疗。我们相信，我们的结果不仅将促进对关节炎发病机制的理解，而且 也有助于开发治疗类风湿关节炎的新疗法。

Project Summary Rheumatoid arthritis (RA) is a multifactorial disease that involves both a genetic and an environmental component. Approximately 0.5-1% of the population in the U.S. is afflicted with RA. Current treatments, including biologics such as anti-tumor necrosis factor-α, are effective, but only in about 50-60% of RA patients. In addition, their use is associated with severe adverse effects, such as immune suppression that renders RA patients more vulnerable to infections. In order to develop better therapeutics, a more comprehensive understanding of the pathogenesis of RA is required. Our lab has previously shown that there is a dynamic change in gene expression at different phases of adjuvant-induced arthritis (AA) in Lewis rats. Furthermore, a traditional Chinese medicine celastrus and its bioactive component celastrol can suppress AA. However, their mechanisms of action have not been fully defined. Micro-RNAs (miRNAs) are short (~19-22 nucleotides long), non-coding RNA sequences that repress the activity of specific genes. These short sequences either inhibit translation or cause degradation of the target messenger RNA (mRNA). We hypothesize that miRNAs play a vital role in the development and progression of autoimmune arthritis, and that specific miRNAs can also serve as biomarkers for this disease. Moreover, we suggest that celastrus/celastrol mediates its effect in part by regulating the levels of specific miRNAs. Aim 1. To define the miRNA profiles of arthritic Lewis rats at early and late phases of AA, and identify significantly altered and relevant miRNAs as well as their potential target genes that are involved in arthritis pathogenesis. Aim 2. To identify specific miRNAs whose expression is significantly altered following treatment of arthritic rats with celastrus or its bioactive component celastrol, and to define the role of those miRNAs in mediating the anti-arthritic activity of these herbal complementary and alternative medicine (CAM) products. Using total RNA purified from the draining lymph node cells and the synovial-infiltrating cells of Lewis rats following an arthritogenic challenge, with or without treatment with celastrus/celastrol, we plan to use miRNA microarray to identify miRNAs of interest. To further validate the selected miRNA, we will transfect a cell line with miRNA mimic/antagonist to over-/under-express, respectively the miRNA of interest. Then we will employ qRT-PCR, western blotting, and immunochemistry to determine their effects on target genes associated with arthritis pathogenesis as well as their control by herbal CAM therapy. We believe that our results would not only advance the understanding of arthritis pathogenesis, but also help develop novel therapeutics for RA.