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

项目摘要 风湿性关节炎（RA）是一种多因素疾病，涉及遗传和环境 成分在美国，大约0.5-1%的人口患有RA。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，但只有约50-60%的RA患者。 此外，它们的使用与严重的副作用有关，例如使RA发生的免疫抑制。 患者更容易受到感染。为了开发更好的治疗方法， 需要了解RA的发病机制。我们的实验室先前已经证明， 刘易斯大鼠实验性关节炎（AA）不同阶段基因表达的变化。而且有 中药南蛇藤及其活性成分雷公藤红素可抑制AA。但他们的 作用机制尚未完全确定。微小RNA（miRNAs）很短（约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 翻译或引起靶信使RNA（mRNA）的降解。我们假设miRNAs发挥了 在自身免疫性关节炎的发展和进展中起着至关重要的作用，并且特定的miRNAs也可以 作为这种疾病的生物标志物。此外，我们认为，南蛇藤/雷公藤红素介导其作用的一部分， 调节特定miRNAs的水平。目标1。明确关节炎刘易斯大鼠早期的miRNA谱， 和晚期阶段的AA，并确定显着改变和相关的miRNA以及它们的潜在靶点， 基因参与关节炎发病机制。目标2.为了鉴定特定的miRNAs， 在用南蛇藤或其生物活性组分雷公藤红素治疗关节炎大鼠后显著改变，和 为了确定这些miRNAs在介导这些草药补充剂的抗关节炎活性中的作用， 替代医学（CAM）产品。使用从引流淋巴结细胞中纯化的总RNA， 致关节炎激发后刘易斯大鼠的滑膜浸润细胞，用或不用 为了研究南蛇藤/南蛇藤醇，我们计划使用miRNA微阵列来鉴定感兴趣的miRNA。为了进一步验证 选择的miRNA，我们将用miRNA模拟物/拮抗剂分别检测过表达/低表达的细胞系， 我们感兴趣的miRNA。然后，我们将采用qRT-PCR、蛋白质印迹和免疫化学来确定 它们对与关节炎发病机制相关的靶基因的作用以及它们通过草药CAM的控制 疗法我们相信，我们的研究结果不仅将促进对关节炎发病机制的理解， 也有助于开发治疗RA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.