Characterization and Targeting of MiR-24 Network in Colitis

项目总结 MicroRNAs是一种非编码的小RNA分子，与不同的 人类炎症性疾病。最近的证据表明，对microRNA表达的操纵可能 对不同病因的炎症性疾病具有治疗潜力。我们和其他人已经确认 在炎症性肠病(IBD)中，microRNAs被解除调控；然而，它们的作用和功能 IBD在病理生理学中的重要性还不是很清楚。我们的初步证据显示miR-24 溃疡性结肠炎(UC)的2个队列的结肠活检组织中表达特异性上调，但不是 克罗恩病(CD)患者或患有其他胃肠道疾病(肠易激综合征、结直肠癌)的患者。 此外，在活动期疾病UC患者的结肠活检组织中，miR-24的表达高于 UC缓解期患者。尽管已经发现几个microRNAs在不同的 胃肠道(GI)疾病，我们的结果表明miR-24在UC中特异性地去调节， 提示其在UC发病机制中具有重要作用。MicroRNA领域中的一个关键问题是鉴定 它们的直接基因靶标。在这里，我们遵循了一种无偏见的综合计算和分子分析 并发现miR-24可能通过调节由一个基因网络组成的基因网络参与UC的发病 通过直接靶向hNFA的炎症(HFN4A-PPARγ-NF-κB)通路和肠道通透性控制 机制可能是通过直接调控claudin-7，从而影响ERK和MMPs信号通路。因此，这些 数据表明miR-24调节炎症和上皮通透性，这是UC的两个关键方面 发病机制。另一方面，HNF4a和/或claudin-7增强的miR-24的抑制作用增强 表达，表明存在反馈环路网络。这是第一个描述 MicroRNA作为炎症反应和肠道通透性的变阻器，通过调节 反馈环网。在这里，我们将检验miR-24网络发挥重要作用的新假设 通过调节炎症和肠道通透性机制在UC病理生理学中的作用。基于 强有力的初步发现以下目标将解决这一总体假设。目标1将评估 MIR-24-HNF4a网络在控制UC炎症反应中的作用目标2将描述 MiR-24影响肠上皮通透性的分子机制。使用人类UC患者组织将 确认目标1和目标2中的发现与UC的相关性。目标3将检查miR- 24抑制，通过给予一种新的化学稳定的miR-24抑制剂(UCDI-1)，在小鼠模型中 实验性结肠炎(体内)和UC患者新鲜分离的结肠外植体(体外)。理解 MiR-24分子网络不仅将为miR-24在UC病理生理学中的作用提供新的见解， 但它也可能决定UCDI-1抑制miR-24在UC中的治疗潜力。

PROJECT SUMMARY MicroRNAs are small non-coding RNA molecules that have been implicated in the pathogenesis of different human inflammatory diseases. Recent evidence indicates that manipulation of microRNA expression could have therapeutic potential in inflammatory diseases of different etiologies. We and others have identified that microRNAs are deregulated in Inflammatory Bowel Disease (IBD); however their role and functional importance in IBD pathophysiology is not well understood. Our preliminary evidence revealed that miR-24 expression is specifically up-regulated in colonic biopsies from 2 cohorts of ulcerative colitis (UC), but not Crohn's disease (CD) patients or patients with other GI diseases (irritable bowel syndrome, colorectal cancer). Furthermore, miR-24 expression is higher in colonic biopsies from UC patients with active disease relative to UC patients in remission. Although several microRNAs have been found to be deregulated in different gastrointestinal (GI) diseases, our results demonstrate that miR-24 is specifically deregulated in UC, suggesting an important role in UC pathogenesis. A key question in the microRNA field is the identification of their direct gene targets. Here, we have followed an unbiased integrative computational and molecular analysis and found that miR-24 is potentially involved in UC pathogenesis by regulating a gene network consisting of an inflammatory (HFN4A-PPARγ-NF-κB) circuit by targeting directly HNFA, and an intestinal permeability control mechanism, by modulating directly claudin-7, which affects the ERK and MMP signaling pathways. Thus, these data suggest that miR-24 regulates inflammation and epithelial permeability, both key aspects of UC pathogenesis. On the other hand, suppression of HNF4A and/or claudin-7 reinforced miR-24 increased expression, suggesting the presence of a feedback loop network. This is the first evidence describing a microRNA to act as a rheostat of the inflammatory response and intestinal permeability, through regulation of a feedback loop network. Here, we will examine the novel hypothesis that the miR-24 network plays an important role in UC pathophysiology by regulating inflammatory and intestinal permeability mechanisms. Based on strong preliminary findings the following aims will address this overall hypothesis. Aim 1 will evaluate the role of miR-24 – HNF4A network on controlling the inflammatory responses in UC. Aim 2 will characterize the molecular mechanism of miR-24 on intestinal epithelial permeability. Use of human UC patient tissues will confirm the UC relevance of the findings in aims 1 and 2. Aim 3 will examine the therapeutic potential of miR- 24 inhibition, by administering a novel chemically-stable miR-24 inhibitor (UCDI-1), in mouse models of experimental colitis (in vivo) and in freshly isolated colonic explants from UC patients (ex vivo). Understanding the miR-24 molecular network will not only provide novel insights on the role of miR-24 in UC pathophysiology, but it may also determine the therapeutic potential of miR-24 inhibition by UCDI-1 in UC.