microRNA miR-147 Dampens Alveolar Epithelial Inflammation During ARDS

项目摘要 该拨款申请的主要目标是识别保护肺泡的微小RNA（miRNA）靶点 急性肺损伤（ALI）时上皮细胞过度炎症。ALI的特征是急性呼吸道感染， 在非心源性肺水肿的情况下失败。急性呼吸窘迫综合征（ARDS） 在人类身上。目前的应用集中于鉴定可靶向抑制肺泡炎的miRNA。 上皮炎症 为了确定抑制肺泡炎症的miRNA靶点，我们将小鼠暴露于呼吸机诱导的肺 损伤（VILI）和分离的肺泡上皮细胞。微阵列分析将miR-147鉴定为主要候选者。 验证性研究表明，miR-147在ALI期间通过体内机械通气诱导， 通过体外将肺泡上皮细胞暴露于周期性机械拉伸。miR-147水平的体内升高 在ALI期间，尽管中性粒细胞或巨噬细胞耗尽，但仍然持续。此外，我们还确定了一个角色， 用于miR-147诱导的低氧诱导型转录因子HIF 1A。miR-147-/-小鼠经历更严重的肺 受伤期间。对miR-147靶点的搜索鉴定了toll样受体衔接分子2（TICAM 2）作为前 炎症靶点与miR-147与TICAM 2表达的联系一致，我们发现肺泡上皮细胞 从miR-147 loxp/loxp SPC Cre+小鼠中分离的Ticam 2显示升高的Ticam 2水平。同样，增加 miR-147 loxp/loxp SPC Cre+小鼠VILI期间的肺水肿通过伴随遗传学方法复苏。 删除Ticam 2。通过含有miR-147的纳米颗粒过表达miR-147在ALI期间具有保护作用。 最后，在ARDS患者中进行的原理验证研究显示，他们的BAL液中miR-147水平升高。因此，我们认为， 我们假设HIF依赖性的miR-147诱导代表了抑制肺部的内源性途径 炎症我们设计了3个具体目标，首先利用体外细胞培养技术研究miR-147的表达， 建模系统（目标1）。目的2是关注ALI的体内研究，其中我们将利用组织特异性 miR-147缺失的方法，并检查TICAM 2 3'端miR-147结合位点突变的小鼠。 非翻译区（这些小鼠的遗传靶向是成功的）。最后，我们将靶向miR-147， 目标3中的ARDS治疗。

Project Summary The main goal of this grant application is to identify microRNA (miRNA) targets that protect the alveolar epithelium from excessive inflammation during acute lung injury (ALI). ALI is characterized by acute respiratory failure in the setting of non-cardiogenic pulmonary edema. It causes acute respiratory distress syndrome (ARDS) in humans. The current application is focused on identifying miRNAs that could be targeted to dampen alveolar epithelial inflammation. To identify miRNA targets that dampen alveolar inflammation, we exposed mice to ventilator-induced lung injury (VILI) and isolated alveolar epithelial cells. A microarray analysis identified miR-147 as a leading candidate. Confirmatory studies demonstrated that miR-147 is induced during ALI by mechanical ventilation in vivo or through in vitro exposure of alveolar epithelia to cyclic mechanical stretch. In vivo elevations of miR-147 levels during ALI persisted despite depletion of neutrophils or macrophages. In addition, we identified a role for hypoxia-inducible transcription factor HIF1A for miR-147 induction. miR-147-/- mice experience more severe lung injury during VILI. A search for miR-147 targets identified toll-like receptor adaptor molecule 2 (TICAM2) as pro- inflammatory target. In line with a link of miR-147 with TICAM2 expression, we found that alveolar epithelia isolated from miR-147loxp/loxp SPC Cre+ mice showed elevated Ticam2 levels. Similarly, increased pulmonary edema during VILI of miR-147loxp/loxp SPC Cre+ mice was resuscitated by concomitant genetic deletion of Ticam2. miR-147 overexpression via miR-147-containing nano-particles was protective during ALI. Finally, proof-of-principle studies in ARDS patients showed elevated miR-147 levels in their BAL fluid. Therefore, we hypothesize that HIF-dependent induction of miR-147 represents an endogenous pathway to dampen lung inflammation. We designed 3 specific aims, where we will first study the expression of miR-147 utilizing in vitro modeling systems (Aim 1). Aim 2 is focused on in vivo studies of ALI, where we will utilize tissue-specific approaches of miR-147 deletion and examine mice with a mutation of the miR-147 binding site in the TICAM2 3' untranslated region (genetic targeting for these mice was successful). Finally, we will target miR-147 for the treatment of ARDS in Aim 3.