Let-7d 在特发性肺纤维化中的抑制作用及其作用。
Inhibition and role of let-7d in idiopathic pulmonary fibrosis.
机构信息
University of Pittsburgh Medical Center, NW 628 MUH, 3459 5th Avenue, Pittsburgh, PA 15261, USA.
出版信息
Am J Respir Crit Care Med. 2010 Jul 15;182(2):220-9. doi: 10.1164/rccm.200911-1698OC. Epub 2010 Apr 15.
RATIONALE
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation.
OBJECTIVES
To determine changes in expression and role of microRNAs in IPF.
METHODS
RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry.
MEASUREMENTS AND MAIN RESULTS
Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells.
CONCLUSIONS
Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).
背景
特发性肺纤维化（IPF）是一种慢性、进行性、通常致命的肺纤维化疾病，其特征为上皮细胞表型和成纤维细胞增殖的深刻变化。
目的
确定 microRNAs 在 IPF 中的表达和作用变化。
方法
将来自 10 个对照和 10 个 IPF 组织的 RNA 在 Agilent microRNA 微阵列上杂交，并通过定量实时聚合酶链反应和原位杂交进行确认。通过染色质免疫沉淀、电泳迁移率变动分析、荧光素酶测定以及转化生长因子-β诱导的 let-7d 表达降低来证实 SMAD3 与 let-7d 启动子的结合。通过免疫组织化学定位 HMGA2，一种 let-7d 的靶标。在小鼠中，通过气管内给予 let-7d 拮抗剂来抑制 let-7d，并通过免疫组织化学、免疫荧光、定量实时聚合酶链反应和形态计量学来确定其作用。
测量和主要结果
包括 let-7d 在内的 18 个 microRNAs 在 IPF 中显著下调。转化生长因子-β下调 let-7d 的表达，并证实了 SMAD3 与 let-7d 启动子的结合。抑制 let-7d 导致多种上皮细胞系中间充质标志物 N-钙粘蛋白-2、波形蛋白和α-平滑肌肌动蛋白（ACTA2）以及 HMGA2 的增加。IPF 肺中 let-7d 明显减少，并且表达 let-7d 的上皮细胞数量与肺功能相关。HMGA2 在 IPF 肺的肺泡上皮细胞中增加。体内抑制 let-7d 导致肺泡隔增厚以及 SFTPC（肺相关表面活性蛋白 C）表达的肺泡上皮细胞中胶原、ACTA2 和 S100A4 表达增加。
结论
我们的结果表明 microRNAs 在 IPF 中具有作用。IPF 中 let-7d 的下调以及该 microRNA 在体外和体内的纤维化作用表明，该 microRNA 在防止肺纤维化中具有关键的调节作用。临床试验在 www.clinicaltrials.gov 上注册（NCT 00258544）。
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