Department of Advanced Preventive Medicine for Infectious Disease, Tohoku University Graduate School of Medicine, 2-1 Seiryoumachi, Aobaku 980-8575, Sendai, Japan.
Respir Res. 2013 Sep 24;14(1):95. doi: 10.1186/1465-9921-14-95.
The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined.
Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR.
The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition.
Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.
由于组织修复异常导致成纤维细胞过度和持续积累，导致特发性肺纤维化等纤维化疾病。最近的报告显示，在特发性肺纤维化过程中 microRNAs 发生了显著变化，并为 microRNAs 在纤维化背景下的肌成纤维细胞分化和上皮-间充质转化中的作用提供了证据。据报道，miR-21 在纤维化过程中成纤维细胞中上调，并通过抑制 Smad7 促进转化生长因子-β信号。然而，miR-21 的表达变化及其在肺纤维化过程中的上皮-间充质转化中的作用尚未确定。
分析盐水或博来霉素处理的 C57BL/6J 小鼠的肺和特发性肺纤维化患者的肺标本。进行酶消化以分离单个肺细胞。通过流式细胞分选分离肺上皮细胞。使用定量 PCR 和原位杂交分析 miR-21 的表达。为了在培养中诱导上皮-间充质转化，将分离的小鼠肺肺泡 II 型细胞在纤维连接蛋白包被的腔室载玻片上培养，存在转化生长因子-β，从而产生增强上皮-间充质转化的条件。为了研究 miR-21 在上皮-间充质转化中的作用，我们用 miR-21 抑制剂转染细胞。从新鲜分离和培养的细胞中分离总 RNA。使用定量 PCR 定量 miR-21 以及肺泡上皮或间充质细胞分化标志物基因的 mRNA。
从博来霉素诱导的肺纤维化模型系统中分离的肺上皮细胞中上皮标记基因的表达降低，而间充质标记基因的表达增加。miR-21 在博来霉素诱导的肺纤维化和人类特发性肺纤维化中分离的肺上皮细胞中显著上调。在增强上皮-间充质转化的条件下，培养的肺泡上皮细胞中 miR-21 也上调。外源性给予 miR-21 抑制剂可防止在诱导上皮-间充质转化的培养条件下培养的原代小鼠肺泡 II 型细胞中波形蛋白和α-平滑肌肌动蛋白的表达增加。
我们的实验表明，miR-21 在肺纤维化过程中在肺上皮细胞中增加，并促进上皮-间充质转化。
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