Background: The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic\r\ndiseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs\r\nduring idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast\r\ndifferentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that\r\nmicroRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta\r\nsignaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in\r\nepithelial-mesenchymal transition during lung fibrosis have not yet been defined.\r\nMethods: Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with\r\nidiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung\r\nepithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using\r\nboth quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated\r\nmouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of\r\ntransforming growth factor-�Ÿ, thus generating conditions that enhance epithelial-mesenchymal transition. To\r\ninvestigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21\r\ninhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs\r\nof genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using\r\nquantitative PCR.\r\nResults: The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased\r\nexpression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased.\r\nMicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis\r\nand human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells\r\nunder the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21\r\ninhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary\r\nmouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition.\r\nConclusions: Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung\r\nfibrosis and that it promotes epithelial-mesenchymal transition.
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