The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.
Department of Advanced Preventive Medicine for Infectious Disease, Tohoku University Graduate School of Medicine, 2-1 Seiryoumachi, Aobaku 980-8575, Sendai, Japan. hkubo@med.tohoku.ac.jp.
Mitsuhiro
M
Yamada
Hiroshi
H
Kubo
Chiharu
C
Ota
Toru
T
Takahashi
Yukiko
Y
Tando
Takaya
T
Suzuki
Naoya
N
Fujino
Tomonori
T
Makiguchi
Kiyoshi
K
Takagi
Takashi
T
Suzuki
Masakazu
M
Ichinose
D000199:Actins / Q000378:metabolism; D000818:Animals; D001761:Bleomycin / Q000009:adverse effects; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008168:Lung / Q000378:metabolism* / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D011658:Pulmonary Fibrosis / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D012313:RNA / Q000235:genetics; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014746:Vimentin / Q000378:metabolism
D000199:Actins; C524940:MIRN21 microRNA, human; C531950:MIRN21 microRNA, mouse; D035683:MicroRNAs; D016212:Transforming Growth Factor beta; D014746:Vimentin; D001761:Bleomycin; D012313:RNA