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

Abstract

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.

Figure 1

The identification and isolation of epithelial, endothelial and mesenchymal cells from mouse lungs. (A) A fluorescence-activated cell sorting strategy. Single lung cells were stained with antibodies against EpCAM, VE-cadherin and CD45. 7-AAD was used for dead cell exclusion. Epithelial, endothelial and mesenchymal cells are identified as CD45^- EpCAM⁺ VE-cadherin^- cells, CD45^- EpCAM^- VE-cadherin⁺ cells and CD45^- EpCAM^- VE-cadherin^- cells, respectively. (B) The expression of mRNAs in isolated lung epithelial (Ep), mesenchymal (Me) and endothelial (En) cells from untreated mice. The mean and SEM of relative mRNA expression are shown (n = 6). The values relative to those of epithelial cells are presented for E-cadherin and SPC. For VE-cadherin, values relative to those of endothelial cells are presented. For vimentin, ZEB1 and ZEB2, values relative to those of mesenchymal cells are presented. The data were analyzed by one-way analysis of variance with a post hoc test (Scheffé’s test). * p < 0.05 versus lung epithelial cells. † p < 0.05 versus lung mesenchymal cells.

Figure 2

miR-21 is increased in lung epithelial cells during experimental bleomycin-induced lung injury. (A) Representative flow cytometric analyses of CD45^- lung cells from saline- and bleomycin-treated mice. (B) The percentage of each cell type within all CD45^- lung cells. Note that bleomycin induced a decrease in the percentage of epithelial cells (Ep) and an increase in that of mesenchymal cells (Me) but no change in endothelial cells (En). (C) The relative expression of mRNA of lung epithelial marker genes (E-cadherin and SFTPC) or EMT marker genes (Vimentin, Zeb1 and Zeb2) is shown. (D) The relative expression of microRNAs is shown. The values are mean ± SEM (n = 6). The values relative to those of the saline-treated epithelial cells are presented. The data were analyzed by Student t test. * p < 0.05 versus epithelial cells in the saline group. † p < 0.05 between saline group and bleomycin group in each cell type. § p < 0.05 versus epithelial cells in the bleomycin group.

Figure 3

Histological analyses for miR-21 expression in mouse and human lung tissues using in situ hybridization. (A-D) In situ hybridization for miR-21 was performed in mouse lung tissues obtained from saline- (A) or bleomycin-treated mice (C, D). Figure 3C and 3D show the areas demonstrating severe and mild fibrotic lesions, respectively. Staining using control probes with a scrambled sequence showed minimal background staining (B). (E-G) In situ hybridization for miR-21 was performed in non-fibrotic lung tissue obtained from patients without fibrotic lung disease (E) and patients with IPF (F, G). Figure 3F and 3G show the areas demonstrating severe and mild fibrotic lesions, respectively. Arrows indicate staining for miR-21. Asterisks indicate fibrotic foci. Arrowheads indicate anthracosis. Bar = 50 μm.

Figure 4

miR-21 is increased in lung alveolar type II cells from idiopathic pulmonary fibrosis (IPF) patients. (A) Representative flow cytometric analyses of CD45^- VE-cadherin^- lung cells for the isolation of human alveolar type II cells (CD45^- VE-cadherin^- EpCAM⁺ T1-α^- cells) from the non-fibrotic lungs of patients without fibrotic lung diseases and the fibrotic lungs of patients with IPF. (B) The relative expression of microRNAs is shown. The values represent the mean ± SEM (n = 3). The values relative to those of the human alveolar type II cells from non-fibrotic lungs are presented. The data were analyzed with Student’s t-test. * p < 0.05 versus the non-fibrotic lung group.

Figure 5

The EMT-inducing culture conditions increase miR-21 expression in mouse lung alveolar epithelial cells. (A) Representative flow cytometric analyses for the expression of pro-SFTPC in isolated mouse alveolar type 2 cells. Note that most of the isolated cells were positive for both EpCAM and pro-SFTPC. (B) Representative immunofluorescence staining for pro-SFTPC in the isolated cells. Most of the isolated cells were positive for pro-SFTPC (left), whereas EPCAM-CD45- cells were negative for pro-SFTPC (right). Scale bars: 20 μm. (C) The EMT-inducing culture conditions (EMT; cultured on fibronectin with TGF-β for 6 days) decrease lung epithelial cell marker gene expression and increase EMT marker gene expression in mouse lung alveolar epithelial cells. The relative expression of mRNA of lung epithelial cell marker genes (E-cadherin and SFTPC) or EMT marker genes (vimentin, α-SMA, Zeb1 and Zeb2) is shown. The values are mean ± SEM (n = 6). mRNA is expressed relative to levels observed prior to culture. The data were analyzed by one-way analysis of variance with a post hoc test (Scheffé’s test). * p < 0.05 versus cells not cultured (NC). † p < 0.05 versus control cultures (Con; cultured on Matrigel/collagen for 6 days). (D) The relative expression of microRNAs is shown. The values are mean ± SEM (n = 6). microRNA expression was relative to the levels observed prior to culture. The data were analyzed by one-way analysis of variance with a post hoc test (Scheffé’s test). * p < 0.05 versus NC. † p < 0.05 versus Con.

Figure 6

miR-21 attenuates TGF-β-induced epithelial-mesenchymal transition in mouse alveolar type II cells. The relative expression of mRNA of lung epithelial cell marker genes (E-cadherin and SFTPC) or EMT marker genes (vimentin, α-SMA, Zeb1 and Zeb2) prior to culture or 6 days after culture in the condition inducing EMT is shown. The values are mean ± SEM (n = 6). mRNA is expressed relative to levels observed prior to culture. NC: cells not cultured. MOCK: cells only treated with transfection reagents. Con siRNA: cells transfected with non-targeting siRNA. miR-200c: cells transfected with synthesized miR-200c. miR-21-i: cells transfected with miR-21 inhibitor. miR-200c & miR-21-i: cells co-transfected with both synthesized miR-200c and miR-21-i. The data were analyzed by one-way analysis of variance with a post hoc test (Scheffé’s test). * p < 0.05 versus NC. † p < 0.05 versus MOCK. § p < 0.05 versus Con siRNA.

Figure 7

Representative phase contrast images of mouse alveolar type II cells cultured under EMT-inducing conditions. Images of the cells 6 days after culture are shown. MOCK: cells only treated with transfection reagents. Con siRNA: cells transfected with non-targeting siRNA. miR-200c: cells transfected with synthesized miR-200c. miR-21-i: cells transfected with miR-21 inhibitor. 200c & 21-i: cells co-transfected with both synthesized miR-200c and miR-21 inhibitor. Note that the cells cultured under EMT-inducing conditions adopted their spindle-like shape with irregular processes, whereas cells transfected with synthesized miR-200c and/or miR-21 inhibitor adopted a cobblestone-like appearance and had granules (lamellar bodies). Scale bars: 20 μm.