All animal experiments were approved by the Tohoku University Animal Experiment Ethics Committee and were performed in accordance with the Regulations for Animal Experiments and Related Activities at Tohoku University.
Patients and preparation of tissue samples
Human lung tissue was obtained from patients who underwent lung resections at the Department of Thoracic Surgery at Tohoku University Hospital (Aobaku, Sendai, Japan) or at Ishinomaki Red Cross Hospital (Hebita, Ishinomaki, Japan). Non-fibrotic lung tissue specimens were obtained from 3 patients who underwent surgery for lung cancer. The specimens were resected from portions distal to the cancerous lesions, and histopathological examination confirmed that these non-fibrotic tissues did not contain any lesions, including those associated with cancer, fibrosis, emphysema or inflammatory changes. Fibrotic lung tissue specimens were obtained from 3 patients who underwent open lung biopsy or lung resection because of lung cancer. The patients were clinically diagnosed as having IPF based on pathological examinations of the fibrotic lesions. This study was approved by the Ethics Committees at Tohoku University School of Medicine and the Ishinomaki Red Cross Hospital. All subjects gave informed consent.
The animal model of pulmonary fibrosis
Seven- to eight-week-old male C57BL/6 mice were used in our experiments. C57BL/6 mice were purchased from CLEA Japan (Yokohama, Japan). All mice were housed in a specific pathogen-free facility and were maintained under constant temperature (24°C), humidity (40%), and light cycle (8:00 A.M. to 8:00 P.M.) conditions, with food and water provided ad libitum. To induce pulmonary fibrosis, mice were treated intratracheally with bleomycin hydrochloride (Nippon Kayaku, Tokyo, Japan) on day 0 as described in our previous study . Briefly, mice were anesthetized with ketamine via intraperitoneal injection and were then instilled with 0.04 mg of bleomycin hydrochloride in 100 μl of saline through a 27G needle inserted between the cartilaginous rings of the trachea. The lungs were harvested 14 days after instillation for further analyses.
The preparation of single lung cells from whole lung
Single lung cells from mice were isolated as previously described with some modifications . Briefly, mice received an overdose of inhaled halothane, and their lungs were perfused with PBS via the right ventricles. The PBS-perfused lungs were isolated with other mediastinal organs. The Dispase II solution (Roche Applied Science, Mannheim, Germany; final concentration, 2.0 U/mL) was instilled into the lungs through the trachea, which was then ligated with a silk suture. After incubation at 37°C for 50 min, the lungs were separated from the other mediastinal organs. The lungs were then thoroughly minced and digested in PBS containing 0.1% collagenase (Roche Applied Science) and 0.01% deoxyribonuclease I (Sigma-Aldrich, St. Luis, MO) at 37°C for 20 min. The cells were then suspended in red blood cell lysis buffer to remove red blood cells (Sigma-Aldrich) and were subsequently washed with PBS. The cells were then centrifuged and re-suspended in PBS.
Flow cytometry and the sorting of lung component cells
We used the following antibodies: Alexa Fluor 647-conjugated anti-mouse EpCAM antibody (clone G8.8, Biolegend, San Diego, CA); phycoerythrin (PE)-conjugated anti-mouse VE-cadherin antibody (clone VECD1, Biolegend); and fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD45 antibody (clone 30-F11, Biolegend). To discriminate between live and dead cells, we used 7-amino actinomycin D (7-AAD; eBioscience, San Diego, CA). The antibodies were incubated with the samples for 30 min at 4°C and the samples were then washed. We re-suspended the cells in 2% FBS/PBS and labeled dead cells with 7-AAD. We sorted live and single-cell-gated subpopulations based on their staining patterns using EpCAM, VE-cadherin and CD45 with a FACS Aria II Cell Sorter and FACS Diva ver 6.1 (BD Biosciences, San Jose, CA). FACS analyses were conducted using FCS Express 3 software (De Novo Software, Los Angeles, CA). The sorted epithelial (CD45-, EpCAM+ cells), endothelial (CD45-, VE-cadherin+ cells) and mesenchymal cells (CD45-, EpCAM-, VE-cadherin- cells) were collected in DMEM/10% FCS/Penicillin/Streptomycin/amphotericin B (Life Technologies, Grand Island, NY) for further analyses.
Isolation of alveolar epithelial type II cells from human lung tissues
Human lung cells were isolated as previously described , and human ATII cells were isolated as previously described with some modifications . Briefly, we used phycoerythrin-conjugated anti-human EpCAM antibody, Alexa Fluor 647-conjugated anti-human T1α antibody, and FITC-conjugated anti-human VE-cadherin antibody. To discriminate between live and dead cells, we used 7-amino actinomycin D. We sorted live and single-cell-gated subpopulations based on their staining patterns for EpCAM, T1α, and VE-cadherin expression using a FACS Aria II Cell Sorter and FACS Diva, version 6.1 (BD Biosciences).
Total RNA purification and the quantification of microRNAs and mRNAs
Total RNA containing microRNAs was purified from the cells using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). A miScript Reverse Transcription Kit was used for reverse transcription of microRNAs and mRNAs into cDNA. The microRNAs were quantified by real-time PCR using an miScript Primer Assay (Qiagen). mRNAs were also quantified by real-time PCR using a QuantiTect Primer Assay (Qiagen). The specific primer sets for quantification were purchased from Qiagen as follows: MS00001827 for miR-200c; MS00011487 for miR-21; MS00033740 for RNU6B snRNA (a constitutively expressed housekeeping control); QT00121163 for E-cadherin; QT00109424 for Surfactant protein C (SP-C); QT00110467 for VE-cadherin; QT00159670 for Vimentin; QT00140119 for alpha-smooth muscle actin (α-SMA); QT00105385 for ZEB1; QT00148995 for ZEB2 (E-cadherin repressors); QT01658692 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a constitutively expressed gene).
In situ hybridization (ISH)
In situ hybridization was performed according to manufacturer’s protocol provided in the MicroRNA ISH Buffer and Controls Kit (Exiqon, Woburn, MA), with some modifications. Briefly, human lung tissues were inflated with 10% formalin solution and embedded in paraffin. Mouse lung tissues were inflated with 4% paraformaldehyde solution and embedded in paraffin. Slides were deparaffinized and incubated with proteinase-K for 10 min at 37°C and washed with PBS. The hybridization mixture contained 5 nM double-DIG LNA™ microRNA probe for miR-21 or scramble-miR as the negative control (Exiqon) and was applied and hybridized for 1 h at 53°C. Slides were washed in SSC buffer and incubated with RNase solution (20 μg/ml, Wako Pure Chemical Industries, Osaka, Japan) for 30 min at 37°C. Slides were washed in SSC buffers and incubated with blocking solution containing goat serum for 15 min at RT. Sheep anti-digoxigenin-POD (poly) Fab fragments (at 1:500 in antibody diluent, Roche) were applied and incubated overnight at 4°C. Slides were then washed and incubated with biotin-SP-conjugated AffiniPure goat anti-horseradish peroxidase (1:200) at 37°C for 30 min (Jackson ImmunoResearch). Finally, the slides were washed and labeled with Streptavidin-AP (Roche). NBT/BCIP solution (Roche) was used for detection. After washing in water and counter-staining with Nuclear Fast Red, the slides were dehydrated and mounted for observation by microscopy.
The isolation and culture of murine alveolar type II cells
Murine alveolar type II cells were prepared from 7-week-old male C57BL/6 mice by a modification of the method of Corti et al. . Mice were anesthetized with a ketamine-xylazine-atropine mixture by intraperitoneal injection. The abdominal cavity was opened, and mice were exsanguinated by cutting the inferior vena cava and the left renal artery. The diaphragm was cut, and the chest plate and thymus were removed. With the use of a 23G needle on a 10-ml syringe, the lungs were perfused with 10 ml of 0.9% saline via a right ventricle. The trachea was isolated and cannulated with a 20-gauge iv catheter that was secured by a ligature. Two ml BD Dispase solution (BD Bioscience) was rapidly instilled through the cannula into the trachea followed by 0.5 ml low melting point agarose (Sigma-Aldrich) that was preheated to 45°C. The lungs were immediately covered with ice for 2 min to allow the agarose to solidify. After this incubation, the lungs were removed from the animals and incubated in 1 ml BD dispase for 45 min (25°C) before being placed on ice until the next step. The lungs were subsequently transferred to a 100 mm culture dish containing 10 ml of 25 mM HEPES-buffered DMEM/10% FCS/Penicillin/Streptomycin/amphotericin B (Life Technologies) and 100 U/ml DNase I (Sigma-Aldrich). The lung tissue was gently teased from the bronchi by curved forceps and was then gently swirled for 10 min. The resulting cell suspension was filtered sequentially through 100 μm and 40 μm cell strainers (BD Biosciences). The single lung cells were collected by centrifugation at 1200 rpm for 10 min at 4°C. The cells were re-suspended in PBS with 0.5% BSA and 2 mM EDTA and were then incubated with MACS Mouse CD45 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 4°C. The cells were then washed before proceeding to magnetic separation by autoMACS (Miltenyi Biotec) to deplete CD45+ cells. The collected CD45- cells were then incubated with an anti-FITC-conjugated anti-mouse EpCAM antibody (Biolegend) for 10 min at 4°C. After washing, the cells were incubated with MACS anti-FITC MicroBeads (Miltenyi Biotec) for 15 min at 4°C. After incubation and washing, the cells were separated magnetically by autoMACS (Miltenyi Biotec) to collect the CD45- EpCAM+ cells. The collected cells were assessed by immunostaining for EpCAM and pro SP-C. Greater than 99% of the cells were positive for EpCAM and >96% of cells were typically positive for pro-SP-C. Viability was >95%.
The cells were cultured on chamber slides that were coated with Matrigel-rat tail collagen (70:30 vol/vol) to maintain the mouse type II cell phenotype in vitro  or were cultured in human fibronectin pre-coated chamber slides (BD Biosciences) under EMT-inducing culture conditions. The cells were maintained in SAGM (Lonza, Basel, Switzerland) without hydrocortisone and containing 5% charcoal/dextran-treated FBS (Invitrogen, Carlsbad, CA) and 10 ng/ml human recombinant KGF (Rocky Hill, NJ) in a 37°C, 5% CO2 incubator as described. For the EMT-inducing culture conditions, 4 ng/ml human recombinant TGF-β was added to the media. The cells were analyzed on culture day 6.
The transfection of microRNA mimics and/or inhibitors into murine alveolar type II cells
We transfected synthesized miR-200c (Qiagen) and/or a miR-21 inhibitor composed of single-stranded, modified RNAs that specifically inhibit miR-21 function (miScript miRNA Inhibitor; Qiagen) into isolated mouse alveolar type II cells using HiPerFect Transfection Reagent (Qiagen). Control oligonucleotides (Qiagen) were transfected into negative control samples. To optimize transfection conditions, we used AllStars Hs Cell Death Control siRNA (Qiagen), which targets essential cell survival genes. The transfection efficiency was measured by observation of the level of cell death after the transfection of this siRNA. The transfection conditions that resulted in more than 80% cell death when compared with transfection with control oligonucleotides was used in the experiments. After transfection, the alveolar type II cells were cultured on fibronectin-coated chamber slides with SAGM supplemented with 5% charcoal/dextran-treated FBS, 10 ng/ml KGF and 4 ng/ml TGF-β, conditions that enhance EMT. The transfections of the microRNA and/or the miR-21 inhibitor were repeated on day 3. The cells were harvested on day 6.
Data presentation and statistical analysis
Unless otherwise noted, all data presented are expressed as the means ± standard error of the means (SEM). Statistical analyses were performed using Statistica software (StatSoft Inc., Tulsa, OK, USA). The data were assessed for significance using an unpaired t-test for comparisons between the two groups or by ANOVA with Scheffé’s post hoc method for multiple comparisons. Statistical significance was defined as p < 0.05.