Isolation and characterization of mouse MSCs from bone marrow and cell culture
Mouse mesenchymal stem cells (mMSCs) was isolated from bone marrow and cultured in DMED medium (Hyclone) supplemented with 10% exosome-free fetal bovine serum (FBS; Gibco) as previously described . The cells were passaged with 0.25% trypsin containing 0.02% ethylenediaminetetraacetic acid until they reached 90% confluency. A purified population of mMSCs with spindle-shaped morphology was obtained 3 weeks after initial culture. To analyze the multipotential, mMSCs were cultured in appropriate induction medium, and the osteogenic, adipogenic, and chondrogenic differentiation was stained in Alizarin Red, Oil Red, and Alcian Blue, respectively, according to the manufacturers’ instruction. Flow cytometry was used to analyze the phenotypic characteristics of mMSCs. The cells were stained with mouse conjugated monoclonal antibodies, including CD44-APC (103012, BioLegend, USA), CD34-PE (128609, BioLegend, USA), CD11b-PE (101207, BioLegend, USA), CD90.2-PE (105311, BioLegend, USA), and Sca-1-PE (108107, BioLegend, USA). And identical concentrations of the corresponding IgG isotype antibodies were used as negative control (BD biosciences, CA). A minimum of 10000 events were acquired on a FACS instrument (BD bioscience, CA). FlowJo software was employed to analyze the results. The mouse alveolar epithelial cells MLE-12 were purchased from ATCC (Manassas, VA) and routinely maintained in RPMI 1640 medium (Hyclone, UT) with 10% exosome-free FBS and 1% pen/strep (Gibco). Cells were routinely maintained in a humidified incubator containing 5% CO2 at 37 °C. Exosome-free PBS were prepared by pelleting the exosomes by ultracentrifugation at 100000g for 3 h at 4 °C, and the resulting supernatant was filtered through a 0.2-μm filter.
Isolation and characterization of exosomes
mMSCs were cultured with 10% exosome-free FBS, and cell culture medium were harvested every 2 days, and differentially centrifuged at 300g for 10 min, 2000g for 15 min and 10000g for 30 min to remove floating cells and cellular debris. Then the supernatants were filtered through a 0.22-μm filter and ultracentrifuged again at 100000g at 4 °C for 1 h (Beckman Coulter, Inc., USA). The exosome-enriched pellet was washed with 10 ml of 1 × PBS and pelleted again by ultracentrifugation at 100000g at 4 °C for 1 h. The resulting pellet was suspended in 1 × PBS for whole exosome application. The size distribution of exosomes was measured by nanoparticle tracking analysis (NTA) according to the operating instructions (Nano Sight NS300, Malvern, United Kingdom). The exosomal surface markers CD63, TSG101, and CD9 (#EXOAB series, System Biosciences), as well as the negative marker GM130 (ab187514, Abcam, USA), were detected via western blot assay. Additionally, exosome morphology was visualized by transmission electron microscopy (Hitachi, Japan) as previously described.
Cell radiation and treatment
MLE-12 cells were seeded into the 6-well plates. When MLE-12 cells grew at about 70% confluency, a total of 40 μg mMSCs exosomes were added into each well directly, and PBS was used as control. 24 h later, MLE-12 cells with or without exosomes treatment were subjected to a single dose of 8 Gy radiation via a 300 KV X-ray machine (HITACHI, Japan) at the room temperature. Then the cells were harvested for the following experiments at 48 h post-irradiation. To block the exosome generation, GW4869 (Sigma, USA) was added into mMSCs for 48 h at a final concentration of 5μM. Next, MLE-12 cells were incubated with the above medium one day before radiation exposure, and followed by collection as mentioned above.
Mouse model with radiation-induced lung injury and treatments
Seven-week-old C57BL/6 mice were maintained under standard laboratory conditions for 1 week prior to treatment. Mice were divided into two groups (16 mice/group) and anesthetized with 10% choral hydrate before their whole thorax exposed to a single dose of 14 Gy radiation. The treated animals received a i.v. (tail vein) dose of exosomes (200 μg per mouse suspended in 400 μl PBS) 2 h before radiation, then repeated once two weeks until sacrifice. Exosomes naïve mice (control group) only received PBS via the same route. Mice were humanely sacrificed at 1 w, 4 w, 8 w, and 12 w to collect serum and lung samples for further experiments. Efforts were made to ensure the animals suffered minimally. All animal experiments were approved by the Institutional Animal Experiment Committee at 920th Hospital.
Histology, Immunohistochemistry and Masson’s trichrome staining
The left lung tissue in each mouse was fixed in 10% formalin for at least 48 h. After which, they were dehydrated with a series of acetone, and embedded in paraffin. The samples were sliced into 4-μm slices and stained with hematoxylin and eosin (H&E), or using a Masson’s trichrome Kit (Sigma-Aldrich, USA) to detect collagen. For immunohistochemistry (IHC) staining, samples were blocked with 3% hydrogen peroxide for 30 min and then incubated at 4 °C overnight with primary antibodies against E-cadherin (#3195, cell signal technology, USA), Vimentin (#5741, cell signal technology, USA) at a dilution of 1:100. After washing, the sections were incubated at 37 °C with appropriate biotinylated secondary antibody (Zhongshan, China). The sections were observed with the light microscope (Olympus, Japan), and the protein expression was analyzed as previously described .
The collagen concentration was measured by Hydroxyproline (Hyp) assay Kit (Solarbio life science, China) according to the manufacturer’s protocol. Some lung tissue samples were hydrolyzed in lysis buffer solution at 110 °C for 2h. Absorbance of colored products was measured at 560 nm. The Hyp content was calculated as micrograms of Hyp per milligram of wet weight (μg/mg).
Enzyme-linked immunosorbent (ELISA) assay
At each time-point post-irradiation, cardiac puncture was performed to obtain the whole blood. The blood was allowed to clot at room temperature for 2 h and centrifuged at 3000 g 4 °C for 15 min. The clear top layer was collected, and the levels of interleukin-1β (IL-1β, ab197742, Abcam, USA) and interleukin-6 (IL-6, ab222503, Abcam, USA) were measured using a commercial ELISA kit following the manufacturer’s protocol. The OD value of each well was measured at 450 nm via a Microplate Reader (Thermo Fisher Scientific Inc., USA) and calculated at the linear portion of the curve.
Exosomes labeling and confocal microscopy
Exosomes isolated from mMSCs were pre-treated with the green lipophilic fluorescent dye PKH67 according to the instructions (Sigma-Aldrich, USA). Subsequently, the fluorescently labelled exosomes with a final concentration of 10 μg/mL were added into MLE-12 cells. After incubation for the indicated time points, MLE-12 cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Then the cells were stained with mounting medium containing 4’, 6-diamidino-2-phenylindole (DAPI; Invitrogen, USA) for 5 min. After staining, cells were visualized under confocal laser-scanning microscopy (Olympus, Japan).
Mature mmu-miR-466f-3p mimic, mmu-miR-466f-3p inhibitor, or mmu-antagomir-466f-3p (for in-vivo assay), and individual scrambles were purchased from RuiBoBio Company (Guangzhou, China). The CDS sequence of c-Met without 3’UTR was cloned into the pcDNA-3.0 plasmid to construct the pcDNA-c-Met vector. And siRNA against c-MET (Invitrogen) was used to knockdown c-MET expression. When cells reached 50% confluency, miRNA mimics (50 nM) or inhibitor (100nM) was transfected with Lipofectamine 3000 Kit (Invitrogen, USA) and Opti-MEM serum-free medium (Invitrogen, USA) according to the manufacturer’s instructions. 48 h after transfection, cells or conditional medium were harvested for the following experiments.
Transmission electron microscopy (TEM)
Cells or exosomes were fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1% mol/l phosphate buffer, followed by 1% OSO4 overnight, dehydrated with a series of acetone, embedded and solidified. Then, the samples were sliced into 50-nm ultrathin sections to observe the intracellular structures via TEM HT7700 (Hitachi, Japan) as described previously .
Microarray analysis of miRNAs
The miRNA profile between irradiated MLE-12 cells with or without mMSCs-Exos treatment was performed at KangCheng Biotechnology Corporation (Shanghai, China). Agilent Mouse miRNA microarray (Agilent Technologies, USA) was used in the analysis. According to the manufacturer’s protocol, miRNAs were labeled and hybridized with miRNA complete Labeling and Hybridization kit. Data normalization and processing were performed using Quantile algorithm, Gene Spring Software 12.6 (Agilent Technologies, USA). The differential expression of miRNAs was performed via the Pearson’s correlation analysis with Cluster 3.0 and TreeView software, and the differentially expressed genes (DEGs) were identified to have at least |logFC| > 2, p value < 5% in expression.
Quantitative RT- PCR
According to the manufacturer’s protocol, total RNA was extracted from cells and exosomes via TRIZOL and TRIZOL LS reagent, respectively (Invitrogen, USA). Complementary DNA (cDNA) was synthesized using the Reverse Transcription Kit (Takara, Japan) under the following conditions:42 °C for 1 h and then 95 °C for 5 min. Prior to isolation of exosome RNA, C. elegans cel-miR-39 (0.25 nM) standard RNA (RiboBio, China) was added to each sample as a spike-in control. PCR reactions were performed in triplicate on the ABI Prism 7900 (Applied Biosystems, USA) using the SYBR PCR Master Mix (Takara, Japan). The sequences of primers were presented in Table S1. The relative expression of individual genes was analyzed by the 2-△△Ct method through normalizing to U6 or cel-miR-39.
Western blot assay
Protein was extracted from cells by a protein extraction reagent (Roche, Switzerland), and measured by a BCA protein kit (Pierce Biotechnology, USA). 40-μg protein for each sample was electrophoresed in 10-15% SDS gel, then transferred to nitrocellulose membranes. After blocked with 5% BSA solution at room temperature for 1 h, membranes were incubated with primary antibodies at a dilution of 1/1000 at 4 °C overnight, including E-cadherin (#3195, cell signal technology, USA), Vimentin (#5741, cell signal technology, USA), Snail (#3879, cell signal technology, USA), c-MET (ab216330, Abcam, USA), total AKT (ab38449, Abcam, USA), p-AKT (ab8805, Abcam, USA), GSK3β (ab227208, Abcam, USA), p-GSK3β (#9322, cell signal technology, USA), or GAPDH (ab181602, Abcam, USA). Then, followed with secondary antibody (1:5000) at 37°C for 1h. The signals were detected by ECL Kit (Pierce Biotechnology, USA), and were analyzed by Image pro plus software.
For immunofluorescence staining, cells cultured in six-well plates were fixed in 4% paraformaldehyde for 20 min, washed with PBS, and next permeabilized with 0.1% Triton X-100 for 30 min at room temperature. Then cells were blocked with 5% bovine serum albumin for 1 h, and incubated with primary antibodies against E-cadherin (#3195, cell signal technology, USA), Vimentin (#5741, cell signal technology, USA), and Snail (Bioss, bs-1371R, CHINA) at dilutions of 1:200 at 4 °C overnight. After washing, the sections were incubated with fluorescent secondary antibodies at room temperature for 30 min. Nuclei were counterstained with DAPI, and the slides were observed via a confocal laser-scanning microscope (Olympus, Japan).
Luciferase reporter assay
The predicted miR-466f-3p-binding sequences in c-Met 3’-UTR was amplified by PCR and inserted into pmirGLO vector (Promega, USA) to construct luciferase reporter vector (pmirGLO-c-Met-wt). Similarly, the potential binding sites of miR-466f-3p in the above sequences were mutated by Quickchange Mutagenesis Kit (Agilent Technologies, USA) to construct mutant vectors, labeled as pmirGLO-c-Met-mut. H293T cells were seeded into 12-well plates at a density of 1×105 cells/well. 24 h later, miR-466f-3p mimics or scrambles were co-transfected with recombinant wide-type or mutant vectors by Lipofectamine 3000 (Invitrogen, USA). The empty pmirGLO vector was transfected as control. The luciferase activities were standardized to the value of the co-transfected group with an empty vector and scrambles.
The data were presented as means ± standard deviation (SD.) of three independent experiments. All statistical analysis were performed by GraphPad Prism software. The differences among control and experimental groups were measured by Two-tailed student’s t-tests, whereas ANOVA calculated differences among multiple groups. P-value < 0.05 was considered to be statistically significant.