Human bone marrow-derived mesenchymal stem cells (BMMSCs) were purchased from ScienCell, (CA, USA, catalog number:7500) and maintained in mesenchymal stem cell medium, consisting of basal medium, 5% of fetal bovine stem serum, 1% mesenchymal stem cell growth supplement and penicillin/streptomycin solution (MSCM, ScienCell, catalog number:7501). The passages from 5th to 8th were used in all the experiments.
CD4+ naïve T cells isolation and in vitro induction of Treg cells differentiation
The splenocytes were washed with PBS for 3 times and the mouse naïve CD4+ T cell isolation kit was used to isolate the naïve CD4+ T cells according to the manufacturer’s instruction. In Brief, CD4+ activated/memory T cells and non-CD4+ T cells were depleted by indirect magnetic labeling using a cocktail of biotin-conjugated antibodies against various markers including CD8a, CD11b, CD11c, CD19, CD25, CD45R, CD49b, CD105, anti-MHC class II, Ter-119 and TCR γ/δ followed by addition of antibiotin microbeads. 5 µg/ml plate-bound anti-CD3, 2 µg/ml anti-CD28, 2 ng/ml TGFβ and 100 Unit/ml IL-2 were used to activate the isolated cells (1×106 cells/well). The cells were stained with FITC-conjugated anti-CD45RA and APC-conjugated anti-CD4 antibodies. After washing, the cells were fixed and made permeable using permeabilization buffer and then stained with PE-labeled anti-Foxp3 antibody. The tests were detected by flow cytometry.
RNA extraction and qRT-PCR
The extraction of total RNA and the analysis of qRT-PCR were performed according to the previous description . We used TRIZOL reagent (Thermofisher, USA) to extract total RNA by in cells and tissues. Taqman probes (Applied Biosystems, USA) were used to quantify miRNAs. Briefly, 1 µg of total RNA was transcribed to cDNA using AMV reverse transcriptase (Takara, Japan) and a RT primer. The reaction conditions were: 16 oC for 30 min, 42 oC for 30 min and 85 oC for 5 min. Real-time PCR was performed using a Taqman PCR kit on an Applied Biosystems 7300 sequence detection system (Applied Biosystems, USA). The reactions were performed in a 96-well plate at 95 oC for 10 min, followed by 40 cycles of 95 oC for 10 sec and 60 oC for 1 min. U6 was used as the internal control.
Western blotting analysis
Cells were lysed in 1% n-octyl-p-D-glucopyranoside (OG) buffer (20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% OG, 1 mM EDTA, 10 g/ml leupeptin, 2 g/ml aprotinin, 1 mM PMSF). The total protein density was determined using bicinchoninic acid (BCA) protein assay kit (synthgene, China).The protein was then incubated overnight with the following primary antibodies at 4°C: CD9, CD63 and TSG101 (1:500, SBI, USA), POSTN (1:1500, Abcam, USA), GAPDH (1:2000, Abcam, USA). GAPDH served as a loading control. After incubation with the corresponding second antibodies, protein bands were quantified using Image J Software.
In vitro mineralization assay
Mesenchymal stem cell osteogenic differentiation medium (MODM, Sciencell, Sandiego, CA, USA) was used to induce osteogenic differentiation of BMMSCs. After 3 days of induction, alkaline phosphatase (ALP) activity was assayed with an ALP activity kit according to the manufacturer’s protocol (Sigma- Aldrich). To detect mineralization, cells were induced for 2 weeks, fixed with 70% ethanol and stained with 2% Alizarin Red (Sigma-Aldrich). To quantify the calcium mineral density, Alizarin Red was destained with 10% cetylpyridinium chloride (CPC) in 10 mmol/L sodium phosphate for 30 minutes at room temperature. The calcium concentrations were determined by an absorbance measurement at 562 nm on a multiplate reader and compared to a standard calcium curve with calcium dilutions in the same solution. The final calcium level in each group was normalized to the total protein concentration detected in a duplicate plate.
Exosome isolation and labeling
Exosome-depleted FBS was used in the following experiments to avoid the impact of exosomes. FBS was depleted of exosomes by ultracentrifugation at 1×106 g at 4°C for 16 h (Beckman Coulter Avanti J-30I, USA). After being incubated for 48–72 h, the culture medium was harvested and exosomes were isolated by ultracentrifugation. Briefly, cell culture medium was sequentially centrifuged at 300 g for 10 min, 2,000 g for 15 min, and 12,000 g for 30 min to remove floating cells and cellular debris. These were then passed through a 0.22-µm filter. The supernatant was further ultracentrifuged at 1×106 g for 2 h at 4°C, washed in phosphate-buffered saline (PBS), and submitted to a second ultracentrifugation in the same conditions. Exosomes were quantified with bicinchoninic acid (BCA) method. Exosomal protein was measured by BCA protein assay kit (synthgene, China). Purified exosomes were fluorescently labeled using PKH67 (Sigma, USA), according to the protocol.
Transmission electron microscope
Exosomes were precipitated and immediately fixed in 2.5% glutaraldehyde at 4°C for the electron microscope observation. After fixation, specimens were processed through dehydration in gradient alcohol, and infiltrated in epoxy resin and then embedded. The ultrathin sections were stained with uranyl acetate and lead citrate, and were observed under transmission electron microscope (TEM) (JEM-1010; JEOL,
NanoSight tracking analysis (NTA)
Isolated exosomes were analyzed using Nanosight LM10 system (Nanosight Ltd, Navato, CA) equipped with a blue laser (405 nm). Nanoparticles were illuminated by the laser and their movement under Brownian motion was captured for 60 seconds and the video recorded was subjected to NTA using the Nanosight particle tracking software to calculate nanoparticle concentrations and size distribution.
For exosome tracking experiments, PKH67 membrane dye (Sigma) was used to label exosomes. Labeled exosomes were washed in 10 ml of culture medium, collected by ultracentrifugation (1×105g, 2h) and re-suspended in culture medium. Exosome labeling with PKH67 (Sigma) was performed following the manufacturer’s procedures.
Luciferase reporter assay
pMIR-POSTN‑3'‑UTR‑WT (or pMIR-H19‑3'‑UTR‑WT) as well as pMIR- POSTN‑3'‑UTR‑MUT (or pMIR-H19‑3'‑UTR‑MUT) luciferase reporter plasmids were constructed by Synthgene Biotech (Nanjing, China). The sequences that could binding to miR-154-5p were partly mutated and inserted into the reporter plasmid in order to identify the binding specificity. The implementation method refers to previous study . Briefly, BMMSCs cells were seeded in a 24 well plate until reaching 60% confluence. Each well was co-transfected with luciferase reporter plasmids (0.5µg) and RNA mimics (100 pmol) using Lipofectamine 2000 (Thermofisher, USA) according to the manufacturer's protocol. The luciferase activity was measured after 48 h of transfection, by using the Dual-Luciferase Reporter Assay (Promega, Shanghai, China) according to the manufacturer's instructions and normalized to Renilla signals.
RIP assay was performed using an RNA Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions with an anti-Ago2 antibody (2 µg; Millipore) and normal mouse IgG as an NC. qPCR was performed using Taqman Universal PCR Mix as described above.
A Pierce™ Magnetic RNA-Protein Pull-Down Kit (ThermoFisher, USA) was used to perform RNA pull-down assays according to the manufacturer’s instruction. Biotinylated H19 RNA was synthesized by Synthgene (China). In brief, 50 pmol biotinylated RNA were incubated with 50 µl prewashed streptavidin-agarose beads for one hour at 4°C for each assay. Then, RNA-bound beads were incubated with lysates from BMMSC cells cytosolic/nuclear extracts and eluted proteins were detected by western blot.
All experiments were repeated three times and the data are presented as the mean ± standard deviation using SPSS 18.0 (SPSS, inc.). One-way ANOVA and post hoc Dunnett's T3 test were performed in order to compare the differences among and between groups, respectively. P < 0.05 was considered to indicate a statistically signifcant result.