Mice
The animal experiments were conducted in strict conformity with the guidelines of Animal Welfare Committee of Health Science Center in Peking University (LA2019019). Male, 5-week-old BALB/c-nu/nu nude mice (Charles River, Wilmington, MA, USA) were randomly assigned to 3 groups (n = 6 per group) and maintained with specific pathogen-free conditions.
Cell lines
The hASCs isolated from three separate donors were purchased in ScienCell Research Laboratories (Carlsbad, CA, USA). For each donor, the in vitro cell experiments were performed at least three times individually. For proliferation culture, cells were maintained in proliferation medium (PM), containing Dulbecco’s modified Eagle medium (Thermo Fisher Scientific, Rockford, IL, USA), 1% (v/v) penicillin/streptomycin (Thermo Fisher Scientific) and 10% (v/v) fetal bovine serum (ExCell Bio, Shanghai, China). When the cells reached 70-80% confluence, osteoinduction was performed by adding osteogenic medium (OM), which contained the above culture medium for promoting proliferation, 100 nM dexamethasone (Sigma-Aldrich), 0.2 mM L-ascorbic acid (Sigma-Aldrich) and 10 mM β-glycerophosphate (Sigma-Aldrich). The cell culture conditions were 37°C with 5% CO2 and 100% relative humidity.
Lentivirus transfection
Recombinant lentiviruses carrying green fluorescent protein (GFP)-tagged plasmid vectors of negative control (NC), miR-137 overexpression (miR-137), miR-137 knockdown (anti-miR-137), NOTCH1 shRNA (anti-NOTCH1), LSD1 shRNA (anti-LSD1), and BMP2 shRNA (anti-BMP2) (Additional file 3: Table S1) were produced and packaged by GenePharma (Suzhou, Jiangsu, China). Lentiviral transfection of hASCs were conducted at a multiplicity of infection of 100 for 24 h with the presence of 5 mg/mL polybrene, and then selected by 1 μg/ml puromycin (Sigma-Aldrich). The transfection rates of lentiviruses were estimated by counting the number of GFP-tagged cells and total cells with an inverted fluorescence microscope (TE2000-U, Nikon, Tokyo, Japan).
Alkaline phosphatase (ALP) staining and quantification
After 7 days of culture in PM or OM, the hASCs were used for ALP staining and activity test according to the published protocol [10]. ALP staining was operated following the BCIP/NBT staining kit (Beyotime, Shanghai, China) instructions. For the quantitative tests of ALP activities, cells were washed with phosphate buffer saline (PBS) and 1% Triton X-100 (Solarbio, Beijing, China), then scraped in Milli-Q water and subjected to three cycles of freezing and thawing. By employing the BCA method and the pierce BCA protein assay kit (Thermo Fisher Scientific), total protein was read at 562 nm and computed with a bovine serum albumin standard curve according to the manufacturer’s protocol. Afterwards, ALP activity was detected at 520 nm applying an alkaline phosphatase assay kit (Jiancheng, Nanjing, Jiangsu, China) and finally normalized to the total protein concentrations of cells.
Alizarin red S (ARS) staining and quantification
After 14 days of culture in PM or OM, the hASCs were applied to detect the matrix mineralization. Following being fixed with 95% ethanol for 30 min, cells were soaked in 1% ARS staining solution (pH 4.2; Sigma-Aldrich, St. Louis, MO, USA) for 20 min at room temperature. To assess the degree of mineralization, stained areas of each well were separately dissolved in 100 mM cetylpyridinium chloride (Sigma-Aldrich) for 1 h and the absorbances were detected at 562 nm. Finally, the relative ARS intensity was normalized to the total protein concentrations of cells.
RNA extraction, reverse transcription, and quantitative real-time polymerase chain reaction (qRT-PCR)
After 3, 7, and 14 days of culture in PM or OM respectively, total RNA of cells was isolated with TRIzol (Invitrogen, Carlsbad, CA, USA) and synthesized into the first-strand cDNA using a reverse transcription system (Takara, Tokyo, Japan). All the transcripts were quantified using the FastStart universal SYBR green master (ROX) (Roche, Indianapolis, IN, USA) and a 7500 real-time PCR detection system (Applied Biosystems, Foster City, CA, USA). Relative expression levels of mRNA and miRNA were normalized to GAPDH mRNA and U6 snRNA, respectively. The sequences of the primers employed were listed in Additional file 4: Table S2.
Western blotting
The hASCs were rinsed with ice PBS three times and immersed in RIPA buffer (HuaxingBio, Beijing, China) mixed with protease inhibitor cocktail (HuaxingBio). The pierce BCA protein assay kit (Thermo Fisher Scientific) was used to determine the protein concentration. A 25 μg sample of protein was added and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then followed by transfer to the polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Strips on the membranes were blocked with 5% nonfat dry milk (BioRuler, Danbury, CT, USA) for 1 h at room temperature, incubated overnight at 4°C with primary antibodies at a dilution of 1:1000, and then for 1 h at room temperature with goat anti-rabbit secondary antibodies labeled with horseradish peroxidase (ZSGB-BIO, Beijing, China; ZB-2301) at a dilution of 1:10000. The primary antibodies used were as follows: anti-GAPDH (ZSGB-BIO; TA-08), anti-NOTCH1 (Cell Signaling Technology, Beverly, MA, USA; 3608S), anti-HES1(Abcam, Cambridge, UK; ab108937), anti-LSD1 (Cell Signaling Technology; 2139S), anti-BMP2 (Abcam; ab14933), anti-SMADA4 (Abcam; ab40759) and anti-RUNX2 (Cell Signaling Technology; 12556). Relative band intensities were measured with the ImageJ software.
Dual-luciferase reporter assay
The 3' untranslated region (3' UTR) alignments of the target regions in NOTCH1 were predicted by TargetScan and RNA22. Reporter vectors were constructed based on the previous method [32]. The 3' UTR sequences of NOTCH1, which contained the possible binding sites of miR-137, were PCR amplified and then inserted into pEZX-MT06 vectors (GeneCopoeia, Rockville, MD, USA) to create NOTCH1-WT (wild-type NOTCH1) luciferase reporter plasmids. Mutated forms were generated by site-directed mutagenesis (GeneCopoeia) and named NOTCH1-MT (mutant-type NOTCH1) luciferase reporter plasmids. For luciferase assay, the hASCs were planted on 24-well culture plates with a density of 5 × 104/well and co-transfected with 1 µg NOTCH1-WT or -MT plasmids, 100 nM NC or miR-137 mimics, and lipofectamine 3000 (Invitrogen). The luciferase activities were examined by a dual-luciferase reporter assay system (Promega, Madison, WI, USA) 48 h later, and standardized to renilla luciferase activity for each transfected well.
Heterotopic osteogenesis examinations in vivo
After the transfection with NC, miR-137 and anti-miR-137 lentiviruses, hASCs of the third passage were maintained in PM for 1 week, collected and incubated with auto-setting calcium phosphate cement (ACPC; Rebone, Shanghai, China) for 1 h at 37°C. Then the hASC-ACPC mixtures were transplanted subcutaneously to the dorsal regions of nude mice (n = 6 per group) for the analyses of heterotopic bone formation in vivo 8 weeks later. After being collected and fixed in 4% paraformaldehyde, the samples were photographed with soft X-ray. The radiograph was obtained by applying a Senograph 2000D molybdenum-rhodium twin target X-ray apparatus (GE, Fairfield, CT, USA). The radiation distance is 20 cm and the radiographing conditions were 22.0 kV, 35.0 mA. For histological evaluation, the specimens were decalcified in 10% ethylene diamine tetraacetic acid solution (pH 7.4) for 14 d, embedded into paraffin, then sliced into 5 μm-thick sections before the subsequent hematoxylin and eosin (HE) staining and Masson trichrome staining. The rabbit anti-OCN primary antibodies diluted to 1:100 (Servicebio, Wuhan, Hubei, China; GB11233) were used for immunohistochemical (IHC) staining.
Statistical analysis
Data and statistical analyses were conducted with SPSS Statistics 20.0 software (IBM, Armonk, NY, USA). All data were shown as mean ± standard deviation (SD) of three individual experiments. For the comparison of two or multiple groups, Student's t-test or one-way analysis of variance (ANOVA) combined with Tukey’s test were applied, respectively. A two-tailed test with p value < .05 was indicated as statistically significant.