Isolation and culture of the hBMSCs
This study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (Qingdao, China). Bone marrow samples were collected from three patients with fracture of the femoral neck (age 45, 47, and 52 years old) during total hip arthroplasty (THA) surgery conducted at the Department of Orthopedics at the Qingdao University Affiliated Hospital. All donors provided written informed consent. Cells were isolated and purified from the bone marrow tissue using density gradient centrifugation, as previously described [19], and were then cultured in Dulbecco modified Eagle’s medium (DMEM, Solarbio, Beijing, China) containing 10% (v/v) fetal bovine serum (FBS, Gibco, Thermo Scientific, Australia) and 100 units/ml penicillin-streptomycin (Solarbio, Beijing, China) in a 5% CO2 atmosphere at 37°C. The cells were passaged at a 1:2 ratio after they had reached 90% confluency. Cells at passage 3 were used in all experiments.
Phenotypes of the hBMSCs
Flow cytometric analysis was used to assess the expressions of the surface markers on the hBMSCs using an Apogee A50-MICRO flow cytometer (Apogee, UK), as described below. In brief, cells were digested using trypsin, centrifuged, and resuspended in cold phosphate-buffered saline (PBS) containing 1% FBS. After the concentration was adjusted to 1x106 cells/ml, the cell suspension was incubated with the following antibodies: 20 µL of anti-CD34‑PE, 20µL of anti-CD45‑PE, 5 µL of anti-CD73-FITC, and 2 µL of CD90‑FITC (BD Biosciences, USA), respectively, for 30 minutes in dark at 37°C. After the cells were washed three times with cold PBS, 100 µL of the single-cell suspension was used for the flow cytometric analysis. Untreated cells were used as a negative control.
Assessment of the morphology of the apoptotic cells
Cells at passage 3 were grown in 24-well plates and were treated with 10-6 mol/L Dex for 10 days. From day 1 to day 10, a chromatin dye Hoechst 33342/PI Kit (Solarbio, Beijing, China) was used to assess the morphology of the apoptotic cells, following the manufacturer’s instructions. Apoptotic cells, which showed morphological characteristics, such as chromatic agglutination, karyopyknosis, and nuclear fragmentation, were identified and counted under a fluorescent microscope. Necrotic cells emitted a red hyperfluorescence and a blue hyperfluorescence. In addition, Hoechst 33342/PI staining assay of the hBMSCs treated with various concentrations of Dex (10-8 mol/L, 10-7 mol/L, and 10-6 mol/L) was also performed for 7 days. The hBMSCs were treated with the solvent of Dex as the control. The experiment was performed in triplicate.
Flow cytometric analysis to determine apoptosis
Cells at passage 3 were grown in 6-well plates and treated with 10-6 mol/L Dex for 10 days. From day 1 to day 10, a Annexin V-PE/7-AAD Kit (BD Biosciences, USA) was used to analyze the percentage of apoptotic cells using a Apogee A50-MICRO flow cytometer (Apogee, UK), as recommended by the manufacturer, and at least 104 cells were analyzed in each sample. In addition, flow cytometric analysis of the hBMSCs treated with various concentrations of Dex (10-8 mol/L, 10-7 mol/L, and 10-6 mol/L) was also performed for 7 days, as mentioned above. The hBMSCs were treated with the solvent of Dex as the control. The experiment was performed in triplicate.
Flow cytometric analysis of the cell cycle
The effects of various concentrations of Dex (10-8 mol/L, 10-7 mol/L, and 10-6 mol/L) on the cell cycle of the hBMSCs was evaluated using a Cell Cycle Detection Kit (Solarbio, Beijing, China), as recommended by the manufacturer. In brief, cells at passage 3 were grown in 6-well plates and treated with Dex. Then, the hBMSCs at the logarithmic growth phase were collected and used to determine the cell cycle using flow cytometry (Apogee A50-MICRO, UK). FlowJo version 10 (BD, USA) was used to analyze the data, and at least 104 cells were analyzed in each sample. The hBMSCs were treated with the solvent of Dex as the control. The experiment was performed in triplicate.
Cell proliferation assay
The effect of Dex on hBMSC proliferation was evaluated using crystal violet assay. In brief, cells at passage 3 were grown in 96-well plates at an initial density of 2×104 cells/well and treated with 10-6 mol/L Dex for 7 days. Then, the cells were fixed using 4% paraformaldehyde for 10 min, and stained with 0.25% crystal violet solution for 30 min. After washing three times, the cells were observed under an inverted phase-contrast microscope and then 10% acetic acid was added to dissolve the crystal violet. Subsequently, crystal violet content was quantified at 570 nm using a microplate reader (Tecan, Austria). The experiment was performed in triplicate.
β-galactosidase (β-GAL) activity assay
The effect of Dex on the senescence of the hBMSCs was evaluated using β-GAL activity assay. In brief, cells at passage 3 were grown in 6-well plates and treated with 10-6 mol/L Dex for 10 days. From day 1 to day 10, cells were collected and counted, and then β-galactosidase (β-GAL) activity was detected using a β-GAL activity Kit (Solarbio, Beijing, China), as described by the manufacturer. Absorbance at 400 nm was measured using a microplate reader (Tecan, Austria), and β-GAL activity (nmol/h /104 cell) was calculated, as recommended by the manufacturer.
Osteogenic and adipogenic differentiation of the hBMSCs
For osteogenic differentiation, after 60% confluency was reached, the hBMSCs were cultured in an osteogenic induction medium that contained complete medium supplemented with 50 μg/ml ascorbic acid (Solarbio, Beijing, China), and 10 mmol/L β-glycerophosphate (Solarbio, Beijing, China), with Dex (10-8, 10-7, and 10-6 mol/L) for 14 days [20]. The osteogenic induction medium was replaced every 2 days. The hBMSCs were treated with the solvent of Dex as the control.
For adipogenic differentiation, after 80% confluency was reached, the hBMSCs were cultured in an adipogenic induction medium that contained complete medium supplemented with 500 μmol/L isobutylmethylxanthine (Solarbio, Beijing, China), 100 μmol/L indomethacin (Solarbio, Beijing, China), and 10 μg/ml insulin (Solarbio, Beijing, China), with or without Dex (10-8, 10-7, 10-6 mol/L) for 4 days. Then, the adipogenic induction medium was changed to a maintenance medium that contained the complete medium supplemented with 10 μg/ml insulin and 5 μmol/L pioglitazone (Solarbio, Beijing, China), with or without Dex (10-8, 10-7, and 10-6 mol/L) for 10 days [20]. The medium were replaced every 2 days. The hBMSCs were treated with the solvent of Dex as the control.
Alizarin red S (ARS) staining assay
The osteogenic differentiation of hBMSCs after treatment with the osteogenic induction medium was evaluated using ARS staining assay. In brief, the cells were fixed using 4% paraformaldehyde for 20 min, and stained with 0.2% ARS solution 15 min. After washing three times, mineralization nodule formation in the cells was observed under an inverted phase-contrast microscope. The experiment was performed and analyzed in triplicate.
Oil Red O (ORO) staining assay
The adipogenic differentiation of the hBMSCs after treatment with the adipogenic induction medium was assessed using ORO staining assay. In brief, the cells were fixed using 4% paraformaldehyde for 20 min, and stained with ORO solution (Solarbio, Beijing, China) for 15 min. After washing three times, lipid droplet formation in the cells was observed under an inverted phase-contrast microscope. The experiment was performed and analyzed in triplicate.
Western blotting analysis
The protein expression levels of the osteogenic markers (BSPII and Runx-2) and adipogenic markers (PPAR-γ and CEBP-α) were detected using western blotting analysis. Briefly, cells at passage 3 were grown in 6-well plates and treated with various concentrations of Dex (10-8 mol/L, 10-7 mol/L, and 10-6 mol/L) for 48 h. The hBMSCs were treated with the solvent of Dex as the control. Total proteins from the hBMSCs were extracted using a RIPA Lysis Buffer (Solarbio, Beijing, China) and a protein loading buffer (EpiZyme, Shanghai, China) heated at 95°C after total protein density was determined using a BCA Protein Detection Kit (Solarbio, Beijing, China). The protein samples were separated using SDS-PAGE and electrotransferred onto a PVDF membrane (Merck-Millipore, France). Then, the membrane was blocked, and in order incubated with primary antibodies and secondary antibodies. After visualization using ECL-PLUS reagents (Merck-Millipore, France), the target bands were scanned using a BioSpectrum Imaging System (UVP, USA). The integrated density was used to quantify the results of the western blotting analysis using ImageJ software (vesion 1.52u), and the results were normalized using GAPDH.
The primary antibodies, including rabbit anti-human BSPII, Runx-2, PPAR-γ, and CEBP-α antibodies, were purchased from Cell Signaling Technologies (Danvers, USA). The primary antibody for rabbit anti-human GAPDH and all secondary antibodies were purchased from Elabscience (Shanghai, China). All antibodies were diluted in the antibody dilution (Boster Biological Technology, Shanghai, China) at an appropriate ratio specified by the manufacturer.
Microarray Assays
The hBMSCs were cultured in complete DMEM containing 10% FBS and 100 units/ml of penicillin-streptomycin along with 10-6 mol/L Dex (Dex-induced group, Dex) or with the solvent of Dex (control group, Control). After treatment for 7 days, total RNA from was extracted from the hBMSCs in the two groups using a RNAiso plus kit (Takara Bio Inc., Kusatsu, Japan), following the manufacturer’s instructions. The purity and concentration of the RNAs were assessed at OD260/280 using a spectrophotometer (NanoDrop ND-1000). Total RNA was reverse-transcribed into cDNA, which was labeled using a fluorescent dye (Cy5 and Cy3-dCTP) and hybridized with the Agilent human lncRNA+mRNA Array V4.0 designed with four identical arrays per slide (4×180K format). The microarrays were washed and then scanned using a G2565CA Microarray Scanner (Agilent). The lncRNA+mRNA array data were analyzed for data summarization, normalization, and quality control using GeneSpring software V13.0 (Agilent). A fold change of ≥ 2.0 or ≤ 2.0 and a P value (t-test) of < 0.05 were used as threshold values to select differentially expressed lncRNAs and mRNAs. The experiment was performed and data were analyzed in triplicate.
Bioinformatics Analysis
DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/) was used to conduct Gene ontology (GO) and pathway enrichment analyses. GO enrichment analysis was performed to identify the functions of the differentially expressed genes between the two groups, including the biological processes, cellular components, and molecular functions involved. Pathway enrichment analysis was performed using Reactome, KEGG, PID, PANTHER, BioCarta, and BioCyc. Furthermore, the coding-non-coding gene co-expression (CNC) network was constructed based on the correlation analysis between mRNA and lncRNA expression (Pearson correlation coefficients > 0.99 or ≤ 0.99). A P value of < 0.05 was considered to indicate statistical significance.
Quantitative Real-Time PCR (qRT-PCR)
Differentially expressed mRNAs and lncRNAs were selected at random to confirm the results of the microarray assays using qRT-PCR. This was conducted in addition to the confirmation of the mRNA expression levels of osteogenic markers (BSPII and Runx-2) and adipogenic markers (PPAR-γ and CEBP-α). In brief, total RNA was obtained from the hBMSCs in the two groups using the RNAiso plus kit (Takara Bio Inc., Kusatsu, Japan), and then reverse transcribed into cDNA using a PrimeScript RT reagent kit (Takara Bio Inc., Kusatsu, Japan). qRT-PCR was performed on a Roche LightCycler 480 Detection System (Roche, Switzerland), using the SYBR Premix Ex Taq II kit (Takara Bio Inc., Kusatsu, Japan), following the manufacturer’s instructions. All forward and reverse primers used for the genes were provided by the Ribobio Corporation (Guangzhou, China) and are listed in table 1. The relative expression level of each gene was evaluated using the 2-△△Ct method and normalized to GAPDH. The experiment was performed and data were analyzed in triplicate.
Statistical Analysis
SPSS 19.0 software (IBM, Armonk, NY, USA) was used to conduct all statistical analyses. One-way analysis of variances (ANOVA) was performed to compare data between more than three groups, and the unpaired t test was performed to compare data of two groups. All data are presented as mean ± SD, and a P value of < 0.05 was considered to indicate statistical significance. All charts were constructed using GraphPad Prism 8 software (GraphPad, CA, USA).