Cell line and osteogenic differentiation induction medium
The hBMSCs were purchased from Fuyuanbio Co., Ltd (Shanghai, China). The cells were passaged and the third generation of hBMSCs was taken for the following experiment. The osteogenic differentiation induction medium: DMEM (Invitrogen, USA) containing 10 µM dexamethasone, 50 µM vitamin C, 10 mM β-glycerol sodium phosphate, and 10% FBS.
The hBMSCs was cultured in a 6-well culture plate at a concentration of 5×103/ml. After 24 h, they were transfected with miRNA-153-3p mimic, inhibitor and the corresponding negative control (NC) (ThemoFisher, USA) with lipofectamine 2000 (ThemoFisher, USA) according to the transfection instructions, the cells were incubated in 5% CO2, 37°C incubator and a blank control group was set up. After 5 h, the transfection medium was replaced with DMEM, and the cells were cultured for another 8 h, and then the transfected cells were taken for the following experiment.
Cells viability assay
The cell counting kit-8 (CCK-8; Beyotime Biotechnology, China) was used to detect the viability of the transfected hBMSCs according to the manufacturer instructions, and the OD value was read at 450 nm. The untransfected hBMSCs were as the control group.
Cells proliferation ability assay
After fixing the cell sample with 4% paraformaldehyde, washed it with PBS solution 3 times, then blocked it with 5% BSA at room temperature for 1 h, then incubated it with rabbit anti-Ki67 primary antibody (1:800; Abcam, UK) at room temperature for 6 h. Then the cell sample were washed 3 times with PBS solution and incubated with Alexa Fluor® 488 goat anti-rabbit secondary antibody (1:1000; Abcam, UK) at room temperature for 3 h. After washing 3 times with PBS solution, the cells were counterstained the cell nucleus with Hoechst 33342 dye at room temperature for 30 min. The positive cells were observed under a fluorescence microscope, the positive cells number was calculated under 10 randomly different fields, and the percentage of Ki67-positive cells = Ki67-positive cell number/Hoechst-positive cell number.
Osteogenic differentiation culture
The hBMSCs transfected with miRNA-153-3p mimic, inhibitor, mimic NC or inhibitor NC were cultured in a 24-well culture plate at a concentration of 5×105/ml with DMEM medium in a 37°C, 5% CO2 incubator. When about 80% confluence, the culture medium was replaced with the osteogenic differentiation induction medium for 14 days. The untransfected hBMSCs were cultured as the control group.
miRNA-153-3p expression level assay
The total RNA in cells was extracted by TaqMan miRNA isolation kit (Applied Biosystems, USA), then the expression of mature miR-153-3p was measured with the TaqMan miRNA assay and TaqMan Universal PCR Master Mix (Applied Biosystems, USA). Reaction conditions: 94°C 4 min→94°C 30 s→56°C 30 s→72°C 30 s, cycle 40 times. U6 was used as the internal reference. The qRT-PCR relative quantitative method was used to analyze the experimental result. The miRNA-153-3p expression level was calculated with 2−△△Ct method.
Alizarin red staining
After 14 days of osteogenic differentiation, the cell samples were fixed with 4% paraformaldehyde. After washing 3 times with PBS, the cell were incubated with 2% alizarin red staining solution (Beyotime, China) for 10 min at room temperature. Some of cells in each group were observed under an inverted microscope, the cell mineralization of other cells in each group were quantified with alizarin red extracted with 100 mM cetylpyridinium chloride solution (Sigma, USA), and the OD value was read at 570 nm.
Alkaline phosphatase (ALP) activity assay
After 14 days of osteogenic differentiation, the cell samples were washed with PBS. The cells were lysed with 1% Triton X-100 for 15 min, centrifuged at 10,000 g for 5 min, and the supernatant were collected for ALP activity assay which was detected with ALP Assay Kit (Beyotime Biotechnology, China) according to the manufacturer instructions, and the OD value was read at 405 nm.
Western blot analysis
After 14 days of osteogenic differentiation, the total protein of cells was extracted using RIPA buffer (Beyotime, China). Briefly, 10 µg of total protein from each sample was electrophoresed and transferred to PVDF membrane, and the PVDF membrane was blocked with 5% BSA. Then the PVDF membrane was successively incubated with rabbit anti-RUNX2 primary antibody (1:800, Abcam, UK), mouse anti-Collagen I primary antibody (1:800, Abcam, UK) or mouse anti-β-actin primary antibody (1:1000, Abcam, UK) and IRDye 800-conjugated affinity-purified goat anti-rabbit second antibody (1:4000, Rockland Immunochemicals, USA) or IRDye 700-conjugated affinity-purified goat anti-mouse second antibody (1:4000, Rockland Immunochemicals, USA). The quantity of protein bands were analyzed with Odyssey laser scanning system (LI-COR Inc., USA).
Luciferase reporter gene experiment
The QuickChange site-directed mutagenesis kit (Stratagene, USA) was used to mutate the core sequence of 3'UTR of RUNX2. The wild-type (WT) and mutated (MU) sequence were cloned into pMIR-Report luciferase vector to obtain RUNX2 WT-luc and RUNX2 Mu-luc. The RUNX2 WT-luc, RUNX2 Mu-luc, miR-153-3p mimic (ThemoFisher, USA) and the corresponding NC were transfected into HEK293 cells. The cells were incubated in a saturated humidity, 37°C, 5% CO2 incubator for 48 h, and then were lysed and analyzed with a dual‑luciferase reporter assay system (Promega Corporation, USA) according to the manufacturer instructions.
The data obtained in this research were statistically analyzed with SPSS 22.0 software. The data obtained in this study were all measurement data and conform to the normal distribution, which were presented as mean ± standard deviation (M ± SD). Comparison among groups was tested by independent t test or one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.