Human BMSCs (hBMSCs) were harvested from the femoral heads of patients undergoing total hip arthroplasty, as reported in a previous study (18). Ethical approval was obtained by the Office of Research Ethics at the Shanghai Sixth People's Hospital of Shanghai Jiao Tong University (Shanghai, China). hBMSCs were cultured in complete medium consisting of alpha minimal essential medium (MEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen; Thermo Fisher Scientific, Inc.), 100 units/ml penicillin and 10 µg/ml streptomycin. The culture medium was replaced with fresh medium every 3 days. Cells were passaged at ~90% confluence, and cells at passages 3–6 were used in the experiments. To induce osteogenic differentiation, hBMSCs were cultured in osteogenic induction medium, which consisted of complete medium, 10 mM β-sodium glycerophosphate, 50 µg/ml L-ascorbic acid and 10 nM dexamethasone. The following graded concentrations of VK₂: 10⁻⁵ M (VK⁻⁵), 10⁻⁶ M (VK⁻⁶) and 10⁻⁷ M (VK⁻⁷), were used to treat hBMSCs (18).

Following culture in osteogenic medium for 21 days, hBMSCs were rinsed with PBS, fixed with 4% paraformaldehyde for 20 min and subsequently stained with 40 mM alizarin red working solution for 10 min. Following this, cells were rinsed twice with PBS, and images were captured using an inverted phase contrast microscope.

hBMSCs were seeded at 10⁵ cells/well in a 6-well plate. After 24 h, the culture medium was replaced with fresh MEM containing 10% FBS, and 5 µg/ml polybrene (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), the cells were transduced with 5×10⁶ TU/ml lentiviral vector (Shanghai GenePharma Co., Ltd., Shanghai, China) carrying miR-133a (miR-133a-control; sequence, TTT GGT CCC CTT CAA CCA GCTG) or a miRNA negative control (miRNA negative; sequence, TTC TCC GAA CGT GTC ACGT) using the four plasmid system. Briefly, this system involves the co-transfection of a recombinant virus plasmid encoding for a transfer vector with three kinds of auxiliary packaging vector into 293T cells. The supernatant containing the virus particles become concentrated following 72 h, and then the high-titer lentivirus concentrate is obtained within 293T cells. The medium was replaced with complete culture medium 24 h after transduction. The transduction efficiency was assessed 96 h following transduction and cells were selected with 2 µg/ml puromycin until colonies of stably transduced cells were formed.

Following culture in osteogenic medium, total RNA was isolated from cells using TRIzol^® reagent; 1 µg RNA was reverse-transcribed with the EasyScript One-step gDNA Removal and cDNA Synthesis supermix (Beijing Transgen Biotech Co., Ltd., Beijing, China) in a total volume of 20 µl, according to the manufacturer's protocol. Following this, qPCR was performed using SYBR^® Green reagents (Beijing Transgen Biotech Co., Ltd.) for 40 cycles under the following conditions: Denaturation at 95°C for 30 sec, annealing at 95°C for 5 sec and extension at 60°C for 30 sec. A 65–95°C solubility curve was constructed with ABI Prism 7900 (Applied Biosystems; Thermo Fisher Scientific, Inc.). All values were normalized to those of GAPDH, and the fold change method (2^−ΔΔCq) was used for analysis (19). The primers used for the amplification of target mRNAs are listed in Table I.

For the detection of mir-133a, miRNAs were isolated using the EasyPure miRNA kit (Beijing Transgen Biotech Co., Ltd.) according to the manufacturer's protocol. qPCR analysis of mir-133a was performed using the SYBR Green RT-qPCR kit (BioTNT Co., Ltd., Shanghai, China). The stem-loop RT primer for miR-133a was 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAGCTGGT-3′, and qPCR primers were: Forward, 5′-TTTGGTCCCCTTCAAC-3′ and reverse, 5′-TCAACTGGTGTCGTGG-3′. U6 served as a control with primers as described previously (20). The thermocycling conditions were as follows: Denaturation at 95°C for 5 min, annealing at 95°C for 5 sec and extension at 60°C for 30 sec, for a total of 40 cycles. A 65–95°C solubility curve was constructed, and the value of mir-133a expression was normalized to that of U6 using the 2^−ΔΔCq method.

Total protein extracted with Cell Lysis buffer (#9803; Cell Signaling Technology, Inc., Danvers, MA, USA) from osteogenic-induced hBMSCs was quantified using a Bicinchoninic Acid (BCA) kit (Thermo Fisher Scientific, Inc.). Following this, total protein was denatured by heating at 95°C for 5 min, mixed with loading buffer, and 30 µg proteins were separated by SDS-PAGE on 10% acrylamide gels. Proteins were subsequently transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% dried skimmed milk, and incubated with rabbit anti-human GAPDH (1:2,000; #2118; Cell Signaling Technology, Inc., Danvers, MA, USA) and rabbit anti-human Runx2 (1:1,000; ab48811; Abcam, Cambridge, MA, USA) antibodies at 4°C overnight and subsequently incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000; #7074; Cell Signaling Technology, Inc.) at 37°C for 1 h. Following chemiluminescence with a commercial assay (Pierce™ ECL Western Blotting substrate, #32106, Thermo Fisher Scientific, Inc.), protein bands were visualized by Odyssey scanner (LI-COR Biosciences, Lincoln, NE, USA). The bands were quantified using Quantity One software (version 4.52; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and normalized to GAPDH.

hBMSCs were lysed with 0.2% Triton-X 100 and centrifuged at 14,000 × g for 10 min at 4°C,, and the supernatant was detected with an ALP assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol. Absorbance values were measured at a wavelength of 520 nm using the iMark Microplate Absorbance reader (Bio-Rad Laboratories, Inc.) and normalized against the protein concentration determined with the use of a BCA kit.

ALP staining was performed using a 5-Bromo- 4-chloro-3-indolyl Phosphate/Nitro Blue Tetrazolium ALP Color Development kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocol. Briefly, cells were rinsed with PBS, fixed with 4% paraformaldehyde for 20 min and stained with the ALP Color Development kit for 30 min. Images were captured using an inverted phase contrast microscope.

Following incubation of cells for 1 week, the osteogenic medium was removed and the cultures were incubated with MEM for 24 h. Concentrations of OCN in the medium were determined using an ELISA kit (#MK128, Takara Bio, Inc., Otsu, Japan). The values were normalized against the total protein concentration determined using a BCA kit.

SPSS software (version, 20.0; IBM SPSS, Armonk, NY, USA) was used to analyze the values in each group. All experiments were performed in triplicate, and data are expressed as the mean ± standard deviation. One-way analysis of variance followed by Tukey's post hoc test was performed to determine statistical significance. P<0.05 was considered to indicate a statistically significant difference.

To detect miR-133a expression during osteogenic differentiation, RT-qPCR was performed on samples collected at days 0, 1, 3, 7, 14 of differentiation. Osteogenic differentiation was confirmed by Alizarin red S staining (Fig. 1). Compared with undifferentiated cells at day 0, miR-133a expression levels rapidly declined during osteogenic induction to 0.05-fold after 14 days (P<0.05; Fig. 2A). By contrast, the expression levels of Runx2 (Fig. 2B), ALP (Fig. 2C) and OCN (Fig. 2D) mRNA steadily increased in a time-dependent manner, exhibiting a negative correlation with miR-133a expression levels. In addition, the expression of VKORC1 mRNA (Fig. 2E) was inversely correlated with miR-133a expression, as previously reported (14), increasing 13-fold in osteogenic-induced hBMSCs at day 14. These results suggested that the osteogenic differentiation of BMSCs was associated with a reduction in miR-133a expression levels, indicating that miR-133a may be associated with the osteoblast differentiation of mesenchymal cells.

To evaluate the association between miR-133a and VK₂, osteogenic induction of hBMSCs was performed for 1 week in the presence or absence of gradient concentrations of VK₂, and miR-133a expression levels were detected by RT-qPCR. miR-133a expression levels were markedly downregulated by VK₂ treatment compared with the control group (P<0.05), to the greatest extent by 10⁻⁵ M VK₂ (Fig. 3A). In addition, mRNA expression levels of Runx2 (Fig. 3B) and ALP (Fig. 3C) were significantly upregulated by VK₂ treatment (P<0.05), whereas those of OCN were not (P>0.05) (Fig. 3D). ALP and OCN are two late-stage osteogenic differentiation markers of mesenchymal cells, and were therefore further analyzed. Greater ALP activity was detected in cells treated with 10⁻⁵ M and 10⁻⁶ M VK₂ compared with the control (P<0.05; Fig. 3E). OCN concentrations in supernatant were increased by VK₂ treatment, particularly by 10⁻⁵ M VK₂ (P<0.05; Fig. 3F), despite the lack of effect on OCN mRNA expression levels. Detection of Runx2 protein expression levels by western blotting (Fig. 3G) further verified the osteogenic stimulation effect of VK₂ treatment, revealing greater Runx2 protein expression in VK₂-treated cells (P<0.05), except at a concentration of 10⁻⁷ M VK₂, although not significant for 10⁻⁶ M VK₂ (P>0.05) (Fig. 3H). Although the western blotting results were inconsistent with the Runx2 mRNA analyses, which may be associated with the time interval existing between gene and protein expression, these results indicated that VK₂ treatment promoted osteogenic differentiation of hBMSCs, as previously described (18,21), and that the stimulation of osteogenesis may be associated with the inhibition of miR-133a.

To examine whether miR-133a directly regulated the osteogenic differentiation of hBMSCs, miR-133a was overexpressed in hBMSCs via transfection with a lentiviral vector. Compared with cells transfected with the miR-negative control, hBMSCs transfected with miR-133a expressed significantly greater levels of miR-133a, as demonstrated by RT-qPCR (P<0.05; Fig. 4A). To investigate the effect of miR-133a overexpression, the mRNA expression levels of ALP, OCN and Runx2, ALP activity, OCN secretion, and Runx2 protein expression levels were measured in miR-133a-overexpressing hBMSCs incubated with osteogenic induction medium for 7 days. mRNA expression levels of ALP (P>0.05; Fig. 4B), OCN (P>0.05; Fig. 4C) and Runx2 (P<0.05; Fig. 4D) were decreased in miR-133a-overexpressing cells compared with control cells, as were ALP activity (P<0.05; Fig. 4E), OCN secretion (P<0.05; Fig. 4F), ALP staining (Fig. 4G) and Runx2 protein expression levels (P<0.05; Fig. 4H and I), indicating that miR-133a overexpression inhibited the osteogenic differentiation of hBMSCs.

The effect of VK₂ treatment on miR-133a-overexpressing cells was additionally determined. After 7 days of osteogenic induction, VK₂ treatment significantly downregulated miR-133a expression levels in miR-133a-overexpressing hBMSCs (P<0.05; Fig. 4A); the mRNA expression levels of ALP (Fig. 4B), OCN (Fig. 4C) and Runx2 (Fig. 4D) were upregulated by VK₂ treatment. Furthermore, greater ALP activity (Fig. 4E) and increased supernatant OCN concentrations (Fig. 4F), ALP-positive cells (Fig. 4G) and Runx2 protein expression levels (Fig. 4H and I) were detected in VK₂-treated miR-133a-overexpressed cells compared with control cells.

VKORC1 is a key protein involved in the VK cycle, where VK₂ functions to promote the γ-carboxylation of OCN during bone metabolism. The results of a previous (15) and the present study demonstrated that miR-133a expression levels are inversely correlated with VKORC1 mRNA expression levels. To investigate whether VK₂ affects the expression of VKORC1 via miR-133a, osteogenic induction of hBMSCs was performed for 1 week in the absence or presence of gradient concentrations of VK₂, and VKORC1 mRNA expression levels were detected by RT-qPCR. VKORC1 mRNA expression levels were significantly upregulated by VK₂ (P<0.05), particularly at a concentration of 10⁻⁵ M, where levels were 22-fold of those of the control (Fig. 5A). An inverse correlation between miR-133a and VKORC1 was demonstrated, even in the presence of VK₂. In addition, VKORC1 mRNA expression levels were downregulated in miR-133a-overexpressing cells compared with miR-negative control cells (P<0.05), and VK₂ significantly increased VKORC1 mRNA expression levels in miR-133a-overexpressing hBMSCs (P<0.05; Fig. 5B), which further demonstrated that VK₂ positively regulated VKORC1 by downregulating miR-133a expression.