The murine macrophage cell line RAW264.7 was purchased from the American Type Culture Collection and was cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin (100 U/ml)/streptomycin (100 µg/ml) (Hyclone; GE Healthcare Life Sciences) in an incubator containing 5% CO₂ at 37°C. RAW264.7 cells were cultured with 50 ng/ml RANKL (Thermo Fisher Scientific, Inc.) for 3–5 days to induce osteoclastogenesis.

TargetScan (version 7.2; www.targetscan.org/vert_72) and miRTarBase (version 7.0; mirtarbase.mbc.nctu.edu.tw/php/index.php) were used to predict the potential target genes of miR-21. Wild-type (WT) and mutant (MT) binding sites of miR-21 in the 3′-untranslated region (UTR) of Pten were subcloned into pGL3 basic vectors (Promega Corporation), to generate pGL3-Pten-WT and pGL3-Pten-MT, respectively. RAW264.7 cells (1×10⁶ cells/well) were seeded into 6-well plates and were transfected with 10 ng Renilla luciferase plasmid (pRL-TK; Promega Corporation), 10 ng pGL3-Pten-WT or pGL3-Pten-MT, and 25 µM miR-21 mimic (Guangzhou RiboBio Co., Ltd.) or miR-negative control (NC; 5′-CAGUACUUUUGUGUAGUACAA-3′; Guangzhou RiboBio Co., Ltd.) using Lipofectamine^® RNAi Max (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After transfection, the cell were cultured at 37°C with 5% CO₂ for 48 h, a Dual-Luciferase Reporter Gene Analysis system (Promega Corporation) was used to detect the luciferase activity, according to the manufacturer's protocol. With Renilla luciferase used as an internal control, luciferase activity was calculated as firefly fluorescence/Renilla fluorescence ratio.

miR-NC (5′-CAGUACUUUUGUGUAGUACAA-3), miR-21 mimic and miR-21 inhibitor (5′-UCAACAUCAGUCUGAUAAGCUA-3′) were synthesized and obtained from Guangzhou RiboBio Co., Ltd. RAW264.7 cells at the logarithmic phase were seeded into 6-well plates and separated into the following groups: MiR-NC (cells transfected with 25 µM miR-NC), miR-21 mimic (cells transfected with 25 µM miR-21 mimic), miR-21 inhibitor (cells transfected with 25 µM miR-21 inhibitor) and miR-21 mimic + LY294002 [cells transfected with miR-21 mimic and subsequently treated with the PI3K inhibitor LY294002 (10 µM; APExBio Technology) and incubated at 37°C for 48 h]. At 70% confluence, the cells were transfected using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. At 48 h post-transfection at 37°C with 5% CO₂, >60% cells were successfully transfected. Prior to subsequent reverse transcription-quantitative PCR (RT-qPCR), TRAP staining and western blotting experiments, the cells were cultured with 50 ng/ml RANKL for 3 days at 37°C with 5% CO₂ to induce osteoclastogenesis.

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol and was reverse transcribed using the SuperScript First Strand cDNA system (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. PCR amplification was performed using the SYBR Green PCR master mix (Thermo Fisher Scientific, Inc.). Stem-loop RT-qPCR and conventional RT-qPCR were used for quantification of miR-21, and tartrate-resistant acid phosphatase (TRAP; a specific marker of OCs and bone resorption) and Pten, respectively. The primers used in the present study were synthesized by Sangon Biotech Co., Ltd. (Table I). The reaction conditions were as follows: 95°C for 10 min; 40 cycles of 95°C for 15 sec and 58°C for 30 sec; 72°C for 40 sec; and final extension at 72°C for 8 min. The 2^−ΔΔCq method (14) was used to calculate the relative expression levels of miR-21, TRAP and Pten. miRNA and mRNA levels were normalized to the internal reference genes U6 and GAPDH, respectively.

RANKL-induced RAW264.7 cells were seeded at a density of 5×10⁵ cells/well into 24-well plates and cultured for 24 h at 37°C with 5% CO₂. Subsequently, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature. TRAP staining solution was added and incubated at 37°C for 1 h in the dark. The procedure was performed according to the TRAP staining kit protocol (Sigma Aldrich; Merck KGaA). TRAP-positive multi-nucleated cells (TRAP⁺ MNCs) containing >3 nuclei were considered OCs and were observed under an inverted light microscope (magnification, ×200). The number of TRAP⁺ MNCs/well was independently counted under the inverted microscope by two individuals. The average of the three experiments was taken as the number of OCs present.

A pit assay was performed to observe the bone resorption of OCs. Bovine bone slices were generated in the laboratory and were obtained from fresh bovine cortical bone. Bone was purchased from cows that were sacrificed for commercial/consumer purposes. Notably, cows are not an endangered species, and the bones were obtained from a licensed source and did not contain potentially harmful agents (biological and chemical or genetically modified material). Bovine cortical bone was dried, rough-cut and sliced using a low speed diamond saw into pieces ~50 µm thick and 6 mm in diameter. Subsequently, the bone slices were treated in distilled water for ultrasonic cleaning prior to use. At 48 h post-transfection and prior to treatment with RANKL, RAW264.7 cells were seeded onto the bovine bone slices in 24-well plates at 5×10⁵ cells/well and were cultured in routine medium for 24 h at 37°C with 5% CO₂. Subsequently, the medium was changed to routine medium supplemented with 50 ng/ml RANKL for 3 days, and then the bovine bone slices were ultrasonicated at 30 Hz for 10 min at room temperature in 1 mol/l NH₄OH to remove adherent cells. Bone slices were stained with 0.1% toluidine blue at room temperature for 3–5 min and the bone resorption pits were subsequently observed under a light microscope (magnification, ×400). Bone resorption was calculated as pit area/total bone area of each slice, and was calculated using ImageJ software (version 1.8.0; National Institutes of Health).

Total protein was extracted from cells using a Total Protein Extraction kit (Beiing Solarbio Science and Technology Co., Ltd.), according to the manufacturer's protocol. The bicinchoninic acid method was used to measure protein concentration and 40 µg protein/lane was separated by SDS-PAGE on 10% gels and transferred to PVDF membranes (EMD Millipore). PVDF membranes were blocked in TBS containing 0.25% Tween and 5% skimmed milk for 1 h at room temperature. Membranes were incubated with primary antibodies targeted against: Pten (cat. no. ab32199; 1:1,000), Akt (cat. no. ab18785; 1:5,000), phosphorylated (p)-Akt (cat. no. ab38449; 1:1,000), NFATc1 (cat. no. ab25916; 1:500) and GAPDH (cat. no. ab9485; 1:5,000) at 4°C overnight. All primary antibodies were purchased from Abcam. Membranes were then incubated with horseradish peroxidase-conjugated IgG secondary antibodies (cat. no. ab6721; 1:10,000; Abcam) for 1 h at room temperature. ECL detection reagent (Beiing Solarbio Science and Technology Co., Ltd.) was used to observe protein bands and relative protein expression levels were calculated using ImageJ software (version 1.8.0; National Institutes of Health) with GAPDH as an internal reference.

Each experiment was repeated at least 3 times. SPSS software (version 19.0; IBM Corp.) was used to analyze all data. Kolmogorov-Smirnov test was used to confirm that data were normally distributed and data are presented as the mean ± SD. Comparisons among multiple groups were assessed by one-way ANOVA followed by Tukey's post hoc test. An unpaired Student's t-test was used to assess the differences between the two groups in the dual-luciferase assay. P<0.05 was considered to indicate a statistically significant difference.

The mRNA expression levels of the OC marker TRAP were detected in RANKL-induced macrophage RAW264.7 cells at different time points by RT-qPCR, in order to analyze osteoclastogenesis (Fig. 1A). After 3 days of induction, the expression levels of TRAP were significantly increased, and the expression of TRAP was significantly higher on the fifth day compared with on the first and third days. Our preliminary studies detected the background levels of TRAP expression at 0 h and confirmed that they are lower than at the first day of RANKL induction; therefore, the expression of TRAP at 0 h was not recorded in the present study. As presented in Fig. 1B, the expression levels of miR-21 increased progressively during the process of osteoclastogenesis and the change in miR-21 was consistent with the upregulation of TRAP mRNA, indicating that miR-21 was upregulated during osteoclastogenesis in vitro.

Pten promotes the proliferation of T cells and tumor cells, and has a critical role in RANKL-induced OC differentiation (15,16). In the present study, target genes that were potentially regulated by miR-21 were predicted by TargetScan and miRTarBase; Pten, which contains miR-21 binding sites in its 3′UTR, was selected as one of the target genes of miR-21. The results of a dual luciferase reporter gene assay revealed that the luciferase activity of Pten-WT was inhibited by the miR-21 mimic (P<0.05), whereas the luciferase activity of Pten-MT was not affected, thus suggesting that miR-21 directly targeted Pten (Fig. 2A).

In order to verify the effects of miR-21 on Pten, the expression levels of miR-21 and Pten were detected in RANKL-induced RAW264.7 cells post-transfection by RT-qPCR and western blotting (Fig. 2B-D). Compared with the miR-NC group, miR-21 expression was increased, whereas Pten protein expression was decreased in the miR-21 mimic group. Furthermore, the expression levels of miR-21 were downregulated, whereas the protein expression levels of Pten were upregulated in the miR-21 inhibitor group. There was no difference in Pten mRNA expression among the groups. These results suggested that miR-21 negatively regulated Pten at the translational level.

TRAP staining was used to detect osteoclastogenesis of RAW264.7 cells transfected with miR-21 mimic and miR-21 inhibitor, in order to assess the effect of miR-21 on OC differentiation (Fig. 3). The results revealed that the number of OCs was increased in the miR-21 mimic group and markedly decreased in the miR-21 inhibitor group, when compared with the miR-NC group, thus indicating that miR-21 may be crucial for osteoclastogenesis.

The bone resorption assay was used to detect the percentage of bone resorption (Fig. 4). The percentage of bone resorption was significantly higher in the miR-21 mimic group than in the miR-NC group (75.4±10.2 vs. 35.1±7.3%, respectively), and was significantly higher than that in the miR-21 inhibitor group (23.8±5.6%). These results suggested that miR-21 could enhance the bone resorption of OCs.

It has previously been indicated that Pten regulates osteoclastogenesis in RANKL-induced RAW264.7 OC precursors via activating the PI3K/Akt signaling pathway (16). In the present study, the results of TRAP staining and the bone resorption assay revealed that the number of OCs and the percentage of bone resorption in the miR-21mimic + LY294002 group were lower than those in the miR-21 mimic group (Figs. 3 and 4). These findings indicated that miR-21 may regulate osteoclastogenesis and bone resorption via regulating the PI3K/Akt signaling pathway. The present study further detected Pten, Akt, p-Akt and NFATc1 expression in RAW264.7 cells post-transfection, as presented in Fig. 5. The results demonstrated that, compared with the miR-NC group, the protein expression levels of Pten were decreased, and p-Akt and NFATc1 expression was increased in the miR-21 mimic group. Conversely, the protein expression levels of Pten were increased, whereas p-Akt and NFATc1 expression levels were decreased in the miR-21 inhibitor group. Compared with the miR-21 mimic group, the protein expression levels of Pten were upregulated, whereas p-Akt and NFATc1 expression were reduced in the miR-21 mimic + LY294002 group, thus suggesting that the PI3K inhibitor LY294002 strongly reversed the effects induced by the miR-21 mimic. These results indicated that miR-21 may promote osteoclastogenesis and bone resorption via activation of the PI3K/Akt signaling pathway by targeting Pten.