THP-1 human acute monocytic leukemia cells (cat. no. TIB-202; American Type Culture Collection, Manassas, VA, USA) were incubated in RPMI-1640 medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C under 5% CO₂ and saturation humidity. THP-1 cells in the logarithmic stage of growth were seeded (2×10⁵ cells/well) into 6-well plates and co-cultured with 200 ng/ml phorbol-12-myristate-13-acetate (PMA; cat. no. 79346; Sigma-Aldrich; Merck KGaA), 25 ng/ml M-CSF (PeproTech, Inc., Rocky Hill NJ, USA) and 30 ng/ml RANKL (PeproTech, Inc.) to induce their differentiation into osteoclast-like cells, as previously described (2). Cells were cultured for 0, 24, 72 and 120 h, collected and stained by using an Acid Phosphatase, Leukocyte (TRAP) kit (cat. no. 387; Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Subsequently, the TRAP-positive stained cells were photographed using a light microscopy (Nikon Corporation, Tokyo, Japan), and the number of TRAP-positive staining cells were counted and analyzed. Subsequently, the miR-20a expression levels were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) at 0, 24, 72 and 120 h, as described below.

To investigate the influences of miR-20a on the process of monocyte differentiate into osteoclasts, THP-1 cells were co-cultured with 10 ng/ml PMA to induce their differentiation into macrophages (osteoclast precursors) at 37°C for three days (1). Subsequently, the THP-1 cell-derived macrophages were seeded (1×10⁴ cells/well) into 96-well plates and transfected with 100 nM miR-20a mimics or 100 nM miRNA negative controls (miR-NC; GenePharma, Shanghai, China) at 37°C for 6 h using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's guidelines. Untransfected THP-1 cell-derived macrophages were used as a Blank group. At 24 h post-transfection, the transfection efficiency of miRNAs was assessed by determining the miR-20a expression levels by RT-qPCR, using the following primers: Forward, 5′-CCGCTCGTGAAATGTTTAGG-3′, reverse primer, 5′-ATGGAGCCTGGGACGAGA-3′. U6 (forward primer 5′-ATTGGAACGATACAGAGAAGATT-3′; reverse primer, 5′-GGAACGCTTCACGAATTTG-3′) was used as an internal control for normalization based on the study of Zhou et al (15). For RT-PCR, miRNAs was extracted from 2×10⁶ cells by using a mirVana miRNA isolation kit (Applied Biosystems, Foster City, CA, USA) as per the manufacturer's protocol. The cDNA was synthesized by using the Prime Script RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). The RT-PCR was performed using SYBR^® PrimeScript^® miRNA RT-PCR kit (Takara Biotechnology Co., Ltd.) on FTC-3000™ Real-time PCR Cycler (Funglyn Biotech Corp. Ltd., Toronto, ON, Canada). The parameters for RT-PCR were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 30 sec and 60°C for 45 sec.

Following miR-20a mimics transfection, 2×10⁵ cells were seeded into 6-well plates. Cell proliferation was examined at 0, 24, 48 and 72 h using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), following the manufacturer's protocol. Optical density (OD) was detected at 450 nm using a Benchmark microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The rate of cell proliferation was calculated using the following formula: (OD_test-OD_blank)/(OD_control-OD_blank) ×100. All assays were performed in triplicates.

Following miR-20a mimic treatment for 48 h, cells were fixed overnight in ice-cold ethanol (at 4°C). The cells were washed with PBS and incubated with a 1 ml solution comprising 20 mg/ml propidium iodide (PI) and 10 U/ml RNaseA (cat. no. KGA214; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) for 30 min at room temperature. Cells were counted by Accuri C6 Fluorescence-activated cell sorting (BD Biosciences, Franklin Lakes, NJ, USA). Progression of the cell cycle was assayed using the ModFit LT 3.0 software (Verity Software House, Topsham, ME, USA).

Following miR-20a mimic treatment, cells were collected at 48 h. Cell suspensions were prepared by centrifuging cell suspensions at 1,000 × g for 5 min at room temperature. Cells from pellets were re-suspended in PBS and centrifuged at 1,000 × g for 5 min under room temperature, and the supernatant was removed. Subsequently, cells were incubated with Annexin V-Fluorescein isothiocyanate (FITC)/PI, from the Annexin V-FITC/PI Double-staining Apoptosis Detection kit (cat. no. M3021-3; Molecular Biology and Chemical, Shanghai, China) for 15 min away from light, according to the manufacturer' protocol. Cells were analyzed immediately on a BD Accuri C6 flow cytometer equipped with BD Accuri C6 software (BD Biosciences; Becton-Dickinson and Company, Franklin Lakes, NJ, USA). Experiments were performed three times.

Transfected cells were further incubated with M-CSF (25 ng/ml) and RANKL (30 ng/ml) for 3 days (2). To assess the changes of TRAP, NFATc1 and peroxisome proliferator-activated receptor γ (PPARγ) mRNA expression levels, 2×10⁶ cells were collected and treated with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was used for RT-qPCR. The cDNA was synthesized by the Prime Script RT-PCR kit. SYBR-Green-based real time RT-PCR was performed using SYBR^® Premix Ex Taq™ II (Takara Biotechnology Co., Ltd) on FTC-3000™ Real-time PCR Cycler. The parameters for RT-PCR were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 30 sec and 60°C for 30 sec, and finally 72°C for 90 sec. Primers specific for TRAP, NFATc1, PPARγ and GAPDH were synthesized as previously described (16) and as follows: TRAP forward, 5′-GGAGATCAGCTCCAAAGAGATG-3′ and reverse primer, 5′-GGGCAGTCATGGGAGTTCAG-3′; NFATc1 forward, 5′-AGACCGTGTCCACCACCAGC-3′ and reverse primer, 5′-CAGGATTCCGGCACAGTCAAT-3′; PPARγ forward, 5′-TGGCCTCCTTGATGAATA-3′ and reverse primer, 5′-GGCTTGTAGCAGGTTGTC-3′; GAPDH forward, 5′-CATGAGAAGTATGACAACAGCCT-3′ and reverse primer, 5′-AGTCCTTCCACGATACCAAAGT-3′. GAPDH was used as an internal control and for the normalization of TRAP, NFATc1 and PPARγ mRNA levels. Relative mRNA levels were calculated using the 2^−ΔΔCq method (17).

Cells in the Blank and miR-transfected groups were treated with M-CSF and RANKL for three days. Subsequently, the cells (5×10⁶) were lysed using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Inc.). Cell lysates were centrifuged at 12,000 × g for 1 min and 30 µg total proteins, the concentration determined by BCA kit (cat. no. BCA1; Sigma-Aldrich; Merck KGaA) were separated by 8% SDS-PAGE, followed by transfer of separated proteins to polyvinylidene difluoride membranes. Membranes were blocked with Blocking Buffer (1X PBS; 0.1% Tween-20; 5% w/v bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature and incubated with antibodies against TRAP (1:100; cat. no. sc-28204; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), NFATc1 (1:100; cat. no. sc-13033; Santa Cruz Biotechnology, Inc.) or PPARγ (1:100; cat. no. sc-7273; Santa Cruz Biotechnology, Inc.) at room temperature for 1.5 h. Subsequently, the goat anti-mouse IgG secondary antibody (1:10,000; cat. no. 115-035-003; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), goat anti-Rabbit IgG secondary antibody (1:10,000; cat. no. 115-035-003; Jackson ImmunoResearch Laboratories Inc.) and rabbit anti-Goat IgG secondary antibody (1:10,000; cat. no. 33701ES60; Yeasen Corporation, Shanghai, China) was added correspondingly for incubation at 4°C overnight. Protein levels were normalized to those of GAPDH (1:200; cat. no. sc-47724; Santa Cruz Biotechnology, Inc.). Expression levels of protein in each sample were calculated using the Gel-Pro analyzer 3.0 software (Media Cybernetics, Inc., Rockville, MD).

All experiments were performed in triplicate. Data are expressed as mean ± standard deviation, and were analyzed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). Student's t-test was used to analyze the statistical significance between two groups. Data from more >3 groups were analyzed by one-way or two-way analysis of variance, followed by Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

It was observed that the number of TRAP-positive staining cells was significantly increased and some of the THP-1 cells were induced into osteoblasts (P<0.001; Fig. 1A and B). The TRAP-positive cells included both macrophages (osteoclast precursors) and osteoclasts. In addition, miR-20a expression levels were significantly increased at 24 h (P<0.001) and 72 h (P<0.001), but significantly decreased at 120 h (P<0.001) compared with expression levels at 0 h (Fig. 1C). In addition, there was a time-dependent decrease in miR-20a expression levels from 24 to 120 h. These data demonstrated that miR-20a expression was decreased post-differentiation induction. Following transfection with miR-20a mimics into PMA treated THP-1 cells, miR-20a expression levels were significantly increased in cells of by ~40-fold compared with the Blank and miR-NC-transfected groups (P<0.001; Fig. 1D). Since the result in Fig. 1D proved that there was no significant difference between the Blank and miR-NC group, therefore, only the Blank group was used in Figs. 2 and 3.

Results of cell proliferation assays demonstrated that cell proliferation was markedly inhibited in the miR-20a mimics group compared with proliferation in the Blank group at 48 and 72 h (P<0.01 and P<0.001, respectively; Fig. 2A). Furthermore, the percentage of cells in the S phase of the cell cycle was significantly decreased in the miR-20a mimics group compared with the Blank group (P<0.01; Fig. 2B and C), which indicated that cell division was inactive following miR-20a mimics transfection. In addition, the total number of early and late apoptotic cells was significantly higher in the miR-20a mimics group (65.63±2.99%) compared with apoptotic cells in the Blank group (54.3±2.17%; P<0.01, Fig. 2D and E). These results demonstrated that miR-20a may inhibit the growth of PMA-treated THP-1 cells.

As the master regulators of osteoclastogenesis, the mRNA expression levels of TRAP (18), NFATc1 (19) and PPARγ (20) were significantly decreased during the THP-1 cell differentiation process in cells transfected with miR-20a mimics compare with cells in the Blank group (P<0.001; Fig. 3A). In addition, results of western blot analysis confirmed that the protein levels of TRAP (P<0.05), NFATc1 (P<0.001) and PPARγ (P<0.001) were also significantly decreased in the miR-20a mimics group compare with the Blank group (Fig. 3B). These results indicated that miR-20a may regulate osteoclastogenesis by decreasing PPARγ expression during THP-1 cell differentiation process.