The CHON-001 human chondrocyte cell line was obtained from American Type Culture Collection. Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37˚C with 5% CO₂. To establish an in vitro OA model, 5x10⁴ CHON-001 cells were stimulated with recombinant human IL-1β (0, 5, 10 or 20 ng/ml) for 24 h at 37˚C (cat. no. SRP6169; Sigma-Aldrich; Merck KGaA).

miR-106a-5p mimics and negative control (NC) were obtained from Shanghai GenePharma Co., Ltd. CHON-001 cells were seeded into 6-well plates and cultured overnight to ~70% confluence. Subsequently, 5x10⁴ CHON-001 cells were transfected with 10 nM miR-106a-5p mimics or 10 nM NC using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 24 h according to the manufacturer's protocol.

For combination treatment, 5x10⁴ CHON-001 cells were transfected with miR-106a-5p mimics (2, 4, 6 or 10 nM) for 24 h at 37˚C, and subsequently treated with 20 µM BAI (cat. no. 465119; Sigma-Aldrich; Merck KGaA) for a further 24 h at 37˚C. Cells were then stimulated with 20 ng/ml IL-1β for another 24 h at 37˚C to induce the OA phenotype.

CHON-001 cells were seeded into 96-well plates (5x10³ cells/well) and incubated at 37˚C overnight. Following transfection and treatment with BAI and IL-1β, cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, 10 µl CCK-8 solution was added to each well and incubated at 37˚C for 2 h. The absorbance of each well was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

CHON-001 cells were seeded (5x10⁴ cells/well) and cultured to ~80% confluence. Cells were treated with different concentrations of IL-1β (0, 5, 10 or 20 ng/ml) for 24 h 37˚C. Subsequently, early apoptotic, late apoptotic and necrotic cells were assessed using the Annexin V-FITC Apoptosis Staining Detection kit (Abcam) according to the manufacturer's protocol. Briefly, cells were harvested and centrifuged at 626 x g for 5 min at 4˚C. After rinsing twice with PBS, cells were centrifuged at 626 x g for 5 min at 4˚C. Subsequently, cells (1x10⁵) were collected from each group, resuspended in binding buffer, and subsequently stained with 5 µl Annexin V-FITC and 5 µl propidium iodide for 5 min in the dark at room temperature. Within 1 h, cell apoptosis was assessed via flow cytometry using a BD FACSAria flow cytometer (BD Biosciences) and CellQuest Pro software (version 5.1; BD Biosciences).

Following transfection and treatment with BAI and IL-1β, culture supernatants were collected from 6-well plates and then centrifuged for 10 min at 626 x g at 4˚C. Subsequently, cellular IL-6 (cat. no. ES4078) and TNF-α (cat. no. ELK1190) concentrations were determined using ELISA kits (ELK Biotechnology Co., Ltd.) according to the manufacturer's protocol. IL-6 and TNF-α concentrations in cell supernatants (100 µl) were assessed for the following groups: i) Control; ii) 20 ng/ml IL-1β; iii) 20 ng/ml IL-1β + 20 µM BAI; iv) 20 ng/ml IL-1β + miR-106a-5p mimics; and v) 20 ng/ml IL-1β + miR-106a-5p mimics + 20 µM BAI.

To confirm successful transfection of miR-106a-5p mimics, total RNA was extracted from transfected CHON-001 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) Total RNA was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The RT reaction comprised the following: 1 µg RNA, 10 µl reaction solution, 1 µl RT primers and 1 µl dNTPs in a final volume of 15 µl. The mixture was heated at 70˚C for 5 min and then it was quickly placed on ice to cool. Subsequently, 4 µl RT buffer, 1 µl M-MLV Reverse Transcriptase and 1 µl RNase inhibitor were added into the mixture on ice. The RT reactions were subsequently performed as follows: 42˚C for 60 min; 85˚C for 5 min. The following primers were used for RT: U6, 5'-AACGCTTCACGAATTTGCGT-3'; miR-183-5p, 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGTGAATT-3'.

Subsequently, qPCR was performed using GoTaq qPCR Master Mix (Promega Corporation). miRNA expression levels were normalized to the internal reference gene U6 using the U6 snRNA normalization RT-PCR quantitation kit (Shanghai GenePharma Co., Ltd.) and the ABI Prism7700 detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR amplification reactions were subsequently performed as follows: 95˚C for 3 min; and 40 cycles at 95˚C for 10 sec, 58˚C for 30 sec and 72˚C for 30 sec. The following primers were used for qPCR: U6 forward, 5'-GCTTCGGCAGCACATATACTAAAAT-3' and reverse, 5'-CGCTTCACGAATTTGCGTGTCAT-3'; and miR-106a-5p forward, 5'-AAAAGTGCTTACAGTGCAGGTAG-3' and reverse, 5'-GAAAAGTGCTTACAGTGCAGGT-3'. miRNA expression levels were quantified using the 2^-ΔΔCq method (35).

Following transfection and treatment with BAI and IL-1β, total protein was extracted from CHON-001 cells using RIPA buffer (Beyotime Institute of Biotechnology) for 30 min on ice. Total protein was quantified using a BCA protein kit (Thermo Fisher Scientific, Inc.). Proteins (30 µg) were separated via 10% SDS-PAGE (Pierce; Thermo Fisher Scientific, Inc.), transferred onto PVDF membranes and blocked with 5% non-fat milk (diluted in 0.05% TBS-Tween-20) for 1 h at room temperature. Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Rabbit anti-Bax (1:1,000; cat. no. ab32503), rabbit anti-caspase-3 (1:1,000; cat. no. ab2302), rabbit anti-Bcl-2 (1:1,000; cat. no. ab32124), rabbit anti-collagen I (1:5,000; cat. no. ab34710), rabbit anti-collagen III (1:1,000; cat. no. ab184993), rabbit anti-aggrecan (1:1,000; cat. no. ab36861), rabbit anti-MMP-13 (1:5,000; cat. no. ab39012), rabbit anti-MMP-9 (1:1,000; cat. no. ab38898), rabbit anti-Notch1 (1:1,000; cat. no. ab52627), rabbit anti-Hes family BHLH transcription factor 1 (Hes1; 1:1,000; cat. no. ab71559) and anti-β-actin (1:1,000; cat. no. ab8227). The membranes were washed with TBS buffer containing 0.05% Tween-20 for 30 min and incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. ab205718) for 1 h at room temperature. All primary and secondary antibodies were purchased from Abcam. Protein bands were visualized using the Pierce^™ ECL Western Blotting Substrate kit (Thermo Fisher Scientific, Inc.) and the Bio-Rad ChemiDoc Imaging system (Bio-Rad Laboratories, Inc.). Protein expression levels were quantified using Image Lab software (version 5.2.1; Bio-Rad Laboratories, Inc.) with β-actin as the loading control.

Experiments were repeated ≥3 times. Data are presented as the mean ± standard deviation. Statistical analyses were conducted using GraphPad Prism software (version 7; GraphPad Software, Inc.). Comparisons between two groups were evaluated using unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To construct an in vitro OA model, CHON-001 cells were treated with different concentrations of IL-1β (0, 5, 10 and 20 ng/ml) for 24 h. The CCK-8 assay was conducted to assess the effects of IL-1β on chondrocyte viability. IL-1β inhibited CHON-001 cell viability in a dose-dependent manner compared with the control group (0 ng/ml IL-1β), and 20 ng/ml IL-1β inhibited cell viability by ~70%; therefore, 20 ng/ml IL-1β was selected to establish the in vitro OA model in CHON-001 cells (Fig. 1A). The flow cytometry results indicated that 5, 10 and 20 ng/ml IL-1β significantly induced CHON-001 cell apoptosis compared with the control group (0 ng/ml IL-1β), with the 20 ng/ml IL-1β group displaying the highest level of apoptosis among the groups (Fig. 1B). As indicators of a functional cellular OA model, the expression levels of inflammatory cytokines, such as TNF-α and IL-6, were significantly increased by 20 ng/ml IL-1β compared with the control group (Fig. 1D and E). Collectively, the results indicated the successful establishment of an in vitro OA cell model.

CHON-001 cells were cultured with various concentrations of BAI (0, 10, 20, 40 and 80 µM). The CCK-8 assay results indicated that 40 µM BAI significantly reduced cell viability compared with the control group (0 µg/ml BAI), so 20 µM BAI was used for subsequent experiments (Fig. 1A). The effect of BAI on the OA model was assessed by measuring cell viability. BAI dose-dependently reversed IL-1β-induced reductions in cell viability. In addition, CHON-001 cells were transfected with miR-106a-5p mimics and the RT-qPCR results indicated transfection efficiency (Fig. 2C). miR-106a-5p mimics also dose-dependently reversed IL-1β-induced reductions in cell viability (Fig. 2D).

Subsequently, the anti-OA effects of BAI and miR-106a-5p mimics were assessed (Fig. 2E and F). The results indicated that 4 nM miR-106a-5p mimics + 20 µM BAI and 6 nM miR-106a-5p mimics + 20 µM BAI displayed an enhanced protective effect in IL-1β-induced CHON-001 cells compared with 20 µM BAI treatment alone. Therefore, the results suggested that combination therapy may synergistically alleviate IL-1β-induced CHON-001 cytotoxicity; however, some combination strategies may only exhibit additive therapeutic effect in OA.

An in vitro OA model was established and treated with BAI, miR-106a-5p mimics or a combination. The effects of the combination treatment on apoptosis were assessed via flow cytometry. Monotherapy with BAI or miR-106a-5p mimics significantly alleviated IL-1β-induced CHON-001 cell apoptosis (Fig. 3A and B). Moreover, combination treatment with BAI and miR-106a-5p mimics further reversed IL-1β-induced CHON-001 cell apoptosis compared with either treatment alone. Furthermore, the expression levels of apoptosis-related proteins (Bax and active caspase-3) were significantly downregulated in the combination treatment group compared with the BAI and miR-106a-5p mimics monotherapy groups (Fig. 3C, D and F). By contrast, the expression level of the anti-apoptotic protein Bcl-2 was significantly upregulated in the combination treatment group compared with the BAI and miR-106a-5p mimics monotherapy groups (Fig. 3E).

To determine the phenotype of the OA model, the expression levels of collagen I and III, aggrecan, MMP-13 and MMP-9 were detected by western blotting. BAI or miR-106a-5p mimics monotherapy reversed IL-1β-induced aggrecan degradation, but combination therapy was more effective compared with either monotherapy (Fig. 4A and D). Moreover, IL-1β-induced upregulation of collagen I and III was alleviated to a greater extent in the combination treatment group compared with the BAI and miR-106a-5p mimics monotherapy groups (Fig. 4A-C). Similarly, combination treatment reversed IL-1β-mediated upregulation of MMP-9 and MMP-13 to a greater extent compared with the BAI and miR-106a-5p mimics monotherapy groups (Fig. 4A, E and F). In addition, the combination treatment group displayed lower levels of TNF-α and IL-6 compared with the BAI and miR-106a-mimics monotherapy groups (Fig. 4G and H). Collectively, the results indicated that the inflammatory response of the OA model was reversed to a greater degree by combination treatment compared with BAI or miR-106a-5p mimics monotherapy.

IL-1β significantly upregulated active Notch1 expression in CHON-001 cells compared with the control group, but BAI treatment significantly reversed IL-1β-mediated upregulation in the in vitro OA model. By contrast, miR-106a-5p mimics did not inhibit IL-1β-induced upregulation of active Notch1. BAI and miR-106a-5p combination treatment alleviated IL-1β-mediated upregulation of active Notch1 to a greater degree compared with BAI treatment alone (Fig. 5A and B). The protein expression level of Hes1 was also upregulated in the IL-1β group compared with the control group (Fig. 5A and C), which was reversed following treatment with BAI, but not miR-106a-5p mimics. However, the combination of BAI and miR-106a-5p mimics had a greater inhibitory effect on IL-1β-mediated Hes1 upregulation compared with BAI alone (Fig. 5A and C).