A total of 30 paired articular cartilage tissue samples (n=60 samples in total) from 30 OA patients (male, 17; female, 13; age 59.4±4.2 years) and 30 non-OA patients (no history of OA or rheumatoid arthritis; male, 16; female, 14; age 59.1±6.2 years) were collected at the Sixth Hospital of Wuhan (Hubei, China) from March 2016 to March 2017. The present study was approved by the Ethics Committee of the Sixth Hospital of Wuhan, and all patients provided written informed consent.

The murine chondrogenic cell line ATDC5 was obtained from American Type Culture Collection (Manassas, VA, USA). ATDC5 cells were grown in Dulbecco's modified Eagle's medium/Ham's Nutrient Mixture F-12 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 2 mM Glutamine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C with 5% CO₂.

To establish the cell model of OA, ATDC5 cells were treated with 5 µg/ml lipopolysaccharide (LPS; Sigma-Aldrich; Merck KGaA) at 37°C for 5 h. Cells without any treatments were used as the control.

Inhibitor control, miR-195-5p inhibitor, control-small interfering (si)-RNA and the REGγ-siRNA were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). ATDC5 cells were seeded into a 6-well plate (1×10⁶ cells/well) and cultured at 37°C for 24 h. Then, ATDC5 cells were transfected with 100 nM miR-195-5p inhibitor (5′-GCCAAUAUUUCUGUGCUGCUA-3′), 100 nM inhibitor control (5′-CAGUACUUUUGUGUAGUACAA-3′), 2 µl control-siRNA [5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense)], 2 µl REGγ-siRNA [5′-CAGAAGACUUGGUGGCAAATT-3′ (sense) and 5′-UUUGCCACCAAGUCUUCUGTT-3′ (antisense)] or 100 nM miR-195-5p inhibitor+2 µl REGγ-siRNA using Lipofectamine 3000^® regent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. At 24 h post-cell transfection, transfection efficiency was detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

ATDC5 cells (5×10³ cells/well) were cultured in 96-well plates. At 24 h post-transfection with either miR-195-5p inhibitor, inhibitor control or miR-195-5p inhibitor+REGγ-siRNA at 37°C, the cells were treated with LPS for 5 h at 37°C. Cells without LPS treatment were used as the control group. Following stimulation, MTT solution (20 µl/well) was added into the culture medium. Then the plates were incubated for 4 h at 37°C in humidified 95% air and 5% CO₂. Then dimethyl sulfoxide (150 µl) was used to dissolve the purple formazan. Cell proliferation ability was assessed by measuring the absorbance at 490 nm using a Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

To analyze cell apoptosis, flow cytometry was performed with the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis Detection kit (cat. no. 70-AP101-100; Hangzhou MultiSciences Biotech, Co., Ltd., Hangzhou, China). Briefly, following the specific treatments, ATDC5 cells (1×10⁵ cells/well) were collected and washed with cold PBS. Then the cells were stained with FITC-conjugated Annexin V and propidium iodide without light according to the manufacturer's instructions. Finally, the number of apoptotic cells was determined using a flow cytometer (BD Biosciences; Becton-Dickinson and Company, Franklin Lakes, NJ, USA), and FlowJo software version 7.6.1 (FlowJo LLC, Ashland, OR, USA) was used for data analysis.

To detect the levels of IL-1β, IL-6 and TNF-α in cell supernatants, ELISA was performed. Briefly, the cell culture supernatant was collected by centrifugation (1,000 × g at 4°C for 10 min), then the concentrations of the inflammatory cytokines (IL-1β, cat. no. ab100704; IL-6, cat. no. ab100712 and TNF-α, cat. no. ab208348) were determined by ELISA (Abcam, Cambridge, UK) according to the manufacturer's instructions of each kit.

The total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) as per the manufacturer's instructions. Then the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to perform the RT experiment. Amplification conditions for RT experiment were 50°C for 15 min and 85°C for 2 min. Finally, the synthesized cDNAs were analyzed using TaqMan Universal Master Mix II (Applied Biosystems; Thermo Fisher Scientific, Inc.) following the manufacturer's protocols. Amplification conditions were: 10 min at 95°C, followed by 35 cycles of 15 sec at 95°C and 40 sec at 55°C. GAPDH and U6 were used in the present study for normalizing mRNA and miRNA levels. Primer sequences were as following: GAPDH forward, 5′-CTTTGGTATCGTGGAAGGACTC-3′ and reverse, 5′-GTAGAGGCAGGGATGATGTTCT-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; TNF-α forward, 5′-GAACTGGCAGAAGAGGCACT-3′ and reverse, 5′-GGTCTGGGCCATAGAACTGA-3′; IL-1β forward, 5′-TGTGAAATGCCACCTTTTGA-3′ and reverse, 5′-TGAGTGATACTGCCTGCCTG-3′; IL-6 forward, 5′-CCGGAGAGGAGACTTCACAG-3′ and reverse, 5′-CAGAATTGCCATTGCACA-3′; miR-195-5p forward, 5′-GGGGTAGCAGCACAGAAAT-3′ and reverse, 5′-TCCAGTGCGTGTCGTGGA-3′; REGγ forward, 5′-AAGGTTGATTCTTTCAGGGAGC-3′ and reverse, 5′-AGTGGATCTGAGTTAGGTCATGG-3′. Relative gene expressions were calculated by the 2^−ΔΔCq method (20).

The targets of miR-195-5p were predicted using TargetScan bioinformatics software 7.1 (www.targetscan.org/vert_71), and REGγ was revealed to be a potential target of miR-195-5p. To verify this prediction, the wild-type (REGγ-WT) and mutant (REGγ-MUT) 3′UTR of REGγ were cloned into a pmiR-RB-Report^™ dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd.). ATDC5 cells (5×10⁴ cells/well) were co-transfected with 100 ng REGγ-WT or 100 ng REGγ-MUT and 50 nM miR-195-5p mimic (5′-UAGCAGCACAGAAAUAUUGGC-3′) or 50 nM mimic control (5′-UUCUCCGAACGUGUCACGUTT-3′) using Lipofectamine 3000^® regent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. At 48 h post-transfection, the dual-luciferase assay system (Promega Corporation, Madison, WI, USA) was used to determine luciferase activity, which was normalized to Renilla luciferase activity.

Whole cell proteins were extracted from cells (5×10⁵ cells/well) using Radioimmunoprecipitation Assay Lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocols. The protein samples were quantified using the BCA™ Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Western blotting was performed using a Bio-Rad Bis-Tris Gel system (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. Briefly, protein samples (30 µg/lane) were separated by 12% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. Then the membranes were blocked with 5% non-fat milk at room temperature for 1.5 h, incubated with primary antibodies at 4°C overnight, and subsequently incubated with a secondary antibody at room temperature for 4 h. The primary antibodies used in this study were REGγ (cat. no. ab157157; 1:1,000; Abcam), Bcl-2 (cat. no. 4223; 1:1,000), Bax (cat. no. 14796; 1:1,000), β-catenin (cat. no. 25362; 1:1,000), c-Myc (cat. no. 5605; 1:1,000), Cyclin D1 (cat. no. 2978; 1:1,000), p-p65 (cat. no. 3033; 1:1,000) and β-actin (cat. no. 4970; 1:1,000) (all from Cell Signaling Technology, Inc., Danvers, MA, USA). The horseradish peroxidase-conjugated secondary antibody was obtained from Cell Signaling Technology, Inc. (cat. no. 7074; 1:2,000). At the end of this experiment, protein bands were visualized using the enhanced chemiluminescence detection system (Super Signal West Dura Extended Duration Substrate; Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Gel-Pro Analyzer densitometry software (version 6.3; Media Cybernetics, Inc., Rockville, MD, USA) was used for band density quantification.

All experiments were repeated three times. The data were presented as the mean ± standard deviation. Statistical analyses were carried out using SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA). Comparisons between two groups were evaluated by Student's t-test, and comparisons between multiple groups were analyzed using one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered statistically significant.

To determine the miR-195-5p expression level in OA, RT-qPCR was performed. As shown in Fig. 1A, when compared with the control group, the level of miR-195-5p was significantly upregulated in the articular cartilage tissues of patients with OA. In addition, the level of miR-195-5p in the murine chondrogenic cell line ATDC5 was significantly increased following LPS treatment for 5 h in comparison with the control group (Fig. 1B).

Bioinformatics tools on TargetScan (www.targetscan.org) were used to predict the potential targets of miR-195-5p. The results revealed that REGγ was a potential target for miR-195-5p (Fig. 2A). To determine whether miR-195-5p directly modulated REGγ expression via interactions with potential binding sites, a luciferase reporter assay was performed. As shown in Fig. 2B, compared with co-transfection with REGγ-MUT and miR-195-5p mimic, luciferase activity was markedly decreased by co-transfection with REGγ-WT and miR-195-5p mimic. The results indicated that miR-195-5p directly targeted REGγ.

To investigate the effect of miR-195-5p on OA, the present study firstly transfected ATDC5 cells with miR-195-5p inhibitor, inhibitor control, control-siRNA, REGγ-siRNA or miR-195-5p inhibitor+REGγ-siRNA respectively, and the transfection efficiency was detected 24 h following cell transfection. As shown in Fig. 3A, compared with the control group, miR-195-5p inhibitor significantly downregulated the miR-195-5p level in ATDC5 cells. REGγ-siRNA significantly reduced the protein and mRNA levels of REGγ in ATDC5 cells (Fig. 3B and C). In addition, the miR-195-5p inhibitor significantly upregulated the protein and mRNA levels of REGγ, and this upregulation was attenuated by REGγ-siRNA (Fig. 3D and E).

Then, an MTT assay was conducted to determine the effect of miR-195-5p on ATDC5 cell proliferation ability. The data showed that the miR-195-5p inhibitor significantly enhanced the proliferation ability of ATDC5 cells, which was inhibited by LPS treatment, and this enhancement was markedly reversed by REGγ-siRNA (Fig. 4).

The present study further investigated the effect of miR-195-5p on ATDC5 cell apoptosis via flow cytometry. The results indicated that LPS treatment significantly induced ATDC5 cell apoptosis, but this increase in cell apoptosis was inhibited by the miR-195-5p inhibitor. Notably, the results also indicated that REGγ-siRNA significantly eliminated the effect of the miR-195-5p inhibitor on ATDC5 cell apoptosis (Fig. 5A and B). In addition, we the levels of increased Bax and decreased Bcl-2 levels induced by LPS treatment were reversed by the miR-195-5p inhibitor. Furthermore, REGγ-siRNA significantly eliminated the effect of the miR-195-5p inhibitor on the expression of Bcl-2 and Bax in LPS treated ATDC5 cells (Fig. 5C).

The present study then investigated whether miR-195-5p has an effect on the inflammatory response during OA development. The results demonstrated that the mRNA and protein levels of IL-1β, IL-6 and TNF-α were significantly enhanced by LPS stimulation. The miR-195-5p inhibitor significantly reduced the expression levels of IL-1β, IL-6 and TNF-α, and these reductions were reversed by REGγ downregulation (Fig. 6). These results indicated that the miR-195-5p inhibitor prevented the inflammatory response in ATDC5 cells induced by LPS.

Finally, to explore the molecular mechanism underlying the effects of the miR-195-5p inhibitor on ATDC5 cells, the Wnt/β-catenin and NF-κB signaling pathways were analyzed. As shown in Fig. 7, LPS stimulation significantly decreased the protein expression of REGγ, β-catenin, c-Myc and Cyclin D1, and enhanced the phosphorylation of NF-κB [phosphorylated (p)-p65] in ATDC5 cells, while the miR-195-5p inhibitor markedly increased REGγ, β-catenin, c-Myc and Cyclin D1 protein expression and reduced p-NF-κB (p-p65) protein expression. As expected, REGγ-siRNA significantly attenuated the effect of the miR-195-5p inhibitor on REGγ, β-catenin, c-Myc, Cyclin D1 and p-p65 expression in ATDC5 cells.