The murine chondrogenic ATDC5 cell line was obtained from the American Type Culture Collection. The cells were cultured in DMEM: Hams F-12 (1:1; Sigma-Aldrich; Merck KGaA) supplemented with 2 mM glutamine (Sigma-Aldrich; Merck KGaA) and 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) and maintained at 37°C in a humidified incubator containing 5% CO₂. ATDC5 cells were then treated with lipopolysaccharide (LPS; Sigma-Aldrich; Merck KGaA) at different concentrations (0, 1, 2, 4 and 8 µg/ml) at 37°C for 6 h to induce inflammatory injury.

The proliferation of ATDC5 cells was determined using a CCK-8 assay kit (Dojindo Molecular Technologies, Inc.), according to the manufacturers protocol. Cells (5×10³ cells/well) were seeded into a 96-well plate and cultured for 24 h. After treatment with LPS for 6 h, 20 µl CCK-8 solution was added into each well and incubated for another 4 h at 37°C. The absorbance was subsequently detected at a wavelength of 450 nm using a microplate reader.

ATDC5 cells were lysed with cold RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with protease inhibitor. The concentration of protein was measured using the BCA protein assay (Pierce; Thermo Fisher Scientific, Inc.). The proteins (20 µg/lane) were separated using 10% SDS-PAGE, and transferred onto PVDF membranes. Membranes were blocked with 5% skimmed milk at room temperature for 1 h and incubated with the primary antibodies with a dilution of 1:1,000 overnight at 4°C. The antibodies against NRF1 (cat. no. ab137572), Bax (cat. no. ab32503), Bcl-2 (cat. no. ab59348), cleaved caspase-3 (cat. no. ab49822), caspase-3 (cat. no. ab32499), superoxide dismutase 1 (SOD1; cat. no. ab13498), heme oxygenase-1 (HO-1; cat. no. ab13243), NAD(P)H quinone dehydrogenase 1 (NQO-1; cat. no. ab34173) and GAPDH (cat. no. ab9485) were obtained from Abcam. Following incubation with the primary antibodies, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 2 h at room temperature. Bands were visualized using an enhanced chemiluminescence kit (SuperSignal™ West Pico PLUS chemiluminescent substrate; Thermo Fisher Scientific, Inc.). The densitometric analysis was performed using ImageJ software v1.4 (National Institutes of Health).

Total RNA was extracted from ATDC5 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). To detect the mRNA levels of miR-486-5p, the first strand of cDNA was synthesized using the PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd.), according to the manufacturers protocol, and the following temperature conditions: Initial incubation at 37°C for 15 min, followed by an incubation at 85°C for 5 sec. RT-qPCR was performed using a SYBR-Green Prime Script RT-PCR kit (Takara Biotechnology Co., Ltd.), according to the manufacturers instructions. The thermocycler conditions for RT-qPCR were as follows: 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 30 sec. The primer sequences were as follows: miR-486-5p forward, 5-CTCGCTTCGGCAGCACA-3; miR-486-5p reverse 5-ACGCTTCACGAATTTGCGT-3; NRF1 forward, 5- GGAGAGCTTCCCTGCACAGT-3; NRF1 reverse, 5- TTACTTCCATAGCCTGCATTTCC-3; β-actin forward, 5-CCGCGAGCACAGCTTCTT-3; β-actin reverse, 5-CCCACGATGGAGGGGAATAC-3; U6 forward, 5-CTCGCTTCGGCAGCACA-3; and U6 reverse, 5-AACGCTTCACGAATTTGCGT-3. Data were collected and analyzed using 2^−ΔΔCq method (14) and normalized to the internal control U6 and β-actin.

ATDC5 cells were transfected with the miR-486-5p inhibitor (5-CUCGGGGCAGCUCAGUACAGGA-3; 150 nM; Qiagen GmbH) and inhibitor control (miR-NC; 5-UUCUCCGAACGUGUCACGUTT-3; 150 nM; Qiagen GmbH), or transfected with short hairpin RNA (shRNA)1 or shRNA2 targeting NRF1 (100 nM) and its scramble control shRNA (shRNA-NC; empty vector), which were cloned into pSilencer 3.1-H1 puro plasmids (Shanghai GenePharma Co. Ltd.), using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.), according to the manufacturers protocol. The transfection efficiency was detected by RT-qPCR 48 h post-transfection.

The binding site of miR-486-5p on NRF1 was predicted using the miRDB database (www.mirdb.org) and was validated using the luciferase reporter assay. PmirGLO-NRF1-WT or pmirGLO-NRF1-Mut reporter plasmids (Promega Corporation) were co-transfected with the miR-486-5p mimic or miR-NC into ATDC5 cells using Lipofectamine^® 2000 reagent. The luciferase activity was measured using a Dual-Luciferase^® Reporter assay system (Promega Corporation) 48 h post-transfection. The data were standardized to Renilla luciferase activity.

The cell culture medium of ATDC5 cells was collected. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, monocyte chemotactic protein 1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) were determined using ELISA kits (cat. no. 210-TA-005 for TNF-α; cat. no. 201-LB-005 for IL-1β; cat. no. S6050 for IL-6; cat. no. SCP00 for MCP-1; cat. no. 796-IC-050 for ICAM-1; R&D Systems, Inc.) according to the manufacturers instructions.

The cell culture medium of ATDC5 cells was collected. The levels of reactive oxygen species (ROS; cat. no. E004-1-1), malondialdehyde (MDA; cat. no. A003-4-1), SOD (cat. no. A001-3-2) and lactate dehydrogenase (LDH; A020-2-2) were determined using their corresponding assay test kits (Nanjing Jiancheng Bioengineering Institute), according to the manufacturers instructions.

The apoptosis of ADTC5 cells was analyzed using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Sigma-Aldrich; Merck KGaA). Following treatment of LPS, the transfected ATDC5 cells were washed with PBS and incubated with Annexin V-FITC/PI (50 µg/ml) in the presence of RNase A (50 µg/ml; Sigma-Aldrich; Merck KGaA) at room temperature for 30 min in the dark. The apoptotic cells were detected by a FACScan flow cytometer (BD Biosciences) and analyzed using FlowJo software v7.6 (FlowJo LLC).

Statistical analyses were performed using SPSS software (v.17.0; SPSS, Inc.). Data are presented as the mean ± SD of 3 independent experiments. Data were analyzed by the one-way ANOVA followed by the Tukeys post hoc test. P<0.05 was considered to indicate a statistically significant different difference.

Following treatment of ATDC cells with LPS (0, 1, 2, 4 and 8 µg/ml), the cell proliferation was detected using a CCK-8 kit, and the mRNA and protein expression levels of miR-486-5p and NRF1 were detected using RT-qPCR and western blotting, respectively. As shown in Fig. 1A, LPS treatment (2, 4 and 8 µg/ml) significantly decreased the proliferation in a concentration-dependent manner. Furthermore, the mRNA level of miR-486-5p was increased following LPS treatment (2, 4 and 8 µg/ml; Fig. 1B). Protein expression of NRF1 was significantly decreased following LPS treatment, particularly at 4 µg/ml of LPS (Fig. 1C). Therefore, this concentration was chosen for subsequent experimentation. The results revealed that miR-486-5p was upregulated while NRF1 was downregulated in LPS-induced ATDC5 cells. To determine the LPS-induced inflammatory injury in ATDC5 cells, the levels of inflammatory cytokines including TNF-α, IL-1β, IL-6, MCP-1 and ICAM-1 were assayed by ELISA kits. Results in Fig. 1D indicated that LPS induced a significant increase in these inflammatory cytokines, revealing a marked inflammatory injury induced by LPS.

Analysis of the online databases of miRDB revealed that the 3-UTR of NRF1 included a binding site for miR-486-5p (Fig. 2A). A luciferase reporter assay was subsequently performed to validate the interaction between miR-486-5p and NRF1 in ATDC cells. As shown in Fig. 2B, overexpression of miR-486-5p significantly suppressed the activity of the reporter gene, whereas the plasmid containing the mutated sequence did not affect the activity of the reporter gene in ADTC cells. Besides, overexpression of miR-486-5p decreased the expression of NRF1, while inhibition of miR-486-5p increased the expression of NRF1 (Fig. 2C). These results suggested that miR-486-5p could directly target NRF1 and regulate the expression of NRF1 in ATDC5 cells.

The effect of miR-486-5p and NRF1 on LPS-induced ATDC5 cells was investigated. A miR-486-5p inhibitor or shRNA targeting NRF1 was transfected into cells. As shown in Fig. 3A and B, the increased mRNA level of miR-486-5p induced by LPS was significantly decreased following transfection with the miR-486-5p inhibitor. Furthermore, the expression of NRF1 was significantly decreased when cells were transfected with shRNA1 or shRNA2 targeting NRF1. Due to a higher transfection efficacy, shRNA1-NRF1 was used for the further experimentation.

The change of inflammatory cytokines, directly reflecting the degree of inflammatory injury in LPS-induced ATDC5 cells, is presented in Fig. 3C-G. As observed from the results, LPS induction significantly increased the production of TNF-α, IL-1β, IL-6, MCP-1 and ICAM-1. As expected, the production of these inflammatory cytokines was decreased following transfection with the miR-486-5p inhibitor, and co-transfection with shRNA-NRF1 abrogated this effect. These results suggested that the inhibition of miR-486-5p may significantly alleviate LPS-induced inflammatory injury in ATDC5 cells by regulating NRF1.

The effect of miR-486-5p on cell apoptosis in LPS-induced ATDC5 cells was investigated. Flow cytometry analysis revealed an increased apoptosis rate following LPS induction, whereas in the miR-486-5p inhibitor group the apoptotic rate of the cells was decreased compared with the LPS group (Fig. 4A). Additionally, the expression levels of apoptosis-associated proteins were investigated by western blotting. LPS induction significantly decreased the protein expression of Bcl-2, and increased the protein expression of Bax and cleaved caspase-3 (Fig. 4B), indicating that LPS treatment resulted in ATDC cell apoptosis by regulating the expression of apoptosis-associated proteins. Furthermore, the expressions of Bax and cleaved caspase-3 was decreased, and Bcl-2 was increased in the miR-486-5p inhibitor group, suggesting an inhibitory effect of miR-486-5p in LPS-induced ATDC cells. The inhibitory effect of miR-486-5p was decreased by co-transfection with shRNA-NRF1. These results suggested that miR-486-5p could attenuate cell apoptosis in LPS-induced ATDC5 cells by targeting NRF1.

The results presented in Fig. 5A-E demonstrate the changes in oxidative stress markers in the LPS-induced ATDC5 cells. As shown in Fig. 5A-D, LPS induction significantly increased the activity of ROS and the levels of MDA and LDH, and decreased the activity of SOD. Additionally, antioxidant enzymes such as SOD1, HO-1 and NQO-1 were significantly decreased following treatment with LPS, which is indicative of oxidase and anti-oxidase imbalance and may result in oxidative stress injury. Inhibition of miR-486-5p significantly decreased the activity of ROS, decreased the levels of MDA and LDH, and increased the activity of SOD, as well as increased the expression of antioxidant enzymes. These data indicated a decrease in oxidative stress following transfection with the miR-486-5p inhibitor. However, co-transfection with shRNA-NRF1 abrogated this effect. These results suggested that inhibition of miR-486-5p could alleviate LPS-induced oxidative stress in ATDC5 cells by targeting NRF1.