All patients signed informed consent forms prior to the study, and the study received approval from the Institutional Ethics Committee of Nanjing Children's Hospital Affiliated to Nanjing Medical University (Nanjing, China). Human femoral head cartilage tissue was isolated from patients with LCPD (n=20, <14 years old, male 12, female 8) and healthy volunteers (n=20, <14 years old, 12 male, 8 female) from December 2015 to October 2017. LCPD was diagnosed on the basis of ultrasonographic examination and magnetic resonance imaging. Patients with other diseases, such as primary osteoarthritis, ankylosing spondylitis, systemic lupus erythematosus and inflammatory diseases, were excluded. Peripheral venous blood samples were drawn from all patients in the morning prior to surgery, collected into two 4.5-ml Vacutainer sodium citrate anticoagulant tubes (BD Biosciences, Franklin Lakes, NJ, USA) and stored at −80°C until use. Peripheral venous blood samples were centrifuged at 1,000 × g for 10 min at 4°C to obtain serum.

Chondrocytes were isolated from LCPD and healthy control femoral head cartilage tissues via collagenase digestion of cartilage, and cultured in monolayer as described previously (19). Cells of the first passage were used in the experiments of the study. The human cartilage cell line TC28 (immortalized human primary juvenile costal chondrocytes) was purchased from the American Type Culture Collection (Manassas, VA, USA). The two cell lines were propagated as monolayers maintained in Dulbecco's Modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO₂.

TC28 cells were treated with 0.01 mmol dexamethasone (DEX) (cat. no. kf1419; Guangzhou Kafen Biological Technology Co., Ltd., Guangzhou, China) at 37°C for 2 h to establish an in vitro model of LCPD. Cells without any treatment were used as control. Then, the levels of miR-214 and Bax expression were evaluated to characterize the effects of DEX on TC28 cells.

miR-214 mimic (5′CCUGACAAUUAGUAUUU-3′) and mimic control (5′-ACAGGUAGCUGAACACUGGGUU-3′) were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The sequence of Bax was inserted into pcDNA3.1 (Invitrogen; Thermo Fisher Scientific, Inc.); empty vector (control-plasmid) was used as the control. TC28 cells were seeded in 6-well plates (1×10⁶ cells/well), cultured for 24 h and transfected with 100 nM miR-214 mimic, 100 nM mimic control, 1 µg control-plasmid, 1 µg Bax-plasmid or 100 nM miR-214 mimic + 1 µg Bax-plasmid using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The transfection efficiency was determined 48 h later.

Total RNA was extracted from serum, cartilage tissue or cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific); 1 µg of total RNA was reverse transcribed to cDNA using a PrimeScript RT Reagent kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocols. Reaction conditions for reverse transcription were: 50°C for 5 min and 80°C for 2 min. An miRNA-specific TaqMan MiRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for the detection of miRNA expression according to the manufacturer's protocols. SYBR Premix Ex Taq (Takara Bio, Inc.) was used to analyze Bax mRNA expression. U6 small nuclear RNA (U6) and GAPDH expression were used as an internal control for miR-214 and Bax, respectively. The sequences of primers were as follows: miR-214, forward 5′-AGCATAATACAGCAGGCACAGAC-3′, reverse, 5′-AAAGGTTGTTCTCCACTCTCTCAC-3′; Bax, forward 5′-GGCCCACCAGCTCTGAGCAGA-3′, reverse, 5′-GCCACGTGGGCGTCCCAAAGT-5′; GAPDH, forward 5′-TGAACGGGAAGCTCACTGG-3′, reverse, 3′-TCCACCACCCTGTTGCTGTA-5′; and U6, forward 5′-CGCTTCGGCAGCACATATAC-3′ and reverse, 5′-AAATATGGAACGCTTCACGA-3′. RT-qPCR data were analyzed with an ABI 7900 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Amplification conditions for qPCR were as follows: 5 min at 95°C, followed by 35 cycles at 95°C for 15 sec, 40 sec at 55°C, and 72 °C for 1 min. The relative expression levels were calculated using the 2^−ΔΔCq method (20).

The bioinformatics tool miRBase (http://www.mirbase.org) was used to predict the potential targets of miR-214. The results indicated that Bax was a potential target of miR-214. To confirm this prediction, a dual-luciferase reporter assay was performed. A wild type (WT-Bax) and mutant (MUT-Bax) 3′-untranslated regions of Bax were cloned into a pmiR-RB-Report™ dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd., Guangzhou, China). Then, the WT-Bax or MUT-Bax vectors, the miR-214 mimic or mimic control, and the pRL-TK Renilla luciferase reporter (Promega Corporation, Madison, WI, USA) were co-transfected into TC28 cells using Lipofectamine^® 2000. TC28 cells were collected 48 h following transfection and luciferase activity was analyzed using dual-luciferase assay system (Promega Corporation, Madison, WI, USA). Firefly luciferase activity was normalized to that of Renilla.

Proteins from cells, serum and tissues were extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) with Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology, Inc., Danvers, MA, USA). Protein concentrations were determined using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein samples (30 µg/lane) were separated via 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked in 5% non-fat dry milk at room temperature for 1 h and incubated with primary antibodies overnight at 4°C. The primary antibodies used were: Anti-Bax (1:2,000; ab32503, Abcam, Cambridge, UK), anti-Bcl-2 (1:2,000; ab196495, Abcam) and anti-GAPDH (1:5,000; ab9485, Abcam). Membranes were then incubated with a goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (cat. no. ab7090; 1:2,000; Abcam) at room temperature for 3 h. All bands were visualized using enhanced chemiluminescence western blotting detection kits (Merck KGaA). ImageJ 1.38X software (National Institutes of Health, Bethesda, MD, USA) was used to quantify protein expression.

Cell viability was determined by an MTT assay. Following treatment with DEX and transfection with miR-214 mimic, mimic-control, or miR-214 mimic + Bax-plasmid for 48 h, TC28 cells (1×10⁴ cells/well) were seeded in 96-well plates and cultured for 24 h. Subsequently, 20 µl MTT solution (0.5 mg/ml; Sigma-Aldrich; Merck KGaA) was added to each well and the plates were further incubated at 37°C for 4 h. The medium in each well was discarded, and 150 µl dimethyl sulfoxide was added for 30 min. The absorbance at 570 nm was measured using a FLUOstar^® Omega Microplate Reader (BMG Labtech GmbH, Ortenberg, Germany).

An Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection kit [cat. no. 70-AP101-100; MultiSciences (Lianke) Biotech, Co., Ltd., Hangzhou, China] was used to evaluate the apoptotic rate of TC28 cells. Following 48 h since transfection, TC28 cells were collected, washed with PBS and suspended with 5 µl Annexin V-FITC and 5 µl PI for 30 min in the dark at room temperature. A flow cytometer was used to analyze cell apoptosis. And the cell apoptotic rate (early apoptosis and late apoptosis in the right quadrant) was determined using FlowJo software version 7.6.1 (FlowJo LLC, Ashland, OR, USA).

Each experiment was repeated three times. Statistical analysis was performed using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation of at least three independent experiments. Differences between groups were analyzed by Student's t-tests or one-way analysis of variance with Tukey's post hoc tests. P<0.05 was considered to indicate a statistically significant difference.

To determine miR-214 expression in human LCPD cartilage, serum and chondrocytes, samples were collected from 20 patients with LCPD and 20 healthy individuals. As presented in Fig. 1A and B, compared with the healthy group, miR-214 expression was significantly downregulated in femoral head cartilage and serum samples from patients with LCPD compared with the healthy controls. In addition, human primary chondrocytes were isolated from femoral head cartilage tissues from patients with LCPD and healthy controls. The results of RT-qPCR revealed that miR-214 expression was significantly downregulated in human primary chondrocytes from patients with LCPD compared with in healthy controls (Fig. 1C). Furthermore, the expression levels of miR-214 were significantly downregulated in DEX-treated TC28 cells compared with untreated control cells (Fig. 1D).

The bioinformatics tool miRBase (http://www.mirbase.org) was used to predict the potential targets of miR-214. Bioinformatics analyses indicated that Bax was a potential target of miR-214 (Fig. 2A). Subsequently, to determine whether miR-214 directly modulates Bax expression via interactions with potential binding sites, a luciferase reporter assay was performed using TC28 cells transfected with vectors harboring the WT or MUT 3′-UTR of Bax, in the presence or absence of the miR-214 mimic or mimic control. As presented in Fig. 2B, compared with co-transfection with Bax-WT and mimic control, the luciferase activity was significantly decreased following co-transfection with Bax-WT and miR-214 mimic, while Bax-MUT did not. The results indicated that Bax was a target of miR-214.

The expression of Bax was determined in human LCPD cartilage, serum and chondrocytes. As presented in Fig. 3A-C, compared with the healthy control group, the expression level of Bax mRNA was significantly upregulated in femoral head cartilage, serum and primary chondrocytes of patients with LCPD. Meanwhile, compared with the healthy control group, the levels of Bax protein appear to be higher in the femoral head cartilage, serum and primary chondrocytes of patients with LCPD. Furthermore, TC28 cells treated with DEX exhibited significantly elevated Bax mRNA expression compared with untreated control cells and seemingly higher Bax protein levels as well (Fig. 3D).

To further investigate the effects of miR-214 on Bax expression, TC28 cells were transfected with mimic control, miR-214 mimic, control-plasmid, Bax-plasmid, or miR-214 mimic + Bax-plasmid for 48 h. As presented in Fig. 4A, miR-214 mimic significantly increased the expression levels of miR-214 in TC28 cells compared with the control group (Fig. 4A). In addition, TC28 cells transfected with the Bax-plasmid exhibited significantly increased mRNA expression of Bax (Fig. 4B) and seemingly higher Bax protein levels as well (Fig. 4C). Compared with the mimic control group, transfection with miR-214 mimic significantly decreased the mRNA expression of Bax in TC28 cells, which was reversed by Bax overexpression (Fig. 4D). The results of western blot assay suggested that miR-214 mimic decreased the protein levels of Bax in TC28 cells, which may have been reversed by Bax overexpression (Fig. 4E).

MTT assays were performed to detect the viability of TC28 cells, and flow cytometry was conducted to detect TC28 cell apoptosis. As presented in Figs. 5 and 6, compared with the control group, DEX treatment significantly reduced viability of TC28 cells and promoted apoptosis. Conversely, overexpression of miR-214 significantly increased cell viability and inhibited cell apoptosis compared with DEX treatment alone; these effects were reversed by Bax overexpression. Additionally, the protein expression of Bax and Bcl-2 was determined, and the Bcl-2/Bax ratio was calculated. As presented in Fig. 7, compared with the control, the protein expression of Bax increased following DEX treatment, while that of Bcl-2 decreased and the Bcl-2/Bax ratio was reduced. These effects were inhibited by miR-214 overexpression and promoted by Bax overexpression.