- Tissue samples and chondrocyte isolation
This research was approved by the Ethics Committee of Tianjin Hospital, China (2014-008). Discarded cartilage tissues were obtained from 10 normal patients (without OA) undergoing traumatic above-knee amputation and 10 OA patients undergoing total knee replacement surgery, aged 47–78 years. The OA patients were clinically diagnosed to be Kellgren-Lawrence grade 3 based on radiographic examination. All clinical specimens were obtained after patients gave informed consent. The two groups were paired and cartilage samples were matched by age, sex and body mass index.
Chondrocytes were isolated from the articular cartilage of clinical specimens obtained from normal and OA patients. Cartilage samples were minced and digested in 0.15% (w/v) collagenase (CLS-2, Worthington, USA) for 16 h at 37°C, in medium consisting of Dulbecco’s Modified Eagle Medium (DMEM, Gibco, UK) supplemented with 10% fetal bovine serum (FBS, HyClone, USA), 100 U/mL penicillin (Gibco) and 100 μg/mL streptomycin (Gibco). Isolated chondrocytes were washed in PBS and filtered through a 100 μm cell strainer (BD Biosciences, USA). The cells were seeded at high density (1 × 104 cells/cm2) and kept in maintenance medium for 2 days prior to gene expression analysis.
- SW1353 cell culture and transfection
Human chondrosarcoma cells (SW1353) were obtained from the American Type Culture Collection (ATCC). Cells were grown in maintenance medium consisting of DMEM supplemented with 10% FBS at 37°C with 5% CO2.
To silence HOTAIR function, SW1353 cells were transfected with small interfering RNA (siRNA) oligonucleotides targeting HOTAIR or the negative control (50 nM), using Lipofectamine™ RNAiMAX (Invitrogen, USA) according to the manufacturer’s instructions. The gene-specific siRNA is siHOTAIR (5’-GAACGGGAGUACAGAGAGAUU-3’). Transfected cells were kept in maintenance medium for 48 hours prior to further analyses.
To overexpress HOTAIR in SW1353 cells, a HOTAIR expression retrovirus vector was constructed. Full-length HOTAIR was amplified by PCR and cloned into the pBABE retroviral vector (Cell Biolabs, USA) using the primers 5’-GACTCGCCTGTGCTCTGGAGCT-3’ and 5’-TTGAAAATGCATCCAGATTTTT-3’. SW1353 cells were infected with retrovirus containing HOTAIR or negative control (vector) in the presence of 10 μg/mL polybrene (Sigma-Aldrich, USA). The supernatant was removed after 24 hours and replaced with maintenance medium containing 1μg/mL puromycin. Cells were cultured for 48 hours prior to further analyses.
- Analyses of HOTAIR-overexpressing SW1353 cells
A chromatin immunoprecipitation (ChIP) assay was conducted for SW1353-Vector and SW1353-HOTAIR cells by Tianjin Zhongrui Biotechnology Co. Ltd., using EZ-ChIP™ (catalogue number 17-371; Sigma-Aldrich, USA) according to the manufacturer’s instructions. Briefly, sheared crosslinked chromatin was immunoprecipitated with anti-H3K27me3 antibody (catalogue number 07-449, Sigma-Aldrich) and normal rabbit IgG (catalogue number 12-370, Sigma-Aldrich). After chromatin immunoprecipitation, retrieved DNA was detected by standard end-point PCR. The PCR products were analysed by gel electrophoresis and quantitated using GelAnalyzer. Primers for the WIF-1 promoter region are designed according to published methods (59), and the sequences are listed in Supplementary Table 1.
The nuclear and cytoplasmic protein fractions were extracted from SW1353-Vector and SW1353-HOTAIR cells using the NE-PER™ Nuclear and Cytoplasmic Extraction Kit (catalogue number 78833; Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Proteins were detected by Western blot analysis.
- Dual luciferase reporter assay
pGL3-WIF1 Promoter was constructed by inserting PCR product containing 1205bp in the 5’-flanking sequence of the human WIF-1 promoter into the pGL3-basic vector. Together with 40 ng pRL-TK Vector (Promega, USA) containing Renilla luciferase as an internal control, 200 ng pGL3-WIF1 Promoter were transfected into SW1353-Vector and SW1353-HOTAIR cells using Lipofectamine™ 2000 (Invitrogen, USA) according to the manufacturer’s instructions. Transfected cells were cultured in maintenance medium, and luciferase reporter assays were performed 36 hours after transfection. Cells were lysed and the firefly and Renilla luciferase activities in each well were measured using the Dual Luciferase Reporter Assay System (Promega). All measured luciferase activities were normalised to pRL-TK Vector activity, and firefly luciferase activity was normalised to Renilla luciferase activity for each well.
Total RNA was isolated from cells using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, samples were homogenised using the TRIzol reagent and chloroform was added, after which RNA was precipitated using isopropanol. The RNA was resuspended in 20 μL purified water.
Reverse transcription into cDNA was performed using 1 μg total RNA from each sample using PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio, USA) according to the manufacturer’s instructions. Gene expression levels were quantified with SYBR Premix Ex TaqII kit (Takara Bio) using a 7900HT Fast Real-Time PCR System (Applied Biosystems, USA) and normalised to GAPDH. Primer sequences were purchased from Sigma-Aldrich and listed in Supplementary Table 1. Relative gene expression was calculated using the comparative Ct (2−ΔΔCT) method.
Cells were lysed using a cell lysis buffer (Beyotime, China), and protein content was quantified using a BCA assay (Thermo Fisher Scientific). Equal amount of protein from each sample was electrophoresed on 10% (w/v) SDS-PAGE and transferred to PVDF membranes (EMD Millipore). The membranes were blocked with 5% milk in TBST for 1 hour at room temperature, and incubated at 4°C overnight with primary antibodies against WIF-1 (catalogue number sc-373780; Santa Cruz Biotechnology, USA), β-catenin (catalogue number C7082; Sigma-Aldrich), and GAPDH (catalogue number 5174; CST, China) at 1/1000 dilution. Membranes were washed three times for 10 minutes each with TBST, and then incubated for 1 hour at room temperature with secondary antibody conjugated to horseradish peroxidase (HRP) at 1/5000 dilution (anti-rabbit IgG, CST). The blots were visualised using enhanced chemiluminescence reagent (ECL, Thermo Fisher Scientific, USA). The blot intensity was quantified using molecular imaging software (Carestream Health, USA) after normalising to the corresponding loading control.
Data for all experiments were obtained from at least three independent samples, and all results were expressed as mean ± standard deviation. Statistical analysis was performed using the Stata 12.0 statistical software package (StataCorp, USA). One-way ANOVA with Tukey’s multiple comparisons test was used for statistical comparisons. A P-value of 0.05 was considered statistically significant.