Isolation and culture of primary mouse chondrocytes
Primary murine chondrocytes were isolated from newborn mice as described previously . Briefly, 5-day-old C57BL/6 mice were sacrificed by intraperitoneal injection of pentobarbital and then isolated articular cartilage. The isolated articular cartilage was digested with 3 mg/mL collagenase D for 90 min at 37 ℃ under 5% CO2 and 0.5 mg/mL collagenase D overnight at 37 ℃. After centrifugation at 400 g for 10, the supernatant was discarded and cells precipitation was resuspended in DMEM supplemented with 10% fetal bovine serum, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. Then seed chondrocytes on a culture dish at density of 8×103 cells per cm. Change the culture medium after 2 d of culture, and the isolated chondrocytes reach confluence by 6-7 d. Only passage 1 to 3 were used for further experiments.
Induction of OA mouse model
10-week-old C57BL/6J male mice were purchased from Animal Center of the Chinese Academy of Sciences (Shanghai, China) and housed in plastic cages with free access to drinking water and a pellet based diet. Experimental OA model was induced by surgical destabilization of the medial meniscus (DMM) as described previously . Briefly, under general anesthesia, the medial collateral ligament and the medial meniscus of the right knee were resected under a microscope. After surgery, the mice were randomly divided into the following groups: sham group, OA group. 8 weeks after surgery, mice were sacrificed for detection of LincRNA-Cox2 expression.
Quantitative real-time polymerase chain reaction (qRT-PCR)
qRT-PCR was performed to detect the expression of mouse LincRNA-Cox2 and miR-150 in chondrocytes after transfection and IL-1β treatment. Total RNA of chondrocytes was extracted using TRIzlo reagent (Thermo Fisher Scientific, MA, USA). After transcription to cDNA, qRT-PCR was performed by SYBR Green Master Mix (Applied Biosystems, CA, USA) and the setting parameters are as follows: firstly 95 °C for 10 min, then 95 °C for 15 s and 60 °C for 30 s lasting 40 cycles. The cycle threshold (Ct) values were obtained, normalized to the level of GAPDH and compared with the control. Data were quantified using the 2-△△CT method. The primer sequences of mouse LincRNA-Cox2 were as follows: forward 5’-AAGGAAGCTTGGCGTTGTGA-3’; reverse 5’-GAGAGGTGAGGAGTCTTATG-3’.
The proliferation capability of chondrocytes was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT assay). 5×103 cells were seeded into 96-well plates for 24 h, then cells were transfected with si-NC, si-Cox2, inhibitor NC and miR-150 inhibitor, respectively. 48 h after transfection, 20 uLMTT (5 mg/ml) (Sigma, CA, USA) was added, and the cells were incubated for 4 h at 37 ℃ under 5% CO2. Subsequently, the supernatant was discarded and 200 ml DMSO was added. Finally, the OD490 nm value was measured to evaluate the proliferation capability of chondrocytes.
Apoptosis of chondrocytes was determined using Annexin V FITC Apoptosis Detection Kit (BD Bioscience, NJ, USA). After IL-1β treatment and/or relevant transfection, cells were collected, washed with phosphate-buffered saline (PBS) and resuspended in 1 Binding Buffer at a concentration of 1×106 cells/mL. Then 5 µl of annexin V FITC and PI were added. After incubation for 15 min at room temperature in the dark, quantification of apoptotic cells was analyzed by flow cytometry (BD Bioscience, NJ, USA). Data was analyzed using FlowJo software (Tree Star, Ashland, OR).
After the indicated treatment, chondrocytes were collected and washed with PBS, then lysed on ice with RIPA lysis buffer supplemented with 10 mM of PMSF (Beyotime, Nanjing, China) for 15 min. Total protein was quantified using the BCA Protein Assay Kit (Solarbio, Beijing, China). Then, proteins in equal amounts were subjected to 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Bio‐Rad Laboratories, CA, USA). After blocking with 5% skimmed milk for 2 h at room temperature, membranes were incubated with specific primary antibodies against Ki67, PCNA, Bax, Capase-3, Capase-9, GSK-3β, p- GSK-3β (ser9), β-catenin, cynlin D1, c-Myc, MMP-7 and GAPDH (Santa Cruz, CA, USA) overnight at 4 °C. Then membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature, and protein bands were visualized using electrochemiluminescence (ECL) plus (GE Healthcare; Buckinghamshire, England, UK) according to the manufacturer’s instructions. Densitometry analysis of bands was performed using Image J software (National Institutes of Health, Bethesda, MD, USA). GAPDH was used as an endogenous protein for normalization
The miR-150 mimic, miR-150 inhibitor and their negative control (Scramble and anti-NC) were synthesized by GenePharma Co (Shanghai, China). The full-length wide-type LincRNA-Cox2 sequences was constructed into the pEX-2 plasmid (GenePharma, Shanghai, China). An empty pEX-2 plasmid was transfected as a negative control. siRNA specific for LincRNA-Cox2 was constructed into U6/Neo plasmid (GenePharma, Shanghai, China). An empty U6/Neo plasmid with non-targeting sequences was transfected as a negative control (NC). Cell transfections were conducted using Lipofectamine 3000 reagent (Invitrogen, CA, USA) depending on the manufacturer’s descriptions. After 48 h, the transfection efficiency was detected by qRT-PCR.
Dual luciferase activity assay
The 3’UTR target site was generated by PCR and the luciferase reporter constructs with the LincRNA-Cox2 sequences carrying a putative miR-150-binding site into pMiR-report vector were amplified by PCR. Cells were co-transfected with the reporter construct, control vector and miR-150 or scramble using Lipofectamine 3000 (Life Technologies, USA). Reporter assays were done using the dual-luciferase assay system (Promega) following to the manufacturer’s information.
All results were observed from at least three independent experiments. Statistical analysis was carried out using SPSS 19.0 and data were presented as the mean ± SD as indicated. Statistical differences between two groups were determined by two-tailed Student’s t-test. Differences among more than two groups in the above assays were estimated by one-way ANOVA. The linear relationship among levels of LincRNA-Cox2and miR-150 in OA mice was analyzed by Spearman’s correlation coefficient. P< 0.05 was considered statistically significant.