Patients and grouping
The cartilage tissue of OA patients was derived from the knee joints of 30 patients who had undergone total knee arthroplasty. Meanwhile, the healthy cartilage tissues were obtained from 20 patients who had not undergone OA or RA (rheumatoid arthritis). There was no statistical difference in age and gender between OA patients and healthy volunteers. The age and sex of all the samples were matched. All patients voluntarily signed the informed notice. The current study obtained the approval of Ethics Committee of the hospital (ethic vote 198/203).
The femoral articular cartilage of the femur was obtained and cut. Then, it was digested by 0.2% type II collagenase, oscillation resolving for 40min at 37°C, washed by D-Hanks, and diluted in DMEM/F12 (Gibco, USA) combined with 10% FBS, 100 u/ml penicillin (Gibco, USA) as well as 100 mg/ml streptomycin (Gibco, USA). These cells were culture in a saturated humidity incubator (37°C, 5% CO2). The solution was changed every two days until the chondrocytes grew into sheets and covered over 85% confluence 18. The cells of 2 or 3 generations were enrolled for the further investigation.
The MEG3 overexpression vector pcDNA3.1-MEG3, si-MEG3 for MEG3 knockdown, si-EOXO1 used to decrease the expression of FOXO1 (Sangon Biotech, Shanghai, China), miR-361-5p mimics and miR-361-5p mimics NC (Guangzhou Reeber) were prepared, followed by cells transfection according to the instructions of the transfection reagent. All transfections were carried out by Lipfectamine 2000 transfection kit (Invirtrogn, USA) according to the instructions. After 48 hours, the samples were treated with IL-1β (10 ng / ml) for 24 hours. Finally, cells of each group were collected for following experiments.
Real-time fluorescent quantitative PCR
Total RNA (500 ng) from sample of each group was obtained as well as quantified using TRIzol reagent, and cDNA template was synthesized by PrimeScript RT kit (Takara biomedical Technology Co., Ltd., Beijing, China). GAPDH was used as reference (Table 1). The associated conditions were as follows: 95°C for 3 min, 39 cycles at 95 °C for 10 s, 55 °C. Fluorescence signals were collected at the end points of each cycle extension on the ABI7500 platform, followed by the amplification curve investigation. Relative expression of candidate genes were calculated by 2-∆∆CT method 19.
Luciferase reporter assay
The regulatory relation between MEG3 and miR-361-5p was revealed by using StarBase as well as miR-361-5p and FOXO1. A wild type (MEG3-WT) containing the indicated fragment of the putative binding site between MEG3 3’UTR and miR-361-5p was constructed as well as mutant (MEG3-MUT) without the above mentioned fragment. Then, the wild type or mutant were cloned into the pmirGLO reporter vector (Promega, USA). After inoculation of human chondrocytes in 24-well plates (5 × 105/well) for 24 hours, miR-361-5p mimic or mimics NC, and MEG3-WT and MEG3-MUT were co-transfected into human chondrocytes by Lipofectamine 3000 (Thermo Fisher Scientific), followed by dual-luciferase reporter gene assay after 48 hours transfection. Similar to the above steps, the FOXO1-WT and FOXO1-MUT were synthesized, and then, the correlation of miR-361-5p and FOXO1 was determined using dual-luciferase reporter gene assay.
RIP was determined by using a Magna RIPTM RNA kit (Millipore, USA). Briefly, the cultured chondrocytes were further suspended in RIP lysis buffer (Solarbio). Then, cell extract was incubated overnight with RIP buffer containing human anti-Ago2 antibody beads (Millipore) (Input and normal IgG served as controls). Finally, RNA samples were extract using TRIzol reagent, followed by relative enrichment of MEG3/FOXO1 and miR-361-5p detection.
Cultured cells and chondrocytes isolated from treated tissues were treated with protein lysate RIPA buffer to extract the whole proteins. Briefly, the extracted protein was quantified by bicinchoninic acid (BCA) method. A total of 50 g samples was investigated by 10% polyacrylamide gels, and transferred to polyvinylidenefluoride membranes (5% Skim milk/BSA). Samples was treated with primary antibodies including β-Catenin (#19807),MMP-13 (#94808), Collagen II (#34712), PCNA (#13110), Bax (#5023) and Bcl-2 (#4223) antibody (1:1000, Cell Signaling, Boston, USA), as well as Ki67 (ab92742), ADAMTS-5 (ab41037) and Aggrecan (ab36861) (Abcam, Cambridge, MA, USA) 17. Then, samples were treated with the HRP-conjugated secondary antibody (1:5000, #7074; Cell Signaling Technology, Danvers, MA,USA). Finally, the protein was developed with diaminobenzidine (DAB). All experiments were repeated for 3 times.
All cells were placed in 24-well plates (2 × 103 cells/well, 37 °C and 5% CO2). After being incubated for 0, 24, 48, 72 h, a total of 10 μL CCK-8 (Sigma-Aldrich) reagent was added into each well for further incubation. OD490 value was measured by enzyme-linked immunosorbent assay 20.
Flow cytometry assay
Simply, the cells were treated by trypsin supplemented with 200 μL Annexin V-FITC, incubated for 10 min in dark, and then washed with 200 μL PBS and 10 μL PI. Cell apoptosis was then detected by using flow cytometry (Beckman Coulter), followed by the data analysis.
OA rat model construction
Male SD rats (200-250 g) were obtained from the Experimental Animal Center of Taishan Medical College. All rats were anesthetized with intramuscular injection of sodium pentobarbital (0.05 mg/g, Chuangdong Co., Chongqing, China). Then, SD rats were traversed by the medial collateral ligament and destabilized by the medial meniscus (DMM). One week after the operation, si-NC and si-MEG3 (1×109 PFU, 20 μl) were injected into the knee joint of the recipient rat (n = 6 for each group, 20 μL per joint) twice a week for 4 weeks. Eight weeks after the operation, the rats were sacrificed with cervical dislocation method (external force dislocated the cervical vertebra of rat and disconnects the spinal cord from the cerebrospinal cord), and then the knee joints were harvested. All experiment was performed in Experimental Center of Taishan Medical College. This study was approved by the Laboratory Animal Ethics Committee of Taishan Medical College (No. 2019146), and all experiments abided by the guide for the care and use of laboratory animal.
Histological and immunostaining investigation
Cartilage samples was placed in paraformaldehyde (4%), embedded in paraffin and cut into sample slice (5μm/slice). The cartilage destruction was evaluated by using the Safranin ‘O’ staining. Histological scores were measured according to the International Osteoarthritis Research Association (OARSI) grading system, which ranging from 0 (normal) to 6 (>80% represented the cartilage loss). Scores were determined from multiple serial sections of the knee joint of each mouse.
The SPSS 18.0 (Chicago, IL, USA) was selected as the software for data analysis. Meanwhile, all the data in current investigation were represented as the mean ± standard deviation (SD). Significant differences between two groups were assessed using Student’s t-test, while least significant difference between means (LSD)-t multiple comparison test was processed for more than two groups. Spearman analyses were performed to identify the correlations of miR-361-5p and MEG3 or FOXO1. P < 0.05 was considered as statistically significant 21.