Patients and grouping
The cartilage tissue of OA patients came from the knee joints of 30 patients who had undergone total knee arthroplasty. Meanwhile, the healthy cartilage tissue came from 20 patients who had not undergone OA or RA (rheumatoid arthritis). All patients voluntarily signed the informed notice. The current study obtained the approval of Ethics Committee of the hospital (ethic vote 198/203).
Cell culture
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℃, washed by D-Hanks, and diluted in DMEM/F12 (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS), 100 u/ml penicillin (Gibco, Rockville, MD, USA), 100 mg/ml streptomycin (Gibco, Rockville, MD, USA). Then 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 more than 85% of the wall of the bottle 22. The cells of 2 or 3 generations were enrolled for the further investigation.
Cell transfection
PcDNA3.1-MEG3 overexpression vector, si-MEG3 (Sangon Biotech, Shanghai, China), miR-361-5p mimics and miR-361-5p mimics NC (Guangzhou Reeber) were constructed, followed by cells transfection according to the instructions of the transfection reagent. Based on different transfection, osteoarthritic chondrocytes were divided into Blank group, pcDNA3.1-NC group, pcDNA3.1-MEG3 group, si-NC group, si-MEG3 group, pcDNA3.1-NC + mimics NC group, pcDNA3.1-MEG3 + mimics NC group, pcDNA3.1-NC + miR-361-5p mimics group and pcDNA3.1-MEG3 + miR-361-5p mimics group. All transfection was carried out by Lipfectamine 2000 transfection kit (Invirtrogn,USA) according to the instructions. Then, the cells transfected with pcDNA3.1-NC and pcDNA3.1-MEG3 were cultured for 48 h by adding 10 μmol/l of Wnt/β-catenin signaling pathway inhibitor XAV939 (Tocris Bioscience), which was named as pcDNA3.1-NC +XAV939 group and pcDNA3.1-MEG3+ XAV939 group respectively. After 48 hours of transfection, cells were stimulated 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 extracted and quantified using TRIzol reagent, and cDNA template was synthesized by reagent kit (invitrogen, San Diego, USA) according to the manufacturer's instructions. GAPDH was used as reference. The primers were shown in Table 1. The reaction 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, followed by the amplification curve investigation. Relative expression of candidate genes were calculated by 2-∆∆CT method 23.
Luciferase reporter assay
The regulatory relation between MEG3 and miR-361-5p was predicted based on StarBase (http://starbase.sysu.edu.cn/). A wild type (MEG3-WT) or mutant (MEG3-MUT) fragment of the MEG3 3'UTR containing miR-361-5p was synthesized. Then, the wild type or mutant sequences were cloned into the pmirGLO reporter vector (Promega, Madison, WI, USA). After inoculation of human chondrocytes in 24-well plates (5 × 105/well) for 24 hours, miR-361-5p mimic or miR-361-5p NC, MEG3-WT or MEG3-MUT were co-transfected into human chondrocytes by Lipofectamine 3000 (Thermo Fisher Scientific). Finally, the luciferase intensity was measured by Dual Luciferase Reporter Assay Kit (Promega, E1910, WI, USA) 48 hours after transfection.
RIP assay
RIP was determined by using Magna RIPTM RNA binding protein immunoprecipitation kit (Millipore, Bedford, MA, USA). Briefly, the cultured chondrocytes were collected and 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). The next day, the magnetic beads were incubated with proteinase K. Total RNA was subsequently isolated from the extract using TRIzol reagent. Finally, relative enrichment of MEG3 and miR-361-5p was determined by RT-qPCR analysis.
Western blot
Cultured cells were lysed with 100 μl/50mL protein lysate RIPA. Briefly, the extracted protein was quantified by bicinchoninic acid (BCA) method. A total of 50 g proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% polyacrylamide gels, and transferred to polyvinylidenefluoride membranes. After blocked with 5% Skim milk/BSA, the membrane was incubated with primary antibodies including β-catenin (#8480), Non-phospho (Active) β-Catenin (#19807), MMP-1 (#54376), MMP-13 (#94808), cyclinD1(#2922), c-Myc (#9402), 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) at 4°C overnight 21. The β-actin was used as internal control. After washed by TBST for three times, the membranes were incubated with horseradish peroxidase-labeled sheep anti-rabbit secondary antibody (1:5000, Biyuntian biotechnology) at room temperature for 1 h. Finally, the protein was stained with diaminobenzidine (DAB) kit. All experiments were repeated for 3 times.
CCK-8 assay
All cells were seeded in 24-well plates (2 × 103 cells/well) and incubated at 37 °C with 5% CO2. After being incubated for 0, 24, 48, 72 h, a total of 10 μL CCK-8 (Sigma-Aldrich) were added to each well and then incubated for 4 h. OD490 value was measured by enzyme-linked immunosorbent assay 24.
Flow cytometry assay
Flow cytometry was performed to detect the apoptosis of transfected cells. Simply, the cells digested by trypsin was mixed with 200 μL Annexin V-FITC, incubated for 10 min in dark, and then washed with 200 μL PBS and 10 μL PI. Cell cycle progression was subsequently monitored based on flow cytometry (Cell Lab Quanta, Beckman Coulter) and the data were analyzed using Multi-Cycle AV software (Phoenix Flow Systems, SanDiego, CA).
OA rat model construction
Animal experiments were carried out according to the guidelines for animal experiments in our hospital. Male Sprague-Dawley (SD) rats (200-250 g) were purchased from the Experimental Animal Center of Shandong University. All rats were anesthetized with intramuscular injection of sodium pentobarbital (0.05 mg/g, Chuangdong Co., Chongqing, China). The experimental OA of 10-week-old SD rats was traversed by the medial collateral ligament and destabilized by the medial meniscus (DMM). One week after the operation, si-NC, si-MEG3 (1×109 PFU, 20 μl) was 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 were in accordance with the guide for the care and use of laboratory animals established by United States National Institutes of Health (Bethesda, MD, USA).
Histological analysis and immunostaining
Rat cartilage was fixed in 4% paraformaldehyde, embedded in paraffin and cut into sample slice (5μm/slice). In order to assess the extent of cartilage destruction, Safranin ‘O’ staining was performed. Histological scores were performed 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.
Statistical Analysis
Data were analyzed using version 18.0 (SPSS, Chicago, IL, USA) and 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. P < 0.05 was considered as statistically significant 25.
All rats were anesthetized with intramuscular injection of sodium pentobarbital (0.05 mg/g, Chuangdong Co., Chongqing, China).
All rats were sacrificed with cervical dislocation method (external force dislocated the cervical vertebra of rat and disconnects the spinal cord from the cerebrospinal cord).