Healthy specific pathogen-free Sprague-Dawley (SD) rats (weighing 200 ± 20g) were purchased from Vital River Laboratory Animal Technology (Beijing, China). Rats were fed in a clean animal room (21 ± 3 ℃; relative humidity, 50±10%) with free access to food and drink under a 12 h dark/light cycle. Rats were randomly assigned into two main groups: (1) OA group: rats underwent anterior cruciate ligament transection surgery to destabilize the joints and induce OA. (2) Sham group: a similar incision was made in the right joint capsule of the rat. On the next day, adenovirus-mediated CRNED overexpression vector (Ad-CRNED) or DACT1 interference vector (sh-DACT1) were injected into the OA rat model via tail vein. The volume of adenovirus fluid injected at one time was 0.05-0.1 mL/10 g per body weight, and the dosage of adenovirus solution was 2×109 PFU per rat.
All animal experiments were approved by the Second Affiliated Hospital of Xi’an Jiaotong University Animal Ethics Committee and were performed in accordance with the Committee’s guidelines.
Cell Differentiation and Culture
ATDC5 cell line was purchased from Riken BioResource Center (Tsukuba, Japan). Undifferentiated ATDC5 cells were cultured in DMEM/F12 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (Gibco, Rockville, MD) and 1% P/S (penicillin/streptomycinat) at 37 °C in 5% CO2. When the cell confluence reaches about 75%, 1% insulin-transferrin-selenite (ITS) is added to the medium, and then the medium was changed every two days to induce the cells to differentiate into chondrocyte-like cells. Cells were used for the subsequent experiments after 2 weeks of differentiation in culture.
Adenovirus-mediated CRNED, DACT1 and p300 overexpression vectors were constructed using full-length sequences of CRNED, DACT1 and p300. The adenovirus vectors containing small hairpin RNA (shRNA) sequence targeting CRNED (sh-CRNED), DACT1 (sh-DACT1), p300 (sh-p300) or negative control vector (sh-NC) were amplified and cloned by GeneChem (Shanghai, China). All transfection was performed using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).
Cells were seeded into 96-well plates and incubated at 37 ℃ under 5% CO2 for 24 h. After treatment, 10 μL of MTT reagent was added into each well, and then cells were incubated at 37℃ for 4-6 h. After that, the supernatant was discarded and 100 μL of DMSO was added into each well. The absorbance of each well was measured at a wavelength of 450 nm after thecrystals were sufficiently dissolved.
Apoptosis analysis was carried out by using Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Multisciences, Shanghai, China). In brief, the treated cells were washed with pre-cooled PBS and cell suspension was prepared. Next, 100 μL of cell suspension was incubated with 5 μL of Annexin V-FITC and 5 μL of PI in the dark for 20 min. Cell apoptosis was detected by using BD Accuri flow cytometer (BD Biosciences).
The concentrations of IL-6, IL-8, and TNF-α in cell supernatant were measured using ELISA kits (R&D Systems, USA) according to the manufacturer’s instructions. The absorbance of each well at the wavelength of 450 nm were measured. The experiment was repeated three times to obtain the mean value.
Total protein extracted from cells and rat cartilage tissues was lysed with RIPA lysis buffer. CelLytic™ NuCLEAR™ Extraction Kit (Sigma, St. Louis, MO, USA) was used to extract nuclear proteins. Protein concentrations were determined by using the BCA Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein were separated on 10% SDS/PAGE gels and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking in 5% skim milk at room temperature for 2 h, membranes were incubated at 4 ℃ overnight with primary antibodies including anti-ADAMTS-5 (ab41037, 1:250, Abcam, Cambridge, UK), anti-MMP13 (ab51072, 1:1000, Abcam), anti-Collagen 2 (ab188570, 1:1000, Abcam), anti-Aggrecan (ab3778, 1:1000, Abcam), anti-β-catenin (ab68183, 1:500, Abcam), anti-DACT1 (ab42547, 1:1000, Abcam), anti-c-Myc (ab32072, 1:1000, Abcam) and anti-Cyclin D1 (ab16663, 1:200, Abcam). Then, membranes were incubated with HRP-conjugated specific secondary antibodies for 2 h at room temperature. Protein band signals were measured using enhanced chemiluminescence reagents (Beyotime). Image J software was used to analyze the gray level of bands.
Total RNA was extracted from cells and rat cartilage tissues using TRIzol kit (Invitrogen, Carlsbad, CA, USA). 1 μg of RNA was reverse transcribed to cDNA using a Reverse Transcription Kit (Takara, Dalian, China). Synthesized cDNAs were used for RT-qPCR by using SYBR Premix Ex Taq Kit (Takara). The thermal cycling conditions were as follows: 3 minutes at 95 °C, 35 cycles for 15 seconds at 95 °C, 30 seconds at 60 °C, and 15 seconds at 72 °C. The relative mRNA levels were normalized to GAPDH and calculated by using 2-ΔΔCT method. DACT1: 5’-GGA AGA GGA CAG GCT TGG AAA C-3’ (S) and 5’-GTC CCA TTG TTC AGA GAA GGT ATC-3’ (R). CRNED: 5’-TGA AGG AAG GAA GTG GTG CA-3’ (S) and 5’-TCC AGT GGC ATC CTA CAA GA-3’ (R).
Chromatin Immunoprecipitation (CHIP) Assay
ChIP assay was carried out using the EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore, Burlington, MA) in accordance with the manufacturer’s protocol. In brief, cells were incubated with formaldehyde for 10 min to produce DNA-protein crosslinks. Next, the cell lysates were sonicated into 200-1000 bp chromatin fragments and immunoprecipitated with anti-H3K27ac (ab4729, Abcam) and anti-p300 (ab2832, Abcam).
RNA Immunoprecipitation (RIP) Assay
RIP assay was performed by using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore). In brief, the treated cells were washed by ice-cold PBS and lysed in a radioimmu-noprecipitation buffer for 30 minutes at 4 °C. Next, the whole cell lysate was incubated with A/G sepharose beads coupled with anti-DACT1 (Abcam) or normal human IgG (Millipore) antibodies. The precipitated RNA was analyzed by qPCR.
RNA pull-down Assay
The transcribed RNA was biotin-labeled with Biotin RNA Labeling Mix (Ambio Life), treated with RNase-free DNase I (Roche, Indianapolis, IN, USA), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). 1 mg of whole-cell lysates were incubated with 3 μg of purified biotinylated transcripts at room temperature for 1 h. And then the complex was isolated with streptavidin agarose beads (Invitrogen, San Diego, CA, USA). The eluted proteins were measured by Western blot analysis.
Hematoxylin and Eosin (H&E) and Safranin O-fast Ereen Staining
The knee joints were fixed with formalin, decalcified with 0.5 M EDTA at pH 8.0 for 4 weeks, and dehydrated and embedded in paraffin. The tissue sections were cut into 4-6 μm thickness along the sagittal direction. Next, H&E and Safranin O-Fast Green staining was performed. At least five visual fields of each picture were selected to assess the severity of OA by using the Osteoarthritis Research Society International (OARSI) scoring system .
Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-end Labeling (TUNEL) Assay
Paraffin sections of knee joint cartilage tissues were stained using TNUEL (Roche, Basel, Switzerland) method in accordance with the manufacturer's protocol. After staining, normal cells showed purple-blue, and apoptotic cells showed yellow-brown. TUNEL-positive cells were evaluated (×100) in six randomly-selected fields, and the average number of apoptotic cells was calculated.
All data in this study were analyzed using SPSS 22.0 software (IBM Corp., Armonk, NY). Data were presented as mean ± standard error of mean (mean ± SEM). Differences between two groups were analyzed by using Student’s t test. And comparisons among multiple groups were measured by using one-way analysis of variance (ANOVA). The level of significance was set at *P < 0.05.