Experimental pigs and sample collection
Pigs are very similar to humans in the aspects of anatomical structure, physiology, biochemical, metabolism, and the development mechanism of diseases. The genetic spectrum is also similar to the human gene spectrum. Therefore, using pigs as the experiment model can fully meet the research process of various human pathological development and physiology analyses needs. Earlier studies have shown that Diannan small-ear (DSE) pigs are good cartilage-defect animal models (18). Therefore, we employed DSE pigs in our present study. Twelve DSE pigs (40 ± 3 days) with similar weaning weights were raised in commercial cages, and bred under the same nutritional and 12:12 light/dark cycle at room temperature. They were offered an ad libitum water. All the animals were provided by Kunming medical university, and the animal experiments were carried out by the Guide to Nursing and Use of Experimental Animals and approved by the Ethics Review Committee of Animal Experiments of Kunming Medical University (kmmu20221858). All animal methods are reported by ARRIVE guidelines.
The DSE pigs were divided into Control and Model groups randomly. The pigs in the Model group underwent the Hulth procedure in the right knee joint following the detailed method reported by Jia et al. (19). To establish a joint injury model in the short term, pigs were released from the cages and driven to run back and forth on the 30-m-long road for 10–15 min every three days.
At 6–8 months (15 − 20kg), all the DSE pigs were executed after inhalation anesthesia by isoflurane, and the samples of blood, joint liquid, and tissues at the joint were harvested and stored at − 80°C for further use. Some of the tissues were fixed by 4% polymerization formaldehyde.
Isolation of pig PBMSCs
The cells in bone tissues were collected and treated with PBS rinse and centrifugation. PBMSCs were isolated from pigs in the Model group according to the previous literature (18, 20). PBMSCs were cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) at 37 ℃, 5% CO2 cell culture incubator for 0 days (NC), 7 days (Induced-7d), and 14 days (Induced-14d), IL-1β, TNF-α, and IL-6 stimulation group (Inflammation), Induced for 14 days and then stimulated with IL-1β, TNF-α, and IL-6 (Inflammation + Induced-14d) groups.
Vector construction and transfection
BMSCs cells induced for 14 days were seeded into 6-well plates with 6×105 cells per well and incubated overnight. Cells were transfected with overexpression plasmid of miR-181a, circZCCHC14, FTO, and vector. The small interfering (si) RNA of FTO (si-FTO) and its negative control (NC) were synthesized by GenePharma (Shanghai, China). All the transfections were conducted following the instruction of Lipofectamine 2000 (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 80–90% confluent. Two days after transfection, cells were harvested for the following experiments.
Real-time quantitative PCR (qPCR)
Total RNA in peripheral blood and joint lipids were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The expression levels of circZCCHC14, FTO, collagen type II alpha 1 chain (COL-2A1), aggrecan (ACAN), GREM1, and miR-181a were measured using qPCR. The primers were designed and synthesized by Sangon (Shanghai, China; Table S1). SYBR Green PCR master mix (Applied Biosystems, cat. #4309155) was used to perform qPCR. Then, the productions were conducted on an ABI 7900 system (Foster City, CA, USA) and calculated using the 2–ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as the reference gene. All experiments were tripled.
Methylated RNA immunoprecipitation qPCR (MeRIP-qPCR)
The MeRIP-qPCR was performed as previously described (21). The Magna methylated RIP (MeRIP) kit (Millipore, cat. #CR203146) was employed. Cells were harvested and then centrifugated at 1,500 rpm at 4℃ for 5 min. After removing the supernatant, cells were mixed with RIP lysis buffer and incubated with the lysate. m6A antibody (5 µg) containing magnetic beads was administrated for a rotation of 30 min. The beads were eluted using RIP buffer twice. After rotation overnight, the beads were washed using a high-salt buffer. The RNA enrichment was calculated by qPCR.
Western blotting
The western blotting analysis was performed on cells and transfections. The proteins in cells were lysis by RIPA lysis buffer (#R0278, Sigma) and quantified by BCA Protein Assay Kit (Beyotime, Jiangsu, China). The standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method was used. The membranes were blocked with 5% non-fat milk and incubated with the primary antibodies of YTH N6-methyladenosine RNA binding protein (YTHD) F1 (YTHDF1; # ab220162, 1:1000, Abcam), YTHDF2(#ab220163, 1:1000, Abcam), YTHDF3(#ab220161, 1:1000, Abcam), YTHDC1(#ab259990, 1:1000, Abcam), YTHDC2(#ab220160, 1:1000, Abcam), methyltransferase-like 3 (METTL3) (#ab195352, 1:1000, Abcam), METTL14(#ab220030, 1:1000, Abcam), WTAP(#ab195380, 1:1000, Abcam), FTO(#ab126605, 1:20000, Abcam), COL-2A1(#ab188570, 1:1000, Abcam), ACAN(#ab3778, 1:1, Abcam), GREM1(#ab231065, 1:5, Abcam), bone morphogenetic protein 2 (BMP2; # ab214821, 1:1000, Abcam), and GAPDH (#ab9485, 1:2500; Abcam). Then, the PVDF membranes were incubated with an anti-rabbit HRP-conjugated secondary antibody (1:5000). The bands were observed under an ECL kit (Millipore, Germany).
Alcian blue staining
Firstly, the cells are cleaned with PBS for 10 min twice, fixed with 10% formaldehyde for 20 minutes, and buried with paraffin. Then, the sections were dewaxed, distilled, and soaked with Alley’s new blue stain solution (Sigma, A3157) for 30 min. Finally, the sections were washed three times and observed under the microscope.
Bioinformatics Analysis
Before the function investigation and verification of the FTO m6A-forming and demethylase enzymes on circZCCHC14, bioinformatic analyses were performed. The potential m6A modification sites and the m6A-forming and demethylase enzymes of circZCCHC14 were predicted in m6Avar and SRAMP databases.
RNA pull-down analysis
RNA pull-down assay was conducted to determine the interaction between circZCCHC14 and FTO. Briefly, the FTO or FTO-Mut probe was synthesized and biotinylated by GenePharma (Shanghai, China). RNA pull-down assay was carried out using the Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the protocol. The Streptavidin-coupled magnetic beads were incubated with an FTO probe (Servicebio, China). Cells were lysed in a specif ic lysis buffer (Ambion, Austin, TX), treated with magnetic beads, and then washed with precooled lysis buffer and salt buffer solution. In the end, the RNA-binding protein complexes were washed and eluted for real-time qPCR analysis.
RNA immunoprecipitation (RIP) assay
One EZ-Magna RIP kit (Millipore, Billerica, MA, USA) was used to detect the interaction between circZCCHC14 and FTO according to the manufacturer’s introduction. Generally, the transfected cells were treated with RIP buffer and incubated with anti-Argonaute 2 (Ago2; 1:50; #ab186733) overnight. IgG (1:100; ab109489; Abcam) employed as control. Then, the samples were digested with proteinase K, and TRIzol-chloroform was employed to extract the immunoprecipitated RNA. Finally, the RNAs precipitated by Ago2 or IgG were detected by qPCR.
Statistical analysis
GraphPad Prism 8 (San Diego, CA, USA) was employed for the statistical analysis. Biorender (Biorender-Javashuo) was used to draw the summary diagram. One-way ANONA comparison analysis was performed on groups. All the experiments were tripled. Data are exhibited as mean ± standard deviation (SD). P < 0.05 was considered a significant difference.