Data collection

OA-related datasets were searched from the freely available GEO database (www.ncbi.nlm.nih.gov/geo). The GSE169077 dataset containing knee cartilage samples from 5 normal subjects and 6 OA patients was gained. Analysis of DEGs was implemented using the GEO2R tool attached to the GEO. A screening of DEGs was conducted based on the criteria with |log2 (FC)|> 1 and p < 0.05.

Clinical articular cartilages

Knee articular cartilages (n = 25) were collected from OA patients who underwent total knee replacement surgery at the Ganzhou People’s Hospital. Control cartilage samples were taken from trauma patients who had never suffered from OA or rheumatoid arthritis, with an age matched to OA patients. After the operation, these knee articular cartilages were stored in liquid nitrogen immediately. All cartilage donors had provided written informed consent. Approval for this study was obtained from the Research Ethics Committee of the Ganzhou People’s Hospital.

Cell culture

Human chondrocytes CHON-001 (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s Modified Eagle Medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone) and 1% antibiotics (Solarbio, Beijing, China) at 37 °C with 5% CO2.

Cell transfection

Short hairpin RNA (shRNA) specific for targeting CA12 (sh-CA12) or WTAP (sh-WTAP), an overexpression plasmid for CA12 (oeCA12), and their corresponding negative controls (sh-NC, oe-NC) were purchased from GenePharma (Shanghai, China). Transient transduction was undertaken using Lipofectamine 3000 (Invitrogen) following the directions provided by the manufacturer. Lentiviral particles were generated using a lentiviral packaging system consisting of psPAX2 (Addgene, Watertown, MA, USA) and PMD2.G (Addgene). The infection of host cells with lentiviral particles was performed in the presence of 6 µg/mL polybrene (Solarbio). Stable transduced cells were then selected with puromycin (Solarbio) for a fortnight. After transfection, chondrocytes were treated with 10 ng/mL of IL-1β (Solarbio) for 24 h.

RNA extraction and RT-qPCR

The MolPure Cell/Tissue DNA kit (Yeasen, Shanghai, China) was utilized to extract total RNA from clinical articular cartilages and cultured chondrocytes in line with the operating instructions provided by the manufacturer. After examination of RNA concentration, reverse transcription was undertaken with a reverse transcription kit (Takara, Dalian, China). The complementary DNA was then amplified to detect relative WTAP and CA12 mRNA levels by qPCR using a SYBR Green Master Mix (Vazyme, Nanjing, China) following the thermocycler conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. Quantification of fold change was made using the 2−ΔΔCt method. Primer sequences are registered in Table 1.

Table 1 Primers sequences used for qPCRAntibodies and western blot analysis

Clinical articular cartilages and cultured chondrocytes were homogenized in RIPA buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail. Protein samples were quantified using the BCA protein assay kit (TIANGEN, Beijing, China). Protein samples (approximately 30 μg) were separated on 8–12% SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% nonfat milk, the membranes were immunoblotted with the primary antibodies overnight at 4 °C and subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Lastly, visualization of the blots was accomplished using enhanced chemiluminescence substrate reagent (Tanon, Shanghai, China). Primary antibodies used: CA12 (ab195233, 1/10000; Abcam, Cambridge, MA, USA), WTAP (ab195380/ab155655, 1/1000; Abcam); anti-Bax (ab32503, 1/2000; Abcam), anti-Cleaved caspase-3 (ab32351, 1/5000; Abcam), and anti-MMP13 (ab51072/ab39012, 1/1000; Abcam), anti-COL2A1 (ab34712, 1/2000; Abcam), anti-aggrecan (ab3778, 1/1000; Abcam), and anti-GAPDH (ab8245, 1/5000; Abcam).

Cell counting kit-8 (CCK-8) assay

Chondrocytes were seeded in 96-well plates (4 × 103 cells) and cultured overnight, followed by application of IL-1β (10 ng/mL) for 24 h. The CCK-8 reagent (10 µL; Solarbio) was added and incubated for 2 h. Measurement of the OD value at 450 nm was conducted by a microplate reader (BioTek, Winooski, Vermont, USA).

Flow cytometry assay

After incubation for 48 h, the cells were collected and trypsinized and washed with PBS. Subsequently, cells were suspended and stained with the Annexin V-FITC Apoptosis Detection Kit (Beyotime) as per the instructions supplied by the manufacturer. Cell apoptosis was monitored by a FACScan flow cytometer (BD Bioscience, San Jose, CA, USA).

ROS analysis

To measure ROS production, we labeled chondrocytes with a DCFH-DA fluorescent probe (Solarbio). The chondrocytes were taken for frozen sections, and incubated in 10 μmol/L DCFH-DA for 30 min at 37 °C. Observation of stained cells was carried out with a fluorescence microscope (Nikon, Tokyo, Japan). Image J software was used for quantitative fluorescence analysis.

Measurement of OxS-related indicators

The chondrocytes were lysed and centrifuged (4 °C, 3000 rpm) for 15 min, followed by the collection of the supernatants. The activities of superoxide dismutase (SOD) and catalase (CAT) as well as the intracellular malonaldehyde (MDA) were assessed using the corresponding kits (Jiancheng Bio., Nanjing, China; Beyotime) in terms of the manufacturer’s directions.

Enzyme-linked immunosorbent assay (ELISA)

Interleukin (IL)−6 and IL-1β levels released from cultured cells were assessed with specific ELISA kits (Elabscience, Wuhan, China) in according to the kit instructions.

RNA stability analysis

Transfected chondrocytes were treated with IL-1β and actinomycin D (5 μg/mL) for 0, 3, 6, 9, 12 h. After extraction of total RNA, CA12 mRNA levels were analyzed by RT-qPCR.

RNA immunoprecipitation (RIP) assay

Briefly, IL-1β-treated chondrocytes were lysed with RIPA lysis buffer (Beyotime). Cell lysates were immunoprecipitated with an anti-WTAP or anti-IgG antibody at 4 °C overnight. The captured products were obtained for the assessment of CA12 mRNA levels. Execution of the RIP assay was done as per the manufacturer’s instructions using the Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Millipore, Billerica, Massachusetts, USA).

Methylated RIP (MeRIP)-qPCR

For the meRIP assay, a methylated RNA immunoprecipitation kit (BersinBio, Guangzhou, China) and an RNA immunoprecipitation kit (RiboBio, Guangzhou, China) were applied. Isolation of total RNA from IL-1β-treated chondrocytes was undertaken, followed by fragmentation to approximately 300 bp using an ultrasonic cell disruptor. Then, the fragmented RNA was co-immunoprecipitated with an anti-m6A antibody at 4 °C for 4 h, followed by incubation with protein A/G beads for 1 h and eluting by proteinase K to acquire the m6A-modified RNA. RNA samples were subjected to RT-qPCR analysis to analyze CA12 abundance.

Experimental animals

Male C57BL/6 mice (8-week-old; Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were maintained at 21 °C ± 2 °C on a 12-h light/dark cycle and with food and water provided ad libitum. All experimental protocols were performed in compliance with the Institutional Animal Care and Use Committee of the Ganzhou People´s Hospital. Posttraumatic OA mouse models were constructed by destabilization of the medial meniscus (DMM) surgery [19]. After anesthesia, mice in the Sham group underwent a sham operation via an incision in the medial capsule of the knee joint (n = 6). Twelve C57BL/6 mice were separated into 2 groups: DMM and DMM + sh-WTAP.

Adeno-associated virus 9 containing sh-WTAP (sh-WTAP) and its control virus (WZ Biosciences, Jinan, China) was administered to C57BL/6 J mice (10 µl, 1 × 1012 vg/ml) by intra-articular injection. Three weeks later, the DMM surgery was performed to establish posttraumatic OA mouse models. The knee joints of the mice were collected for subsequent experiments at the eighth week after surgery.

Histological analysis

The knee joint specimens were fixed in 10% zinc-buffered formalin overnight, paraffin-embedded, and sectioned into 4-μm sections. After deparaffinization with xylene and gradient hydration, the sections were stained with hematoxylin and eosin (HE) (Beyotime) or safranin O/fast green (Biosharp, Hefei, China) following the corresponding instructions.

Statistical analysis

All experiments were repeated at least 3 times and the results are presented as the mean ± standard deviation (SD). Data were processed using GraphPad Prism (GraphPad, La Jolla, CA, USA). Comparison of the two groups was done using t-test. Comparisons of multiple groups were conducted using one-way or two-way ANOVA followed by Tukey’s post hoc test. Statistical significance was set at p < 0.05.