Animals and care

The experimental animals used in this study were approved by the Animal Protection and Utilization Committee of the Second Hospital of Shanxi Medical University (Taiyuan, China) (2021010,DW2022063)and strictly adhered to the Guidelines for the Protection and Use of Laboratory Animals issued by the China Animal Science Council. A total of 18 Sprague Dawley (SD) rats and 30 mices with the Col2a1-cre-ERT2 /GFPf1/fl -RFP-miR-1 gene were housed in specific pathogen-free (SPF) barrier facilities under controlled temperature and humidity with alternating 12-h light and dark cycles. Experimental animals were fed SPF-grade chow and sterile drinking water. The behavior and health status of rats and mice were monitored regularly during the experiment. Animals were anesthetized with sodium pentobarbital.

miR-1 small nucleic acid drugs

miR-1 small nucleic acid drugs (miRNA agomir) were supplied by the Guangzhou RIBOBIO Corporation (Guangzhou, China). miRNA agonists regulate target mRNA levels by mimicking endogenous miRNAs.

Animals modeling

Eighteen 8-week-old SD rats were equally divided into three groups: sham-operated, negative control, and experimental groups. Only the joint capsule was cut in the sham-operated group, while the negative control group and experimental group underwent anterior cruciate ligament transection (ACLT). One week after surgery, 50 µL saline was injected into the joint cavity of the sham-operated group, 50 µL agomiR-1 control was injected into the negative control group, and 50 µL agomiR-1 small nucleic acid drug was injected into the experimental group [14, 15]. The rats were euthanized and the articular cartilage tissue was removed from each group of SD rats 8 weeks after modelling. The Col2a1-cre-ERT2 /GFPf1/fl -RFP-miR-1 gene mice were randomly divided into experimental and control groups and the experimental mice were injected with tamoxifen (Sigma Aldrich, USA), 100 uL, at a concentration of 20 mg/mL for 5 d at 8 weeks, while the control mice were injected with only an equal amount of corn oil. Fifteen days later, the experimental and control groups were simultaneously underwent destabilization of medial meniscus (DMM) to induce OA in the knee of mice [16]. After 8 weeks of modeling, the mice were euthanized and the articular cartilage was removed.

Immunohistochemistry

Paraffin specimens were serially sectioned 5 μm thick, followed by immunohistochemical staining to detect the expression levels of target proteins, followed by the primary antibody: rabbit anti-mouse anti-MMP-13 (1:100 cat. no. bs-10581R, Bioss).

TMT-based proteomics of knee cartilageExtraction and digestion of cartilage proteins from knee joints

Mouse knee cartilage tissue was well ground in liquid nitrogen, followed by the addition of phenol extraction solution and centrifugation. The upper layer was added to 5 times the volume of pre-cooled 0.1 M ammonium acetate-methanol solution. After centrifugation, the precipitate was washed with methanol, followed by removal of the methanol with acetone and centrifugation. The sample lysate was added to dissolve the precipitate and the protein concentration was determined using a BCA protein assay kit (PC0020, Solarbio, Beijing, China). DTT at a concentration of 5 mM was added to the sample, incubated for 30 min and the same volume of iodoacetamide was added, followed by acetone at a ratio of 1:6. The sample was centrifuged and the precipitate was added to 100 µL TEAB (200 mM) and digested overnight with 1/50 of the sample mass of trypsin.

TMT labeling and high-performance liquid chromatography (HPLC) fractionation

After tryptic digestion, peptides were reconstituted in TEAB (pH6.5)and labeled using a TMT kit (90066B, ThermoFisher, Shanghai, China). The labeled peptides were fractionated using high-performance reverse liquid chromatography (HPLC) system (Thermo Fisher Scientific, Waltham, MA, USA). The peptides were graded on an Agilent Zorbax Extend - C18 narrow diameter column (Agilent Technologies Co. Ltd, Beijing, China) and dried under vacuum.

LC-MS/MS analysis

The peptides were sampled at a flow rate of 300 nL/min onto a pre-column Acclaim PepMap100 100 μm × 2 cm (RP-C18, Thermo Fisher Scientific) and then separated using an analytical column (Acclaim PepMap RSLC, 75 μm × 15 cm; RP-C18, Thermo Fisher). The peptides were analysed by Thermo Scientific Q Exactive mass spectrometer (Thermo Fisher Scientific) and the intact peptides were detected using Orbitrap at 70000 resolution.

MS/MS spectrum collection is completed using a data-dependent positive ion mode for high-energy collision fragmentation with MS/MS resolution set to 17500.

Raw data analysis

Proteome Discoverer 2.4 (Thermo Fisher Scientific) was used to identify and analyze the MS/MS raw data. Carbamidomethyl (C), TMT6plex (N⁃ term) were selected as fixed modifications and oxidation (M) as variable modifications; up to 2 missed cuts were allowed for the digested peptide fragments, and the error range was controlled within 10 ppm for the parent ion and 0.02 Da for the peptide ion fragments. The error range is within 10 ppm for the parent ion and 0.02 Da for the peptide ion fragment.

Bioinformatics analysis

Candidate differentially-expressed proteins (DEPs) were identified using 1.2-fold change and p < 0.05 thresholds, followed by analysis of DEPs using bioinformatics tools. Heat maps and volcano plots of differentially expressed proteins were plotted using R software [17]. Principal component analysis (PCA) of differential proteins was subsequently performed using SPSS (v.26.0) software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed proteins was conducted using the “clusterProfiler” and “DOSE” tools of R software [18,19,20]. Protein-protein interaction network analysis was performed using STRING online, and visualization of the network was performed using Cytoscape (v.3.6.0) software [21, 22].

Primary chondrocyte isolation, culture, and RT-qPCR

Remove the mouse knee cartilage tissue with a blade, frozen it in liquid nitrogen, and ground, and RNA was extracted using the RNAeasy™ Animal RNA Isolation Kit with Spin Column (R0026,beyotime,shanghai,china) according to the manufacturer’s instructions. A portion of the samples were reverse transcribed into cDNA using the HyperScript™III miRNA 1st strand cDNA Synthesis Kit (by stem-loop) (R601, EnzyArtisan, Shanghai, China). The RNA was subsequently reverse transcribed into cDNA and the expression of the target gene in the cDNA was detected by RT-qPCR as previously described [23]. The qPCR conditions were as follows: preincubation of samples at 95 °C for 30 s, and then 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 45 s, and dissolving at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. miRNA-1 qPCR conditions were as follows: Preincubation of samples at 95 °C for 30 s, and then 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and dissolved at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s.Primer sequences for RT-qPCR were synthesized by Shanghai Biotech Shanghai China, and the sequences are shown in Table 1.

Table 1 Primers for used for RT-qPCRStatistics

The data is expressed as mean ± standard deviation and statistically analyzed using SPSS software (version 22.0) and Graph Prism software (version 8.0). Results were compared with an independent t-test., where * represents P < 0.05 and is statistically significant.