Patient samples

A total of 71 human OA cartilages and 33 normal cartilages were obtained from individuals undergoing knee arthroplasty and trauma patients without the history of OA or rheumatic arthritis, respectively. OA was diagnosed according to the American College of Rheumatology criteria [15]. All cartilage samples were obtained from the medial side of the knee joint. The specimens were further processed for histological examination and were categorized according to the modified Mankin scoring system [16]. Of them, three cartilage specimens of each group (pathological vs. control) were randomly selected for miRNA solexa sequencing. The Ethics Committee of our institution approved this study protocol, and written informed consent was obtained from each participant.

MicroRNA-sequencing analysis

The Illumina HiSeq 2000 platform (San Diego, CA, USA) was employed to purify three OA patients and three normal controls for miRNA-sequencing analysis, following the manufacturer’s directions. The OA and control samples showed homogeneity in the selection of demographic factors, including gender, age, and body mass index. MiRNA sequencing was conducted utilizing miRBase 21.0. For quality control and library preparation for Illumina sequencing, a minimum quantity of 500 ng of total RNA was necessary. Following the purification of tiny RNA molecules (less than 30 nucleotides) using PAGE purification, a pair of solexa adaptors were ligated to their 5′ and 3′ ends. The amplification of small RNA molecules was conducted employing adaptor primers for a total of 17 cycles. Subsequently, fragments with an estimated length of 90 bp, which included both the small RNA and adaptors, were extracted from an agarose gel. The DNA that had undergone purification was utilized for the purpose of cluster creation and subsequent sequencing analysis. The image files created by the sequencer were then subjected to processing in order to provide data of high digital quality. Following the masking of adaptor sequences and elimination of contaminated reads, the clean reads underwent computational analysis.

Cell culture and transfection

Chondrocytes were extracted as described before [17]. Primary human chondrocytes were acquired from human OA cartilage (damaged medial femoral condyle) and normal controls. The articular cartilage of the medial femoral condyle in mouse knee with OA was dissected employing a surgical microscope, with the objective of precisely isolating the cartilage while avoiding the underlying subchondral bone. Dissected articular cartilage was subjected to enzymatic digestion to acquire the primary chondrocytes. In brief, PBS was utilized to wash the articular cartilage that underwent dissection, and then it was subjected to a 15-min incubation at 37 °C in trypsin-ethylenediaminetetraacetic acid (EDTA). This was followed by utilizing 2 mg/mL collagenase at 37 °C for 2-h digestion in Dulbecco’s modified Eagle’s medium treated with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin in a 5% CO2 atmosphere. Throughout the culturing time, cells were maintained at 37 °C in an environment of 5% CO2 and 95% air, with medium changes occurring every 2–3 days. The identical medium as OA chondrocytes was employed to preserve the C28/I2 cells.

Human chondrocytes and C28/I2 cells underwent transfection with miR-653-5p mimics labeled or unlabeled with Cy3 utilizing the Silencer® siRNA Labeling Kit (AM1636), miR-653-5p inhibitor, and their negative controls (Thermo Scientific Dharmacon ®) at 50 nM utilizing Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Life Technologies, CA, USA). Then, the cells were utilized for the subsequent investigations at 48 h (normal chondrocytes) or 72 h (OA chondrocytes) following the transfection. The establishment of OA cells of C28/I2 cell line used the method of IL-1 (1.5 ng/L, Sigma Aldrich, Mo, USA) stimulation. The cells were employed for subsequent experiments after stimulating the cells with IL-1 for one day.

RNA isolation, cDNA synthesis, and quantitative real-time PCR analysis (qRT-PCR)

MiRNeasy Mini Kit (Qiagen, Valencia, CA, USA) was utilized to isolate the total RNA from cartilage tissues and cultivated cells following the manufacturer’s directions. Nanodrop (Thermo Scientific, Waltham, MA, USA) and Bioanalyzer (Agilent Inc., Santa Clara, CA, USA) were employed to detect the quantity and quality of RNA. TaqMan microRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) in a final volume of 15 µl was employed to generate cDNA (16 °C for 30 min, 42 °C for 30 min, 85 °C for 5 min, and hold at 4 °C). The experimental procedure included conducting all reactions in triplicate utilizing a 7500 real-time system (Applied Biosystems, CA, USA). The reactions were performed under specific conditions, which included a first denaturation step for a duration of 10 min at 95 °C, 40 cycles of denaturation at 95 °C for 15 s, and subsequent annealing/extension for a period of one minute at 60 °C. The comparative Ct (ΔΔCt) technique (2−ΔΔCt with logarithm transformation) was utilized to conduct data analysis. The specific primers are as follows: miR-653-5p: 5′-GTGTTGAAACAATCTCTACTG-3′ and 5′-TCCACGACACGCACTGGATACGAC-3′, U6 snRNA: 5′-GTGCTCGCTTCGGCAGCACAT-3′ and 5′-TACCTTGCGAAGTGCTTAAAC-3′.

5-Ethynyl-2′-deoxyuridine (EdU) assay

EdU assay with a Kit of EdU Staining Proliferation and Alexa Fluor 555 (Beyotime, Shanghai, China) was employed to detect Cell proliferation depending on the direction of the manufacturer. Concisely, chondrocytes were introduced into 24-well plates at a density of 2 × 105 per well. The plates were then incubated at 37 °C in 5% CO2. Following this, a concentration of 50 µM of EdU was administered to each well for 2 h. Following that, Hoechst 33,258 (Beyotime, Shanghai, China) was utilized to stain the cells. The fluorescence microscope (Olympus, Japan) was used to evaluate the ratio between EdU-positive cells and total Hoechst 33,258-positive cells.

Senescence-associated β-galactosidase (SA-β-Gal) assay

The SA-β-Gal staining was conducted employing the cell senescence β-galactosidase staining kit (Beyotime Biotechnology, China), based on the guidelines provided by the manufacturer. In a concise manner, the cells underwent a washing process utilizing PBS and were subsequently fixed with a solution comprising 2% paraformaldehyde and 0.2% glutaraldehyde for 5 min. Then, the cells underwent a washing procedure and were exposed to a staining solution containing SA-β-Gal for 16 h at 37 °C. Following the incubation, the cells underwent a process of washing, and a Nikon Eclipse Ni-U microscope was utilized to image the cells.

Fluorescence in situ hybridization (FISH)

A locked nucleic acid probe with complementarity to miR-653-5p was labeled with 5′ and 3′-digoxigenin, and Exiqon (Woburn, MA, USA) was utilized for synthesis. The chondrocytes from OA patients and normal controls were employed for FISH detection. After seeding the cells on glass slides to complete the sample preparation, FISH Tag RNA Multicolor Kit (F32956, Alexa Fluor™ dye combination, Invitrogen, USA) was utilized for the detection based on the manufacturer’s direction.

A PNA probe (Panagene, F1006) was utilized to conduct Telomere FISH analysis. Chondrocytes were seeded onto glass slides in six-well culture plates and subjected to 2 h incubation at 37°. The adhered cells then received KCl buffer treatment to induce swelling, fixed in a solution of methanol and acetic acid (3:1), rehydrated in PBS, and subsequently, 4% formaldehyde was utilized for fixation. Dehydration was achieved by a sequential application of ethanol concentrations. The slides were then exposed to a hybridization mixture comprising 10 mM NaHPO4 (pH 7.4), 10 mM NaCl, 70% formamide, and 20 mM Tris’s buffer (pH 7.5). To denature the chromosomal DNA, the slides were positioned for 5 min on an 80 °C heating block. Subsequently, the PNA probe was applied to the slides and subjected to 2 h incubation at room temperature. After a comprehensive cleaning process, the slides were prepared for examination by being mounted with Vectashield mounting media that included 4,6-diamidino-2-phenylindole (DAPI) (Vector Labs). Subsequently, a confocal microscope (Carl Zeiss, Oberkochen, Germany) was employed to analyze the slides.

Mitochondrial transmembrane potential was measured by FISH using the JC-1 probe. JC-1 monomers were green fluorescence, and JC-1 mitochondrial aggregates were red fluorescence. This FISH test was conducted utilizing a mitochondrial membrane potential assay kit (Beyotime Biotechnology, China). For JC-1 staining, 1 × 106 cells were subjected to 10 min incubation with 10 mg/ml JC-1 37 °C, and a confocal microscope (Carl Zeiss, Oberkochen, Germany) was utilized to analyze the cells for red and green fluorescence.

3′-Untranslated region (UTR) cloning and luciferase assay

To generate the wild-type (WT) IL-6 3′UTR-Luc reporter plasmid (IL-6 3′UTR), we amplified a fragment of the IL-6 gene’s 3′UTR, which contained the predicted miR-653-5p binding location, via PCR. Subsequently, the fragment that underwent amplification was cloned downstream of the firefly luciferase gene in the psi-CHECKTM-2 vector (Promega, Madison, WI) utilizing XhoI and NotI enzymes (Thermo Fisher Scientific). To create constructs with mutations in the putative miR-653-5p binding location of the WT IL-6 3′UTR, the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, CA, USA) was employed to conduct site-directed mutagenesis. Following the PCR, a 20 µL portion of the reaction mixture underwent digestion with DpnI at 37 °C for one hour. Subsequently, a 10 µL fraction was subjected to transformation into DH5 alpha Escherichia coli in order to generate the mutant construct plasmids. The confirmation of the authenticity of all constructions was achieved via the process of sequencing (Cosmogenetech, Seoul, Korea). In the luciferase examination, human primary chondrocytes and C28/I2 cells were evenly seeded and distributed at a concentration of 3000 cells per well in a 96-well plate. The cells were subjected to co-transfection with either the WT or mutant-type IL-6 3′UTR-Luc reporter plasmid, along with either the miR-control or miR-653-5p, employing Lipofectamine PLUSTM reagent (Invitrogen). Cell lysates were obtained two days post-transfection, and luciferase activity was quantified employing the Dual-Glo Luciferase Assay kit (Promega, WI, USA), depending on the guidelines of the manufacturer. The firefly luciferase activity was employed to standardize the luciferase activity.

Western blotting

The Western blot analysis was conducted in accordance with established protocols. In this study, proteins were subjected to separation on a 10% SDS-PAGE gel. Subsequently, the separated proteins were transferred onto PVDF membranes (Amersham, Buckinghamshire, UK). These membranes were then subjected to a blocking step employing 5% non-fat dried milk for a duration of 2 h. Following the blocking step, the membranes were incubated with primary antibodies for a period of 12 h. The primary antibodies employed in this investigation included the following: anti-IL-6 antibody (1:1000, Cell Signaling Technology, #12,912), anti-COL2A1 antibody (1:500, Abcam, ab34712), anti-MMP13 antibody (1:1000, Santa Cruz Biotechnology, sc-515,284), anti-p16INK4a antibody (1:1000; Abcam, ab270058), anti-β-actin antibody (1:1000, Cell Signaling Technology, #4970), anti-p-STAT3 antibody (1:1000, Santa Cruz Biotechnology, sc-293,059), anti-STAT3 antibody (1:1000, Santa Cruz Biotechnology, sc-8019), anti-p-JAK1 antibody (1:1500, Abcam, ab138005), and anti-JAK1 antibody (1:1000, Abcam, ab133666). Following the washing step in TBST (composed of 10 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Tween 20), the membranes were subjected to a 2-h incubation with horseradish peroxidase conjugated anti-rabbit or anti-mouse antibody (1:2000; Abcam, ab205718, ab205719). The process of normalization was conducted utilizing blotting on identical membranes using an antibody against β-actin. The quantification of relative expression was performed employing Quantity One program, specifically version 4.52 (Bio-Rad).

Establishment of OA Model and Mir-653-5p treatment

The C57BL/6J mice were kept in a controlled environment with a 12-h light/dark cycle. They were provided unlimited access to standard mice food and water. Subsequently, a surgical intervention was conducted in order to produce a model of experimental OA in mice that were 10 weeks old [18]. The procedure included the use of a surgical microscope to accomplish destabilization of the medial meniscus (DMM) in the right knee joints under general anesthesia. In addition, sham procedures were conducted by surgically opening and exposing the right knee structures, followed by the subsequent closure of the articular capsule and skin incision without any disruption to the joint tissue.

For miR-653-5p treatment of experimental OA, 10 µL (200 nmol) volume agomiR-653-5p, antagomiR-653-5p, or their negative controls (GenePharma, China) were administrated into the knee joint employing a 33G needle and a micro-syringe (Hamilton). The mice were treated with their first injection 7 days after DMM. Subsequently, the second and third injections were administered during the second and third weeks after DMM, respectively. The mice were euthanized at the end of an 8-week treatment and then underwent histopathological investigation. The Institutional Animal Care and Use Committee of the University of Southeast authorized all experiments on mice in our investigation procedure.

Immunohistochemistry (IHC) and histochemical staining

The cartilage specimens were subjected to fixation in a solution containing 4% paraformaldehyde, followed by decalcification employing a solution containing 10% EDTA. The dehydrated specimens underwent embedding in paraffin, followed by the cutting of pieces of 5 mm in thickness. The slides underwent quenching in a solution containing 3% H2O2 in methanol, followed by three rinses in PBS following the processes of deparaffinization and rehydration. The slides were subsequently exposed to antigen retrieval employing trypsin at 37 °C for 20 min. Following three rinses in PBS, the slides were subjected to a 30-min incubation with a blocking reagent. The slides were subjected to incubation at 4 °C overnight with primary antibodies [MMP3 (1:500, Santa Cruz Biotechnology, sc-21,732); TNF-α (1:1000, Abcam, ab27026)]. The sections next received successive treatment with a biotinylated secondary antibody and a streptavidin-peroxidase conjugate and developed employing a DAB Substrate Kit for peroxide (Vectastain Universal ABC Kit; Vector). The negative control segments were subjected to incubation with an IgG isotype control.

The tissues underwent embedding in paraffin, followed by sectioning and subsequent staining with hematoxylin-eosin and Safranin O/fast green. The decalcified cartilage specimens were subjected to staining with Safranin O and then assessed employing the osteoarthritis research society international (OARSI) grading method with scores ranging from 0 to 6 [19].

Immunofluorescent analysis

The cultured cells received treatment with a fixative solution consisting of 4% paraformaldehyde. Subsequently, they were permeabilized using a PBS solution comprising 0.25% Triton X-100 for 10 min at room temperature. Nonspecific binding sites were blocked using goat serum. The cells were subjected to overnight incubation with primary antibodies at 4 °C. The main antibodies utilized in the investigation included anti-COL II antibody (1:100, Sigma Aldrich, AB2031), anti-aggrecan antibody (1:1000, Santa Cruz Biotechnology, sc-33,695), anti-MMP13 antibody (1:2000, Abcam, ab39012), anti-ADAMTS5 antibody (1:1000, Abcam, ab246975), anti-IL-6 antibody (1:1000, Cell Signaling Technology, #12,912), anti-p21 antibody (1:100, Abcam, ab109520), and anti-p16INK4a antibody (1:1000; Abcam, ab270058). The cells underwent three rinses with PBS and were then treated with goat anti-rabbit IgG (H&L) combined with either Alexa Fluor 555 (1:100, Abcam, ab150078) or Alexa Fluor 488 (1:500, Abcam, ab150077). Following the washing procedure, the nuclei were then subjected to counterstaining with DAPI (Invitrogen) for 5 min. A confocal microscope (Carl Zeiss, Oberkochen, Germany) was utilized to visualize the fluorescence.

Statistical analysis

For statistical analysis, the GraphPad Prism 7 program (GraphPad Software, San Diego, CA, USA) was utilized. Normal distribution of the data was verified employing the Shapiro-Wilk test. Comparison of statistical variations between the two experimental groups was detected by two-tailed unpaired Student’s t-test (for parametric data). Statistical analysis was utilized to compare multiple groups with one-way analysis of variance (ANOVA) followed by Tukey’s post hoc, Sidak’s post hoc or Bonferroni’s post hoc test. A P value of < 0.05 was considered statistically significant.