Patients and tissue samples
Cartilage samples were obtained from 16 OA patients (7 men and 9 women, age range 54-72 years) who underwent total knee replacement at the Affiliated Nanping First Hospital of Fujian Medical University. Normal articular cartilage samples were isolated from 10 donors (5 men and 5 women, age range 45–67 years), who underwent trauma without a history of OA or rheumatoid arthritis. All cartilage samples were immediately frozen and stored in liquid nitrogen. The diagnosis of OA patients was made according to the criteria of the American College of Rheumatology. OA severity was graded using weight-bearing anteroposterior radiographs of the affected knee.
Cell culture and treatment
Human primary chondrocytes were isolated from cartilage tissues as previously described [24]. In brief, cartilage tissues were firstly digested with 0.25% trypsin (Invitrogen, Carlsbad, CA, USA) for 30 min, followed by 0.2% type II collagenase (Invitrogen) for 4 h at 37°C. The 293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The obtained chondrocytes and 293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA), with 10% fetal bovine serum (Invitrogen), 1% penicillin-streptomycin (Sigma, St. Louis, MO, USA), and 1% l-glutamine (Invitrogen) at 37°C and 5% CO2 in a humidified atmosphere. The chondrocytes were stimulated with 10 ng/ml of IL-1β (Sigma-Aldrich, St. Louis, MO, USA) for 12 h.
Cell transfection
Lentiviral particles expressing sh-circPRKCH or HGF were designed and synthesized by GenePharma (Shanghai, China). The miR-145 mimics, miR-145 inhibitor, and negative controls were purchased from RiboBio (Guangzhou, China). When cells grew to 60% confluence, the transfection was performed using Lipofectamine 2000 reagent (Invitrogen). Cells were harvested after 48 h. After at least 1 week of viral infection, stable cell clones could be selected with antibiotics (puromycin, 2–5μg/ml, Sigma).
MTT assay
Cell viability was assayed by MTT assay. The cultured chondrocytes were inoculated in 96-well plates (1×105 cells/well). Then, 20 μl (500 μg/ml) MTT solution (Sigma) was added to each well at 37°C for 4 h. After MTT removing, cells were mixed with 150 μl of dimethyl sulfoxide (DMSO, Sigma) for 10 min. A microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to detect the optical density at a wavelength of 490 nm.
Flow cytometry assay
Cell apoptosis was detected using the Annexin V-FITC/propidium iodide (PI) apoptosis kit (Invitrogen). Briefly, chondrocytes were cultured for 48 h, digested using trypsin without EDTA (Thermo Fisher Scientific), and washed with PBS. The cells were collected and adjusted to a cell density of 1 × 106 cells/ml. Then, cells were stained with Annexin V-FITC and PI for 15 min. At last, a BD FACS flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect the fluorescence intensity.
Dual-luciferase reporter assay
Artificially synthesized circPRKCH wild-type and mutant-type (namely circPRKCH-WT/MUT) and HGF 3′-UTR wild-type and mutant-type (HGF 3′-UTR-WT/MUT) constructs containing the putative binding sites of miR-145 (GenePharma, Shanghai, China) were inserted into psiCHECK2 vectors (Promega, Madison, WI, USA). Lipofectamine 2000 (Invitrogen) was then used to co-transfect miR-145 mimics or NC mimics with wild-type or mutant constructs into 293T cells. Forty-eight hours after transfection, luciferase activity was detected using the luciferase reporter gene analysis system (Promega).
Animal experiments
Specific-pathogen-free (SPF) grade male C57BL/6 mice (8 weeks old; 18–22 g) were purchased from SJA Laboratory Animal Co., Ltd (Hunan, China, n=20), which were housed in ventilated racks at 21–22 °C with a 12-h light/12h dark cycle. Sterile food and water were provided. OA was induced by injecting monosodium iodoacetate into the right knee joint of mice. Briefly, after deep anesthetization with isoflurane (2–4 %), mice were injected with monosodium iodoacetate (Sigma-Aldrich) (3 mg in saline). Control mice were injected with an equivalent volume of PBS. As described previously [17], the mice were randomly divided into four groups: control, OA, OA+sh-NC, and OA+sh-circPRKCH (n=5 each group). Lentiviral particles expressing shRNA targeting circPRKCH were injected into the mouse tail vein. The OA mice in the sh-NC group were injected with empty lentiviral particles in the same way. After 8 weeks of operation, all mice were sacrificed by CO2 inhalation. Left knee joint tissues were separated and processed for further experiments.
Histological examination
The cartilage tissue specimens were fixed in 4% paraformaldehyde for 24 h. The specimens were dehydrated and embedded in paraffin. Tissue blocks were then sliced into sections (5 μm thick). After that, the sections were deparaffinized in xylene and rehydrated with graded ethanol. Then, the sections were stained with hematoxylin for 5 min, followed by immersing in 1% acidic ethanol (1% HCl in 70% ethanol) for 20 s and rinsing with distilled water for 2 min. Subsequently, the sections were stained with eosin solution for 3 min, dehydrated, and mounted. The slices were photographed with an Olympus light microscope (Olympus, Tokyo, Japan).
To assess cartilage destruction, each section was stained with Safranin O-Fast Green solution (Sigma) for 4 min. After being washed in distilled water, sections were dehydrated in graded ethanol and mounted from xylene. Pathological changes were observed under a microscope (Olympus).
RNA extraction and real-time quantitative PCR
Trizol reagent (Invitrogen) was used to extract total RNA. PrimeScript™ RT Kit (Takara, Kyoto, Japan) was used for reverse transcription of circPRKCH and HGF. Reverse transcription of miR-145 was conducted using a Mir-XTM miRNA First Strand Synthesis Kit (Takara). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, for circPRKCH and HGF) and U6 small nuclear RNA (U6, for miR-145) were used as internal controls. The qPCR was conducted using the SYBR Green PCR Kit (Takara). Relative quantitative analysis of gene expression was calculated by the standard 2-∆∆Ct method. The following primers: circPRKCH F: 5′-GTCTACCCTACCTGGCTCCAT-3′, R: 5′-CATTGCAAATCCCCTCCTTGC-3′; miR-145 F: 5′-TCCAGTTTTCCCAGGAATCCCT-3′, R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′; HGF F: 5′-AAATCCTCGAGGGGAAGAAG-3′, R: 5′-AGCCCTTGTCGGGATATCTT-3′; GAPDH F: 5′-CATCATCCCTGCCTCTACTGG-3′, R: 5′-GTGGGTGTCGCTGTTGAAGTC-3′; U6 F: 5′-CTCGCTTCGGCAGCACA-3′, R: 5′-AACGCTTCACGAATTTGCGT-3′. The data were analyzed using the standard 2-∆∆Ct method. The experiment was performed in triplicate.
Western blot assay
Cells were lysed and protein was extracted using RIPA buffer (Invitrogen). In addition, the BCA protein assay kit (Beyotime Bio, Shanghai, China) was used to detect the concentration of protein. Equal amounts of protein (30 μg) samples were separated using 10% SDS-PAGE gels and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk, the membranes and the following primary antibodies were incubated overnight at 4°C: anti-HGF (EPR12230, 1:1000, Abcam, Cambridge, MA, USA), anti-MMP-3 (EP1186Y, 1:1000, Abcam), anti-MMP-13 (1: 2000; Abcam), anti-Aggrecan (6-B-4, 1:1000, Abcam), anti-Collagen II (EPR12268, 1:1000, Abcam), anti-c-MET (EPR19067, 1:1000, Abcam), anti-p-c-MET (EP2367Y, 1:1000, Abcam), and GAPDH (6C5, 1:2000, Abcam). GAPDH protein was used as the inner control. After washing, the membranes were incubated with the HRP-conjugated secondary antibody at 1:2000 dilution for 1 h at room temperature. The resulting bands were detected with an ECL detection system (Millipore).
Statistical analysis
Graphpad Prism 7.0 software (Graphpad, La Jolla, CA, USA) was executed to analyze all data. In this study, each experiment was repeated independently at least three times. Data were presented as mean ± standard deviation (SD). Statistical differences between the two groups were assessed by Student’s t test. In addition, a one-way analysis of variance (ANOVA) followed by Tukey post hoc was carried out for multiple group comparisons. When P < 0.05, it was considered to be statistically significant.