Source of organization

The articular cartilage tissues of OA patients (n = 22) (8 males and 14 females, age range 65–77 years) in Wujin Hospital Affiliated with Jiangsu University were selected, and the normal tissues were taken from the normal articular cartilage of amputees (n = 22) (10 males and 12 females, age range 61–75 years) due to accidental injuries. All patients gave informed consent. And this experiment was supported by the Ethics Committee of Wujin Hospital Affiliated with Jiangsu University.

Cell culture and transfection

Human chondrocytes (CHON-001) were offered by Nanjing Kebai Biotechnology Co., Ltd. (Nanjing, China). Cells were cultured in DMEM medium (Gibco, Carlsbad, CA, USA) containing 10% FBS (Gibco) at 37℃ with 5% CO2. In this, 10 ng/mL IL-1β was used to stimulate CHON-001 cells for 24 h to construct OA cell models [24, 25].

Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used for cell transfection. Circ_0044235 overexpression vector (oe-circ_0044235), miR-375 mimic or inhibitor (anti-miR-375), small interfering RNA against PIK3R3 (si-PIK3R3) and their controls were obtained from Geneseed (Guangzhou, China).

Quantitative real-time PCR (qRT-PCR)

The total RNA was extracted using TRIzol reagent (Invitrogen) and reversely transcribed into cDNA using cDNA synthesis Kit (Takara, Dalian, China). QRT-PCR was performed using SYBR Green (Takara). Relative expression was calculated by 2−ΔΔCt method with GAPDH or U6 as internal reference. Primers were shown in Table 1.

Table 1 Primer sequences used for qRT-PCRActinomycin D (ActD) and RNase R assays

ActD (2 mg/mL; AAT Bioquest, Sunnyvale, CA, USA) was used to treat CHON-001 cells for indicated times. Then, the RNA was isolated, and qRT-PCR was performed to measure circ_0044235 expression and linear CDC27 mRNA expression.

3 U/µg RNase R (Geneseed) was used to dispose RNA for 30 min. Then, qRT-PCR was used to detect circ_0044235 expression and linear CDC27 mRNA expression.

Cell counting kit-8 (CCK-8) assay

After transfection and treatment for 48 h, the cells were trained with 10 µL CCK-8 solution for 4 h according to the instructions of CCK-8 Kit (Solarbio, Beijing, China). The absorbance at 450 nm was monitored by the microplate reader.

Flow cytometry

After transfection 48 h, CHON-001 chondrocytes were digested by trypsin. The chondrocytes were stained by Annexin V-FITC and PI staining solution (Beyotime, Shanghai, China) for 15 min. Flow cytometry was used to measure the chondrocyte apoptosis rate.

Western blot

The total protein was extracted by RIPA lysis buffer (Beyotime), and then resolved by 10% SDS-PAGE and transferred to PVDF membranes. The membrane was sealed with 5% skim milk powder solution. The membrane was incubated with primary antibody against Bax (1:1,000, ab32503, Abcam, Cambridge, CA, USA), Bcl-2 (1:1,000, ab32124, Abcam), cleaved-caspase-3 (1:1,000, ab2302, Abcam), MMP-13 (1:3,000, ab39012, Abcam), Collagen II (1:1,000, ab34712, Abcam), Aggrecan (1:1,000, ab36861, Abcam), PIK3R3 (1:1,000, H00008503-A01, Abnova, Taiwan, China), or GAPDH (1:2,500, ab9485, Abcam) and cultivated at 4℃ overnight. Then, the membrane was fostered with Goat anti-rabbit or mouse IgG (1:50,000, ab205718 or ab205719, Abcam) at room temperature for 90 min, and ECL luminescent solution (Beyotime) was added for development. The relative protein expression level was calculated using GAPDH as internal reference.

Enzyme-linked immunosorbent assay (ELISA)

According to the manufacturer’s instructions, TNF-α and IL-6 in supernatant were detected by human TNF-α and IL-6 Elisa kits (Hangzhou Lianke Biotechnology Co., Ltd., Hangzhou, China).

Dual-luciferase reporter assay

The wild-type or mutant-type vectors of circ_0044235 or PIK3R3 (circ_0044235 WT/MUT or PIK3R3 3’UTR WT/MUT) were constructed using psiCHECK2 reporter vector. The above vector was co-transfected with miR-NC and miR-375 mimic into CHON-001 chondrocytes, and luciferase activity was estimated according to the instructions of Dual-Luciferase Reporter Gene Assay Kit (Beyotime).

RNA immunoprecipitation (RIP) assay

RIP kit (Millipore, Billerica, MA, USA) was used to examine the binding of circ_0044235 or PIK3R3 to Ago2 protein. CHON-001 cells were collected and lysed, cell extract was incubated with antibody for co-precipitation with magnetic beads-coupled Ago2 antibody or IgG antibody (Millipore). After RNA was extracted, qRT-PCR was used for the detection of circ_0044235, miR-375 and PIK3R3 expression.

Statistical analysis

These results were expressed as the mean ± SD. GraphPad Prism 8.0 was used for statistical analysis, and differences were analyzed by student’s t-test or analysis of variance. P < 0.05 was a significant difference.