OA patients and controls
Research subjects of this study included 60 OA patients (40 males and 20 females) and 60 healthy controls (40 males and 20 females). All patients and controls provided informed consent. All patients were admitted to Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from May 2018 to May 2020. Age range of OA patients was 41 to 60 years, with a median of 51 years. Age range of healthy controls was 41 to 60 years, with a median of 52 years. All patients were excluded from initiated therapy with 3 months before admission and other severe clinical disorders. No recurrent cases were enrolled, and all patients were newly diagnosed cases. Among the 60 OA patients, hip was affected in 31 cases and knee was affected in 29 cases. Healthy controls showed normal physiological functions after systemic physiological exams at the aforementioned hospital. Ethics Committee of aforementioned hospital approved this study.
Synovial fluid and chondrocytes
Prior to therapy, a syringe was used to extract synovial fluid (2ml) from the affected sites of patients. To perform control experiment, synovial fluid was also collected from the corresponding sites of healthy controls (31 cases of hip and 29 cases of knee). Liquid nitrogen storage was performed prior to the subsequent assays.
Primary chondrocytes from Sigma-Aldrich (Cat# 402OA-05A, USA) were included in this study to serve as the cell model of OA. These chondrocytes were from an adult with OA. Cell culture was performed following manufacturer’s instructions. In cases of LPS treatment, cells were cultivated in medium supplemented with 0, 2, 4, 6, 8 and 10 µg/ml LPS for further 48h prior to the subsequent experiments.
Vectors, siRNAs and transfections
With pcDNA3.1 vector (Invitrogen) and pcDNA3.1(+) CircRNA Mini Vector (Addgene) as backbone, expression vectors of circZNF652 and PTEN were constructed, respectively. SiRNA negative control (NC) and circZNF652 siRNA were purchased from Invitrogen. Chondrocytes (108) at around 80% confluence were subjected to expression vector (1μg) or NC miRNA (40 nM) transfection using lipofectamine 2000 (Invitrogen). NC experiments were performed by transfecting the same amount of empty vector or NC siRNA into the same number of cells. In all transfections, control (C) cells were untransfected cells. In fresh medium cells were cultivated in fresh
medium for 48h prior to the subsequent assays.
Isolation of RNA from synovial fluid and chondrocytes was performed using RNAzol (Sigma-Aldrich), followed by genomic DNA removal performed by DNase I digestion at 37°C for 2h. RNA integrity was analyzed by 5% urea-PAGE gel electrophoresis.
RNA purifty was analyzed by measuring OD 260/280 rations. RNA samples with an OD 260/280 ratio close to 2.0 (pure RNA) were subjected to reverse transcriptions using SS-IV-RT system (Invitrogen). SYBR® Green Quantitative RT-qPCR Kit (Sigma-Aldrich) was used to perform all qPCRs with GAPDH as an internal control to determine the expression of circZNF652 and PTEN mRNA. Three technical replicates were included in each reaction. Ct value normalizations were performed using 2-ΔΔCT method.
PIPA solution (Invitrogen) and BCA assay (Invitrogen) were used to extract and quantify protein from chondrocytes. In boiling water RNA samples were incubated for 12 min to achieve protein denaturation. Electrophoresis was performed using 10 % SDS-PAGE gel, followed by using PVDF membranes to transfer proteins. Following blocking in 5% fat-free milk for 2h, GAPDH (1: 1600, ab37168, Abcam) and PTEN (1: 1600, ab31392, Abcam) primary rabbit antibodies were used to incubate membranes overnight at 4 °C. After that, anti-rabbit IgG-HRP (1:1600, MBS435036, MyBioSource) secondary antibody was used to incubate membranes for 2h. ECL (Sigma-Aldrich) was used to develop signals and Image J v1.46 software was used for data normalizations.
Cell apoptosis analysis
Chondrocytes with transfections were subjected to cell apoptosis analysis through cell apoptosis assay. A 6-well cell culture plate was used to cultivate chondrocytes (20,000 cells per well) in medium containing 10 µg/ml LPS for further 48h at 37°C. Following 0.25% trypsin digestion, propidium iodide (PI, Dojindo, Japan) and Annexin V-FITC (Dojindo, Japan) staining was performed, followed by flow cytometry to analyze cell apoptosis.
Expression levels of circZNF652 and PTEN mRNA in synovial fluid samples from both OA patients (n=60) and healthy controls (n=60) were expressed as average values of three technical replicates and unpaired t test was used for data comparison. Data of multiple cell transfection groups and LPS treatment groups was expressed as mean +/- SD values of three biological replicates, and ANOVA Tukey’s test was used for data comparisons. Correlations were analyzed by Pearson’s correlation coefficient. P<0.05 was deemed statistically significant.