OA patients and healthy controls
This study enrolled 52 OA patients (20 males and 32 females, severe stages) and 52 healthy controls (20 males and 32 females) at Affiliated Hospital of Guizhou Medical University between March 2019 and June 2020. This study was approved by Ethics Committee of this hospital. Age ranges of patients and control were both 52 to 66 years, with a median of 59 years. All OA patients were diagnosed for the first time and no severe clinical disorders were diagnosed. Initiated therapy was excluded from the patients. All healthy controls showed normal physiological functions during systemic physiological health test. All patients signed informed consent. According to affected sites, the 52 patients included 20 cases of knee, 14 cases of hip, and 18 cases of feet.
Treatment and synovial fluid preparation
Synovial fluid (about 1.0 ml) was extracted from the affected sites of patients and the corresponding sites of controls prior to therapy. All patents were treated with acetaminophen and physical therapy. Synovial fluid (about 1.0 ml) was also extracted from the affected sites of patients at 1 and 2 months after the initiation of therapy. All synovial fluid samples were kept in liquid nitrogen storage prior to subsequent assays.
Two types of chondrocytes
Chondrocytes derived from an adult OA patient (402OA-05A) and a healthy control (402-05A) were both from Sigma-Aldrich. Chondrocyte growth medium (PromoCell) was used to cultivate cells at 37°C, 5% CO2 and 95% humidity.
Transfections and dual luciferase reporter assay
CYTOR expression vector was constructed using pcDNA3.1 vector (Invitrogen) as backbone. Mimic of miR-10a-5p and miRNA negative control (NC) were the products from Sigma-Aldrich. Two types of chondrocytes were transfected with either 1µg CYTOR expression vector or 50 nM miRNA using lipofectamine 2000 (Invitrogen). NC cells (empty vector- or NC miRNA- transfected cells) and Control (C, untransfected cells) cells were included.
CYTOR luciferase vector was established with pGL3 luciferase vector (Promega) as backbone. Lipofectamine 2000 (Invitrogen) was used to co-transfect CYTOR luciferase vector+NC miRNA (NC group) or CYTOR luciferase vector+miR-10a-5p mimic (miR-10a-5p group), and luciferase activity was measured at 48h later.
RNA isolation and process
Trizol reagent (Invitrogen) was used to isolate total RNA from both synovial fluid samples and chondrocytes, followed by genomic DNA removal performed by incubating with DNase I (Invitrogen) for 2h. Electrophoresis performed using 5% urea-PAGE gels was used to check RNA integrity. OD 260/280 ratios were determined to reflect RNA purity and a ratio close to 2.0 indicates pure RNA.
RT-qPCRs
PrimeScript RT Reagent Kit (Takara Bio) was used for the preparation of cDNA samples with total RNA samples as template. QuantiTect SYBR Green PCR Kit (QIAGEN) was used to perform all qPCRs with 18S rRNA as an internal control to analyze the expression of CYTOR. Expression of miR-10a-5p was analyzed by The All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia) with U6 as an internal control. Each PCR was performed in three technical replicates and 2−ΔΔCt method was used to normalize Ct values of target genes to corresponding internal controls.
Cell apoptosis assay
Two types of chondrocytes were subjected to cell apoptosis assay at 48h post-transfection. In a 6-well cell culture plate, chondrocytes (20,000 cells in 2ml medium per well) were cultivated for further 48h. After that, cells were washed with ice-cold PBS and resuspended in binding buffer. Following Annexin-V FITC and propidium iodide (PI) staining, FACSCalibur instrument was used to analyze cell apoptosis.
Statistical analysis
Unpaired t test was used to compare the expression levels of CYTOR and miR-10a-5p between patients and controls. Heatmaps were plotted using Heml 1.0 software to present the changes in the expression levels of CYTOR and miR-10a-5p during treatment. Data of cell transfection experiments was expressed as mean±SD values of three biological replicates and ANOVA Tukey’s test was used for data comparison. P<0.05 was statistically significant.