OA patients and controls
Research subjects of this study included 60 OA patients (20 males and 40 females, 55 to 71 years, 63.0 ± 5.3 years) and 60 healthy controls (20 males and 40 females, 56 to 71 years, 63.3 ± 5.1 years) who were admitted to Jiujiang University Clinical Medical College, Jiujiang University Hospital between March 2016 and June 2019. This study passed the review of Ethics Committee of aforementioned hospital. OA patients’ inclusion criteria: 1) patients who were diagnosed for the first time; 2) no therapies were initiated before admission. OA patients’ exclusion criteria: 1) other clinical disorders were diagnosed; 2) recurrent OA. The 60 OA patients included 27 cases of stage III and 33 cases of stage IV. According to the affected sites, there were 31 knee-affected cases and 29 hip-affected cases. The diagnosis of OA was performed through conventional methods, such as joint fluid analysis and X-ray imaging. Controls were enrolled to match the age and gender distributions of OA patients. All participants were informed of experimental design of this project. All patients and controls signed informed consent.
Synovial fluid
Before the initiation of therapies, extraction of synovial fluid from the affected sites was performed on all patients. To match OA patients, extraction of synovial fluid from knee was performed on 31 controls and extraction from hip was performed on the rest 29 cases. A liquid nitrogen sink was used to store the samples before use.
Chondrocytes and transient transfections
Primary chondrocytes (402OA-05A) from an OA adult were purchased from Sigma-Aldrich (USA) and were cultivated under conditions of 37 °C with 5% CO2 in Chondrocyte Growth Medium (PromoCell). Cells were harvested at passage 5 to 7 to perform following experiments.
CASC2 expression vectors were constructed using pcDNA3.1 vector (Sangon) as backbone. Synthesis of miR-93-5p mimic and miRNA negative control (NC) was performed by Sangon. Lipofectamine 2000 Transfection Reagent (Invitrogen) was used to transfect 50 Nm miRNA (miRNA NC as NC group) or 10 Nm vector (empty vector as NC group) into 106 cells. Control© cells were untransfected cells. The interval between following experiments and transfections was 24 h.
Luciferase reporter assay
CASC2 full length cDNA was cloned into pGL3 plasmids (Promega). Through the aforementioned methods, cells were transfected with CASC2 vector+ miR-93-5p mimic or CASC2 vector+miRNA NC. Luciferase activity was measured by Dual Luciferase Reporter Assay Kit (Promega Corporation) using cells harvested at 24 h post-transfection.
RNA samples and qPCR
RNAiso Plus kit (Takara) was used to extract total RNA from synovial fluid specimens and in vitro cultivated cells. To harvest miRNAs, 85% was used to precipitate and wash RNA samples. To remove genomic DNA, all RNA samples were digested with DNA eraser at 37 °C for 1 h. In cases of LPS treatment, chondrocytes were treated with LPS at a concentration of 0, 200, 500 and 100 ng for 24 h before use.
Total RNA reverse transcriptions (RTs) were performed using PrimeScript RT Master Mix (Takara) with total RNA as template to synthesize cDNA samples. With cDNA samples as template, qPCR mixtures were prepared using SYBR Premix Ex TaqTM II (Takara, Japan) to measure the expression levels of CASC2. 18S rRNA was used as the endogenous control of CASC2.
To measure the expression levels of mature miR-93-5p, polyadenylation, RTs and qPCR mixtures were performed using All-in-One™ miRNA qRT-PCR Reagent Kit (Genecopoeia). U6 was used as the endogenous control of miR-93-5p.
PCR reactions were performed in 3 replicates and fold-changes of gene expression were calculated using 2−ΔΔCt method.
Cell apoptosis analysis
Cells were harvested at 24 h post-transfection and cell suspensions (3 × 105/ml) were prepared using non-serum cell culture medium. Cells were seeded onto a six-well plate with 2 ml cell suspension per well, followed by the addition of 1 μg/mL LPS. Following cell culture under aforementioned methods for 24 h, Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific) was used to detect apoptotic cells. FACSCalibur flow cytometer was used to perform flow cytometer and data were analyzed by CellQuest software (BD Biosciences).
Western-blot
RIPA solution (Invitrogen) was used to was used to isolate proteins from chondrocytes. Proteins were denatured in boiling water for 10 min and were separated by 10% SDS-PAGE gel electrophoresis. PVDF membranes were used to transfer proteins and blocking was performed in fat-free milk (5% in PBS) at room temperature for 90 min. Primary antibodies included rabbit anti-human cleaved caspase 3 (ab49822, 1:1000; Abcam) and GAPDH (ab9485, 1: 1000, Abcam) at 4 °C for 15 h, followed by incubation with IgG-HRP goat anti-rabbit secondary antibody (MBS435036, 1:1000, MyBioSource) at room temperature for 2 h. Signals were produced using Amersham ECL Western Blotting Detection Reagent (GE Healthcare) and were normalized using Image J v1.46 software.
Statistical analysis
Means ± standard deviation (SD) was used to express the data of 3 independent biological replicates involved in each experiment. Unpaired t test was used to compare 2 groups. Exploration of differences among multiple groups was performed using ANOVA (one-way) and Tukey test. Correlations were analyzed by linear regression. p < 0.05 indicated statistically significant differences.
