2.1. Research subjects and collection of synovial fluid
First Hospital of Qiqihar City Ethics Committee approved this study. At this hospital, this study enrolled a total of 62 OA patients (knee OA) and 62 healthy controls. Both OA and control group included 22 males and 40 females, and the age was 57 to 71 years (62.8+/-5.9 years). All OA patients were excluded from recurrent cases, any initiated therapy and other clinical disorders. All OA patients and healthy controls signed informed consent. Prior to therapy, synovial fluid (1.0 ml) extraction from affected sites of OA patients and the knee of the healthy controls was performed.
2.2. Synoviocytes and transient transfections
Synoviocytes (type B, primary cells) were purchased from Sigma-Aldrich (Cat. 408OA-05A). These cells are derived from an adult OA patient. Synoviocytes were cultivated in human synoviocyte media (Cell applications). Cell culture conditions were 5% CO2, 37°C and 95% humidity. In cases of LPS treatment, synoviocytes were treated with LPS at dosages of 0, 2, 4, 6, 8 and 10 µg/ml for 48h prior to the subsequent analysis.
Overexpression of circCTNNA1 and miR-29a was reached in synoviocytes by transfecting pcDNA3.1-circCTNNA1 vector (Invitrogen) or miR-29a (Invitrogen) using Lipofectamine 2000 (Invitrogen). NC experiments were transfections of NC miRNA or empty pcDNA3.1 vector. Untransfected cells were used as control (C) cells. Expression vectors and miRNAs were first mixed with transfection reagent to form transfection mixtures, followed by incubation with cells for 6h. After that, cells were washed with fresh medium. Prior to the subsequent assays, cells were cultured for 48h.
2.3. Preparation RNA samples
RNAzol (Sigma-Aldrich) was used for the isolation of total RNAs from both synoviocytes and samples of synovial fluid from both OA and control group. DNase I (Invitrogen) was used to remove genomic DNA. After that, RNAs were separated through 5% urea-PAGE electrophoresis to analyze RNA integrity. Ratios of OD260/280 were determined to analyze RNA purity and a ratio close to 2.0 indicated pure RNA samples.
Preparation of cDNA samples was performed using 5000ng total RNA per reaction. With 18S rRNA as an endogenous control, qPCRs were performed using SYBR Green Master Mix (Bio-Rad) to determine the expression of circCTNNA1.
The expression of mature miR-29a was performed by performing poly (A) addition, followed by miRNA reverse transcriptions and qPCRs with poly (T) was reverse primer. All steps were completed using All-in-One™ miRNA qRT-PCR Detection Kit* (GeneCopoeia). U6 was used as the internal control of miR-29a.
Primer sequences were: 5’-TGCGTAGACAGCTCCGCAAAG-3’ (forward) and 5’-ATCAATTTGTTGGCATGTTC-3’(reverse) for circCTNNA1; 5’-TAACCCGTTGAACCCCATT-3’ (forward) and 5’-CCATCCAATCGGTAGTAGC3’(reverse) for 18S rRNA. Forward primer of miR-29a was ACTGATTTCTTTTGGTG. MiR-29a reverse primer and U6 primers were from the kit. PCR reaction conditions were: 95°C for 3min, followed by 40 cycles of 95°C for 10s and 58°C for 45s. The method of 2−ΔΔCt was used to normalize the Ct values.
2.5. RNA-RNA interaction prediction and RNA pull-down assay
The interaction between circCTNNA1 and miR-29a was predicted using an online program named IntaRNA2.0. The long sequent was circCTNNA1 and the short sequent was miR-29a. Other default parameters were used.
Biotinylated NC (Bio-NC) and circCTNNA1 RNA (Bio-circCTNNA1) were synthesized by Sangon biotech (Shanghai, China). Bio-NC and Bio-circCTNNA1 were transfected into synoviocytes through the aforementioned methods. The transfected cells were transfected in fresh medium for 48h, followed by the preparation of cell lysis. After that, streptaviden magnetic beads (Invitrogen) were used to pull down Bio-NC and Bio-circCTNNA1, followed by RT-qPCRs to determine the expression of miR-29a.
2.6. Western blot
RIPA solution and BCA assays (Invitrogen) were used to isolate and quantify protein samples. Protein samples were denatured and subjected to electrophoresis using 5% urea-PAGE gel. PVDF membranes were used to transfer proteins, followed by incubation with caspase antibody (ab2302, Abcam) and anti-rabbit IgG-HRP secondary antibody (1:1000, MBS435036, MyBioSource). Signals were produced with ECL Western Blotting Substrate (Thermo Fisher Scientific).
2.7. Apoptosis assay
Synoviocytes with transfections were transferred to a 6-well cell culture plate with 2ml medium containing 40,000 cells per well. Three replicate wells were set for each experiment. LPS was added to reach 10 µg/ml and cells were cultivated under the aforementioned methods for 48h. After that, Annexin-V FITC and propidium iodide (PI) staining was performed in dark for 20 min. After that, cell apoptosis was analyzed by flowing software.
2.8. Statistical analysis
Comparisons between Control and OA groups were performed by unpaired t test. ANOVA (one-way or two-way) Tukey’s test was used to compare multiple groups. Correlations were analyzed by Pearson’s correlation coefficient analysis. P < 0.05 was statistically significant.