Ethics Statement
Our studies about human participators have been approved by the Ethics Committees of the Second Xiangya Hospital of Central South University. In accordance with the ethics committee guidelines, written and informed consent was acquired from all patients or consultants.
Cell culture and treatment
The human osteosarcoma cell lines SaoS-2, U2OS, HOS, MG63 and human normal osteoblasts hFOB1.19 were acquired from China Center for Type Culture Collection (CCTCC, Shanghai, China). OS cells were maintained in Dulbecco's modified Eagle's medium, Eagle's minimal essential medium, DMEM growth medium, and McCoy's 5A medium mixed with 10.0% fetal bovine serum (FBS) with 1% antibiotic (Gibco, Rockville, MD, USA) in a humidified at 37°C in a 5% CO2 humidified environment,while hFOB1.19 cells were maintained at 34 °C in a cell incubator with 5% CO2 humidified environment.
Patient Samples
All samples from 25 osteosarcoma patients between 2013 and 2019 in the Second Xiangya Hospital of Central South University. Normal specimens were obtained from tissues adjacent to osteosarcoma tissues. Once the osteosarcoma or noncancerous tissue had been isolated, samples were all frozen in liquid nitrogen and kept at -80°C until RNA extraction.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA in tissues and cell lines was extracted with RNA isolation kit (Invitrogen, Carlsbad, CA, USA). DNA was obtained by reverse transcription with reverse transcription kit (Hitachi, Ltd., Tokyo, Japan). qRT-PCR was employed on the RT-PCR system (StepOneTM, Applied Biosystems, Darmstadt, Germany) with SYBR Premix Ex Taq II (TaKaRa, Dalian, China). U6 or GAPDH was taken as internal references. Relative expression was calculated by the 2-△△Ct method. The primers were exhibited in Table 1.
Actinomycin D (ActD) treatment
The half-life of RNAs were assessed by the addition of actinomycin D (ActD, 5 mg/ml) into the cell culture medium. Total RNA was extracted at 0, 4, 8, 16 and 24 h after ActD treatment. Real-time qPCR against GAPDH was performed to assess the half-lives of circPARD3 and PARD3 mRNA.
RNase R treatment
HOS cells and U2OS cells were treated with or without RNase R (3 U/μg; USA) at 37°C for 10 min. Phenol-chloroform extraction was performed to purified RNA from the mixture. Then, the RNA was subjected to cDNA synthesis and qPCR analysis. CircPARD stability was determined by detecting circPARD expression by qPCR.
Subcellular fractionation assay
OS Cells were resuspended for 10 min on ice in 500μl CLB buffer. Homogenization was performed by mixing with a 1 ml needle 15 times on ice. Then, 50 μl of 2.5 M sucrose was added to restore isotonic conditions. Centrifugation was performed at 6300 g for 5 min in a able to p centrifuge at 4 °C. The pellet was washed and resuspended in TSE buffer and then centrifuged at 4000 g for 5 min in a tabletop centrifuge at 4 °C until the supernatant was clear. The resulting supernatant was discarded, and the pellet was nuclei. The supernatant was sedimented at 14000 rpm in a tabletop centrifuge. The resulting pellet was membranes and the supernatant contained cytoplasmic contents.
Cell transfection
Transient transfection was carried out using the Lipofectamine 3000 reagent (Thermo Fisher Scientific) in this study. siRNAs targeting circPARD3 (si-circPARD3: sense: 5′-CACCUG UAGACAGGUAAAUAC-3′, antisense: 5′-GUAUUUACCUGUCUACAGGUG-3′), siRNAs targeting SKIL (si-SKIL: sense: 5′-CCGGGUAAUAGGAAGAGGAAGUUAU-3′, antisense: 5′-AUAACUUCCUCUUCCUAUUACCCGG-3′), negative control siRNAs (si-NC), miR-1294mimics, miR-1294 inhibitor, and miRNA-NC were synthesized by GenePharma (Shanghai, China). Overexpression vectors for CircPARD3 WT and CircPARD3 MUT were constructed (BersinBio, Guangzhou, China). Overexpression SKIL plasmids were established (Thermo Fisher Scientific).
Cell Counting Kit-8 (CCK-8) Assays.
The HOS and U2OS cells were seeded in the 96-well plates and cultured. 10 mL of CCK-8 solution (Dojindo, Japan) was added to each well after the cells were transfected for 0, 24, 48, 72 and 96 h. The CCK-8 solution was subsequently incubated with the cells for another 4 h. The optical density value of each well was measured using a microplate reader.
Colony Formation Assays.
Transfected cells were seeded at a density of 600 cells/ well into a 12-well plate and then cultured for 8 days. Then, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 min and stained with a 0.5% crystal violet solution for 10 min. The colonies were counted and imaged under a microscope.
Wound-healing assays.
The transfected cells were seeded in six-well plates. The cells were scratched with a 200-µl pipette tip when the cell confluency reached 90%. Representative images of cell migration were captured by photographing 10 high-power fields at 0 and 48 h after injury. Remodeling was measured as the diminishing distance across the induced injury area normalized to the 0 h control and expressed as a relative migration rate.
Transwell Assays.
Based on the manufacturer’s instructions, 1x104 cells in serum-free medium were seeded into the upper chamber of 24-well transwell chamber (Corning, NY, USA) for migration detection, as well as 1x105 cells into upper chamber pre-coated with Matrigel for invasion detection. 10% FBS þ RPMI medium was added into the lower chamber. After 24h, the chamber was fixed with 4% paraformaldehyde and stained using 0.05% crystal violet (Beyotime, Beijing, China). The number of invasive cells was counted in five randomly selected microscopic views and photographed.
RNA immunoprecipitation
Transfected HOS cells were lysed and incubated with RIPA buffer containing magnetic beads conjugated with anti-AGO2 antibody (1:1000, Abcam). Anti-IgG was used as control. Samples were treated with Proteinase K, and immunoprecipitated RNA was collected and tested via RT-PCR.
Dual‑luciferase reporter assay
The binding sites of miR-1294 in circPARD3 were predicted by the Circinteractome database. The sequences of circPARD3 and mutant circPARD3 (within the binding sites to miR-1294) was inserted into the pcd-control vector for the construction of the wild type (WT) and mutant (MUT) luciferase reporter vectors for circPARD3, respectively. The binding sites of SKIL in miR-1294 were predicted by the Targetscan database. HOS and U2OS cells were cotransfected luciferase reporter vectors and miR-NC or miR-1294. The dual luciferase reporter assay kit (Promega) was employed to assess the luciferase intensities of the luciferase reporter vectors in HOS cells.
Western blot
Cells were harvested and lysed in RIPA lysis buffer (Vazyme, China). The concentration of total protein was determined by a BCA protein assay kit (Vazyme, China). All proteins were transferred into polyvinylidene difluoride (PVDF) membrane, and the membrane was blocked with 5% solution of skimmed milk powder for 1 h at room temperature. The membrane was incubated with the indicated primary antibodies at 1:1000 and incubated overnight. The corresponding secondary antibody (at 1:10,000 dilution) was incubated for 40 min at room temperature. The bands were visualized using enhanced chemiluminescence reagents. The primary antibodies were used as follows: SKIL (0.5 μg/ML, Abcam) and β-actin (1:3000, Abcam).
Xenograft assay
NOD/SCID mice (8 weeks old) were randomly divided into two groups (n = 5). NOD/SCID mice were inoculated subcutaneously with HOS cells (1 × 107 per tumor) pre-transfected with control vector or pcd-circPARD3. Tumor volume was measured and calculated every week. Five weeks later, all mice were euthanized and the tumor tissues were weighed and collected. The experiment was permitted by the Animal Research Committee of the Second Xiangya hospital Affiliated to Central South University.
Statistical Analysis
Graphpad Prism 8.1.0 (GraphPad, USA) was used to perform the statistical analysis in this study. Comparisons between two groups were analyzed using Student’s t-test. Comparisons among more than two groups were performed using one-factor analysis of variance followed by the Bonferroni’s post hoc tests. The correlation between circPARD3 and miR-1294 was evaluated using Spearman’s rank correlation test. P-values of 0.05 or less were considered statistically significant. All data were presented as the mean± standard deviation (SD).