Patients and specimens
Thirty specimens obtained from osteosarcoma patient were used to detect the expression of hsa_circRNA_102049 using quantitative real-time polymerase chain reaction (q-PCR). The specimens were provided by the Guangdong Province Hospital of Traditional Chinese Medicine, Zhuhai Branch. None of the patients had undergone chemotherapy or radiotherapy, and had provided written informed consent prior to surgery. For proper specimen preservation, osteosarcoma tumors and normal adjacent specimens were frozen in liquid nitrogen and stored at -80 °C immediately after resection. The diagnosis and clinical stage of patients with osteosarcoma were classified according to the Tumor Node Metastasis (TNM) Classification of Malignant Tumors (sixth edition) prescribed by the Union for International Cancer Control (UICC) by two experienced pathologists (Yang et al. , 2020). The study protocol was approved by the Clinical Research Ethics Committee.
Human osteosarcoma cell lines (MG-63, U2OS, 143B, and HOS) and normal human fetal osteoblast cell line (hFOB1.19) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO2.
Lipofectamine 3000 (Invitrogen) was used for transfection experiments according to the manufacturer’s instructions. Small interfering RNAs (siRNAs) were purchased from Beijing Qingke Biotechnology Co., Ltd. For RNA interference, siRNA duplexes targeting hsa_circRNA_102049 were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd. The sequences of the siRNAs used were as follows: siRNA F: 5′-AGC AUG AAG AAC UAC AGA ACG-3′ and siRNA R: 5′-UUC UGU AGU UCU UCA UGC UGG-3′. The scrambled sequence was used as a negative control (NC).
RNA extraction and q-PCR
Total RNA was extracted from osteosarcoma tumors, normal adjacent specimens, and cell lines using the Tiangen kit. The assay was performed according to the manufacturer’s instructions. GAPDH expression level was used as an internal control. The relative mRNA expression of each sample was analyzed using the ΔΔCt method. Primers were purchased from Beijing Qingke Biotechnology Co., Ltd. The sequences of the primers used were as follows: ZO-1 F: 5′-ACA AAC AGC CCT ACC CAT CTC G-3′, ZO-1 R: 5′-CTT CAA AAC GTG GAA AGT ACC CT-3′; E-cadherin F: 5′-TCA CGC TGT GTC ATC CAA CGG-3′, E-cadherin R: 5′-GAC AGA CCC CTT AA AGA CCT CC-3′; N-cadherin F: 5′-TAC AGA CAT GGA AGG CAA TCC C-3′, N-cadherin R: 5′-ACG TCA TGG CAG TAA ACT CT-3′; and α-SMA F: 5′-GCA TCA TCA CCA ACT GGG ACG ACA-3′, α-SMA R: 5′- CTC TTC AGG GGC AAC ACG AAG CTC-3′.
Cell proliferation assay
Cell proliferation was determined using a CCK8 kit according to the manufacturer’s instructions. Osteosarcoma cell lines (U2OS and 143B) transfected with NC or siRNA were seeded in 96-well plates (4 × 103 cells/well) and cultured for different durations (0, 24, or 48 h). CCK8 solution (20 µL) was added to each well, and cells were incubated at 37 °C for 4 h. Finally, the optical density for each well was measured using a microplate reader at 450 nm. Each treatment was repeated three times.
To detect cell proliferation, a 5-ethynyl-2′-deoxyuridine (EDU) assay was performed according to the manufacturer’s instructions. The method of plating transfected cells was the same as described for the CCK8 method. EDU reagent (10 µL) was added to the cells in each well of the 96-well plate, and incubated at 37 °C for 2 h. The medium was discarded, and the cells were fixed with paraformaldehyde for 30 min and washed three times with phosphate-buffered saline (PBS). The cells were sequentially treated with penetrant, staining reaction solution, etc., and photographed under a confocal microscope.
For the wound-healing assay, U2OS and 143B cells transfected with the NC or siRNA were seeded on 6-well plates, 4 × 105 cells per well, and cultured for 24 h. Each treatment was repeated three times. A scratch was created in the cell monolayer growing in each well and cells were then incubated in a serum-free medium. The closure of the scratch at different time points ( 0 and 24 h) was evaluated using Professional software (Toup View).
Transwell invasion assay
Transwell chambers (8 µm, 24-well insert; Corning, Lowell, MA, USA) were used to observe osteosarcoma cell invasion (U2OS and 143B). Briefly, cells were transfected with the NC or siRNA. Medium (500 µL) containing 10% fetal bovine serum was added to the lower chamber, and 4 × 103 cells in 500 µL of serum-free medium were added to the upper chamber. Cells were cultured at 37 °C for 24 h. Next, the cells were fixed with paraformaldehyde for 30 min and washed with PBS. Non-migrating cells were removed with a mask and stained with 1% crystal violet for 30 min. The other steps were the same as those used in the scratch assay.
Western blot (WB)
Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) was used to extract total protein. A BCA protein assay kit was used to determine the total protein concentration. Proteins were resolved by performing SDS-PAGE using 10% resolving gels, and the proteins were transferred to nitrocellulose membranes for WB analysis. After blocking the non-specific binding sites in 5–10% nonfat milk for 1 h, the membrane was incubated with primary antibody for 4 h at 4 °C and then with horseradish-conjugated secondary antibody for 4 h at room temperature. An ECL kit was used to detect the intensity of immunofluorescence reaction and the band density was normalized to that of GAPDH.
All data are presented as the means ± standard deviation (SD). SPSS ver.20.0 (IBM Corporation, SPSS, Chicago, IL, USA) was used for the statistical analysis of all results. The p-values were calculated using two-tailed Student's t-test and Pearson's χ2 test. Results with p < 0.01 were considered significant.