Tissue samples
Fifty paired OS tissues and adjacent tissues were collected simultaneously from patients who underwent complete resection in Affiliated Jinhua Hospital, Zhejiang University School of Medicine. ShRNAs for circFNDC3B were designed and synthesized by GenePharma (Shanghai, China) and the target sequences were listed in Table S1 in Supplementary File 1. The histological diagnosis was confirmed by two independent pathologists according to the criteria defined by the World Health Organization. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Affiliated Jinhua Hospital, Zhejiang University School of Medicine (2023 − 199).
Cell culture
Human OS cell lines (U2OS, Saos-2, HOS, MG63 and 143B) and human osteoblast hFOB1.19 cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). OS cells were maintained in RPMI-1640 medium (HyClone, South Logan, UT, USA) supplemented with penicillin, streptomycin and 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) in a humidified 37℃ incubator containing 5% CO2. hFOB1.19 cells were cultured in DMEM/F12 media (GIBCO) supplemented with 0.3mg/ml G418 and 10% fetal bovine serum in a humidified 34℃ incubator containing 5% CO2.
Real-time quantitative reverse transcriptase PCR (qRT-PCR)
Total RNA was isolated by TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA). Nuclear and cytoplasmic RNA were separated using a Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada). cDNA was synthesized with PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). qRT-PCR assays were performed with SYBR Green PCR Kit (Takara) and specific primers presented in Table S2 in Supplementary File 1. The 2−ΔΔCT method was adopted to analyze the relative gene expression with GAPDH served as an internal control. The back-splicing junction of circFNDC3B of the PCR products was verified by Sanger sequencing.
Overexpression plasmids, shRNAs and transfection
Lipofectamine 3000 (Invitrogen) was used to transfect overexpression plasmids and shRNAs into OS cells. Full circFNDC3B sequence was constructed into a pcDNA3.1 (+) CircRNA Mini vector (Addgene, MA, USA) to generate the circFNDC3B overexpression plasmids. RNA binding motif protein 47 (RBM47), Insulin-like growth-factor-2 mRNA binding protein (IGF2BP1) or FNDC3B cDNAs were constructed into a pcDNA3.1 (+) vector to generate the overexpression plasmids. ShRNAs for circFNDC3B were designed and synthesized by GenePharma (Shanghai, China) and the target sequences were listed in Table S3 in Supplementary File 1. ShRNAs for RBM47, IGF2BP1 and FNDC3B were purchased from Santa Cruz Biotechnology (sc-89082-SH, sc-40695-SH, sc-78339-SH, Dallas, Texas, USA). Overexpression or knockdown efficiency was assessed by qRT-PCR assay.
CCK8 Assay
OS cells were seeded in 96-well plates, and 10 µl of CCK8 solution (Beyotime Biotechnology, Shanghai, China) was added at each time point after transfection. The absorbance at 450 nM was detected by a microtiter plate reader after incubation.
5-Ethynyl-2’-deoxyuridine (EdU) assay
OS cells were seeded in 6-well plates and transfected with overexpression plasmids or shRNAs for 48 hours. EdU assays were conducted using a Cell-Light EdU Apollo567 In Vitro Kit (Ribobio, Guangzhou, China). Cells were incubated with 50 µM EdU buffer at 37°C for 2 h and Apollo dyeing reaction solution at room temperature for 30 min, followed by staining the nuclei with 4,6-diamidino-2-phenylindole (DAPI). Images were acquired using a fluorescence microscope to assess DNA replication activity via EdU-positive rates of cells.
Wound-healing assay
OS cells were seeded in 6-well plates and 10 µL pipette tips were used to scrape a straight scratch in the single-cell layer. The cells were then cultured with serum-free RPM-1640 medium for 24 h. Images of the wounds were captured at 0 h and 24 h after injury at the same wound location. ImageJ software was applied to calculate the wound-healing rate to evaluate the relative cell migration ability.
Transwell invasion assay
Transwell invasion assays were conducted using transwell chambers (Corning, NY, USA) pre-coated with diluted Matrigel (Corning). OS cells transfected for 48 h were harvested and re-suspended in 200 µl serum-free RPM-1640 medium. The cell suspension was added to the upper chamber and complete medium (supplemented with 10% FBS) was added to the lower chamber. After 24 h, cells invading through the membrane were fixed with methanol followed by staining with 0.1% crystal violet. Five random fields per chamber were selected to count the invaded cell numbers and calculate the average. This assay was repeated three times independently for further statistical analysis.
In vivo xenograft assay
The study for the animals was approved by the Experimental Animal Welfare and Ethics Committee of Affiliated Jinhua Hospital, Zhejiang University School of Medicine (Approval No. AL-JHYY202344). Lentivirus was used to stably overexpress circFNDC3B and RBM47 and silence FNDC3B in MG63 cells, as well as to silence circFNDC3B in 143B cells. Four male BALB/c mice (four-week-old) were subcutaneously injected with OS cells (1×107). Tumor volume was recorded every week by the 0.5 × length × width2 method before the animals sacrificed by cervical dislocation.
RNA fluorescence in situ hybridization (RNA-FISH)
RNA-FISH assay was performed using Alexa Fluor 594-labeled oligonucleotide probes specific for circFNDC3B junction sequence and Fluorescent In Situ Hybridization kit (RiboBio) according to the instructions provided by the manufacturer. Briefly, OS cells were fixed with 4% paraformaldehyde and treated with 0.5% Triton X-100. Then, cells were hybridized with circFNDC3B probe at 37°C overnight in a hybridization chamber. DAPI was chosen for labeling cell nuclei and the images were acquired with a fluorescence microscope.
Immunofluorescence
Cells seeded on coverslips were fixed with 4% paraformaldehyde, treated with 0.5% Triton X-100, and incubated with antibodies against RBM47 (PA5-52282, Invitrogen) or IGF2BP1 (712138, Invitrogen) at 4°C overnight. Then, cells were incubated with goat anti-rabbit IgG-FITC antibody, followed by staining the nuclei with DAPI, at room temperature in the dark. The images were acquired with a fluorescence microscope.
RNA binding protein immunoprecipitation (RIP) assay
RIP assay was carried out with an EZMagna RIP kit (Millipore, Billerica, MA, USA). Briefly, OS cells were lysed with RIP buffer and the lysates were incubated with magnetic beads conjugated with antibodies against RBM47 (orb630577, biorbyt, Wuhan, China), IGF2BP1 (712138, Invitrogen) or Normal Rabbit IgG (#2729, Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After the sample was digested with proteinase K, the immunoprecipitated RNA was isolated and analyzed by qRT-PCR assay.
RNA stability analysis
To analyze the stability of RNA, first, RNA transcription of OS cells was blocked by treating with 10 µg/mL actinomycin D (Sigma-Aldrich, St. Louis, USA). Then, qRT-PCR assay was conducted to detect the leftover circFNDC3B and FNDC3B mRNA at different time points.
Statistical analysis
All data from at least three independent experiments were presented as mean ± standard deviation (SD) and analyzed by the SPSS software (version 18.0, IBM, Chicago, IL, USA) or GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA). Student’s t-test or one-way ANOVA analysis was performed to compare the difference between groups. P value < 0.05 was regarded as statistically significant.