Fig. 1

CircECE1 validation and expression in osteosarcoma tissue and cells. a. RIP-seq results indicate that CircRNAs interact with c-Myc. The red arrow indicates CircECE1. b. Total RNAs were isolated from the specimens of patients with OS and from chondroma tissues for use in real-time PCR. Tumor samples exhibited significantly higher levels of CircECE1 compared to those in the chondroma tissue. Data represent the mean ± SD (n = 10). c. CircECE1 expression was higher in human OS pulmonary metastasis than in primary OS tissues. Representative images are provided (400× magnification). d. CircECE1 expression in hFOB1.19 and osteosarcoma (OS) cell lines (OS-732, 143B, HOS, SJSA-1, and MG-63) was evaluated by qRT-PCR. Data represent the mean ± standard deviation (SD) (n = 3). * P < 0.05. e. Schematic illustration reveals the ECE1 exon 2–4 circularization that forms CircECE1 (red arrow). The presence of CircECE1 was validated by RT–PCR and subsequent Sanger sequencing. The red arrow represents “head-to-tail” CircECE1 splicing sites. f. The presence of CircECE1 was validated in U2OS and 143B osteosarcoma cell lines by RT–PCR. Divergent primers amplified circECE1 in cDNA but not in genomic DNA. GAPDH was used as a negative control. g. The expression of CircECE1 and ECE1 mRNA in 143B cells treated with or without RNase R was detected by real-time PCR. The relative levels of CircECE1 and ECE1 mRNA were normalized to the value measured in the mock treatment. Data represent the mean ± SD (n = 3). * P < 0.05. h. RNA fluorescence in situ hybridization (FISH) revealed that CircECE1 was predominantly localized within the cytoplasm. CircECE1 probes were labeled with Alexa Fluor 488, and nuclei were stained with DAPI. Scale bar = 50 μm