Fig. 1

circCDYL2 is highly expressed in nasopharyngeal carcinoma and associated with poor prognosis. A. Schematic diagram of the structure of circCDYL2. Sanger sequencing confirmed that circCDYL2 is formed by reverse splicing of exon 2 of CDYL2 pre-mRNA, with a length of 592 nt. B. RT-qPCR was used to detect the relative abundance of circCDYL2 and CDYL2 mRNA in HNE2 and CNE2 cells after actinomycin D (1 μg/ml) treatment for 0, 8, 16, or 24 hours. C. RT-qPCR was performed to examine the expression of circCDYL2 and CDYL2 mRNA in HNE2 and CNE2 cells after RNase R treatment. D. Fluorescence in situ hybridization was used to determine the cellular localization of circCDYL2, showing distribution in both the cytoplasm and the nucleus. Scale bar = 20 μm. E. Cellular localization of circCDYL2 was assessed using nuclear-cytoplasmic fractionation followed by RT-qPCR, confirming the presence of circCDYL2 in both cytoplasm and nucleus. F. The expression of circCDYL2 was detected by RT-qPCR in fresh nasopharyngeal carcinoma (NPC) biopsy tissues (n = 45) and control nasopharyngeal epithelium (NPE) samples (n = 23). The expression of circCDYL2 was significantly upregulated in NPC tissues. G. In situ hybridization was used to detect circCDYL2 expression levels in 203 archived paraffin-embedded tissues, including 56 adjacent nasopharyngeal epithelium tissues. Results showed that circCDYL2 was highly expressed in 67% (136/203) of NPC tissues compared to 12.5% (7/56) in nasopharyngeal epithelium (NPE) tissues. Representative in situ hybridization results for nasopharyngeal epithelium and NPC tissues are shown; 20×, scale bar = 50 μm; 40×, scale bar = 20 μm. H. The overall survival time of NPC patients with high circCDYL2 expression is shorter than that of patients with low circCDYL2 expression, with a five-year survival rate of 48.55% vs 71.32%. HR = 2.38, p < 0.001. I. The expression of circCDYL2 was significantly higher in the radiotherapy resistance group (NPC_R, n = 48), compared with that in the radiotherapy-sensitive group (NPC_S, n = 38). All these NPC patients underwent conventional radiotherapy. Patients who did not experience recurrence or metastasis within 5 years after radiotherapy were defined as the radiotherapy-sensitive group (NPC_S, n = 38), while those who experienced recurrence or metastasis within the first 2 years after initial radiotherapy were considered the radiotherapy resistance group (NPC_R, n = 48). Data were represented as mean ± SD. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001