Fig. 1

Characterization and clinical significance of circPDE5A. A Heatmap clustering displaying all dysregulated circRNAs. Rows represent circRNAs, and columns represent tissues. B Volcano plot comparing the fold change in circRNA level in three pairs of ESCC tissues versus adjacent normal tissues. Significantly up-regulated circRNAs are indicated by red dots, while significantly down-regulated circRNAs are indicated by green dots. C Venn diagram illustrating the selection of low-level circRNAs with protein-coding potential in ESCC. Selection criteria include the presence of internal ribosome entry site (IRES), open reading frame (ORF), and N6-methyladenosine (m6A) in predictions from circPrimer, circBank, and TransCirc databases. D RT-qPCR analysis of relative levels of circDDX3Y and circPDE5A in a normal esophageal epithelial cell line and various ESCC cell lines. E Genomic locations of PDE5A and circPDE5A. Sanger sequencing confirmed the reverse splice site of circPDE5A. F PCR and nucleic acid electrophoresis for determining the presence of circPDE5A using convergent and divergent primers in cDNA and gDNA, respectively. G RNA-FISH detection of subcellular localization of circPDE5A in ECA109 and TE1 cells. Bar represents 5 μm. H Representative images of RNA-FISH detection of circPDE5A level in ESCC tissues compared to adjacent normal tissues. Bar represents 100 μm. I Fold change of circPDE5A level in 100 pairs of ESCC tissues versus adjacent normal tissues. J RT-qPCR analysis to detect the relative levels of circPDE5A in 100 pairs of ESCC tissues versus adjacent normal tissues. K–M RT-qPCR analysis of the relative levels of circPDE5A in ESCC tissues with different T (K), N (L), and TNM (M) stages. N Kaplan–Meier analysis of the correlation between circPDE5A level and overall survival (OS) in patients with ESCC. ***P < 0.005