Fig 2: CCDC6 increased microsatellite instability and unfavorable prognosis in GC patients(A) Analysis of the relative expression levels of CCDC6 in human GC and adjacent normal tissues of TCGA sequencing database (Wilcox test and paired Wilcox test). (B) CCDC6 expression was also upregulated in our collected GC tissues compared with the paired adjacent noncancerous tissues by qPCR analysis. (C) We conducted Spearman’s correlation analysis to describe the correlation of CCDC6 gene expression levels with the MSI score of GC patients. (D) The correlation analysis of CCDC6 expression with OS of GC patients was conducted with Kaplan-Meier plotter. (E–H) Correlation analyses of CCDC6 expression with OS of GC patients with stage I, stage II, stage III, or stage IV disease. (I) Correlation analysis of CCDC6 expression with survival after chemotherapy in GC patients. GC patients were split into two groups with high and low expression of CCDC6 based on the autoselected best cutoff provided by KM plotter.

Fig 3: Knockdown of circDNA2 reduced CCDC6 expression and inhibited the proliferation of GC cells in vitro(A) Schematic diagram of the siRNA sequence for the backsplicing junction site of circDNA2. (B) The transfection efficiency was detected by quantitative real-time PCR assays 48 h after transfection of the overexpression plasmid or the siRNA of circDNA2. (C) The CCDC6 level was determined by quantitative real-time PCR assays in the circDNA2 or si-circDNA2 transfection group relative to the corresponding NC transfection group. (D–F) Cell proliferation of AGS and SGC-7901 GC cells transfected with circDNA2 knockdown or overexpression was comprehensively evaluated by CCK-8, EdU, and colony formation experiments; scale bar, 50 μm. The data are represented as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 4: Identification of miR-149-5p as a potential miRNA for circDNA2 sponging(A) We screened out hsa-miR-149-5p and hsa-miR-375, which can target the CCDC6 gene 3′ UTR and be sponged by circDNA2. (B) Predicted consequential pairing of miRNAs with the CCDC6 gene in 372 GC tissues from TCGA database. (C) The relative expression levels of miR-149-5p in GC tissues and adjacent normal tissues were analyzed using TCGA RNA-seq data. (D) Pearson correlation analyses between miR-149-5p and CCDC6 expression were performed for GC tissues and adjacent normal tissues using TCGA data. (E) The predicted binding site of circDNA2 and miR-149-5p. (F) Dual-luciferase reporter assays were used to determine whether there was a direct binding site between circDNA2 and miR-149-5p. (G and H) Coprecipitation and detection of circDNA2 and miR-149-5p in AGS and SGC-7901 cell lysates with RNA pulldown and quantitative real-time PCR assays. (I) FISH analysis revealed that circDNA2 colocalized with miR-149-5p in the cytoplasm of GC tissue cells. Scale bars, 50 μm. (J) Mutual regulation of circDNA2 and miR-149-5p indicated by quantitative real-time PCR. (K) RNA FISH assays for a human GC tissue microarray. (L and M) The relative expression levels and correlation of circDNA2 and miR-149-5p in GC and pair-matched adjacent normal tissues. The data are represented as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.