Fig. 3

CircHAS2 as a miR-1244 sponge to modulate CCNE2 expression in CRC. A Heatmap showing differentially expressed miRNAs in SW620 cells treated by either vehicle control or anlotinib (10 µM) for 24 h. B The relative expression of circHAS2 in SW620 cells was analyzed by RIP assay with AGO2 and IgG antibodies. C Relative miRNAs expressions were verified after indicated treatment in SW620 cells. D The RIP assay was used to detect the relative expression of circHAS2 in SW620 cells with indicated treatment. E The RIP assay was used to detect the expression of AGO2 protein in SW620 cells with indicated treatment. F Representative images of the FISH assay revealed the colocalization of circHAS2 and miR-1244 in the cytoplasm of SW620 cells with the target probe labeled with Cy3 and nuclei stained with DAPI (scale bar: 20 μm). G Luciferase activity of circHAS2 in SW620 cells was verified with co-transfected Luc circHAS2 WT or MUT and miR-1244 mimics or inhibitor. H Relative miRNA expressions were confirmed after anlotinib treatment (L: 5 µM, H: 10 µM) in SW620 for 24 h. I Prediction of potential downstream target genes for miR-1244 using RNA sequencing, TargetScan, miRDB, and miRWalk. J Relative CCNE2 expressions were verified after indicated treatment in SW620 cells. K Overall survival curves of 70 CRC patients based on CCNE2 expression levels in our center. L The correlation between circHAS2 and CCNE2 was determined by Pearson correlation analysis from our center. M Luciferase activity of CCNE2 in SW620 cells was verified with co-transfected Luc CCNE2 WT or MUT and miR-1244 mimics or inhibitor. N Representative Western blotting images in SW620 cells with indicated treatments for CCNE2, CDK2, p53, and p21 proteins. GAPDH was used as an internal control. Statistical significance in two-group experiments was evaluated using a two-sided Student’s t-test. One-way analysis of variance was employed for multiple-group comparisons. Survival analysis was performed using the Log-rank method. Data are represented as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance