Fig. 3.

circSLC43A1 reduces EMT, cell migratory ability, invasiveness, and tumor growth by directly downregulating miR-1226-5p expression. A Schematic diagram of the selection of candidate circRNAs that directly inhibit the expression of miR-1226-5p. B The expression levels of four candidate circRNAs, selected using the circbank database, were compared in radiosensitive or resistant HCT116 and SW480 cells. C circSLC43A1 was quantified by amplification of cDNA of circSLC43A1 in SW480 cells using divergent and convergent primers. D After RNase R treatment in HCT116, the mRNA levels of circSLC43A1 and SLC43A1 were determined using qRT-PCR. E The expression level of miR-1226-6p was measured when circSLC43A1 was overexpressed in HCT116 RR cells. F After co-transfecting cells with miR-1226-5p mimic and the wild-type (WT) or mutant-type (Mut) vector of circSLC43A1, the binding of the two factors was confirmed in HCT116 cells using dual luciferase assay. G-I circSLC43A1 was overexpressed alone or together with miR-1226-5p in HCT116 and SW480 cells, respectively. G TWIST, SNAIL, N-cadherin, and ZEB1 protein expression was detected by Western blot analysis. Transwell migration (H) and matrigel-coated invasion assays (I) were applied to the indicated cells. J HCT116 RR overexpressing miR-1226-5p alone or together with circSLC43A1 were injected subcutaneously into the right flank of Balb/c nude mice (n = 4; 1 × 10⁷ cells/mouse). At 2 weeks after injection, tumor tissues were harvested from sacrificed mice. A whole image of the tumor (left) and the volume of the tumor (right) were quantified and displayed in a graph. Scale bar 1 cm. qRT-PCR were normalized to GAPDH and U6. β-Actin was used for normalization in Western blot analysis. The experiment was repeated with triplicates and representative Western blotting images are shown. The data are presented as the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t test, and One-way ANOVA followed by bonferroni comparison test