Fig. 6

CDX1 is a target of miR-155-5p. A CDX1 is predicted as a target of miR-155-5p by comparing the lists of downregulated mRNAs obtained from RNA sequencing results with databases (miRDB and TargetScan). B The expression levels of CDX1 protein and mRNA in KM12C and KM12L4 were compared by western blot and RT-qPCR analyses. C The expression level of CDX1 in primary and liver metastatic colon cancer tissues was determined by RT-qPCR. D Following transfection of KM12C and KM12L4 cells with pre-miR-155-5p and anti-miR-155-5p respectively, the levels of CDX1 protein and mRNA were assessed by western blot and RT-qPCR analyses, respectively. E, F Direct interaction of miR-155-5p with the 3’UTR of CDX1 mRNA, Ago2 RIP (E), and luciferase activity assays (F) were performed. G–I Following transfection of KM12C cells with siRNAs targeting circPPFIA1-L or circPPFIA1-S, the expression levels of CDX1 protein and mRNA (G), the enrichment of CDX1 mRNA in Ago2 RIP (H), and luciferase assay (I) were performed as described above. J, K To examine whether overexpression of circPPFIA1s increases CDX1 expression, KM12C cells were transfected with blank or overexpression vectors. The expression levels of CDX1 protein and mRNA were determined by western blot and RT-qPCR, respectively (J). The effect of circPPFIA1s overexpression on the interaction between miR-155-5p and CDX1 mRNA was examined by Ago2 RIP (K). L To examine whether the inhibition of miR-155-5p restores downregulation of CDX1 by knockdown of circPPFIA1s, western blot analysis was performed. M, N Following the transfection of KM12C cells with CDX1 siRNA, the efficacy of CDX1 siRNA was examined by western blot and RT-PCR (M). The effect of CDX1 silencing on the metastatic potential of KM12C cells was checked by transwell invasion and migration assays (N). Statistical analyses were performed using the Student’s t-test using three independent experiments (*p < 0.05). All data represent mean ± standard variation (SD)