Fig. 5

CNIH4 is a downstream target of the circUCK2(2,3)–miR-149-5p axis. A Heat map of downregulated genes in PLC/PRF/5 cells with circUCK2(2,3) knockdown. B Venn plot of downregulated genes and potential miR-149-5p targets. C Dual luciferase assays were performed in 293T cells transfected with miR-149-5p mimics together with reporter vectors carrying the 3′UTR of CNIH4 or SMAGP. D and E qRT–PCR to determine expression changes of CNIH4 and SMAGP in SNU182 cells with circUCK2(2,3) downregulation (D) and in SNU398 cells with circUCK2(2,3) overexpression (E). F and G qRT–PCR analysis of CNIH4 mRNA levels in 70 paired HCC and peritumor tissues in the Jin-Ling cohort. H Tumor versus peritumor expression ratio of CNIH4 in the Jin-Ling cohort. I and J Expression correlation between circUCK2(2,3) and CNIH4 in the Jin-Ling cohort (I) and in the Xin-Hua cohort (J). K and L Western blotting of CNIH4 protein levels in SNU182 cells with circUCK2(2,3) knockdown (K) and in SNU398 cells with WT or 149-mut circUCK2(2,3) overexpression (L). M and N IHC staining of CNIH4 in PLC/PRF/5 tumor xenografts with circUCK2(2,3) knockdown (M), and in SNU398 tumor xenografts with WT or 149-mut circUCK2(2,3) overexpression (N). O qRT–PCT to determine knockdown efficiency of CNIH4. P CCK8 assays in PLC/PRF/5 cells with CNIH4 knockdown. Q Wound healing assays in PLC/PRF/5 cells with CNIH4 knockdown. Statistical analyses were performed using paired Student’s t-tests (**p < 0.01; ***p < 0.001; ****p < 0.0001). siNC negative control of siRNA, si-1 siRNA#1, si-2 siRNA#2, siCNIH4 siRNA targeting CNIH4, NC negative control, 3’UTR 3' untranslated region, VC vector control, OE overexpression, 149-mut circUCK2(2,3) mutant with mutated miR-149-5p binding site, WT wild type, PT peritumor, HCC hepatocellular carcinoma, luc2 firefly luciferase, hRluc renilla luciferase, neo neomycin