Fig. 2.

miR-224 targets BTRC and regulates its expression in protein level but not in mRNA level. a MiRecords predicted that there existed a seed region with the complementary base pairing (bold) between miR-224 and the 3′UTRs of BTRC, and this seed region in pmirGLO BTRC 3′UTR-WT was mutated into the sequences (Italic with underline) to reconstruct the plasmid of pmirGLO BTRC 3′UTR-MUT. WT: wild type, MUT: mutant. b The dual-luciferase activity of pmirGLO-BTRC-3′UTR-WT and pmirGLO-BTRC-3′UTR-MUT in HEK293T cells after co-transfection with miR-224 mimic or NC showed the interaction between miR-224 and 3′UTR of BTRC. ****p < 0.0001, n.s. = no significance. c The expression of BTRC in HCT-116 and DLD-1 cells transfected with miR-224 inhibitor or negative control (NC) in mRNA level by RT-qPCR, normalized to negative controls. GAPDH was used as an endogenous control. n.s. = no significance. d Representative images (left) and the relative quantification (right) of the protein expression levels of BTRC in HCT-116 and DLD-1 cells transfected with miR-224 inhibitor or negative control (NC), normalized to negative controls by western blot. Western blot bands were quantified by ImageJ and GAPDH was used as an endogenous control. *p < 0.05