Figure 5. ZEB1 and ZEB2 were direct targets of miR-144-3p. (A) Venn diagram showed direct targets of miR-144-3p in four public database and crucial EMT-TFs for CRA. Only two potential targets ZEB1 and ZEB2 were presented in all the four database and EMT-TFs. (B) The predicted sequences of miR-144-3p binding sites within the 3’-UTR of ZEB1 and ZEB2 including the wild-type (WT) or mutant (Mut) binding site were shown. (C) Luciferase reporter assay showed that overexpression of miR-144-3p significantly repressed luciferase activity in 293T cells transfected with WT binding site of ZEB1 and ZEB2 3'-UTR. (D&E) miR-144-3p inhibited ZEB1 and ZEB2 expression. (D) qRT-PCR results showed mRNA expression levels of ZEB1 and ZEB2 in LovomiR-144-3p mimic cells, HCT116 cellsmiR-144-3p inhibitor and matched control cells. Data were analyzed by using 2-ΔΔCt method. (E) Western blot results showed protein expression of ZEB1 and ZEB2 in LovomiR-144-3p mimic cells, HCT116 cellsmiR-144-3p inhibitor and matched control cells. (F) qRT-PCR analysis of ZEB1 and ZEB2 mRNA expression level in 30 CRATs. Pearson correlation analysis was applied to examine the correlation between miR-144-3p expression and ZEB1 or ZEB2 mRNA expression. (G, H) miR-144-3p promoted OVOL1/2 expression. (G) Western blot analysis of protein expression of transcription factors OVOL1/2. miR-144-3p mimic induced the expression of OVOL1/2 in Lovo cells and miR-144-3p inhibitor reduced OVOL1/2 expression in HCT116 cells. (H) miR-144-3p mimic induced mRNA expression of OVOL1/2 in Lovo cells and miR-144-3p inhibitor reduced OVOL1/2 expression in HCT116 cells. TF, transcription factor. ***, P < 0.001.