Figure 5. miR-375-3p directly targets YAP1 and SP1. (A) Predicted binding regions between wild-type (wt) or mutant (mut) 3′-UTRs of YAP1/SP1 and miR-375-3p. The sequences formatted in bold red represent the mutant miR-375-3p binding sites in YAP1 or SP1 3′ UTR. (B) Luciferase reporter assay showed the decreased luciferase activity in miR-375-3p-overexpressed cells (HCT116/FU and HCT8/FU) for 3′ UTR wild-type constructs. The luciferase activity was normalized to Renilla luciferase. (C, D) The mRNA expression levels of YAP1/SP1 in parental cells (HCT116, HCT8) and 5FU-resistant cells (HCT116/FU, HCT8/FU) were analyzed following transfections of miR-375-3p mimics or inhibitors into the four cell lines. (E) YAP1 and SP1 protein expression levels were detected by western blot in CRC parental cell lines (HCT116 and HCT8) and 5FU-resistant cell lines (HCT116/FU and HCT8/FU). (F) Western blot was performed to analyze the protein expression levels of YAP1 and SP1 not only in 5FU-resistance cell lines (HCT116/FU and HCT8/FU) overexpressed miR-375-3p, but also in CRC parental cell lines (HCT116 and HCT8) inhibited miR-375-3p. Simultaneously with 5FU treatment or not. (G) Representative images of tumor lumps in HCT116/FU-xenograft that were stained with YAP1 and SP1 by IHC. *P < 0.05, **P < 0.01 and ***P < 0.001.