Figure 5

miR-199b-5p binds directly to the 3′-UTR of Snai1 and ZEB1 mRNAs. (a) In silico analyses predicted a highly conserved binding site for miR-199b-5p in the 3′-UTR of Snai1 and ZEB1 mRNAs across species. (b) The luciferase reporter plasmids containing the wild-type 3′-UTR or mutant 3′-UTR of Snail and ZEB1 with the putative binding sites for miR-199b-5p. (c) The direct binding of miR-199b-5p to 3′-UTR of Snai1 and ZEB1 mRNAs was detected by a dual luciferase activity assay. The luciferase reporter plasmids and β-galactosidase expression plasmids were co-transfected into HEK293 or HCEnC-21T cells with either miR-199b-5p mimic, miR-199b-5p mimic negative control (Neg Ctrl), miR-199b-5p inhibitor, and miR-199b-5p inhibitor negative control (Neg Ctrl). Luciferase activity was measured by the dual luciferase reporter assay system. Relative luciferase activities were calculated by normalizing firefly luciferase activity to β-galactosidase activity in the same sample to correct for transfection efficiencies. Data are represented as the mean ± SEM (n = 3; p < 0.05). (d) The effect of miR-199b-5p on the expression of Snail and ZEB1. HCEnC-21T cells were transfected with miR-199b-5p mimic or miR-199b-5p inhibitor with their corresponding negative controls. 48 hours post-transfection, total RNA was extracted and mRNA expression levels of Snail and ZEB1 were quantified by qRT-PCR. Data are represented as the mean ± SEM (n = 3; p < 0.05).