Fig. 5

Ccnd1 and Fzd6 are repressed by H19X-encoded miRNAs. A The gene structures of Ccnd1 and Fzd6 are shown with binding sites for H19X-encoded miRNAs in the 3′ UTR. Ccnd1 mRNA expression was significantly repressed by mimic treatment of miR-322-5p or miR-503-5p, and not miR-450b-5p. Fzd6 mRNA levels were reduced by approximately 40% upon mimic treatment of miR-450b or miR-542-3p (n = 3). B The CCND1 protein levels decreased with miR-322-5p and miR-503-5p mimic treatment. The quantified signal intensities in the western blotting are presented as a bar graph (n = 2). C The positions and sequences of miRNA binding sites in the 3′ UTR of Ccnd1 and Fzd6 are presented (upper panel). The seed region of miRNA is highlighted in red and is base paired with the matched sequence of each target mRNA. The binding activity of miRNAs to the predicted sites in 3′ UTR was measured using a dual-luciferase reporter assay (n = 3). The luciferase activity was reduced by transfection of the corresponding miRNA when the reporter contained the 3′ UTR fragment of Ccnd1 or Fzd6. Data are presented as mean ± SEM. ***p < 0.005, **p < 0.01, *p < 0.05