Fig. 3.

CREB1 is a target of miR-1, miR-28-A, and miR-296-3p. A, Schematic representation of the 3′-UTR sites of the CREB1 gene targeted by miR-1, miR-28-A, and miR-296-3p. B, Immunoblots of the CREB1 and vinculin proteins, used as loading control. One representative out of three independent experiments. The lower panel represents the mean densitometric analysis of three independent experiments. Proteins were extracted from PC Cl 3 cells transfected with scrambled oligonucleotide, miR-548c, miR-1, miR-28-A, and miR-296-3p (50 nmol/ml) alone or in combination (mix miR) and collected after 48 h. C, qRT-PCR analysis of CREB1 mRNA in the same samples shown in B. Relative expression values indicate the relative change in CREB1 mRNA expression levels between miR-treated and scrambled oligonucleotide-treated cells, normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The bars represent the mean ± se (n = 3); P < 0.05 vs. scrambled control. D, Relative luciferase activity in PC Cl 3 cells transiently transfected with wild-type and mutant constructs for miRNA seed sequences and with miR-1, miR-28-A, and miR-296-3p oligonucleotide and a control nontargeting scrambled oligonucleotide. The relative activity of firefly luciferase expression was standardized to a transfection control, using Renilla luciferase. The bars represent the mean ± se (n = 3); P < 0.05 vs. the scrambled oligonucleotide. E, qRT-PCR analysis of AREG, NR4A2, and CCNA2 specific mRNA in PC Cl 3 cells transfected with miR-1, miR-28-A, miR-296-3p, or the scrambled oligonucleotide (50 nmol/ml). Relative expression values indicate the relative change in gene expression levels between miR-treated and scrambled oligonucleotide-treated cells, normalized with GAPDH. The bars represent the mean ± se (n = 3); P < 0.05 vs. scrambled control.