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. 2016 Sep 12;44(22):10603–10618. doi: 10.1093/nar/gkw788

Figure 3.

Figure 3.

miR-24-3p's targeting sites are located at Oct4, Nanog, Klf4 and c-Myc 3′UTRs, whereas miR24-2-5p's sites are localized at Klf4 and c-Myc 3′UTRs. (A) Schematic representation of luciferase reporter constructs containing mutations in miR-24-3p target sites in Oct4-3′UTR, Nanog-3′UTR, Sox2-3′UTR, Klf4-3′UTR and c-Myc-3′UTR. WT, wild-type; M, mutant. (B) The effect of miR-24-3p mimic on reporter activities of WT Oct4-3′UTR, Nanog-3′UTR, Sox2-3′UTR, Klf4-3′UTR, c-Myc-3′UTR and their mutants. The reporter constructs, together with miR-24-3p mimic, were transfected into HEK293T cells. Mock indicates non-transfected cells, and vector denotes a reporter plasmid without any 3′UTR. Firefly luciferase activities were normalized to an internal transfection control (Renila luciferase). (C) The effect of endogenous miR-24-3p on the reporter activities of Oct4-3′UTR, Nanog-3′UTR, Sox2-3′UTR, Klf4-3′UTR and c-Myc-3′UTR. mESCs (5 × 105 cells) were first electroporated with 30 μg of the shPRMT7-7 plasmid. Twelve days later, PRMT7-depleted mESCs were co-transfected with both WT luciferase-3′UTR (or luciferase-mutant 3′UTR) reporters and control LNA (or LNA-miR-24-3p [a strong anti-sense inhibitor of miR-24-3p]). Cells were harvested 2 days after transfection. (D) Schematic representation of luciferase reporter constructs bearing mutations in miR-24-2-5p target sites in Oct4-3′UTR, Nanog-3′UTR, Sox2-3′UTR, Klf4-3′UTR and c-Myc-3′UTR. WT, wild-type; M, mutant. (E) The effect of miR-24-2-5p mimic on reporter activities of WT Oct4-3′UTR, Nanog-3′UTR, Sox2-3′UTR, Klf4-3′UTR, c-Myc-3′UTR and their mutants. Data are presented as the mean ± SD of three independent experiments. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) indicate statistically significant changes.