Figure 1

Screening for miRNAs that directly target the 3′ UTR of NR4A2.

(a) A reporter plasmid containing the 3′ UTR of NR4A2 downstream from a firefly luciferase (Fluc) gene was used to identify miRNAs that putatively regulate NR4A2, relative to an internal Renilla luciferase (Rluc) control gene. (b) 293T cells were transfected with the NR4A2 3′ UTR (WT 3UTR) reporter construct for 24 h. Transfected cells were reseeded in 96-well plates and reverse transfected with 75 individual cancer-relevant miRNAs. After 48 h of transfection, a Dual-Glo luciferase assay was performed, the ratio of Fluc/Rluc was calculated and the log2 fold change was determined for each miRNA (n = 3), relative to a transfection control (pSIF, n = 9) and presented as a waterfall plot. The Fluc/Rluc value for pSIF was set as 1. Statistical significance was calculated using a one-way ANOVA and Dunnett’s test for multiple comparisons between pSIF and the indicated miRNAs. (c) A schematic of the predicted miR-34 seed region in the NR4A2 3′ UTR (WT 3UTR). This predicted miR-34 binding site in the NR4A2 3′ UTR was mutated and the resulting loss of complementarity is shown (34mut). (d) WT 3UTR or 34mut reporter constructs were cotransfected with pSIF or the indicated miR-34 isoforms into HCT116 colorectal carcinoma cells for 72 h, after which a Dual-Glo luciferase assay was performed. The fold change of the Fluc/Rluc ratio with respect to pSIF was calculated (the Fluc/Rluc value of pSIF for each reporter transfection group was set as 1) and the statistical significance of the relation between pSIF and miR-34a or miR-34c was determined using a two-way ANOVA and Tukey’s test for multiple comparisons (****P ≤ 0.0001; n.s.P > 0.05). Statistically significant changes in WT 3UTR and 34mut for each miRNA transfection are indicated by ^####(P ≤ 0.0001).