ANKRD46 is a direct target of miR-21 in BC cell lines. (a) Predicted alignment of miR-21 with the target site derived from ANKRD46 and EIF4A2 3' UTR, determined with the software miRBase Targets V5 and miRNAMap, respectively. Note the seed matches at the 5' end of miR-21 (grey boxes) and the mutated nucleotides (underlined). (b) Luciferase assays show that miR-21 directly repress ANKRD46 mRNAs through 3' UTR interactions. Part of the 3' UTRs of wild-type ANKRD46 (478-bp length) and EIF4A2 (194-bp length), or the mutations were cloned into pMIR-REPORT vector (Applied Biosystems), downstream of luciferase. These vectors were then cotransfected with synthetic miR-21 (pre-miR-21) or miR-control in 293T cells, and luciferase activity was quantified. The graph shows the percentage of remaining luciferase activity calculated by normalizing the miR-21 expression values on the miR-control values. (c) Western blot to assay ANKRD46 and EIF4A2 after miR-21 knockdown, with GAPDH as equal loading control, followed by densitometric analysis. Cells were treated and harvested as described in Figure 4b. Data in (b) and (c) are mean + SD (n = 3). * P < 0.05. WT, wild-type; Mut, mutant.