Figure 2

Identification of direct miR-16 targets by luciferase reporter screening. (A) Schematic description of the hypothetical duplexes formed by interactions between miR-16 and the wild-type (WT) or mutant (MUT) 3′-UTRs of CDS2, PRDM4 and MAP7. For WT 3′-UTRs, perfect base pairing between the “seeds” (the core sequence that encompasses the first 2–8 bases of the mature miRNA) and cognate targets was noted. For MUT 3′-UTRs, the sequence that interacts with the 2–8 bases of miR-16 were mutated. The predicted minimum free energy values of each hybrid are indicated, and the miR-16 binding sequences at the 3′-UTR of CDS2, PRDM4 and MAP7 are highly conserved across species. (B) The identification of direct miR-16 targets by luciferase reporter screening. Firefly luciferase reporters containing the CDS2, PRDM4 or MAP7 3′-UTRs were co-transfected with pre-miR-16 or pre-miR-control into A549 cells. Luciferase reporters containing the 3′-UTRs of PPP1R11, CHUK, LAMP2 and SLC35A4 served as negative controls. At 24 h post-transfection, cells were assayed using luciferase assay kits. The results are presented as the mean ± SD of three independent experiments (** p < 0.01; *** p < 0.001). (C) Direct recognition of the 3′-UTRs of CDS2, PRDM4 and MAP7 by miR-16. Firefly luciferase reporters containing either WT or MUT CDS2, PRDM4 or MAP7 3′-UTRs were co-transfected with pre-miR-16, pre-miR-control, anti-miR-16 or anti-miR-control into A549 cells. At 24 h post-transfection, cells were assayed using luciferase assay kits. The results presented are the mean ± SD of three independent experiments (*** p < 0.001).