Figure 5

Verification of PRDM1 as a direct target gene of miR-223. (A) The complementarity between miR-223 and its 3 conserved putative binding sites in the PRDM1 3′-untranslated region (UTR) is highlighted in bold between different species. (B) Luciferase reporter assays were performed in 293 T cells that were co-transfected with miR-223 mimic and wild type pmirGLO expression-PRDM1-3′UTR (WT) reporter plasmid or mutant pmirGLO expression-PRDM1-3′UTR reporter plasmids harbouring point mutations in the target sites for miR-223 (Mut1, Mut2, Mut3, Mut1 + 2, Mut1 + 3, Mut2 + 3, and Mut1 + 2 + 3). Mimic Negative Control was used as a negative control (NC). Firefly luciferase activity was normalised relative to Renilla luciferase activity. Transfection of the miR-223 mimic resulted in a marked decrease in luciferase activity in the WT group compared to the NC group (48.08%). Mutations in each of the putative target sites or combined mutations restored luciferase activity to varying degrees: 74.87% for Mut1, 85.21% for Mut2, 74.84% for Mut3, 90.76% for Mut1 + 2, 87.55% for Mut1 + 3, 81.15% for Mut2 + 3, and 94.51% for Mut1 + 2 + 3. Data are presented as mean ± SE of 4 independent experiments. (C) Two nucleotides in the middle of each target site were mutated to generate different mutant luciferase reporters.