HSP90B1is a direct target of miR-223. (A) 3′untranslated region (3′UTR) of HSP90B1 (263 nt length) with a predicted binding site for miR-223 at 204–210 nt (grey box). The figure shows the mature miR-223 sequence (hsa-miR-223) aligned with HSP90B1 3′UTR wild type (WT, up), and with the polymorphism (VAR, below). The seed region is shown in bold. The rs2307842 polymorphism (in grey) disrupts the putative binding site for miR-223 by deleting the last three nucleotides of the seed region. (B) Luciferase reporter assays to confirm targeting of HSP90B1 3′UTR by miR-223. Ectopic miR-223 expression inhibits the wild-type but not the variant HSP90B1 3′UTR reporter activity in HEK293 cells. Cells were co-transfected with miR-223 precursor/negative control (NC) miRNA and with either wild-type (WT) or variant (VAR) HSP90B1 3′UTR reporter construct. Luciferase activity assay was performed 24 h after transfection. The columns represent normalized relative luciferase activity by means with 95% confidence intervals from 4 independent experiments (Mann–Whitney test, *P < 0.05). (C) and (D) Ectopic miR-223 expression reduced both HSP90B1 mRNA (C) and protein (D) expression in H929 cell line (WT) but not in MM1S (VAR). Cells were transfected with miR-223 precursors and negative controls. After 24 h, cells were analyzed for HSP90B1 expression by qRT-PCR (C) and western blot (D). The data shown are representative of 3 independent experiments (Mann–Whitney test, *P < 0.05).