C/EBPα-p42 up-regulates miR-223 and leads to down-regulation of E2F1 protein expression. (A-B) K562-C/EBPα-p42-ER cells and K562-ER cells (A) and K562-C/EBPα-p30-ER cells and K562-C/EBPα-BRM2-ER cells (B) were induced with β-estradiol (5μM) for respective time points. Total RNA was analyzed by quantitative real-time RT-PCR with oligos for miR-223. Data are represented as mean ± SD from 3 independent experiments. *P < .05; **P < .01. (C) K562-C/EBPα-p42-ER cells and K562-ER cells were induced with β-estradiol (5μM) for respective time points. Total protein was analyzed by Western blot analysis with anti-E2F1 antibody. Values below the gel image indicate the down-regulation (fold) of E2F1 protein level normalized to β-tubulin.

E2F1 is a direct target of miR-223. (A) Schematic representation of the E2F1 transcript. Predicted miR-223 binding site is depicted. The numbers (+749 to +754) represent the nucleotides (relative to the E2F1 termination codon) that are predicted to base pair with the miR-223 seed sequence. (B) Sequences of the predicted miRNA-223 binding sites in human, mouse, and rat genomes. Highly conserved nucleotides are shown in gray. (C) Schematic representation of luciferase constructs used for reporter assays. (D) Luciferase assays in 293T cells transfected with vectors shown in panel C and miR-223 and control oligonucleotides (mimic). Bars represent luciferase activity for the corresponding vectors. Data are represented as mean ± SD from 3 independent experiments. **P < .01. (E-F) Western blot analysis of E2F1 on whole-cell lysates from sorted Ly6G^pos bone marrow neutrophils from wild-type (WT) and miR-223 knockout (KO) animals¹¹ (E), on whole-cell lysates from U937 cells treated with 20 nm of control and miR-223 oligonucleotides (mimic; F).

miR-223 functions as a tumor suppressor in acute myeloid leukemia. (A) Quantitative real-time RT-PCR for miR-223 was carried out using bone marrow cells derived from AML patients. Values were normalized with U6. Cord blood (CB) and peripheral blood (PB) and AML with normal karyotype (nk), with complex karyotype (ck), with FLT3 mutation (FLT3 mut), and with CEBPA mutation (CEBPA mut). Data are represented as mean from 3 experiments. (B) U937 cells were transfected with lentiviral control or lenti-223 vectors. Two days later, total RNA was isolated and analyzed for miR-223 by quantitative real-time RT-PCR. Data are represented as mean ± SD from 3 independent experiments. **P < .01. (C) Growth curve of U937 cells transfected with lentiviral control or lenti-223 vectors. Data are represented as mean ± SD from 3 independent experiments. **P < .01. (D) Analysis of cell-cycle distribution during miR-223 lentiviral overexpression by flow cytometry. Representative FACS data from U937 cells transfected for 2 days with control and miR-223 lentiviral vector. (E) Flow cytometry of BrdU/PI-stained U937 cells transfected with lentiviral control or lenti-223 vectors from a representative experiment. Two days after lentiviral transfection, cells were labeled with BrdU for 45 minutes. Cells were then fixed, stained with FITC-labeled anti-BrdU antibody and PI, and analyzed by flow cytometry to determine the cell-cycle distribution. Bivariate analysis of the incorporation of BrdU (BrdU FITC, vertical axis) and the DNA content (FL3, PI staining, horizontal axis) was performed.

E2F1 functions as a repressor of the miR-223 gene. (A) Schematic representation of the different miR-223 promoter constructs used for reporter assays. (B-D) Luciferase reporter assays were performed in U937 cells using indicated reporters and E2F1 (B) and C/EBPα (C-D). Bars represent promoter activity for the corresponding vectors. Data are represented as mean ± SD from 3 independent experiments. *P < .05; **P < .01. (E-F) Chromatin derived from K562-C/EBPα-p42-ER cells (E) and AML blast cells (F) was immunoprecipitated with anti-E2F1, anti-C/EBPα, and immunoglobulin G antibodies. Recovered DNA was PCR amplified with primers specific for the E2F1 binding amplicon (oligo 1) and C/EBPα binding amplicon (oligo 2). (G) U937 cells were transfected with control or E2F1 vector. Total RNA was analyzed by quantitative RT-PCR with oligos for miR-223 (top) and with oligos for E2F1 (bottom). Data are represented as mean ± SD from 3 independent experiments. *P < .05; **P < .01.

Schematic representation of a model for the role of miR-223, C/EBPα, and E2F1 in normal granulopoiesis and in AML. During granulopoiesis (top panel), C/EBPα binds and transactivates miR-223 promoter, which in turn leads to E2F1 repression and inhibition of cell-cycle progression resulting in myeloid differentiation. When C/EBPα is deregulated by various mechanisms in AML (bottom panel), transactivation of miR-223 is inhibited, which results in accumulation of E2F1. Overexpressed E2F1 binds to miR-223 promoter and inhibits miR-223 transcription through a negative feedback loop resulting in myeloid cell-cycle progression and block of differentiation.