MicroRNA-223 regulates granulopoiesis but is not required for HSC maintenance in mice

MIR233 is genetically or epigenetically silenced in a subset of acute myeloid leukemia (AML). MIR223 is normally expressed throughout myeloid differentiation and highly expressed in hematopoietic stem cells (HSCs). However, the contribution of MIR223 loss to leukemic transformation and HSC function is largely unknown. Herein, we characterize HSC function and myeloid differentiation in Mir223 deficient mice. We show that Mir223 loss results in a modest expansion of myeloid progenitors, but is not sufficient to induce a myeloproliferative disorder. Loss of Mir223 had no discernible effect on HSC quiescence, long-term repopulating activity, or self-renewal capacity. These results suggest that MIR223 loss is likely not an initiating event in AML but may cooperate with other AML associated oncogenes to induce leukemogenesis

Loss of <i>Mir223</i> is not associated with alterations in HSC number or function.

<p>(A) Representative plots showing the gating strategy to identify multipotent progenitors (MPP, CD34⁺Flt3⁺), CD34^-KLS (CD34^-Flt3^-KLS), or CD150⁺CD48^-KLS cells. (B) Progenitor data for 8–12 week old wild-type (WT) or pooled male and female Mir223 deficient mice (223KO). (C) Representative plots showing cell cycle analysis of CD150⁺CD48^-KLS cells. (D) CD150⁺CD48^-KLS cell cycle data. (E) Wild-type or <i>Mir223</i> deficient bone marrow (Ly5.2) was mixed at a 1:1 ratio with wild-type (Ly5.1) competitor bone marrow and transplanted into irradiated recipients (Ly5.1/5.2). Shown is the percentage of Ly5.2 donor cells (n = 9–10 mice from two independent experiments). (F) Lineage distribution in the bone marrow 24 weeks after transplantation. (G) Serial transplants were performed with either Ly5.2 wild-type or <i>Mir223</i> deficient donor marrow. The percentage of donor Ly5.2 cells in the blood 6–8 weeks after transplantation is shown. (n = 4–5 mice per cohort at each time point). All data represent the mean ± SEM. *p<0.05, **p<0.005, ***p<0.001.</p

Loss of <i>Mir223</i> is associated with minimal perturbations of basal granulopoiesis.

<p>(A) Peripheral blood from male and female wild-type and <i>Mir223</i> deficient mice (223KO) was analyzed at 8–12 weeks of age. (B) Absolute number of neutrophils (PMN, Gr-1^HiCD115^-), monocytes (Mono, Gr-1^INTCD115⁺), B cells (B220⁺), and T cells (CD3e⁺) in peripheral blood. (C) Bone marrow mononuclear cell count (BMMNC). (D) Representative plots showing the gating strategy to identify mature neutrophils, granulocyte precursors (Pre, Gr-1^INTCD115^-) and monocytes. (E) Bone marrow lineage and granulocyte precursor data. (F) Representative plots showing the gating strategy to identify megakaryocyte-erythroid progenitors (MEP, CD34^-FcγR^-), common myeloid progenitors (CMP, CD34⁺FcγR^-) or granulocyte-macrophage progenitors (GMP, CD34⁺FcγR⁺); data are gated on lineage^-c-kit⁺sca-1^- cells. (G) Progenitor data.</p

Loss of <i>Mir223</i> is associated with an increased stress granulopoiesis response.

<p>(A) Wild-type and <i>Mir223</i> deficient mice (223KO) were treated with 150 mg/kg of 5-FU and neutrophil counts were followed for 35 days (n = 8–10 mice per cohort from two independent experiments). (B) Representative plots showing Ki-67 and DAPI staining of GMPs. Harvested on day 14 following 5-FU. (C) Shown is the percentage of GMPs in the G2/M/S phase of the cell cycle. All data represent the mean ± SEM. *p<0.05, **p<0.005, ***p<0.001.</p

Neutrophils counts are normal in aged <i>Mir223</i> deficient mice.

<p>(A and B) Wild-type and <i>Mir223</i> deficient (223KO) mice at least 6 months old were analyzed for (A) peripheral blood or (B) bone marrow neutrophil counts. The absolute number of neutrophils (PMN, Gr-1^HiCD115^-) is shown. All data represent the mean ± SEM.</p

No gender specific alterations in hematopoiesis are present in <i>Mir223</i> deficient mice.

<p>(A and B) Peripheral blood from 8–12 week old wild-type and (A) female <i>Mir223</i>^-/Y or (B) male <i>Mir223</i>^-/- mice was analyzed at 8–12 weeks of age. The absolute number of peripheral blood mononuclear cells (PBMNC), neutrophils (PMN, Gr-1^HiCD115^-), monocytes (Mono, Gr-1^INTCD115⁺), B cells (B220⁺), and T cells (CD3e⁺) are shown. All data represent the mean ± SEM. *p<0.05.</p