Oncomir miR-125b regulates hematopoiesis by targeting the gene Lin28A - PubMed (original) (raw)

Oncomir miR-125b regulates hematopoiesis by targeting the gene Lin28A

Aadel A Chaudhuri et al. Proc Natl Acad Sci U S A. 2012.

Abstract

MicroRNA-125b (miR-125b) is up-regulated in patients with leukemia. Overexpression of miR-125b alone in mice causes a very aggressive, transplantable myeloid leukemia. Before leukemia, these mice do not display elevation of white blood cells in the spleen or bone marrow; rather, the hematopoietic compartment shows lineage-skewing, with myeloid cell numbers dramatically increased and B-cell numbers severely diminished. miR-125b exerts this effect by up-regulating the number of common myeloid progenitors while inhibiting development of pre-B cells. We applied a miR-125b sponge loss of function system in vivo to show that miR-125b physiologically regulates hematopoietic development. Investigating the mechanism by which miR-125b regulates hematopoiesis, we found that, among a panel of candidate targets, the mRNA for Lin28A, an induced pluripotent stem cell gene, was most repressed by miR-125b in mouse hematopoietic stem and progenitor cells. Overexpressing Lin28A in the mouse hematopoietic system mimicked the phenotype observed on inhibiting miR-125b function, leading to a decrease in hematopoietic output. Relevant to the miR-125b overexpression phenotype, we also found that knockdown of Lin28A led to hematopoietic lineage-skewing, with increased myeloid and decreased B-cell numbers. Thus, the miR-125b target Lin28A is an important regulator of hematopoiesis and a primary target of miR-125b in the hematopoietic system.

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Conflict of interest statement

Conflict of interest statement: D.B. is a Director of Regulus, a company devoted to commercialization of antimicroRNA therapies.

Figures

Fig. 1.

Fig. 1.

miR-125b overexpression causes an aggressive invasive myeloid leukemia. (A) Infiltration of GFP+ CD45+ and GFP+ CD11b+ cells into the brain. (Upper) Representative flow cytometric plots are shown. (Lower) Average percent GFP+ CD45+ and GFP+ CD11b+ in the brain are shown from three MG and two MG-125b mice. (B) Leukemic cell infiltration into nonhematopoietic organs. Sections from the kidney, lung, and liver were stained with H&E. The normal structures of the MG-125b mouse kidney, lung, and liver are effaced by a dramatic infiltrate of leukemic cells. A representative image for each tissue is shown. The brain, kidney, lung, and liver were harvested from animals 5 mo after bone marrow reconstitution. During the time of harvest, the average percent GFP+ cells in the spleens of MG and MG-125b mice were 49 ± 9.7% and 91 ± 2.9%, respectively. All plots shown depict the mean with SEM. All data are representative of two independent experiments. Inf, infiltrate; G, glomerulus; A, alveolar space; CV, central vein.

Fig. 2.

Fig. 2.

miR-125b overexpression causes a skewing of the hematopoietic system at 7 wk postreconstitution of bone marrow. (A) Flow cytometric analyses of MG and MG-125b spleens were used to quantify the percent of granulocytes (GR-1+ CD19−), macrophages (F4/80+ CD68+), dendritic cells (CD11b+ CD11c+), T cells (CD3ε+ CD19−), and B cells (CD19+ GR-1−). Total leukocyte counts in the spleen were similar between MG and MG-125b mice. (B) The percent of MPPs and total numbers of CMPs/GMPs, MEPs, CLPs, and pre-B cells in the bone marrow were measured by flow cytometry. Horizontal lines represent the means, and each dot represents an individual mouse. Data are representative of two to three independent experiments with four to five mice per group.

Fig. 3.

Fig. 3.

Inhibiting miR-125b function decreases hematopoietic output. Equal numbers of sorted GFP+ MG- and MG-Sponge–transduced progenitor-enriched bone marrow cells (BMCs) were injected into recipient animals. (A) Total numbers of white blood cells, (B) myeloid cells, (C) granulocytes, and (D) pre-erythroid cells were measured by flow cytometry. Data represent mean number of cells per milliliter of blood (SEM shown). P values were calculated using unpaired Student t test. All results are representative of two independent experiments with 9–10 animals per group.

Fig. 4.

Fig. 4.

miR-125b represses Lin28A expression in mouse and human hematopoietic cells. (A) Relative expression of miR-125b candidate targets in miR-125b–overexpressing progenitor-enriched BMCs compared with MG control. Bone marrow cells from 5-FU–treated mice were infected with miR-125b–overexpressing vector, and RNA was subsequently harvested for expression level analysis. The relative expression level was measured by quantitative PCR. (B) 3′ UTR reporter analyses. The 3′ UTR of the indicated genes was cloned into a luciferase reporter and cotransfected with a miR-125b expression vector. Relative luminescence of the luciferase reporters was measured and normalized to an empty vector control. The Picalm 3′ UTR, which contains no miR-125b putative binding sites, serves as a negative control. The 2mer positive control, which consists of two adjacent miR-125b antisense sites, is also shown. (C) Relative luminescence of the Lin28A 3′ UTR reporter cotransfected with either MG or miR-125b sponge into 293T cells. (D) Protein expression of Lin28A transduced with either MGP empty vector or MGP expressing miR-125b-1, miR-125b-2, or a miR-125b seed mutant in K562 cells. Protein levels were obtained by Western blot, and β-actin was included as a loading control. All data shown represent mean with SEM of two to three independent experiments. *P < 0.02, **P < 0.01.

Fig. 5.

Fig. 5.

Lin28A overexpression inhibits hematopoiesis. Recipients were transplanted with progenitor-enriched BMCs transduced with MIG-Lin28A or MIG. Five weeks postreconstitution, the number of (A) white blood cells, (B) myeloid cells, (C) granulocytes, and (D) erythrocytes per milliliter in the blood were measured by flow cytometry. We did not observe an increase in B cells in the MIG-Lin28A animals. Data are pooled from two independent groups of MIG-Lin28A animals, each with seven to eight animals, and compared with MIG (six animals). Data shown graphically represent the mean and SEM.

Fig. 6.

Fig. 6.

Inhibition of Lin28A increases the number of myeloid cells but decreases B cells. (A) Flow cytometry of progenitor-enriched bone marrow transduced with MG-Lin28A shRNA or MG control vector was performed, and the percent GFP+ cells is indicated. (B) Lin28A mRNA expression was obtained by quantitative PCR on the samples shown in A, and the results were normalized to L32. Representative of two independent experiments with four samples per group. (C) Total myeloid, (D) granulocytes, and (E) B cells were measured from blood 5 wk postreconstitution of bone marrow with seven to eight animals per group. Data shown represent mean and SEM. P values are indicated.

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