miR-9 upregulation leads to inhibition of erythropoiesis by repressing FoxO3

Abstract

MicroRNAs (miRNAs) are emerging as critical regulators of normal and malignant hematopoiesis. In previous studies of acute myeloid leukemia miR-9 overexpression was commonly observed. Here, we show that ectopic expression of miR-9 in vitro and in vivo significantly blocks differentiation of erythroid progenitor cells with an increase in reactive oxygen species (ROS) production. Consistent with this observation, ROS scavenging enzymes, including superoxide dismutase (Sod2), Catalase (Cat), and glutathine peroxidase (Gpx1), are down-regulated by miR-9. In addition, miR-9 suppresses expression of the erythroid transcriptional regulator FoxO3, and its down-stream targets Btg1 and Cited 2 in erythroid progenitor cells, while expression of a constitutively active form of FoxO3 (FoxO3-3A) reverses miR-9-induced suppression of erythroid differentiation, and inhibits miR-9-induced ROS production. Thus, our findings indicate that aberrant expression of miR-9 blocks erythropoiesis by deregulating FoxO3-mediated pathways, which may contribute to the ineffective erythropoiesis observed in patients with hematological malignancies.

Figure 1

Forced expression of miR-9 inhibits G1ER cell differentiation. (A) qRT-PCR analysis of miR-9 precursor expression in Migr1 and Migr1-MiR-9 transduced G1ER cells. Hprt gene was used for normalization. (B) Representative images of the benzidine staining of GFP⁺ G1ER cells expressing EGFP or miR-9/EGFP. Differentiation of the cells was induced with β-estrodial for two days. (C) The diagram shows the average percentage of Benzidine positive cells in GFP⁺ G1ER cells infected with control vector and miR-9 in three independent experiments. *P < 0.05.

Figure 2

Ectopic expression of miR-9 blocks differentiation of primary erythroblasts in vitro. (A) qRT-PCR analysis of miR-9 expression in Migr1 and Migr1-miR-9 transduced E14.5 fetal liver cells. (B) Representative histograms of flow cytometric analysis of erythroblasts at day 1 and day 4 after infection with vector and miR-9 retrovirus. The switch from growth medium to differentiation medium at day 2 promoted erythroblasts differentiation. (C,D) Histograms of average ratio of R1, R2, R3, R4 and R5 in erythroblasts at day 1 and day 4. The experiments were performed in triplicate and repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Forced expression of miR-9 inhibits differentiation and reduces the frequency of cell death of early erythroid progenitor cells. Day 14.5 fetal liver cells were infected with Migr1 vector or Migr1-miR-9, and EGFP+ cells sorted and plated on methylcellulose medium. (A) Histograms showing the number of BFU-E colonies. (B) Histograms showing the number of CFU-E colonies. (C) The representative BFU-E colonies formed by Vector and MiR-9 infected fetal liver cells. (D) Representative image of benzidine staining of cells from BFU-E colonies. *P < 0.05; ***P < 0.001. (E) Representative histograms of flow cytometric analysis of apoptotic and dead cells in vector or miR-9 infected(GFP+) fetal liver cells two days after induction of differentiation in vitro. (F) The percentage of DAPI+ cells in vector or miR-9 infected fetal liver cells. Experiments were repeated three times. **P < 0.01.

Figure 4

miR-9 inhibits erythroblast cell differentiation in vivo. (A) The histogram shows the percentage of Ter119+ cells in gated EGFP+ cells in vector and miR-9 chimeric mice. To generate the chimeric mice, the Lin^- primary bone marrow cells were infected with retrovirus expressing miR-9 or control vector, followed by transplanted into lethally-irradiated wildtype recipient mice. (B) Flow cytometric analysis of the frequencies of erythroblasts in bone marrow cells from the representative vector and chimeric mice. (C) Histogram shows the average frequencies of erythroblasts in vector and miR-9 chimeric mice (means ± SD, n = 5–7). *P < 0.05; ***P < 0.001.

Figure 5

Ectopic expression of miR-9 increases production of ROS in fetal liver cells and G1ER cells. (A) Representative histograms show ROS levels in GFP + fetal cells infected with Migr1 or Migr1-miR-9. Top panel, untreated cells; bottom panel, cells treated with NAC. (B) Summary of ROS levels in Migr1 and Migr1-miR-9 transduced fetal liver cells without treatment (left panel) or with NAC treatment (right panel). (C) qRT-PCR analysis of the expression of ROS scavenging enzymes in vector or miR-9 infected fetal liver cells. Experiments were repeated three times. (D) Representative histograms show ROS levels in GFP+G1ER cells infected with Migr1 or Migr1-miR-9. Top panel, untreated cells; bottom panel, cells treated with NAC. (E) Summary of ROS levels in Migr1 and Migr1-miR-9 transduced G1-ER cells without treatment (left panel) or with NAC treatment (right panel). (F) qRT-PCR analysis of the expression of ROS scavenging enzymes in vector or miR-9 infected G1ER cells. *P < 0.05.

Figure 6

Forced expression of FoxO3 reverses miR-9-mediated inhibition of erythroid progenitor cell differentiation and induction of ROS production. (A) qRT-PCR analysis of the expression of FoxO3, and its known down-stream targets in GFP+ fetal liver cells (left panel) and G1ER cells (right panel) transduced with vector or miR-9. (B) Representative histograms of flow cytometric analysis of GFP+ fetal liver cells transduced with Vector, miR-9, FoxO3-3A and FoxO3A/miR-9. (C) Histograms show the average percentage of cells in different stages of erythroid blast differentiation from these transduced fetal liver cells. (D) ROS levels in fetal liver cells transduced with vector, miR-9, FoxO3a and FoxO3a/miR-9. The experiment was performed in triplicate and repeated 2–3 times. *P < 0.05; ***P < 0.005.