Figure 5.

miR-99 functionally suppresses human AML differentiation. (A) Normalized expression levels of miR-99a, miR-99b and miR-100 by quantitative PCR using miRNA TaqMan probes in human HSPCs, including hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), and megakaryocyte-erythroid progenitors (MEPs). Expression was normalized to sno-R2. Data represent mean ± SEM and are representative of five independent experiments. (B) The colony-forming capacity of CD34⁺ human cord blood cells is reduced after miR-99 KD. CD34⁺ cells were transduced with lentiviral anti–miR-99 or scramble control. GFP⁺ cells were isolated and cultured in complete methylcellulose, and the colonies were scored after 14 d. Data represent mean ± SEM (Student’s t test; n = 3) and are representative of two independent experiments. (C) Flow cytometric evaluation of myeloid differentiation marker expression on MonoMac6 AML cells 5 d after transduction with anti–miR-99 or scramble control. Data represent mean ± SEM (Student’s t test; n = 3) and are representative of three independent experiments. (D) Wright–Giemsa stains of cytospin preparations of MonoMac6 cells 8 d post-transduction with lentiviral anti–miR-99 or Scr reveals induction of differentiation upon miR-99 KD (bars, 25 µm). (E) Overview of the xenotransplantation experiment performed on MonoMac6 AMLs. Cells were transduced with anti–miR-99 or Scr. After 48 h, GFP⁺ cells were flow sorted, and 800,000 cells were transplanted into sublethally irradiated NSGs. BM was analyzed 4 wk after the transplant. (F) miR-99 KD reduces the of GFP⁺ engraftment of MonoMac6 cells in the BM of recipients 4 wk post-transplantation. Data represent mean percentage ± SEM (Student’s t test; n = 4) and are representative of two independent experiments. (G) Representative histogram and aggregated data from flow cytometric evaluation of CD14 expression on GFP⁺ xenografted cells in the BM of the recipient animals 4 wk post-transplantation. Data represent mean percentage ± SEM (Student’s t test; n = 4) and are representative of two independent experiments. (H) Flow cytometry analysis of three AML patient samples after miR-99 KD. Patient samples were transduced with anti–miR-99 or scramble control and analyzed for the expression of differentiation markers 5–8 d later. Data represent mean percentage ± SEM (Student’s t test; n = 2) and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.