Enforced Expression of Mir-125b Blocks Granulocytic Differentiation by Simultaneous Repression of Multiple Targets.

Abstract 1554

Granulopoiesis is a multistep process controlled by a complex system of cytokines and transcription factors that modulate expression of downstream genes and mediate proliferation and differentiation signals. Recent findings demonstrate that miRNAs may provide an additional level of control. We used the 32D murine myeloid progenitor cell line as a model system to study G-CSF-induced granulocytic differentiation. Based on miRNA-expression analyzed by microarray and miR-qRT-PCR, we identified several miRNAs (including miR-34a-c, -125b, -155, 181b, 223, 291a, 370) potentially involved in regulation of granulocytic differentiation. To define the role of individual miRNAs, stable gain- and loss-of-function phenotypes were generated using lentiviral gene transfer of pre-miRNAs and antagomiRs, respectively. We found that enforced expression of miR-125b in undifferentiated 32D myeloid precursors resulted in a complete block of granulocytic differentiation upon G-CSF treatment and partially protected the cells from IL-3 withdrawal-induced cell death. The pivotal role of miR-125b in myeloid differentiation was demonstrated in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with the chromosomal translocation t(2;11)(p21;q23) resulting in miR-125b over-expression [Bousquet M, et al. JEM 2008]. Furthermore, clonogenic assays of primary Lin^- cells revealed that miR-125b cultures generated more and larger colonies as compared to miR-control, indicating a proliferative advantage of miR-125b over-expressing progenitor cells. Correspondingly, enforced miR-125b expression in murine bone marrow has recently been shown to induce a lethal myeloproliferative disorder (MPD) in transplanted mice [O'Connell RM, et al. PNAS 2010]. However, the molecular mechanisms mediated by miR-125b in hematopoietic cells remain largely unknown. By utilizing different miRNA-target prediction programs including Target Scan, DIANA microT v 0.3 and RNA22, we identified Bcl-2 family members Bak1, Mcl-1, Bmf and Puma as putative targets of miR-125b. We confirmed diminished Bak1 protein expression in 32D cells (reduction by ∼40%); however, lentivirus-mediated RNAi targeting Bak1 to the similar level as induced by enforced miR-125b expression resulted only in a delay of cell death in the presence of G-CSF but not in granulocytic differentiation block. Further computational analysis revealed two putative miR-125b binding sites in 3’ UTR of Stat3, the principal Stat protein activated following G-CSF treatment and robustly involved in myeloid differentiation. Specific binding of miR-125b to Stat3 3’UTR was validated by luciferase reporter assay and confirmed by western blotting (reduction by ∼30-40%). We found that shRNA-mediated strong reduction of Stat3 protein (reduction by ∼80%) enables G-CSF dependent cell proliferation (with a delay of some days), and induces a complete block of cellular differentiation. On the contrary, a mild reduction of Stat3 protein expression(by ∼30–40%), as induced by enforced expression of miR-125b, resulted only in a slight delay of cell death but not in block of granulocytic differentiation. Interestingly, simultaneous reduction of both Bak1 and Stat3 by combinatorial RNAi to similar levels as observed in the presence of enforced miR-125b expression, cooperatively delays cell death upon G-CSF treatment as compared to knock-down of Bak1 or Stat3 solely. Since miR-125b has approximately 500 predicted target genes and neither mild knock-down of Bak1 nor Stat3 alone resulted in a block of granulocytic differentiation, we may either miss an additional/unknown important target or the miR-125b-induced phenotype requires simultaneous repression of more targets in addition to Stat3 and Bak1. Nevertheless our study provides experimental evidence that miR-125-mediated phenotypes arise from mild and simultaneous down-regulation of multiple targets/signalling pathways. Therefore the precise and quantitative analysis of miRNA-targets is required to evaluate the safety and benefit of eventual miRNA-based therapeutic strategies to modulate complex cellular phenotypes.