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Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways

Ewa Surdziel, Maciej Cabanski, Iris Dallmann, Marcin Lyszkiewicz, Andreas Krueger, Arnold Ganser, Michaela Scherr and Matthias Eder

Abstract

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-, maturation-, and disease-specific miRNA expression has been described, miRNA-dependent phenotypes and miRNA-regulated signaling in hematopoietic cells are largely unknown. Combining functional genomics, biochemical analysis, and unbiased and hypothesis-driven miRNA target prediction, we show that lentivirally over-expressed miR-125b blocks G-CSF–induced granulocytic differentiation and enables G-CSF–dependent proliferation of murine 32D cells. In primary lineage-negative cells, miR-125b over-expression enhances colony-formation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression, respectively. However, gene-specific RNAi reveals that this reduction, alone and in combination, is not sufficient to block G-CSF–dependent differentiation. STAT3 protein expression, DNA-binding, and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression, indicating miR-125b–mediated reduction of one or more STAT3 cofactors. Indeed, we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/miR-125b cells. Interestingly, gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells.

  • Submitted June 4, 2010.
  • Accepted February 13, 2011.
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