Deciphering the Role of Mir-99∼125 clusters in the Hematopoietic System

Abstract 213

Three human miR-99∼125 clusters, each containing one miR-99/100, let-7 and miR-125 family member in identical polycistronic configuration, are located within or next to long non-coding RNA (lncRNA) host genes on distinct chromosomal regions. The cluster miRNAs are highly expressed in hematopoietic stem and progenitor cells (HSPCs). The individual importance for stem cell homeostasis and differentiation of let-7 and miR-125 was extensively studied. However, genomic redundancy and high phylogenetic conservation of this ncRNA ensemble suggest a functionally linked role on gene regulatory networks, which remained elusive. Here we describe a synthetic phenotype pattern of the miR-99∼125 clusters on the hematopoietic system.

Expression profiling in CD34⁺-HSPCs (n=2) and megakaryocytes (n=1) from healthy donors, in sorted leukemic blasts from patients with Down syndrome (DS) acute megakaryoblastic leukemia (AMKL; n=5), DS-transient leukemia (n=4), non-DS-AMKL (n=3), acute myeloid leukemia (AML FAB M5; n=2) and in various cell lines (CMK, CMY, Meg01, M07e [all megakaryoblastic leukemia], K562, KG1α, NB4, THP1 and Jurkat) indicated an enrichment of mature miR-99∼125 miRNAs and their lncRNA host genes in acute megakaryoblastic leukemia. Quantification of primary miRNA/mRNA transcripts indicated that intronic miR-99∼125 miRNAs are produced in one primary transcript with their lncRNA host genes C21ORF34 and MIR100HG. 5'RACE revealed the transcription start sites of those transcripts, which allowed mapping of phylogenetically conserved transcription factor binding sites in their promoter regions. Subsequent chromatin immunoprecipitation (ChIP) and luciferase reporter assays confirmed the binding and positive regulation by the leukemogenic transcription factor and stem cell regulator HOXA10.

Retroviral overexpression of miR-99∼125 cluster miRNAs in cord blood (CB)-HSPCs showed a recurrent phenotype. The tricistron (miR-99a/let-7c/miR-125b-2) and miR-125b-2 alone accelerated proliferation during megakaryocytic, neutrophilic and monocytic in vitro differentiation, while let-7c and miR-99a/let-7c blocked proliferation. Likewise, common myeloid (CMP) and megakaryocytic/erythroid progenitors (MEP) were expanded upon tricistron (CMP: 1.5-fold; MEP: 2-fold increase relative to control) and miR-125b-2 (CMP: 1.6-fold; MEP: 2.4-fold increase relative to control) overexpression and reduced upon let-7c (CMP: 2.1-fold; MEP: 2.5-fold reduction relative to control) and miR-99a/let-7c (CMP: 2.2-fold; MEP: 3-fold reduction relative to control) overexpression. Inversely, during induced megakaryocytic differentiation stable inhibition of miR-125b using sponge-technology (SP-miR125) or shRNA-mediated downregulation of the C21ORF34 host gene conferred a selective growth disadvantage (SP-miR125: 1.6-fold; shC21ORF34: 1.7-fold reduction relative to control) and inhibited colony-formation of AMKL cell lines (SP-miR125: 1.4-fold; shC21ORF34: 2-fold reduction relative to control) and CB-HSCs (SP-miR125: 1.7-fold; shC21ORF34: 3.7-fold reduction relative to control). Ectopic expression or stable inhibition of miR-99a alone had no effect in any assay.

Collectively we deciphered the functional linkage of miR-99/100, let-7 and miR-125, which are produced from one primary transcript with their lncRNA host genes. The stem cell regulator HOXA10 acts upstream of this ncRNA ensemble. Our study creates a microRNA interaction network, wherein miR-125b dominates let-7 to induce proliferation and expansion of CMPs and MEPs, favoring megakaryoblastic leukemia. This suggests an epistatic circuit regulating stemness and developmental fate.