Abstract
MicroRNAs (miRNAs, miRs) are short non-coding post-transcriptional regulators of gene expression in normal physiology and disease. Acute myeloid leukemia is characterized by accumulation of malignantly transformed immature myeloid precursors, and differentiation therapy, used to overcome this differentiation blockage, has become a successful therapeutic option. The human HL-60 acute leukemia cell line serves as a cell culture model for granulocytic maturation, and dimethyl sulfoxide (DMSO) incubation leads to its differentiation towards neutrophil-like cells, as assessed by biochemical, functional and morphological parameters. DMSO-induced HL-60 cell differentiation constitutes an excellent model to examine molecular processes that turn a proliferating immortal leukemic cell line into mature non-proliferating and apoptosis-prone neutrophil-like end cells. By performing genome-wide miRNA profiling and functional assays, we have identified a signature of 86 differentially expressed canonical miRNAs (51 upregulated; 35 downregulated) during DMSO-induced granulocytic differentiation of HL-60 cells. Quantitative real-time PCR was used to validate miRNA expression. Among these differentially expressed canonical miRNAs, we found miR-125a-5p upregulation and miR-17-92 cluster downregulation acted as major regulators of granulocytic differentiation in HL-60 cells. Enforced expression of miR-125a-5p promoted granulocytic differentiation in HL-60 cells, whereas miR-17-92 ectopic expression inhibited DMSO-induced HL-60 granulocytic differentiation. Ectopic expression of miR-125a-5p also promoted granulocytic differentiation in human acute promyelocytic leukemia NB4 cells, as well as in naïve human primary CD34+-hematopoietic progenitor/stem cells. These findings provide novel molecular insights into the identification of miRNAs regulating granulocytic differentiation of human leukemia cells and normal CD34+-hematopoietic progenitor/stem cells, and may assist in the development of novel miRNA-targeted therapies for leukemia.
Highlights
Polymorphonuclear neutrophils (PMNs) play an essential role in acute inflammation, are the primary mediators of the innate immune response to invading microorganisms, providing the first-line defense against infection, and have been implicated in the pathogenesis of a number of human diseases [1,2,3,4]
Differentiated HL-60 cells following a 4-day incubation showed no, or very little, apoptosis (1.5%), www.oncotarget.com but this percentage of apoptotic cells was increased to about 25% following a 5-day incubation with dimethyl sulfoxide (DMSO) (Supplementary Figure 1), in agreement with previous reports [21, 29], indicating that DMSO-differentiated behaved as end cells prone to undergo apoptosis, like their physiological mature neutrophil counterparts, and 4-day DMSO treatment was the threshold for the acquisition of the apoptosis-prone phenotype, characteristic of mature neutrophils
We have identified that miR-125a-5p upregulation plays a critical role in the differentiation of human acute myeloid leukemia HL-60 and NB4 cells as well as of normal human CD34+-HPCs towards neutrophils or neutrophil-like cells
Summary
Polymorphonuclear neutrophils (PMNs) play an essential role in acute inflammation, are the primary mediators of the innate immune response to invading microorganisms, providing the first-line defense against infection, and have been implicated in the pathogenesis of a number of human diseases [1,2,3,4]. Mature neutrophils enter the bloodstream and tissues as terminally differentiated non-proliferating cells, which have the shortest lifespan of all circulating leukocytes, previously estimated as about 1 day [5, 6], and recently challenged to be 5.4 days [7], as a result of constitutive and spontaneous apoptosis [8]. These proapoptotic mature neutrophils are constantly renewed through a sustained generation of neutrophils by the bone marrow at impressive numbers (1011 cells per day in a normal adult) through a highly controlled, yet incompletely understood process of granulopoiesis [9]. Accumulating evidence has revealed that some cancers have been associated with altered expression of particular miRNAs, and some miRNA signatures have been linked with diagnosis, staging, progression, prognosis and response to treatment in certain tumors [14, 15]
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