Mir-29c and Mir-424 Are Novel Myeloid Differentiation-Associated MicroRNAs in Acute Promyelocytic Leukemia.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. Recent studies showed that they are critically involved in hematopoietic differentiation and function by a coordinating multi-target repression of hematopoiesis-related genes. To identify miRNAs involved in the pathogenesis of acute promyelocytic leukemia (APL), characterized by the t(15;17) translocation, we performed TaqMan Low Density Array-based miRNA expression profiling on blast cells from an APL patient under all-trans retinoic acid (ATRA) treatment. Although recent reports investigated miRNA expression patterns in APL blast cells and cell lines subjected to ATRA in vitro, to our knowledge this is the first study that relies on cells from an APL patient treated with ATRA in vivo. Since the downregulation of the PML-RARA transcript cannot be assessed within a time period of a few days, we monitored effective ATRA treatment by measuring mRNA downregulation of the panleukemic marker Wilms’ tumor (WT)-1. WT1 mRNA levels decreased 64% and 92% at day 3 and 6 upon ATRAtherapy, respectively. Total RNA obtained at diagnosis and at days 3/6 following ATRA therapy were screened for expression patterns of 384 human miRNAs including two endogenous controls, RNU44 and RNU48, for normalization of miRNA expression. Since these controls were regulated upon ATRA treatment, we normalized miRNA expression to miR-93, which showed stable expression in our samples. Consistent with previous in vitro APL miRNA profiling data, the granulocyte-specific miR-223 was induced 6.6-fold at day 6 upon ATRA treatment. For further analysis, we focused on two hematopoietic lineage-specific miRNAs, miR-29c and miR-424 that have not yet been associated with neutrophil development. miR-29c and miR-424 were upregulated 6.5- and 6.0-fold at day 6 in response to ATRA, respectively. Induction of these miRNAs was confirmed by individual real-time RT-PCR assays. Moreover, expression of miR-29c and miR-424 was further investigated in NB4 and HT93 APL cell lines. In both cell lines, miR-424 was upregulated in response to ATRA similar to the patient samples, suggesting a role for miR-424 in granulocytic differentiation in addition to that described in macrophage development. miR-29c, however, showed an upregulation in HT93 but not in NB4 cells implying cell type specific regulation. Additionally, we tested the involvement of miR- 29c in macrophage differentiation of HL60 leukemic cells using phorbol 12-myristate 13-acetate (PMA) as a differentiating agent. Interestingly, miR-29c showed an 8.0- fold upregulation similar to an 8.7-fold induction of miR-424, a known target of the transcription factor PU.1 upon PMA treatment. Based on the similar regulation of miR-29c and miR-424 and the presence of several putative PU.1 binding elements in the miR-29c promoter, we are currently investigating whether miR-29c is a novel transcriptional target of PU.1. A confirmed target of miR-29c is the protein DNA methyltransferase (DNMT 3A and 3B), which is overexpressed in myeloid leukemias. Therefore, induction of miR-29c during myelopoiesis might be needed to target DNMT. In conclusion, we propose a novel association of miR-29c and miR-424 with ATRA-induced neutrophil differentiation.