CRITICAL ROLE OF MICRORNA BIOGENESIS PATHWAY DURING MYELOID CELL FATE DETERMINATION

ABSTRACT Hematopoiesis is a multistage process where a pluripotent self-renewing hematopoietic stem cell (HSC) gives rise to all blood cell lineages. The clonal expansion of hematopoietic precursors blocked at different stages of differentiation characterized acute myeloid leukemia (AML). MicroRNAs (miRNAs) are emerging as constituents of evolutionary highly conserved molecular pathways regulating cell fate decision in several development programs. In particular, in these last years, miRNAs have been involved in the establishment of myeloid differentiation and leukemogenesis. For this reason, it is not surprising that hematological malignancies are characterized by misregulation of either miRNAs expression or protein involved in miRNA biogenesis pathway. In the first part of this project, here we show, that miR26a is downregulated in primary blasts of AML patients and that, during myeloid differentiation of AML cells, it is induced together with a decrease in c-Myc and Ezh2 levels. In addition, increased levels of miR-26a potentiate the antiproliferative effects of 1,25-dihydroxyvitamin D(3) (VitD) and stimulate myeloid differentiation. Moreover, we identified the transcriptional repressor E2F7 as a novel target of miR- 26a. We show that E2F7 significantly promotes cell cycle progression and inhibits monocytic differentiation of AML cells and that the repression of E2F7 by miR-26a contributes to the increased expression of p21CIP1/WAF1 and to the VitD3-induced monocytic differentiation of AML cells. The results of this study identify a novel aspect of VitD3 action in regulating proliferation and differentiation of AML cells through the miR-26a/E2F7/p21CIP1/WAF1 network. In the second part of this project, we show the role of Argonaute 2 (Ago2), a core component of RISC (RNA-induced silencing complex), in human myeloid differentiation. In particular, we observed that Ago2 protein levels are increased during monocyte differentiation of myeloid progenitors, whereas are downregulated during granulocyte differentiation of human leukemic cell lines and freshly isolated blasts from acute promyelocytic leukemia (APL) patients. We found that the Ago2 silencing, lentivirus mediated, alters the correct modulation of transcription factors and microRNAs involved 5 in monocyte cell fate determination, leading to the reduction of VitD-induced monocytic differentiation and subsequently activation by LPS-induced inflammatory response.