MiR-125b regulates cell proliferation and survival in neonatal megakaryocytes

Abstract

Dear Editor, We read with interest the research article by Emmrich et al. [1] on role of miRNAs in the ex vivo generation of platelets from megakaryocytes (MKs). Differentiation protocol was validated using cord blood (CB)and peripheral blood (PB)-derived human hematopoietic stem and progenitor cells. miR-125b plays pivotal role in proliferation and self-renewal of megakaryocyte progenitors[2]. Overexpression of miR-125b increased the number of colony-forming units during in vitro \culture and also produced more platelets (2.5-fold) per MK, showing its effect as oncomiR on megakaryopoiesis [3]. Ailing newborn infants are uniquely predisposed to developing thrombocytopenia. They are affected by disorders such as TAR syndrome and transientmyeloproliferative disorder (TMD). Neonatal MK progenitors are hyperproliferative and generate more MKs per colony but are smaller and less polyploid compared to adult progenitors that generate less platelets per MK [4–7]. The molecular mechanisms for this variation are unknown and thought to be related to the biological differences between them. Recent reports indicate that miRNAs play a crucial role in the regulation of megakaryocytopoiesis. To test the role of miR-125b in megakaryopoiesis, we cultured CD34+ human CB and PB cells in defined serum free medium. After 14 days of culture, >90% of the cells wereMKs (CD41+). miRNA was prepared from these cells and miR-125b levels were measured by qRT-PCR on days 0, 7, 11 and 14 (n=3); the data was analysed by ΔΔ Ct method. Western blot was performed by standard method. miR-125b levels in CB were significantly higher compared to PB-derived MKs, and these differences were consistent at all stages of MK development (0, 7, 11 and 14 days, p <0.05, Fig. 1a). We observed significant (p <0.01) downregulation of miR-125b during different stages of differentiation in CBMKs (Fig. 1b). miR-125b was reported to be overexpressed in TMD and AMKL samples compared to normal MKs [4]. To further support our hypothesis, we checked miR-125b levels in two MK cell lines of different developmental origin, CMK (child) and MEG-01 (adult). Consistent with their developmental origin, we found that miR-125b levels in CMK cells were significantly higher than in MEG-01 cells (∼7.5-fold, Fig. 1c). How exactly miR-125b might contribute to the hyperproliferative phenotype of neonatal MK progenitors is unclear. miRNAs regulate the target gene expression on a post-transcriptional level by binding to the target mRNAs which, in turn, leads to translational arrest. Web-based computational approaches (TargetScan, PicTar and MiRanda) can be used widely in predicting potential targets. Recent report suggests that miR-125b downregulates apoptosisassociated proteins in p53 pathway including BAK1 and TP53INP1, and it blocks the myeloid differentiation in part by targeting CBFβ [1]. miR-125b has been shown to target key proteins regulating apoptosis, innate immunity, inflammation, and hematopoietic differentiation [8]. Here, we show that miR-125b downregulates p53, BAK1 and CDK6 in CB-MKs (Fig. 1d). The high levels of miR-125b expression in CB-MKs might contribute in their rapid proliferation, resulting in increased megakaryocyte numbers; thus, it could be a potential therapeutic target in blood disorders. R. Kandi :R. Undi : R. K. Gutti (*) Hematologic Oncology, Stem Cells and Blood Disorders Laboratory, Department of Biochemistry, School of Life Sciences, University of Hyderabad, (PO) Gachibowli, Hyderabad 500046, AP, India e-mail: guttiravi@gmail.com Ann Hematol (2014) 93:1065–1066 DOI 10.1007/s00277-013-1928-5