Amritapuri, Vallikavu, Kerala 690525
Ravi Gutti, Rambabu Undi, Ravinder Kandi and Itishri Sahu
Hematologic Oncology, Stem Cells and Blood Disorders Laboratory, School of Life Sciences, Department of Biochemistry, University of Hyderabad, Gachibowli, Hyderabad 500 046 (AP), India.
Megakaryocytopoiesis is governed by a complex network of haematopoietic growth factors that regulate the different stages of the process, in which haematopoietic stem cells undergo megakaryocytic lineage commitment, proliferation, maturation, and functional activation to produce platelets. Hyperproliferative progenitors and small, low-ploidyMKs in neonates may be due to the developmental differences in megakaryocytes (MKs) between neonates and adults. The biological relevance of these differences is highlighted by the fact that a number of disorders of megakaryocytopoiesis are restricted to the fetal and neonatal stages of development. The regulatory mechanisms underlying these developmental differences are unknown and the small non-codingmicroRNAs (miRNAs) shown to play a critical role in the regulation of MK development. We hypothesized that miRNAs would be differentially expressed in neonatal and adult MKs, and that these differences would contribute to their biological differences. To test this, we cultured human cord blood (CB) and peripheral blood (PB) CD34+ cells in serum free media with thrombopoietin. After 14 days of culture, > 90% of the cells were MKs (CD41+). miRNA and protein was isolated and expression levels of 88 miRNAs known to be involved in human stem cell differentiation and development were measured using a quantitative PCR-based array kit. Web-based computational approaches (TargetScan, PicTar and MiRanda) were used for putative target prediction and the protein levels were detected using western blot analysis. All samples (n = 4 per group) expressed detectable amounts of all 88 screened miRNAs. TenmiRNAs were expressed at significantly higher levels in CB compared toPBMKs (2 to 21 fold, p < 0.05. We then looked for the target for thisupregulatedmiRNAs in CB MKs. RUNX-1 was a predicted target of five of theupregulatedmiRNAs in CBMKs (miR-9, miR-129-5p, miR-192, miR-215, and miR-370). RUNX1 is an eukaryotic gene and the protein encoded by this gene is a transcription factor, also called AML-1 which is critical for MK maturation. In humans, loss of function mutations in RUNX-1 cause familial platelet disorder with propensity to develop acute myeloid leukemia, an autosomal dominant disorder characterized by quantitative and qualitative platelet defects and a predisposition to develop AML. In addition, RUNX 1 protein levels were approximately 3-fold higher inPB compared to CB MKs. Our results indicate that there are significant differences in the RUNX-1 expression and decreased RUNX-1 levels are consistent with the phenotype of increased proliferation and decreased size andploidy that characterizes neonatal megakaryopoiesis. Low RUNX-1 protein levels in neonatal compared to adult MKs, suggests that its translation is inhibited by those overexpressed miRNAs. We found activation of cyclin D3 transcription and repression of the p21 promoter likely contribute to stimulation of proliferation by Runx1. We believe that Wnt ligands could also stimulate cyclin D and c-Myc expression to favor cell proliferation by stimulating G1 entry from G0 and G1 to S cell cycle progression (Friedman 2009). Epigenetic/transcriptional control, RNA processing, and regulation of protein stability also likely to influence Runx1 activities in developmentalmegakaryocytopoiesis. Further studies to determine whether this contributes to the neonatal MK phenotype are in progress.