Mahin Nikougoftar Zarif
MicroRNAs(miRNAs) are small noncoding RNAs that regulate gene expression by mRNA silencing, consequently have an impact on many biological processes. Previous studies approved the role of miRNAs in megakaryopoiesis. These molecules target specific protein mRNAs. In this study, we analyzed the expression of MAPK, DYRK1A, CDC7, RUNX1 and CDK during megakaryopoiesis. These proteins are targeted for at least two miRNAs which are involved in this pathway. BM derived CD133+ Hematopoietic stem cells (HSCs) were sorted and cultured in cytokines stimulating Mk differentiation media. Differentiation was evaluated by CD42/CD61/CD41 expression and colonogenic capacity in Megacult media. Total RNA was extracted from CD133+HSCs and Megakaryocytic cells. Quantitative Polymerase Chain Reaction (qPCR) was done on synthesized cDNAs. The expression alteraton of target proteins was evaluated by flow cytometry as Mean Fluorescent Intensity (MFI) that shows the antibody reacted proteins. The qPCR results illustrated meaningful down-regulation of MAPK-DYRK1A and CDC7 mRNAs, that are targeted by up-regulated following miRs: miR-22, miR-188, miR-1246, miR-148a, miR-224, miR-486-5p, miR-886 poietic stem cells were collected from three human umbilical cord blood (UCB) samples, and then differentiated to the megakaryocytic lineage and characterized by flow cytometry, CFU-assay and ploidy analysis. Subsequently, microarray analysis was undertaken followed by quantitative polymerase chain reaction (qPCR) to validate differentially expressed miRNA identified in the microarray analysis.
