Dynamic Expression Of MicroRNA-486during Erythroid Differentiaiton Of Hematopoieitc Cells And Its Mechanism

MicroRNAs (miRNAs) are a class of non‐coding single‐stranded RNA molecules of about22nucleotides by the length of the endogenous gene encoding. miRNAs play important roles in the transcriptional and post‐transcriptional regulation of genes, but their expression in hematopoietic stem cell self‐renewal and differentiation cells and their roles,in regulating hematopoiesis are still not clear.MicroRNA‐486(miR‐486) is first identified in the human fetal liver tissue. The human fetal liver is one of the most productive tissues in hematopoietic proliferation and differentiation. MiR‐486is identified by classical methods from the fetal liver tissue. Undoubtedly miRNAs which high exressred by fetal liver cells may be involved in hematopoietic regulation. Genomic analysis showed that the MiR‐486gene is located in introns (8p11.21locus) of Ankyrin (ANK‐1) gene (Gene ID:619554) on chromosome8. ANK‐1is widely expressed in muscle, red blood cells and brain tissue. But the roles of MiR‐486in hematopoieisis regulation remain unclear. The purpose of this study is to observe the dynamic expression of miRNA‐486during erythroid differentiation of hematopoietic cells and its mechanism.We first detect the expression of miR‐486by various mouse tissues including heart, liver and blood by using real‐time quantitative PCR (real‐time PCR) method. It is found that high expression of miR‐486in the blood, muscle and myocardium, and hematopoietic cells and blood cells exress the highest level. Our previous research compared the microRNA profiles of chronic myelocytic leukemia (CML) with normal of CD34+cells. We found that miR‐486highly expressed by CML CD34+cells and is involved in promoting erythroid differentiation. To observe the dynamic expression of MicroRNA‐486during erythroid differentiation of hematopoietic cells, we isolated CD34+cells from human umbilical cord blood and cultured in differentiation medium containing combination of hematopoietic growth factors. We detected erythroid differentiation efficiency of hematopoietic cells by flow cytometry, and miR‐486expression by using RT‐QPCR. The results showed that found miR‐486expression is associated with erythroid differentiation of hematopoietic stem cells. GATA1is recognized as an erythroid specific transcript factor and plays a key role erythroid differentiation and maturation process. Silence GATA1the expression in cord blood CD34+cells by lentiviral‐mediated the shRNA transduction results in down regulation of miR‐486. We found that82%GATA1silencing efficiency leads to lower level of miR‐486levels in CD34+cells. The number of BFU‐E in GATA1silent group set fall to16±5, compared to that of control group set off54±4.89. The result shows that the GATA1silence suppression cord blood CD34+cells erythroid differentiation. These results prove that miR‐486is an erythroid enriched microRNA and plays an important role in erythroid differentiation of hematopoietic stem cells.MicroRNA regulates gene expression and exerts its biological effects through binding3’UTR of target gene. Single microRNA regulates the expression of multiple target genes, and one target gene may also be subject to multiple microRNA regulation. Thus the biological functions of microRNA can be predicted by the target gene analysis. We predicted that miR‐486regulates multiple functional target genes such as FOX1, PTEN, ARID4B et al. We further confirmed that ARID4B is a target gene of microRNA‐486by3‘UTR luciferase reporter assay. The carrier and miR‐486expression vector were co‐transfected HEK293cells, and determination of the activity of the luciferase reporter. The results found that miR‐486can inhibit ARID4B the luciferase reporter activity. MiR‐486group relative fluorescence intensity shows37%decrease compared to control vector (miR‐486:0.631±0.083); These results suggest that miR‐486may play a biological function by inhibiting ARID4B. To sum up, the results of this study can draw the following conclusions: miR‐486is an erythroid enriched microRNA which involved in regulation of hematopoietic differentiation. Its function might be through multiple funcetional target genes such as ARID4B and Foxo1.