MiR-144/451 Inhibits C-Myc To Promote Erythroid Differentiation

BackgroundMature red blood cells originate from hematopoietic stem cells(HSCs)through multiple differentiation stages,including the blast forming unit-erythroid(BFU-E),colony-forming unit-erythroid(CFU-E),orthochromatic,proerythroblast,basophilic erythroblast,polychromatic erythroblast,orthochromatic erythroblast,reticulocyte,and erythrocyte stages.The most unique erythroid differentiation processes in mammals are chromatin condensation of orthochromatic erythroblasts and extrusion of nuclei to give rise to enucleated reticulocytes.These morphological changes ensure maximal hemoglobin production and deformability required for quick circulating in capillaries with a diameter less than that of mature red blood cells.From BFU-E cells to fully functional mature erythrocytes circulating in peripheral blood,the entire differentiation process is precisely regulated and orchestrated by several key transcription factors,including protooncogene c-Myc(here referred to as Myc).Myc is a protooncogene coding a nuclear protein that directly controls over 15%of mammalian genes and is required for cell proliferation and growth.Myc protein is highly expressed in early erythroid progenitors but declines rapidly in the late stage of erythroid cells.However,whether Myc affects erythroid differentiation is still elusive.miRNAs are endogenous genes coding a class of small non-protein-coding RNAs whose length is only 18-25 nucleotide long.miRNAs participate in a various of physiological and pathophysiological functions including normal development,cell growth and differentiation,and tumorigenesis.miR-144/451 is a bicistronic gene locus encoding two highly conserved miRNAs:miR-451 and miR-144.We have previously reported that miR-144/451 is abundantly expressed in erythroid cells.We have also demonstrated that miR-144/451 protects erythroid cells by repressing 14-3-3? and Cab39 in mice and loss of miR-144/451 disrupts erythroid homeostasis due to oxidative stress and apoptosis during erythropoiesis.Whether ablation of miR-144/451 also causes differentiation block during erythropoiesis has not been defined.Methods:miR-144/451 gene knockout model is used for in vivo study.5-fluorouracil(5-FU)was used to eradicate all proliferative hematopoietic precursors.Magnetic beads coated with anti-Ter119 antibodies were used to isolate erythroblasts.Murine G1E,MEL erythroblast cell lines and erythroid precursors isolated from fresh mouse fetal livers are used for in vitro study.Flow cytometry,microarray,Western blot,quantitative real-time PCR,immunohistochemistry and immunocytochemistry are used for analyses of phenotypes.Dual luciferase assay was used to examine the binding activity of miRNAs to their targeted mRNAs.Results:1.H&E staining and immunohistochemical staining for Ter119 cell surface marker presented dramatic accumulation of large nucleated erythroblasts in miR-144/451 gene knockout(KO)bone marrows(BM)compared to wild type(WT)BMs.After a single-dose treatment with 5-fluorouracil(5-FU),which destroys all proliferating hematopoietic progenitors,miR-144/451 KO BM took two days longer when compared to WT controls to generate new reticulocytes in peripheral blood.After 48-hour(h)culture of isolated erythroblasts from embryonic day 14.5(E14.5)mouse fetal livers,the numbers of enucleated cells were reduced by 80%and the nuclear condensation was impaired in KO culture compared to WT cell culture.These findings indicate that an erythroid differentiation block occurred in miR-144/451 KO hematopoietic compartments.2.Gene set enrichment analysis(GSEA)of microarray datasets from BM erythroblasts demonstrated the alteration of Myc-regulated gene networks:Myc-suppressed genes were less transcribed in miR-144/451 KO cells,while Myc-upregulated genes were more transcribed in miR-144/451 KO erythroid precursors.High Myc protein levels(about 1.6-fold)were found in miR-144/451 KO BM erythroid cells compared to WT erythroid cells Erythroblasts from miR-144/451 KO spleens and primary E14.5 FL cells were also found to contain high Myc levels,about 2-fold and 2.4-fold,respectively.Deficiency of miR-144/451 leading to high levels of Myc mRNA and protein was also confirmed by FL erythroblast culture.All these data demonstrate that deficiency of miR-144/451 expression activates the molecular programs regulated by Myc in erythroid precursors3.We used two differentiation-inducible erythroblast cell lines(G1E-JC4 expressing an inducible GATA1-estrogen receptor fusion gene GATA1ER and MEL from murine erythroid leukemic cells)and isolated mouse FL erythroid precursors from E14.5 embryos to find that both Myc mRNA and protein levels were substantially reduced during erythropoiesis.Morphology and Hoechst staining demonstrated that overexpression of MycER chimeric protein in freshly isolated FL erythroblasts and then activation of MycER function by 4-hydroxytamoxifen(4-OHT)dramatically impaired nuclear condensation and enucleation,confirming the requirement of Myc downregulation during erythropoiesis.4.Both morphology and flow cytometric analysis with Hoechst staining revealed that knocking down of Myc expression in erythroblasts from miR-144/451 KO FLs with a shRNA against Myc significantly increased nuclear condensation and enucleation.5.In dual luciferase report assay,where a part of 3'UTR of Myc containing the potential miR-451 binding site was cloned to the downstream of luciferase reporter gene in pGL3-BS vector,overexpression of miR-451 disrupted luciferase expression and reduced enzyme activity by approximately 29-fold,indicating the direct repression of Myc expression by miR-451 binding.Conclusion:1.Depletion of miR-144/451 leads to decreased nuclear condensation and enucleation of erythroid cells during normal development.2.miR-144/451 promotes erythroid differentiation during normal erythropoiesis partially by inhibiting the expression of Myc protooncoprotein.