Document Type : Original Article
Authors
1
Blood Transfusion Research Center, High Institute for Education and Research in Transfusion Medicine, Tehran, Iran;Department of Hematology, School of Para Medicine, Bushehr University of Medical Sciences, Bushehr, Iran
2
Department of Immunology, School of Medicine, Tarbiat Modares University, Tehran, Iran
3
4Department of Hematology, School of Medicine, Tarbiat Modares University, Tehran, Iran
4
Blood Transfusion Research Center, High Institute for Education and Research in Transfusion Medicine, Tehran, Iran
5
5Stem Cell Technology Research Center, Tehran, Iran
Abstract
Objective
MicroRNAs (miRNAs) are small endogenous non-coding regulatory RNAs that control mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role in hematopoietic lineage differentiation. Recent studies have shown that specific miRNAs such as Mir-451 have key roles in erythropoiesis.
Materials and Methods
In this experimental study, murine embryonic stem cells (mESCs) were infected with lentiviruses containing pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors (Gata-1, Klf-1, Epor) and hemoglobin chains (α, β, γ , ε and ζ) genes using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and presence of erythroid surface antigens (TER-119 and CD235a) using flow cytometery. Colony-forming unit (CFU) assay was also on days 14thand 21thafter transduction.
Results
Mature Mir-451 expression level increased by 3.434-fold relative to the untreated mESCs on day 4 after transduction (P < 0.001). Mir-451 up-regulation correlated with the induction of transcriptional factor (Gata-1, Klf-1, Epor) and hemoglobin chain (α, β, γ, ε and ζ) genes in mESCs (P < 0.001) and also showed a strong correlation with presence of CD235a and Ter- 119 markers in these cells (13.084and 13.327-fold increse, respectively) (P < 0.05). Moreover, mESCs treated with pCDH-Mir-451 showed a significant raise in CFU-erythroid (CFU-E) colonies (5.2-fold) compared with untreated control group (P < 0.05).
Conclusion
Our results showed that Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produce artificial red blood cells (RBCs) without the presence of any stimulatory cytokines.
Keywords
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