The Effect of Mir-451 Upregulation on Erythroid Lineage Differentiation of Murine Embryonic Stem Cells

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: Erythropoiesis; Globin Chains; MicroRNAs; Mir-451; mESCs.

Fig.1

A. Transfected HEK293T cells examined by light microscopy and B. Transfected HEK293T cells examined by fluorescent microscopy. Transfection efficiency of murine embryonic stem cells (mESCs) with pCDH-Mir-451 was more than 95% as determined by fluorescent microscopy.

Fig.2

A. Transfected HEK293T cells examined by light microscopy and B. Transfected HEK293T cells examined by fluorescent microscopy. Transfection efficiency of murine embryonic stem cells (mESCs) with pCDH-empty vector was more than 95% as determined by fluores- cent microscopy.

Fig.3

A. Transduced murine embryonic stem cells (mESCs) examined by light microscopy and B. Transduced mESCs examined by fluorescent microscopy. Transduction efficiency of mESCs with pCDH- Mir-451 was more than 60% as determined by flow cytometry for the GFP marker.

Fig.4

Expression analysis (fold changes) of Mir-451 in treated murine embryonic stem cells (mESCs) on day 4. The expression of Mir-451 in the mESCs treated with pCDH-Mir-451 cells was significantly higher than that in mESCs treated with pCDH-empty vector and those untreated. Relative miRNA expression levels were normalized to Snord47 as an internal control. Columns, mean of three different experiments. *; P<0.001 and #; Results were compared with these columns.

Fig.5

A. Expression analysis (fold changes) of transcriptional factors in treated murine embryonic stem cells (mESCs) on day 14 and B. Expression analysis (fold changes) of transcriptional factors in treated mESCs on day 21. Relative transcriptional factors expression levels were normalized to β-Actin as an internal control. Results presented as fold change compared with the control group. Columns, mean of three different experiments. *; P<0.001 and #; Results were compared with these columns.

Fig.6

A. Expression analysis (fold changes) of hemoglobin chains in treated murine embryonic stem cells (mESCs) on day 14 and B. Ex- pression analysis (fold changes) of hemoglobin chains in treated mESCs on day 21. Relative hemoglobin chains expression levels were normalized to β-Actin as an internal control. Results presented as fold change compared with the control group. Columns, mean of three different experiments. *; P<0.05 and #; Results were compared with these columns.

Fig.7

Overexpression of TER-119 in murine embryonic stem cells (mESCs). More than 95% of cells were gated in R1. A. FACS histogram showing transduction efficiency of mESCs with lentiviral vector expressing pCDH-Mir-451, B. FACS histogram showing transduction efficiency of mESCs with lentiviral vector expressing pCDH-empty vector and C. FACS histogram showing transduction efficiency of untreated mESCs. The positive regions were adjusted according to the control isotope antibody reaction.

Fig.8

Overexpression of CD235a in murine embryonic stem cells (mESCs). More than 95% of cells were gated in R1. A. FACS histogram showing transduction efficiency of mESCs with lentiviral vector expressing pCDH-Mir-451, B. FACS histogram showing transduction efficiency of mESCs with lentiviral vector expressing pCDH-empty vector and C. FACS histogram showing transduction efficiency of untreated mESCs. The positive regions were adjusted according to the con- trol isotope antibody reaction.

Fig.9

CFC assay of murine embryonic stem cells (mESCs), A. CFU-E, B. CFU-GEMM and C. CFU-GM with some CFU-E, all observed under an inverted microscope (×100) showing that mESCs generated the colonies. CFU-E; Colony-forming unit-erythroid, CFU-GEMM; CFU- granulocyte, erythroid, macrophage, megakaryocyte and CFU-GM; CFU-granulocyte, macrophage.