Upregulation of miR-17-3p is associated with HbF in patients with β-thalassemia and induces γ-globin expression by targeting BCL11A

Abstract

Background: Large number of microRNAs (miRNAs) have been found to be dysregulated in β-thalassemia, but their roles in β-thalassemia are poorly reported. This study aims to investigate the clinical significance of miR-17-3p in β-thalassemia, and to elucidate its regulatory effect on erythropoiesis and γ-globin expression.

Methods: We collected peripheral blood samples from 17 patients with β-thalassemia (including intermedia and major subtypes) and 17 healthy controls, and the expression levels of miR-17-3p, BCL11 transcription factor A (BCL11A) and γ-globin were detected by qRT-PCR, and their correlations were analyzed. The regulation of miR-17-3p on BCL11A was evaluated in K562 cells by bioinformatics, luciferase reporter gene assay, fluorescence in situ hybridization and Western blotting. Furthermore, the effects on miR-17-3p overexpression and knockdown on erythropoiesis including cell proliferation, cell cycle, cell apoptosis, and erythroid differentiation of K562 cells were assessed by CCK-8, flow cytometry and benzidine blue staining.

Results: The expression of miR-17-3p was upregulated in patients with β-thalassemia, and was positively correlated with fetal hemoglobin (HbF) levels. BCL11A expression was reduced in β-thalassemia patients, and was negatively correlated with miR-17-3p and γ-globin expression. BCL11A was identified as a target gene of miR-17-3p, and was negatively regulated by miR-17-3p. Furthermore, miR-17-3p mediated the upregulation of γ-globin expression in K562 cells through BCL11A. In addition, neither overexpression nor knockdown of miR-17-3p appeared to affect cell proliferation, cell cycle, cell apoptosis or erythroid differentiation of K562 cells in vitro.

Conclusion: The upregulated miR-17-3p is associated with HbF in patients with β-thalassemia. Although miR-17-3p does not affect erythropoiesis, it promotes γ-globin expression by targeting BCL11A, suggesting that miR-17-3p may be a promising miRNA for the treatment of β-thalassemia.

Keywords: BCL11A; Erythropoiesis; HbF; miR-17-3p; β-thalassemia.

Fig. 1

miR-17-3p expression in peripheral blood of patients with β-thalassemia. (A) qRT-PCR analysis of miR-17-3p expression in peripheral blood of patients with β-thalassemia and healthy controls. (B) The expression levels of miR-17-3p were evaluated in the HbF ≥ 10 g/L group and the HbF < 10 g/L group. qRT-PCR: quantitative real-time polymerase chain reaction, HbF: fetal hemoglobin. Error bars represented the means ± SD. ***, P < 0.001

Fig. 2

The potential of miR-17-3p as a monitoring biomarker for patients with β-thalassemia. (A) Distinguishing efficacy of miR-17-3p expression in β-thalassemia patients by ROC curve analysis. (B) Distinguishing efficacy of miR-17-3p in β-thalassemia patients with different levels of HbF. ROC: receiver operating characteristic, AUC: area under the curve

Fig. 3

The expression of BCL11A in patients with β-thalassemia and its correlation with miR-17-3p and γ-globin. (A) Differential analysis of BCL11A mRNA expression in patients with β-thalassemia and healthy controls. (B) Differential analysis of γ-globin mRNA expression in patients with β-thalassemia and healthy controls. (C-E) Pearson correlation analysis of BCL11A, γ-globin and miR-17-3p expression in patients with β-thalassemia. BCL11A: BCL11 transcription factor A. Error bars represented the means ± SD. *, P < 0.05; **, P < 0.01

Fig. 4

BCL11A as a target gene of miR-17-3p. (A) Fluorescent localization of miR-17-3p in K562 cells. (B) Extent of complementarity between miR-17-3p and binding sites present within BCL11A mRNA 3’-UTR. (C-D) Dual luciferase gene reporter assay of HEK-293T cells results show that miR-17-3p could bind to BCL11A mRNA 3’UTR. Expression analysis of BCL11A mRNA (E), and BCL11A protein (F) levels in the miR-17-3p mimics group and NC mimics group. Effect of knockdown of miR-17-3p on the expression analysis of BCL11A mRNA (G), and BCL11A protein (H) levels. Error bars represented the means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05

Fig. 5

Effect of miR-17-3p on γ-globin expression of in K562 cells. Expression analysis of γ-globin mRNA (A) and protein (B) levels in the miR-17-3p mimics group and NC mimics group. Effect of knockdown of miR-17-3p on the expression analysis of γ-globin mRNA (C) and protein (D) levels. Error bars represented the means ± SD. *, P < 0.05; **, P < 0.01

Fig. 6

Effect of miR-17-3p overexpression or knockdown on the proliferation, cell cycle, and cell apoptosis of K562 cells. Changes in proliferation in the miR-17-3p mimics group (A), and the miR-17-3p inhibitors group (B). Effect of miR-17-3p overexpression (C), and miR-17-3p knockdown (D) on cell cycle. Effect of miR-17-3p overexpression (E), and miR-17-3p knockdown (F) on cell apoptosis. Error bars represented the means ± SD. ns, P > 0.05

Fig. 7

Effect of overexpression or knockdown of miR-17-3p on the erythroid differentiation of K562 cells. (A) Effect of miR-17-3p overexpression, and knockdown (B) on erythroid differentiation of K562 cells detected by benzidine blue staining. The effect of miR-17-3p overexpression (C), and knockdown (D) on erythroid differentiation of K562 cells detected by flow cytometry. Error bars represented the means ± SD. ns, P > 0.05