title: Hydroxyurea down-regulates BCL11A, KLF-1 and MYB through miRNA-mediated actions to induce γ-globin expression: implications for new therapeutic approaches of sickle cell disease.
abstract: BACKGROUND
  The major therapeutic benefit of hydroxyurea, the only FDA-approved pharmacologic treatment for sickle cell disease (SCD), is directly related to fetal hemoglobin (HbF) production that leads to significant reduction of morbidity and mortality. However, potential adverse effects such as infertility, susceptibility to infections, or teratogenic effect have been subject of concerns. Therefore, understanding HU molecular mechanisms of action, could lead to alternative therapeutic agents to increase HbF with less toxicity. This paper investigated whether HU-induced HbF could operate through post-transcriptional miRNAs regulation of BCL11A, KLF-1 and MYB, potent negative regulators of HbF. Both ex vivo differentiated primary erythroid cells from seven unrelated individuals, and K562 cells were treated with hydroxyurea (100 μM) and changes in BCL11A, KLF-1, GATA-1, MYB, β- and γ-globin gene expression were investigated. To explore potential mechanisms of post-transcriptional regulation, changes in expression of seven targeted miRNAs, previously associated with basal γ-globin expression were examined using miScript primer assays. In addition, K562 cells were transfected with miScript miRNA inhibitors/anti-miRNAs followed by Western Blot analysis to assess the effect on HbF protein levels. Direct interaction between miRNAs and the MYB 3'-untranslated region (UTR) was also investigated by a dual-luciferase reporter assays.
  
  
  RESULTS
  Down-regulation of BCL11A and MYB was associated with a sevenfold increase in γ-globin expression in both primary and K562 cells (p < 0.003). Similarly, KLF-1 was down-regulated in both cell models, corresponding to the repressed expression of BCL11A and β-globin gene (p < 0.04). HU induced differential expression of all miRNAs in both cell models, particularly miR-15a, miR-16, miR-26b and miR-151-3p. An HU-induced miRNAs-mediated mechanism of HbF regulation was illustrated with the inhibition of miR-26b and -151-3p resulting in reduced HbF protein levels. There was direct interaction between miR-26b with the MYB 3'-untranslated region (UTR).
  
  
  CONCLUSIONS
  These experiments have shown the association between critical regulators of γ-globin expression (MYB, BCL11A and KLF-1) and specific miRNAs; in response to HU, and demonstrated a mechanism of HbF production through HU-induced miRNAs inhibition of MYB. The role of miRNAs-mediated post-transcriptional regulation of HbF provides potential targets for new treatments of SCD that may minimize alterations to the cellular transcriptome.
authors.affiliation: Division of Human Genetics, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, Cape Town, 7925, Republic of South Africa.
authors.forename: Gift Dineo
authors.initials: GD
authors.lastname: Pule
authors.affiliation: Division of Hematology, Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town, Republic of South Africa.
authors.forename: Shaheen
authors.initials: S
authors.lastname: Mowla
authors.affiliation: Division of Hematology, Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town, Republic of South Africa.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Novitzky
authors.affiliation: Division of Human Genetics, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, Cape Town, 7925, Republic of South Africa. ambroise.wonkam@uct.ac.za.
authors.forename: Ambroise
authors.initials: A
authors.lastname: Wonkam
keywords: BCL11A; Human erythroid; Hydroxyurea; KLF-1; MYB; miRNAs-mediated actions; γ-Globin expression