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

摘要

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?