We report in this paper that the DNA-binding drug mithramycin is a potent inducer of γ-globin mRNA accumulation and fetal hemoglobin (HbF) production in erythroid cells from healthy human subjects and β-thalassemia patients. Erythroid precursors derived from peripheral blood were grown in 2-phase liquid culture. In this procedure, early erythroid progenitors proliferate and differentiate during phase 1 (in the absence of erythropoietin) into late progenitors. In phase 2, in the presence of erythropoietin, the latter cells continue their proliferation and mature into Hb-containing orthochromatic normoblasts. Compounds were added on days 4 to 5 of phase 2 (when cells started to synthesize Hb), and cells were harvested on day 12. Accumulation of mRNAs for γ-globin, β-globin, α-globin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were measured by real-time quantitative reverse transcription–polymerase chain reaction (RT-PCR); induction of HbF was analyzed by high-performance liquid chromatography (HPLC) and, at cellular level, by flow cytometry. We demonstrated that mithramycin was able to up-regulate preferentially γ-globin mRNA production and to increase HbF accumulation, the percentage of HbF-containing cells, and their HbF content. Mithramycin was more effective than hydroxyurea, being, in addition, not cytotoxic. This was shown by the lack of cytotoxicity on erythroid and myeloid in vitro primary cell cultures treated with mithramycin at concentrations effective for HbF induction. These results are of potential clinical significance because an increase of HbF alleviates the symptoms underlying β-thalassemia and sickle cell anemia. The results of this report suggest that mithramycin and its analogs warrant further evaluation as potential therapeutic drugs.

In this study we analyzed the microRNA profile of cystic fibrosis (CF) bronchial epithelial IB3–1 cells infected with Pseudomonas aeruginosa by microarray and quantitative RT-PCR, demonstrating that microRNA 93 (miR-93), which is highly expressed in basal conditions, decreases during infection in parallel with increased expression of the IL-8 gene. The down-regulation of miR-93 after P. aeruginosa infection was confirmed in other bronchial cell lines derived from subjects with and without CF, namely CuFi-1 and NuLi-1 cells. Sequence analysis shows that the 3′-UTR region of IL-8 mRNA is a potential target of miR-93 and that the consensus sequence is highly conserved throughout molecular evolution. The possible involvement of miR-93 in IL-8 gene regulation was validated using three luciferase vectors, including one carrying the complete 3′-UTR region of the IL-8 mRNA and one carrying the same region with a mutated miR-93 site. Up-modulation of IL-8 after P. aeruginosa infection was counteracted in IB3–1, CuFi-1, and NuLi-1 cells by pre–miR-93 transfection. In addition, IL-8 was up-regulated in uninfected cells treated with antagomiR-93. Our results support the concept of a possible link between microRNA expression and IL-8 induction in bronchial epithelial cells infected with P. aeruginosa. Specifically, the data presented here indicate that, in addition to NF-κB–dependent up-regulation of IL-8 gene transcription, IL-8 protein expression is posttranscriptionally regulated by interactions of the IL-8 mRNA with the inhibitory miR-93.