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The Regulation of y-globin by microRNAHigh fetal hemoglobin (HbF) attenuates the severity of sickle cell disease (SCD) by interfering with hemoglobin S polymerization; therefore increasing yglobin expression is an effective therapeutic approach for SCD. Variations in HbF levels in sickle cell patients are associated with inherited genetic differences found in genes encoding regulatory factors throughout the genome involved in yglobin gene regulation. Molecular targets such as microRNAs (miRNAs) that modulate gene expression through post-transcriptional mechanisms are under investigation as novel y-globin regulators. The goal of this project is to identify miRNA genes which regulate HbF. First, computational in silico analysis was performed which identified miR-34a as a predicted target in the y-globin gene 3'untranslated region (UTR). Transient and stable expression of miR-34a in K562 cells, demonstrated increased y-globin transcription and HbF synthesis. We observed increased expression ofGATAl, KLFI , CD235a, and the erythropoietin receptor supporting the promotion of erythroid differentiation by miR-34a. The fact that miR-34a activated HbF suggest it targets a negative regulator of y-globin gene expression rather than targeted the 3 'UTR to promote degradation. The levels of total and phosphorylated STA T3 were decreased in the miR-34a stable clones suggesting a role of the y-globin repressor ST A T3, in the mechanism of HbF activation by miR-34a. For the second approach, we analyzed miRNA profiles using reticulocytes isolated from individuals with SCD with low and high HbF levels and identified miR-144 as highly expressed in the low HbF group. miR-144 targets NRF2, a transcription factor that mediates the cellular oxidative stress response and is involved in drug-mediated HbF induction. Primary erythroid progenitors in culture treated with miR-144 mimic had a decrease in the number of HbF positive cells and the levels of NRF2 and HbF protein. Pretreatment with miR-144 inhibitor reversed its negative effect on HbF expression. Further evidence for this novel mechanism of y-globin regulation was collected using KU812 cells treated with hemin to generate a tissue culture model of oxidative stress. The robust activation of NRF2 and HbF induction by hemin was abolished after pretreatment with miR-144 inhibitor. These data support miR- 34a and miR-144, as positive and negative regulators respectively of HbF expression.