Fig 2: MicroRNA29B (MIR29B) mediates HBG reactivation via inhibiting MYB in KU812 cells. (A) Western blot analysis of MYB and HbF protein expression relative to β-actin protein in KU812 cells transfected with increasing concentrations of MYB plasmid DNA (μg). Fold change of (B) MYB mRNA relative to β-actin, (C) MIR29B expression relative to RNU48, and (D) HBG-to-HBB mRNA ratio as a function of HBG/HBG + HBB mRNA expression in KU812 cells following transfection for 48 h with MIR29B mimic (50 or 100 nm) alone or in combination with MYB overexpression plasmid vector (2 μg). (E) Western blot of MYB, HbF, and HbA protein expression with β-actin internal control. (F) Bar graphs showing quantification of the ratio of HbF-to-HbA protein in KU812 cells transfected with MIR29B alone or in combination with MYB. *P < 0.05 **P < 0.005, ***P < 0.0005, and ****P < 0.0001 is statistically significant.

Fig 3: MicroRNA29B (MIR29B) induces HbF in humanized Townes sickle cell mice. (A) Townes SCD mice, which express human α-globin, γ-globin, and βS-globin genes and exhibit phenotypes including chronic hemolysis and sickling similar to human SCD, were surgically implanted with a mini-osmotic pump (Alzet) that continuously released MIR29B mimic at the concentration shown over 28 days. Age-and sex-matched mice (n = 4–10 per group) were treated and blood was collected at weeks 0, 2, and 4 for complete blood counts (CBC) with differential, reticulocytes, %F-cells, and red blood cell sickling under hypoxia conditions. Treatments consisted of 4 mice per group (2 males and 2 females) for MIR29B or SCR mimic at 2, 3, and 4 mg/kg/day and water vehicle and HU (100 mg/kg/day) controls. Tail–vein blood samples were collected in EDTA blood tubes at week 0, 2, and 4 along with weights. Evaluation of the effects of MIR29B on bone marrow hematopoiesis, were conducted by (B) Shown are bar graphs of the percentage of HbF-positive cells (F–cells) in erythroid progenitors isolated from Townes SCD mice and stained with fluorescein isothiocyanate-labeled anti-HbF antibody under the different treatment conditions. (C) Shown are bar graphs of the percentage of sickled cells obtained from freshly collected EDTA blood from sickle mice incubated under 1% hypoxia conditions for 12 h following the treatment conditions. (D) RT-qPCR analysis of MIR29B, MYB, DNMT gene expression and the HBG-to-HBB mRNA ratio as a function of HBG/HBG + HBB in mice treated with MIR29B or Scr control; *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001 is statistically significant.