Program: Oral and Poster Abstracts
Session: 113. Hemoglobinopathies, Excluding Thalassemia – Basic and Translational Science: Poster II
To study the effects of HMGA2 upon erythropoiesis and globin expression, lentiviral constructs were designed for let-7 resistant expression of HMGA2 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2-SPTA1-OE), with a matched empty vector control. Transductions were performed in CD34+ cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Overexpression of HMGA2 was confirmed by Q-RT-PCR (control: below detection limits; HMGA2-SPTA1-OE: 2.51E+04 ± 3.44E+04 copies/ng) and Western blot analyses at culture day 14. Cell counting revealed no significant changes between HMGA2-SPTA1-OE and control (empty vector) transductions at culture day 14. Terminal maturation with loss of CD71 from the erythroblast cell surface and enucleation assessed by thiazole orange staining were analyzed in the control and HMGA2-SPTA1-OE samples at the end of the culture period. Globin genes expression levels were evaluated for HMGA2-SPTA1-OE by Q-RT-PCR. HMGA2-SPTA1-OE caused a significant increase in gamma-globin mRNA expression levels compared to controls (control: 5.02E+05 ± 8.62E+04 copies/ng; HMGA2-SPTA1-OE: 1.45E+06 ± 7.31E+05 copies/ng; p=0.037). Consistent with the increase in gamma-globin mRNA levels, HPLC analyses at culture day 21 demonstrated modest but significant increases in HbF levels in HMGA2-SPTA1-OE compared to controls (HbF control: 5.41 ± 2.15%; HMGA2-SPTA1-OE: 16.53 ± 4.43%; p=0.006).
Possible effect(s) and downstream mechanism(s) triggered by HMGA2-SPTA1-OE were investigated. Q-RT-PCR analyses demonstrated no significant changes in the let-7 family of miRNAs in HMGA2-SPTA1-OE compared to controls. Expression patterns of several transcription factors such as BCL11A, KLF1, SOX6 and GATA1 were investigated by Q-RT-PCR and no significant changes were detected in HMGA2-SPTA1-OE compared to controls. While BCL11A mRNA levels were decreased by HMGA2-SPTA1-OE, the differences did not reach statistical significance (control: 4.26E+02 ± 8.18E+01 copies/ng; HMGA2-SPTA1-OE: 2.84E+02 ± 1.48E+02 copies/ng; p=0.104). However, nuclear BCL11A protein levels assessed by Western analysis were suppressed in HMGA2-SPTA1-OE.
In summary, these results demonstrate that HMGA2, a validated target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in human erythroblasts, and reduces levels of BCL11A protein. These data thus support the notion that suppression of let-7 miRNAs increases fetal hemoglobin, in part, by the targeting of erythroblast HMGA2 mRNA.
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Disclosures: No relevant conflicts of interest to declare.
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