Abstract
Abstract 827
The highly-conserved Lin28 genes regulate cellular metabolism as well as the timing of developmental events and cell fates in multicellular organisms. Lin28 protein acts primarily by negatively regulating biogenesis of let-7 RNA, a microRNA family whose targets include growth-related signaling and transcription factor proteins. Published studies showed significantly increased expression of let-7 in purified adult blood reticulocytes compared to umbilical cord blood reticulocytes (1). This pattern correlates inversely with Lin28B expression. While present in the fetal liver and umbilical cord blood, Lin28B decreased to undetectable levels in adult bone marrow (2). Based upon the association of human ontogeny with hemoglobin switching, Lin28 was explored to identify novel mechanisms for hemoglobin regulation that may be useful for therapeutic application among patients with thalassemia or other hemoglobinopathies.
To study the effects of Lin28B upon erythropoiesis and hemoglobin, ectopic expression of Lin28B was accomplished using retroviral transduction of human CD34+ cells cultivated ex vivo in erythropoietin-supplemented, serum-free cultures for 21 days. All experiments were performed in triplicate using cells from three separate adult volunteers. Lin28B over-expression (Lin28B-OE) was confirmed by Q-RT-PCR (control: 0.14 ± 0.37 copies/ng, Lin28B-OE: 1.8E+04 ± 353.8 copies/ng, p=0.01). Western analyses confirmed protein expression, and confocal microscopy revealed Lin28B predominantly in the cytoplasm of the transduced cells. Proliferation, maturation and morphology assays revealed that Lin28B-OE did not inhibit erythropoiesis when compared to control (empty vector) transductions. Terminal maturation with loss of CD71 from the erythroblast surface and enucleation by culture day 21 was detected in the control and Lin28B-OE samples. Expression levels of globin genes were evaluated upon Lin28B-OE by Q-RT-PCR. Lin28B-OE enhances gamma-globin mRNA expression (control: 5.14E+06 ± 2.6E+06 copies/ng, Lin28B-OE: 1.81E+07 ± 5.82E+06 copies/ng, p=0.038). Protein analysis confirmed the increased expression of gamma-globin. Fetal hemoglobin (HbF) levels were also increased in the Lin28B-OE cultures (control: 5.82 ± 4.54%, Lin28B-OE: 33.63 ± 9.38%; p=0.011). The increased HbF expression was maintained throughout differentiation including enucleated populations of culture-generated erythrocytes.
Possible mechanism(s) for the increased expression of HbF caused by Lin28B-OE were investigated. Q-RT-PCR analyses demonstrated suppression of the let-7 microRNA family with greater-than 70% reductions of let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. Expression patterns of several transcription factors including BCL11A, KLF1, SOX6 and GATA1 were explored. No major changes were detected with the exception of BCL11A. Lin28B-OE caused a 65% reduction in BCL11A expression (control: 3.07E+03 ± 1.5E+02 copies/ng, Lin28B-OE: 1.07E+03 ± 18 copies/ng; p=0.02). Western blot analyses of Lin28B-OE showed a consistent reduction of BCL11A protein. By comparison with Lin28B-OE, separately performed studies of BCL11A knockdown in adult CD34+ cells produced comparable increases in gamma-globin expression, but Lin28B expression in those cells was not affected.
In addition to a more general role in development and metabolism, these experimental results suggest that Lin28B increases fetal hemoglobin and regulates BCL11A in human erythroblasts. Lin28B is thus identified as the first defined link between the regulation of a developmental clock and hemoglobin switching in humans.
Disclosures:
No relevant conflicts of interest to declare.
Recent advances in the management of Thalassaemia: A Review Update