RESUMEN
Hereditary persistence of fetal hemoglobin deletion type-2 (HPFH-2) and Sicilian-δß-thalassemia are conditions described as large deletions of the human ß-like globin cluster, with absent ß-globin chains and a compensatory variable increase in γ-globin. HPFH, in general, may be distinguished from DB-Thalassemia by higher fetal hemoglobin (HbF) levels, absence of anemia and hypochromic and microcytic erythrocytes. MicroRNAs (miRNAs) regulate a range of cellular processes including erythropoiesis and regulation of transcription factors such as the BCL11A and SOX6 genes, which are related to the regulation of γ-globin expression. In this report, a possible association among the overexpression of miRNAs and the expression of the γ-globin gene was analyzed in these two conditions. Forty-nine differentially expressed miRNAs were identified by microarrays in CD34+-derived erythroid cells of two subjects heterozygous for Sicilian-δß-thalassemia, 2 for HPFH-2 and 3 for controls after 13 days of culture. Some of these miRNAs may participate in γ-globin gene regulation and red blood cell function. The BCL11A gene was found to be potentially targeted by 12 miRNAs that were up-regulated in HPFH-2 or in DB-Thal. A down-regulation of BCL11A gene expression in HPFH-2 was verified by quantitative polymerase chain reaction. These data suggest an important action for miRNA that may partially explain the phenotypic differences between HPFH-2 and Sicilian δß-thalassemia and the increased expression of γ-globin in these conditions.
Asunto(s)
Proteínas Portadoras/genética , Hemoglobina Fetal/genética , MicroARNs/genética , Proteínas Nucleares/genética , Factores de Transcripción SOXD/genética , Globinas beta/genética , Talasemia beta/genética , Talasemia delta/genética , gamma-Globinas/genética , Antígenos CD34/metabolismo , Secuencia de Bases , Regulación hacia Abajo/genética , Femenino , Humanos , Masculino , MicroARNs/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras , Análisis de Secuencia de ADN , Eliminación de Secuencia/genética , gamma-Globinas/metabolismoRESUMEN
Increased γ-globin production and consequent fetal hemoglobin (Hb F, α2γ2) formation is an important modulator of the clinical and hematological features of hemolytic anemias, such as sickle cell disease and ß-thalassemia (ß-thal). Hb F genes are genetically regulated, but despite numerous studies, the molecular basis of hemoglobin (Hb) switching is not completely understood. Hereditary persistence of fetal Hb (HPFH) is a consequence of impaired switching in adult life, which results in the continued expression of the γ-globin gene. This study was undertaken to identify genes that could be involved in Hb switching and/or maintenance of elevated Hb F levels. Two libraries were constructed using reticulocytes from normal donors and from Brazilian HPFH subjects. Results suggest that the maintenance of Hb F levels could be associated with some gene/protein expression modifications, such as low expression of KLF1, a transcription factor known to contribute to the regulation and modulation of Hb switching, decreased expression of MIER1, known for the recruitment of chromatin remodeling enzymes, and decreased expression of HOOK3. These data suggest new genes that may play a role in globin gene regulation, γ-globin gene expression and augmentation of Hb F levels, and may represent newly-defined cellular pathways for the control of Hb switching in erythroid cells.
Asunto(s)
Hemoglobina Fetal/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , gamma-Globinas/genética , Hemoglobina Fetal/metabolismo , Biblioteca de Genes , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reticulocitos/metabolismo , Activación Transcripcional , gamma-Globinas/metabolismoRESUMEN
Modulation of intracellular cyclic guanosine monophosphate (cGMP) may characterize a therapeutic target for sickle cell disease (SCD); cGMP-dependent signalling may be important for erythroid foetal haemoglobin induction and exert anti-inflammatory functions in leucocytes. As the inhibition of phosphodiesterases (PDEs), which regulate intracellular cGMP, can result in tissue-specific elevation of cGMP, we studied the gene expressions of cGMP-specific PDEs (-1A, -5A and -9A) in the reticulocytes and neutrophils of healthy controls, steady-state SCD patients and SCD patients on hydroxycarbamide therapy (SCDHC). PDE9A gene expression was found in numerous cell types; however, high expression was found in neutrophils, reticulocytes, CD34(+)-derived erythroid cells and K562 erythroleukaemic cells, indicating a high haematopoietic cell expression. PDE9A gene expression was, however, significantly higher in the reticulocytes and neutrophils of SCD individuals, compared to control cells; Western blotting confirmed the production of PDE9A protein in SCD neutrophils and K562 cells. Inhibition of PDE9A enzyme with the specific inhibitor, BAY73-6691, significantly increased production of the gamma-globin gene (HBG) in K562 cells and reversed the increased adhesive properties of SCD neutrophils. Since elevation of haematopoietic intracellular cGMP may be beneficial in SCD, the relatively limited tissue distribution of PDE9A suggests that it could represent a novel drug target worthy of further study.