Transcription Factors and MicroRNAs Expression Profiles in Reticulocytes From Beta Thalassemia Intermedia and Sickle Cell Anemia Patients,

Abstract 3201

During erythroid differentiation, the expression profile of erythroid-specific genes follows a precise timing that is controlled by a complex network of transcription factors (TFs) and microRNAs (miRNAs) that regulate the expression of a set of target genes. The aim of this study was to analyze the expression profile in sickle cell anemia (SCA) and β-thalassemia (BT) patients of the TFs GATA-1, TAL-1, NFE-2, LDB1, LMO2, EKLF and of the miRNAs 24, 144, 155, 210, 221, 222, 223 and 451, which all of have been previously described in normal erythropoiesis. Reticulocytes were separated from peripheral blood samples of subjects with normal hematological data (n≥8), from untransfused BT IVS-I-6 (T→C) homozygous patients (n=9) and from SCA patients (n=8), without HU therapy. Spherocytosis patient samples were included in the results analysis. Extraction of RNA and miRNA, transcription to cDNAs and qRT-PCR were performed to analyze TFs and miRNAs expressions. In BT patients, statistical analyzes showed that the expression levels of the TFs GATA-1, TAL-1 and NFE-2 were significantly higher (p<0.05) compared to those of normal subjects. GATA-1 is a central mediator of erythroid gene expression and interacts with multiple proteins, such as TAL-1 and NFE-2, and this regulatory network is essential for normal human erythroid maturation in vivo. Many studies have proposed an interaction between these transcriptional mechanisms and some miRNAs, such as 451. MiRNA 451 is coexpressed with miRNA144 in a bicistronic pri-mRNA transcript whose synthesis is directly activated by GATA-1. This study showed that the expression profile of these miRNAs was decreased in BT patients, compared to controls, in agreement with previous studies which demonstrating that miRNA 144/451 deficiency results in erythroid hyperplasia, ineffective erythropoiesis and anemia. In addition to these miRNAs, we also analyzed the expression profile of the miRNAs 24, 155, 210, 221, 222 and 223, which were significantly lower in BT patients (p<0.05). The low expression of these miRNAs could result from oxidative processes in thalassemic red blood cells, due to the imbalance in the synthesis of alpha and beta chains that leads to a decrease in hemoglobin production and promotes free iron accumulation. This event possibly contributes to membrane's lipid peroxidation and may cause miRNA degradation by a toxic microenvironment. In SCA patients, LMO2 and EKLF presented lower expressions (p<0.05) compared to control subjects. The expression of miRNA 221 was significantly lower in SCA patients (p<0.05), while the miRNAs 223 and 451 were significantly overexpressed in this group, compared to control subjects. One biologically relevant miRNA 223 target appears to be LMO2, a critical component of a multiprotein DNA-binding complex required for erythropoiesis. The expression of miRNA 223 was described as antagonist to the expression LMO2, corroborating our results. This study showed that the miRNAs and TFs present significant changes in their expression levels in the reticulocytes of SCA and in BT patients. Taken together these findings suggest that the miRNA post-transcriptional mechanism of erythropoietic TFs regulation could provide insights into phenotypic variation of BT and SCA patients and may contribute to the understanding of molecular pathways of these erythrocyte disorders.

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