Differential Expression of microRNAs and Transcription Factors in Primary Human Erythroblast Culture of β-Thalassemia Intermedia Patients

Erythropoiesis is controlled by a complex interplay of signaling pathways, key transcription factors (TFs), as well as microRNAs (miRs). Although studies have identified miRs and TFs that regulate erythroid commitment and differentiation, the mechanism by which these molecules contribute to erythroid disorders, such as beta thalassemia (BT), is unclear. The aim of this study was to investigate miRs and TFs expression during in vitro erythroid differentiation in normal and ineffective erythropoiesis. Peripheral blood CD34⁺ cells were isolated from a transfusion-independent BT intermedia patient, homozygous for HBB IVS-I-6 (T→C), and cultured in a liquid system stimulating erythropoiesis. Cells were collected on day 10 for quantification of 84 mature miRs using a miRNA PCR Array platform. To minimize interindividual genetic variability, a control sample was collected from a healthy sibling (CON). For validation, CD34⁺ cells from 4 other BT patients and 4 healthy subjects were cultured and collected on days 7, 10, and 13 to measure miRs and TFs expressions during erythroid differentiation.

Results: Data analysis from the miRNA PCR Array showed deregulated expression of 13 miRs in BT compared to CON: miRs 150-5p and 7-5p were decreased (fold change: -3.26 and -2.80) and miRs 32-5p, 96-5p, 155-5p, 146a-5p, 210, 125b-5p, 99a-5p, 125a-5p, 124-3p, 196b-5p, and 130a-3p were increased (fold change: 2.04; 3.72; 3.04; 2.27; 8.42; 2.82; 3.13; 2.48; 5.14; 2.00, and 3.07). The expression profile of ten miRs was successfully validated with the samples on day 10 of BT cultures. In addition, miRs 96-5p, 155-5p, and 210 remained increased on day 13 (P<0.05). Three miRs were down-regulated in BT cultures: 150-5p on days 10 and 13, miRs 24-3p and 144-3p only on day 13 (P<0.05). Seven TFs showed low expression in BT cultures: FOG1 and ALK4 on day 7 (P<0.05), NFE2 and LMO2 on day 10 (P<0.05), and GATA1, TAL1, and KLF1 on days 10 and 13 (P<0.05). Up-regulation of ETO2 occurred on days 10 and 13 (P<0.05), while cMYB, GATA2, LDB1, and PU.1 did not change significantly. Previous studies have shown that up-regulation of ETO2 was associated with low hemoglobin levels and its down-regulation contributes to the activation of TFs involved in GATA1-complex, such as LMO2, FOG1, and TAL1. Based on our preliminary results, the down-regulation of these TFs could be associated with the high expression of ETO2 in BT cultures. Additionally, the low levels of GATA1 may contribute with the decrease of miRs 24-3p and 144-3p, since it is known that this TF directly activates the transcription of these miRs. Our data are also supported by high expression of miRs 96-5p, 146a-5p, and 155-5p previously described in a cell culture model of induced ineffective erythroid maturation. In silico analysis predicted that miR-210 could target two regions of mKLF1, a critical transcription factor that directly activates β-globin and indirectly represses γ-globin gene expression. KLF1 down-regulation observed on days 10 and 13 of BT culture supports a hypothetical miR-210-mKLF1 interaction.

Conclusion: Our data report associations between differentially expressed miRNAs and their possible targets during in vitro beta thalassemic erythroid differentiation. These results corroborate the importance of miRNAs in the hematopoietic process and highlight that further studies of the interaction between TFs and miRs may contribute to a better understanding of the pathophysiology of ineffective erythropoiesis.

Support by FAPESP and CNPq