miR-214 aggravates oxidative stress in thalassemic erythroid cells by targeting ATF4.

Oxidative damage to erythroid cells plays a key role in the pathogenesis of thalassemia. The oxidative stress in thalassemia is potentiated by heme, nonheme iron, and free iron produced by the Fenton reaction, due to degradation of the unstable hemoglobin and iron overload. In addition, the levels of antioxidant enzymes and molecules are significantly decreased in erythrocytes in α- and ß-thalassemia. The control of oxidative stress in red blood cells (RBCs) is known to be mediated by microRNAs (miRNAs). In erythroid cells, microR-214 (miR-214) has been reported to respond to external oxidative stress. However, the molecular mechanisms underlying this phenomenon remain unclear, especially during thalassemic erythropoiesis. In the present study, to further understand how miR-214 aggravates oxidative stress in thalassemia erythroid cells, we investigated the molecular mechanism of miR-214 and its regulation of the oxidative status in thalassemia erythrocytes. We have reported a biphasic expression of miR-214 in ß- and α-thalassemia. In the present study the effect of miR-214 expression was investigated by using miR -inhibitor and -mimic transfection in erythroid cell lines induced by hemin. Our study showed a biphasic expression of miR-214 in ß- and α-thalassemia. Subsequently, we examined the effect of miR-214 on erythroid differentiation in thalassemia. Our study reveals the loss-of-function of miR-214 during translational activation of activating transcription factor 4 mRNA, leading to decreased reactive oxygen species levels and increased glutathione levels in thalassemia erythroid cell. Our results suggest that the expression of activating transcription factor 4 regulated by miR-214 is important for oxidative stress modulation in thalassemic erythroid cells. Our findings can help to better understand the molecular mechanism of miRNA and transcription factors in regulation of oxidative status in erythroid cells, particularly in thalassemia, and could be useful for managing and relieving severe anemia symptoms in patients in the future.

BACH1 regulates erythrophagocytosis and iron-recycling in ß-thalassemia.

Macrophages play essential roles in erythrophagocytosis and iron recycling. ß-thalassemia is characterized by a genetic defect in hemoglobin synthesis, which increases the rate of iron recycling. We previously showed that reduced expression of the BTB and CNC homolog 1 (BACH1) gene leads to increased phagocytosis of abnormal RBCs by activated monocytes. However, the mechanisms underlying this abnormal RBC clearance remained unclear. Herein, the spleen and bone marrow cells of ß-thalassemic mice were examined for erythrophagocytosis CD markers and iron-recycling genes. Higher expression levels of CD47 and CD163 on RBCs and macrophages, respectively, were observed in ß-thalassemic mice than in wild-type cells. The decreased expression of BACH1 caused an increase in Nrf2, Spic, Slc40a1, and HMOX1 expression in splenic red pulp macrophages of thalassemic mice. To investigate BACH1 regulation, a macrophage cell line was transfected with BACH1-siRNA. Decreased BACH1 expression caused an increase in CD163 expression; however, the expression levels were lower when the cells were cultured in media supplemented with ß-thalassemia/HbE patient plasma. Additionally, the iron recycling-related genes SPIC, SLC40A1, and HMOX1 were significantly upregulated in BACH1-suppressed macrophages. Our findings provide insights into BACH1 regulation, which plays an important role in erythrophagocytosis and iron recycling in thalassemic macrophages.

Upregulation of miR-214 Mediates Oxidative Stress in Hb H Disease via Targeting of ATF4.

Thalassemia is a genetic disorder, occurring because of an imbalance in the globin chain production. Oxidative stress in erythroid cells of thalassemia is mainly generated from excess globin chains, by Fenton reaction, leading to hemolysis and ineffective erythropoiesis. Previously, data has shown that microRNAs (miRNAs) are involved in oxidative stress regulation in red blood cells (RBCs). microR-214 has been reported to respond with an external oxidative stress in erythroid cells by modulating activating transcription factor 4 (ATF4). In this study, we illustrated the expressions of miR-214 and ATF4 in Hb H (ß4) disease, and Hb E (HBB: c.79G>A)/ß-thalassemia (ß-thal) reticulocyte samples. Our results showed miR-214 expression was increased in Hb H disease, but not significantly different in Hb E/ß-thal reticulocytes. The ATF4 target was decreased in both thalassemic groups. Moreover, miR-214 expression level positively correlated with the reactive oxygen species (ROS) level, while it was negatively correlated with mean corpuscular volume (MCV), mean corpuscular hemoglobin (Hb) (MCH) and mean corpuscular Hb concentration (MCHC). We suggested that the upregulation of miR-214 correlated with the oxidative stress as well as anemia severity of Hb H disease patients, by suppression of ATF4. Understanding the oxidative pathways in erythrocyte could be useful to manage and relieve the clinical manifestation, such as anemia, in thalassemic patients.