Eini Mohammad, Safarpour Hossain, Miri-Moghddam Ebrahim
Department of Hematology, Faculty of paramedical, Birjand University of Medical Science, Birjand, Iran.
Cellular & Molecular Research Center, Birjand University of Medical Science, Birjand, Iran.
Mol Med. 2025 Apr 8;31(1):132. doi: 10.1186/s10020-025-01184-8.
Despite the established efficacy of hydroxyurea (HU) in increasing fetal hemoglobin (Hb F) levels in patients with intermedia beta-thalassemia (β-thal) and sickle cell anemia, the precise molecular mechanisms underlying these effects remain largely elusive. Understanding these mechanisms is paramount for identifying alternative therapeutic approaches to increase Hb F production while minimizing adverse effects. In this study, we employed weighted gene co-expression network analysis (WGCNA) to investigate the molecular underpinnings of γ-globin switching within GSE90878 dataset. Leveraging this information, we aimed to predict the transcriptome network and elucidate the mechanism of action of HU and Metformin (Met) on this network comprehensively. Through bioinformatic analysis, we identified IGF2BP1 and GCNT2 as key regulators of the γ-globin switching mechanism. To experimentally validate these findings, we utilized the K562 cell line as an erythroid model. Cells were treated with HU (50, 100, and 150 µM) and Met (50, 100, and 150 µM) for 24, 48, and 72 h. The expression levels of the GCNT2, γ-globin, IGF2BP1, miR-199a/b-5p, miR-451-5p and miR-144-3p were quantified using real-time polymerase chain reaction (qPCR). Our results revealed that treatment with HU (150 µM), Met (100 µM), and combination of HU-Met (150/100 µM) significantly increased IGF2BP1 expression by 6.2, 5.3, and 7.1-fold, respectively, after 24 h treatment. Furthermore, treatment with HU (50 µM), Met (50 µM) and HU/Met (50/50 µM) for 24 h led to a 3.3, 1.2, and 5-fold decrease in GCNT2 gene expression, respectively. Notably, the highest levels of γ-globin expression and Hb F production were observed with HU (100 µM), Met (50 µM), and HU/Met (100/50 µM). This study provides compelling evidence that HU and Met significantly enhance γ-globin expression and Hb F production in the K562 cell line. Our findings suggest that these drugs exert their effects by modulating the expression of IGF2BP1 and GCNT2, thus offering valuable insights into potential therapeutic strategies for disorders characterized by low Hb F levels.
尽管羟基脲（HU）在提高中间型β地中海贫血（β-地贫）和镰状细胞贫血患者的胎儿血红蛋白（Hb F）水平方面已证实具有疗效，但其作用的确切分子机制仍 largely 难以捉摸。了解这些机制对于确定替代治疗方法以增加 Hb F 生成同时将不良反应降至最低至关重要。在本研究中，我们采用加权基因共表达网络分析（WGCNA）来研究 GSE90878 数据集中γ-珠蛋白转换的分子基础。利用这些信息，我们旨在预测转录组网络并全面阐明 HU 和二甲双胍（Met）对该网络的作用机制。通过生物信息学分析，我们确定 IGF2BP1 和 GCNT2 为γ-珠蛋白转换机制的关键调节因子。为了通过实验验证这些发现，我们使用 K562 细胞系作为红系模型。细胞分别用 HU（50、100 和 150 µM）和 Met（50、100 和 150 µM）处理 24、48 和 72 小时。使用实时聚合酶链反应（qPCR）对 GCNT2、γ-珠蛋白、IGF2BP1、miR-199a/b-5p、miR-451-5p 和 miR-144-3p 的表达水平进行定量。我们的结果显示，处理 24 小时后，用 HU（150 µM）、Met（100 µM）以及 HU-Met 组合（150/100 µM）处理分别使 IGF2BP1 表达显著增加 6.2、5.3 和 7.1 倍。此外，用 HU（50 µM）、Met（50 µM）和 HU/Met（50/50 µM）处理 24 小时分别导致 GCNT2 基因表达下降 3.3、1.2 和 5 倍。值得注意的是，在用 HU（100 µM）、Met（50 µM）和 HU/Met（100/50 µM）处理时观察到γ-珠蛋白表达和 Hb F 生成的最高水平。本研究提供了令人信服的证据，表明 HU 和 Met 可显著增强 K562 细胞系中的γ-珠蛋白表达和 Hb F 生成。我们的发现表明这些药物通过调节 IGF2BP1 和 GCNT2 的表达发挥作用，从而为 Hb F 水平低的疾病的潜在治疗策略提供了有价值的见解。
