The induction effect of hydroxyurea and metformin on fetal globin in the K562 cell line

Abstract

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.

Keywords: GCNT2; IGF2BP1; Beta-thalassemia; Hemoglobin F; Hydroxyurea; Metformin.

Fig. 1

Flowchart showed preparation, processing, and interpretation of fetal and adult liver gene expression dataset and transcriptome in this study

Fig. 2

Identification of the biological pathway and KEGG analysis of DEGs based on the 65% of genes involved in the oxygen carrier pathway

Fig. 3

Module-trait relationships and enrichment. (a) Each row represents a module eigengene and each column represents fetal globin switching. The numbers in each cell indicate the correlation and p-value; (b) The blue color represents the biological processes and pathways detected. The size of the node indicates the number of genes in each pathway, and the color of the node indicates statistical significance. A darker pathway node indicates greater statistical significance, with a gradient from red (p-value 0.05 − 0.005) to black (p-value < 0.0005)

Fig. 4

Hub genes detection. (a) Blue module features of G.S and M.M which significantly correlated with erythroid Development. Each point represents an individual gene within each module, which is plotted by G.S on the y-axis and MM on the x-axis; (b) Evaluation of similarity between DEGs and Hub genes lists using a Venn diagram. Thirty-one genes that were similar in both lists were then imported to Gene MANIA to construct a co-expression network

Fig. 5

Visualize interactions between the hub-genes in a co-expression blue module using GeneMAINA. The gene relationships indicate that IGF2BP1 and GCNT2 genes play a significant role in the γ-globin switching pathway by influencing the synthesis of the gamma chain. These genes are among the important genes involved in this pathway

Fig. 6

K562 cell differentiation under HU-treatment. (a) cell differentiation assay by CD235 and CD71 in HU treated cells. (b) differentiated cells percentage compare with the control group. HU (100 µM) treated cells were going to differentiation 66% compared to 5.2% in the control group. (**** p-value < 0.0001)

Fig. 7

K562 cells viability under HU treatment. (a) cell viability assayed by Annexin V and PI using flow cytometry in HU treated and control groups. b) the viability in HU-treatment group compared to the untreated group no significant difference observed (p-value > 0.05)

Fig. 8

Hb F percent and γ-globin expression in HU, Met and HU/Met treated cells. (a) Hb F assay using flowcytometery, the F-cell percent in the HU-treated cells (69.5%), Met (67.0%) and HU/Met (81.7%) compare to control group (9.2%). (b) γ-globin gene expression by qRT-PCR in treated cells. (c) the percent of F-cells in the HU, Met and HU/Met treated cells vs. control group (**** p-value < 0.0001)

Fig. 9

IGF2BP1 and GCNT2 expression by qRT-PCR. (a) IGF2BP1 expression level in the HU, Met and HU/Met treatment cells compared to the untrated cells. (b) GCNT2 gene expression in the HU, Met and HU/Met treated cell compare to the control group. (** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001)

Fig. 10

miR199a/b-5p, miR451-5p and miR 144-3p expression. The miR451-5p (a), miR144-3p (b), and miR199a/b-5p (c) expression level in the HU, Met and HU/Met treated cell. (**p-value < 0.01, ***p-value < 0.001, ****p < 0.0001)

Fig. 11

The impact of hydroxyurea and metformin on γ-globin induction involves complex molecular pathways. (a) Upon entering the cell, hydroxyurea enhances the production of ROS and activates P38 through modulation of the PKG pathway, subsequently leading to activation of the RAS-MAPK signaling pathway. This ultimately results in the downregulation of SP1, which in turn increases IGF2BP1 and decreases GCNT2. Consequently, this signaling cascade inhibit the activity of HDAC and DNMT. On the other hand, metformin increases the expression of FOXO3. (b) Additionally, KLF1, activated by BCL11A, displaces the LCR from the beta gene, leading to the suppression of gamma gene expression and a decrease in hemoglobin F production. LIN28 inhibits BCL11A and promotes gamma gene expression. The combined effect of hydroxyurea, including the activation of HDAC and DNMT1, and the inhibition of BCL11A, leads to chromatin remodeling and increased gamma gene expression