Proteomic profiling of circulating ß-thalassaemia/haemoglobin E extra-cellular vesicles reveals that association with immunoglobulin induces membrane vesiculation.

Splenectomised ß-thalassaemia/haemoglobin E (HbE) patients have increased levels of circulating microparticles or medium extra-cellular vesicles (mEVs). The splenectomised mEVs play important roles in thromboembolic complications in patients since they can induce platelet activation and endothelial cell dysfunction. However, a comprehensive understanding of the mechanism of mEV generation in thalassaemia disease has still not been reached. Thalassaemic mEVs are hypothesised to be generated from cellular oxidative stress in red blood cells (RBCs) and platelets. Therefore, a proteomic analysis of mEVs from splenectomised and non-splenectomised ß-thalassaemia/HbE patients was performed by liquid chromatography with tandem mass spectrometry. A total of 171 proteins were identified among mEVs. Interestingly, 72 proteins were uniquely found in splenectomised mEVs including immunoglobulin subunits and cytoskeleton proteins. Immunoglobulin G (IgG)-bearing mEVs in splenectomised patients were significantly increased. Furthermore, complement C1q was detected in both mEVs with IgG binding and mEVs without IgG binding. Interestingly, the percentage of mEVs generated from RBCs with IgG binding was approximately 15-20 times higher than the percentage of RBCs binding with IgG. This suggested that the vesiculation of thalassaemia mEVs could be a mechanism of RBCs to eliminate membrane patches harbouring immune complex and may consequently prevent cells from phagocytosis and lysis.

Up-regulation of microRNA 101-3p during erythropoiesis in ß-thalassemia/HbE.

Ineffective erythropoiesis is the main cause of anemia in ß-thalassemia. The crucial hallmark of ineffective erythropoiesis is the high proliferation of erythroblast. microRNA (miR/miRNA) involves several biological processes, including cell proliferation and erythropoiesis. miR-101 was widely studied and associated with proliferation in several types of cancer. However, the miR-101-3p has not been studied in ß-thalassemia/HbE. Therefore, this study aims to investigate the expression of miR-101-3p during erythropoiesis in ß-thalassemia/HbE. The results showed that miR-101-3p was upregulated in the erythroblast of ß-thalassemia/HbE patients on day 7, indicating that miR-101-3p may be involved with high proliferation in ß-thalassemia/HbE. Therefore, the mRNA targets of miR-101-3p including Rac1, SUB1, TET2, and TRIM44 were investigated to determine the mechanisms involved with high proliferation of ß-thalassemia/HbE erythroblasts. Rac1 expression was significantly reduced at day 11 in severe ß-thalassemia/HbE compared to normal controls and mild ß-thalassemia/HbE. SUB1 gene expression was significantly lower in severe ß-thalassemia/HbE compared to normal controls at day 9 of culture. For TET2 and TRIM44 expression, a significant difference was not observed among normal and ß-thalassemia/HbE. However, the high expression of miR-101-3p at day 7 and these target genes was not correlated, suggesting that this miRNA may regulate ineffective erythropoiesis in ß-thalassemia/HbE via other target genes.

Trienone analogs of curcuminoids induce fetal hemoglobin synthesis via demethylation at Gγ-globin gene promoter.

The reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for ß-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from ß-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG - 53, - 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in ß-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.