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Objective: This study attempted to investigate miR-92a-3p expression in peripheral blood of patients with severe β-thalassemia, and the effect and action mechanism of miR-92a-3p on γ-globin expression and oxidative stress in erythroid precursor cells., Methods: CD34 + hematopoietic progenitor cells (HPCs) were isolated from peripheral blood of healthy volunteers and patients with severe β-thalassemia. The levels of miR-92a-3p, BCL11A, and γ-globin were measured in erythroid precursor cells. High-performance liquid chromatography (HPLC) was used to analyze hemoglobin F (HbF) content. HPCs were induced with erythroid differentiation and erythroid precursor cells were then obtained. The relevance between miR-92a-3p and BCL11A was studied using dual luciferase reporter gene assay, and the correlation between miR-92a-3p and HbF was assayed by Pearson correlation analysis. Reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) in erythroid precursor cells were tested to evaluate oxidative stress. Cell apoptosis was examined by flow cytometry., Results: Remarkably higher expression of miR-92a-3p was observed in erythroid precursor cells. Increased expression of miR-92a-3p resulted in elevated levels of γ-globin, GSH, and SOD, reduced expression of ROS and MDA, and decreased cell apoptosis. BCL11A was identified as a target of miR-92a-3p and to be downregulated by miR-92a-3p. Moreover, BCL11A knockdown alone increased the expression of γ-globin, SOD and GSH, and repressed the levels of ROS and MDA and cell apoptosis, and the following inhibition of miR-92a-3p changed these patterns., Conclusions: Our data indicated that miR-92a-3p might increase γ-globin level and reduce oxidative stress and apoptosis in erythroid precursor cells by downregulating BCL11A.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib shows potent antitumor activity in some non-small-cell lung cancer (NSCLC) cell lines and is approved by the Food and Drug Administration as second and third line treatment for NSCLC. However, the molecular mechanisms by which erlotinib induces apoptosis remain to be elucidated. Here, we investigated the effect of erlotinib on apoptotic signal pathways in H3255 cells with the EGFR(L858R) mutation. Erlotinib induces apoptosis associated with the activation of caspases in a dose- and time-dependent manner. Erlotinib did not alter the expression of apoptotic receptors FAS and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although it induced caspase-8 activation and BID cleavage. In addition, cell death caused by erlotinib was not prevented by coincubation with FAS and TRAIL antagonists, ZB-4 monoclonal antibody and TRAIL/Fc recombinant, suggesting that erlotinib-induced apoptosis is not associated with receptor-mediated pathways. Erlotinib induces loss of mitochondrial membrane potential and release of cytochrome c and second mitochondria-derived activator of caspases/direct IAP binding protein with low pI from mitochondria. Furthermore, erlotinib causes BAX translocation to mitochondria, BAX and BAK conformational changes, and oligomerization. Erlotinib did not induce reactive oxygen species generation, and cotreatment with antioxidants did not alter erlotinib-induced activation of BAX and BAK and apoptosis. However, cotreatment with inhibitors of mitochondrial oxidative phosphorylation significantly blocked erlotinib-induced activation of BAX and BAK and cell death. Benzyloxycarbiny-VAD-fluoromethyl ketone had no effect on erlotinib-induced BAX and BAK activation but effectively prevented apoptosis. Overexpression of BCL-2 caused a significant attenuation of erlotinib-induced cell death, but no effect on BAX and BAK activation. Down-regulation of BAX and BAK gene expression with small interfering RNA led to an effective reduction of erlotinib-induced apoptosis. Our data indicate that activation of BAX and BAK plays a critical role in the initiation of erlotinib-induced apoptotic cascades.