Expression relevance of GATA-1 and miR-451a in erythroid differentiation of K562 cells under hypoxia |
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| Authors: | FU Cheng-bing LIU Fang NIU Zhi-peng HU Cai-yan |
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| Institution: | 1.Research Center for High-Altitude Medicine,;2.Department of Biochemistry, Medical College of Qinghai University, Xining 810000, China |
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| Abstract: | AIM To investigate the expression relevance of GATA binding protein-1 (GATA-1) and microR?NA-451a (miR-451a) in erythroid differentiation of human chronic myeloid leukemia K562 cells under hypoxia. METHODS The K562 cells were divided into 2 groups: normoxia group and hypoxia (1% O2) group, and 40 μmol/L hemin chloride was used to induce K562 cell differentiation for 48 and 72 h. The mRNA expression of γ-globin was detected by RT-qPCR, hemoglobin production was observed by benzidine staining, and flow cytometry was used to detect CD235a expression for verifying erythroid differentiation model. The protein expression of GATA-1 during K562 cell differentiation under normoxia and hypoxia was determined by Western blot. RT-qPCR was used to detect the mRNA expression of GATA-1 and the expression level of miR-451a, and their correlation was analysis. The K562 cells were infected by lentivirus for over-expression or knock-down of GATA-1. Meanwhile, the morphological changes of the cells in the above groups were analyzed by Wright-Giemsa staining method to clarify the erythroid differentiation of K562 cells. The expression miR-451a was detected by RT-qPCR after GATA-1 over-expression or knock-down. REULTS: Under normoxia and hypoxia conditions, the expression levels of γ?-globin and CD235a and the positive rate of benzidine staining at 48 and 72 h were significantly higher than those at 0 h (P<0.05).At 72 h, the expression levels of γ?-globin and CD235a and the benzidine staining positive rate in hypoxia group were significantly higher than normoxia group (P<0.05). The expression of GATA-1 mRNA and miR-451a under hypoxia showed an upward trend during the erythroid differentiation of K562 cells, and was significantly higher than that in normoxia group at 72 h (P<0.05). Correlation analysis showed that the mRNA expression of GATA-1 was positively correlated with miR-451a expression under hypoxia (P<0.01). After over-expression of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the positive rate of benzidine staining, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly higher than those in negative control group (P<0.05). After knock-down of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the benzidine staining positive rate, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly lower than those in negative control group (P<0.05). Compared with negative control group under hypoxia, the expression of miR-451a was significantly increased after GATA-1 over-expression (P<0.05), while the expression of miR-451a was significantly decreased after GATA-1 knock-down (P<0.05). CONCLUSION Hypoxia increases the expression of GATA-1 and then up-regulates miR-451a to promote erythroid differentiation of K562 cells. |
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| Keywords: | Hypoxia GATA binding protein-1 MicroRNA-451a Erythroid differentiation K562 cells |
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