Hu Cai-Yan, Zhang Hui-Jie, Fu Cheng-Bing, Liu Fang
Department of Biochemistry, Medical College of Qinghai University, Xining 810000, Qinghai Province, China.
Department of Biochemistry, Medical College of Qinghai University, Xining 810000, Qinghai Province, China,E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2020 Dec;28(6):2071-2078. doi: 10.19746/j.cnki.issn.1009-2137.2020.06.045.
To investigate the changes of GATA-1 protein expression during erythroid differentiation of K562 cells under hypoxia and how GATA-1 can regulate erythroid differentiation by up-regulating the expression of miR-451a and inhibiting the expression of 14-3-3ζ.
K562 cells were divided into 2 groups: the normoxia group and the hypoxia group, after the induction of hemin for 96 h, the positive cells rate of the benzidine staining, the mRNA expression of γ-globin and the expression of CD235a were detected, and the success of the model was verified. The changes of GATA-1 and miR-451a expression in the above-mentioned 2 groups, the changes of miR-451a expression after over-expressed GATA-1 were detected by Western blot and qRT-PCR. The cells in normoxic group and hypoxia group were divided into negative control group (NC group) and miR-451a over-expression group respectively, and the degree of erythroid differentiation in the four groups was judged according to the corresponding erythroid differentiation indexes, and the expression of 14-3-3ζ was detected by Western blot after over-expressed miR-451a.
The positive cell rate of benzidine staining, mRNA expression of γ-globin and the expression of CD235a after 96 h induction by K562 cells under hypoxia were significantly higher than 0 h, suggesting that the erythroid differentiation model of K562 cells under hypoxia was replicated successfully. The expression levels of GATA-1 protein and miR-451a in the hypoxic group were significantly higher than that in the normoxic group (P<0.05). The expression level of miR-451a in hypoxia group was significantly higher than that in NC group after overexpressed GATA-1 (P<0.05). After over-expressed of miR-451a under hypoxia, the positive cell rate of benzidine staining, the mRNA expression level of γ-globin and the expression of CD235a were significantly higher than those in NC group (P<0.05). The expression level of 14-3-3ζ protein in miR-451a over-expressed group was lower than that in NC group under hypoxia (P<0.05).
Hypoxia can significantly increase the expression of GATA-1 protein, and the increase of GATA-1 expression can up-regulate the expression of miR-451a, thereby inhibiting the expression of 14-3-3ζ protein, which hinders the cell proliferation in erythroid differentiation model of K562 cells and plays an important role in promoting erythroid differentiation.
探讨缺氧条件下K562细胞红系分化过程中GATA-1蛋白表达的变化，以及GATA-1如何通过上调miR-451a的表达和抑制14-3-3ζ的表达来调节红系分化。
将K562细胞分为常氧组和缺氧组，经血红素诱导96 h后，检测联苯胺染色阳性细胞率、γ-珠蛋白mRNA表达及CD235a表达，验证模型成功。采用蛋白质免疫印迹法（Western blot）和实时定量聚合酶链反应（qRT-PCR）检测上述两组中GATA-1和miR-451a表达的变化、过表达GATA-1后miR-451a表达的变化。将常氧组和缺氧组细胞分别分为阴性对照组（NC组）和miR-451a过表达组，根据相应的红系分化指标判断四组的红系分化程度，过表达miR-451a后采用Western blot检测14-3-3ζ的表达。
缺氧条件下K562细胞经96 h诱导后，联苯胺染色阳性细胞率、γ-珠蛋白mRNA表达及CD235a表达均显著高于0 h时水平，提示成功复制了缺氧条件下K562细胞的红系分化模型。缺氧组GATA-1蛋白和miR-451a的表达水平显著高于常氧组（P<0.05）。过表达GATA-1后，缺氧组miR-451a的表达水平显著高于NC组（P<0.05）。缺氧条件下过表达miR-451a后，联苯胺染色阳性细胞率、γ-珠蛋白mRNA表达水平及CD235a表达均显著高于NC组（P<0.05）。缺氧条件下miR-451a过表达组14-3-3ζ蛋白表达水平低于NC组（P<0.05）。
缺氧可显著增加GATA-1蛋白的表达，GATA-1表达增加可上调miR-451a的表达，从而抑制14-3-3ζ蛋白的表达，在K562细胞红系分化模型中阻碍细胞增殖，对促进红系分化起重要作用。
