低氧下miR-451a对K562细胞向红系分化过程的影响

Effect of MiR-451a on Erythroid Differentiation of K562 Cells under Hypoxia

目的:探讨低氧下K562细胞向红系分化过程中GATA-1蛋白的表达变化及GATA-1如何通过上调miR-451a表达抑制14-3-3ζ的表达调控红系分化。方法:将K562细胞分为常氧组及低氧组,经氯化血红素(hemin)诱导96 h后,检测联苯胺染色阳性细胞率、γ-globin的mRNA表达以及CD235a的表达等相应红系分化指标,验证模型是否复制成功;采用Western blot及qRT-PCR检测GATA-1蛋白和miR-451a在上述2组中的表达变化及过表达GATA-1后miR-451a的表达变化;将常氧组和低氧组细胞分别分为阴性对照组(NC组)及miR-451a过表达组,结合相应红系分化指标共同判断4组细胞红系分化程度;过表达miR-451a后通过Western blot检测14-3-3ζ的表达变化。结果:低氧下K562细胞诱导96 h后联苯胺染色阳性细胞率、γ-globin的mRNA表达及CD235a的表达均显著高于0 h(P<0.05),这提示,低氧下K562细胞红系分化模型复制成功。低氧组GATA-1蛋白及miR-451a的表达水平均显著高于常氧组(P<0.05)。低氧下过表达GATA-1后,miR-451a的表达水平较NC组明显升高(P<0.05)。低氧下过表达miR-451a后,联苯胺染色阳性细胞率、γ-globin的mRNA表达水平及CD235a的表达均明显高于NC组(P<0.05)。14-3-3ζ的蛋白表达水平检测结果显示,低氧下miR-451a过表达组14-3-3ζ的表达水平低于NC组(P<0.05)。结论:低氧条件可显著提高GATA-1蛋白的表达水平,GATA-1表达的升高可上调miR-451a,进而抑制14-3-3ζ的蛋白表达,阻碍K562细胞红系分化模型中的细胞增殖过程,发挥促红系分化的作用。

Objective: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.Methods: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 ofγ-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 14-3-3ζwas detected by Western blot after over-expressed miR-451a.Results:The positive cell rate of benzidine staining,mRNA expression ofγ-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 ofγ-globin and the expression of CD235a were significantly higher than those in NC group(P<0.05).The expression level of 14-3-3ζprotein in miR-451a over-expressed group was lower than that in NC group under hypoxia(P<0.05).Conclusion: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 14-3-3ζprotein,which hinders the cell proliferation in erythroid differentiation model of K562 cells and plays an important role in promoting erythroid differentiation.