低氧下K562细胞向红系分化进程中GATA-1与miR-451a表达的相关性研究  

Expression relevance of GATA-1 and miR-451a in erythroid differentiation of K562 cells under hypoxia

作　　者：付成冰 刘芳 牛志鹏 胡彩艳 FU Cheng-bing;LIU Fang;NIU Zhi-peng;HU Cai-yan(Research Center for High-Altitude Medicine,Medical College of Qinghai University,Xining 810000,China;Department of Biochemistry,Medical College of Qinghai University,Xining 810000,China)

机构地区：[1]青海大学医学院高原医学研究中心,青海西宁810000 [2]青海大学医学院生物化学教研室,青海西宁810000

出　　处：《中国病理生理杂志》2020年第4期663-672,共10页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81760333);青海省科技厅(应用)基础研究计划项目(No.2017-ZJ-722)。

摘　　要：目的:探讨低氧下人慢性髓细胞性白血病K562细胞向红系分化进程中GATA结合蛋白1(GATA binding protein-1,GATA-1)与微小RNA-451a(microRNA-451a,miR-451a)表达的相关性。方法:将K562细胞分为常氧组和低氧(1%O2)组,经40μmol/L氯化血红素分别诱导分化至48和72 h。RT-qPCR检测γ-珠蛋白(γ-globin)的mRNA表达,联苯胺染色观察细胞血红蛋白生成情况,流式细胞术检测CD235a的表达水平,验证K562细胞红系分化模型是否复制成功。在K562细胞向红系分化的不同时点,采用Western blot检测两组细胞GATA-1的蛋白表达情况;采用RT-qPCR检测两组细胞GATA-1 mRNA和miR-451a的表达水平并进行相关性分析。检测GATA-1过表达组、干扰组和阴性对照组K562细胞的红系分化指标,同时采用瑞氏-吉姆萨染色法分析上述组别细胞形态学的变化,明确GATA-1过表达和抑制后K562细胞的红系分化情况。RT-qPCR检测GATA-1过表达和抑制后miR-451a的表达变化。结果:两组细胞分化48 h和72 h后,γ-globin表达量、联苯胺染色阳性率和CD235a表达量均显著高于0h(P<0.05),且低氧组72 h的γ-globin表达量、联苯胺染色阳性率和CD235a表达量均显著高于常氧组(P<0.05)。低氧组GATA-1 mRNA和miR-451a的表达水平随红系分化过程均呈现上升趋势(P<0.05),并在72 h均显著高于常氧组(P<0.05)。相关性分析结果显示,低氧下GATA-1 mRNA与miR-451a的表达呈正相关(P<0.01)。GATA-1慢病毒感染K562细胞72 h后,低氧下过表达组γ-globin表达量、联苯胺染色阳性率、CD235a表达量及体积增大、核偏移和核缩小的细胞数均显著高于阴性对照组(P<0.05);72 h干扰组γ-globin表达量、联苯胺染色阳性率、CD235a表达量及体积增大、核偏移和核缩小的细胞数均显著低于阴性对照组(P<0.05)。与阴性对照组相比,低氧下过表达GATA-1后,72 h时miR-451a的表达显著增高(P<0.05);干扰GATA-1后,72 h时miR-451a的表达显著降低(P<0.05)。结论:低氧�AIM:To investigate the expression relevance of GATA binding protein-1(GATA-1)and microRNA-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 40μmol/L hemin chloride was used to induce K562 cell differentiation for 48 and 72 h.The mRNA expression ofγ-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 ofγ-globin and CD235 a 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 ofγ-globin and CD235 a 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 ofγ-globin and CD235 a,the positive rate of benzidine staining,and the cell counts of size

关 键 词：低氧 GATA结合蛋白1 微小RNA-451a 红系分化 K562细胞 

分 类 号：R363.2[医药卫生—病理学] R329.25[医药卫生—基础医学]