干扰ALAS2表达通过下调K562细胞线粒体自噬受体BNIP3L影响红系分化

Knockdown of ALAS2 Affects Erythroid Differentiation by Down-regulating Mitophagy Receptor BNIP3L

目的:探讨下调亚铁血红素合成酶(ALAS2)对线粒体自噬受体BNIP3L的作用及对K562细胞红系分化的影响。方法:通过慢病毒转染干扰ALAS2的表达,实时荧光定量PCR鉴定转染效率。采用流式细胞术检测K562细胞凋亡、细胞分化、线粒体膜电位、活性氧(ROS)水平,氯化血红素诱导K562细胞红系分化;蛋白免疫印迹法检测BNIP3L的表达。免疫共沉淀检测ALAS2与BNIP3L蛋白的相互作用。结果:与病毒空载对照组比较,ALAS2干扰组的BNIP3L mRNA和蛋白表达下降(P<0.01),转录因子GATA1、Nrf2表达下调,活性氧水平降低(P<0.05)。病毒空载对照组线粒体膜电位低于ALAS2干扰组(P<0.05),2组的细胞凋亡率无明显差异。氯化血红素诱导细胞分化96 h,ALAS2干扰组BNIP3L表达增加且高于病毒空载对照组,病毒空载对照组和ALAS2干扰组的CD71^highCD235a^high+CD71^lowCD235a^high细胞比例分别为53.5%和92.9%。ALAS2与BNIP3L蛋白无直接相互作用。结论:细胞内亚铁血红素水平影响BNIP3L表达,这可能与活性氧及转录因子的调节有关。BNIP3L低表达能减少细胞凋亡,而高表达则有利于红系分化。

Objective:To investigate the effect of ALAS2 downregulation on the expression of BNIP3 L and erythroid differentiation in K562 cells.Methods:The expression of ALAS2 was down-regulated by transfection of lentivirus,then quantitative real-time PCR was performed to detect the transfection efficiency.Flow cytometry analysis was applied to evaluate apoptosis of cells,erythroid differentiation,mitochondrial membrane potential and reactive oxygen species(ROS)level.Western blot was used to detect the BNIP3 L expression,Co-immunoprecipitation was performed to analyze the relationship between ALAS2 and BNIP3 L.Results:Compared with sh-NC group,knockdown of ALAS2 induced downregulation of BNIP3 L mRNA and protein expression(P<0.01)and erythroid related transcription factors GATA1,Nrf2 expression,as well as reduction of ROS level(P<0.05).Mitochondrial membrane potential of control(sh-NC)group was lower than that of shALAS2 group(P<0.05),but there was no significant change of cell apoptotic rate in two groups.CD71^highCD235a^high+CD71^lowCD235a^high cells of sh-NC and shALAS2 groups were 53.5%,92.9%at 96 h after hemin induction,respectively.No direct action between ALAS2 and BNIP3 L was observed.Conclusion:The intracellular heme level can affect the expression of BNIP3 L which may be related with the regulation of ROS and transcription factors GATA1 and Nrf2.Higher BNIP3 L facilitates cell differentiation but lower BNIP3 L is favorable for cells survival.