死亡结构域相关蛋白的核-质定位对乳腺癌MCF-7细胞功能的影响

Effect of Subcellular Localization of Death Domain-associated Protein on Cellular Function of MCF-7 Cells

目的研究死亡结构域相关蛋白(death domain-associated protein,DAXX)在乳腺癌细胞MCF-7中的表达及核-质定位,并探讨这两者对MCF-7细胞功能的影响。方法通过构建LV-DAXX-RNAi-GFP干扰与LV-DAXX-GFP过表达慢病毒质粒,转染MCF-7细胞,敲低和过表达DAXX。采用核-质分离法与免疫荧光法验证DAXX在MCF-7细胞中的表达与核-质定位,流式细胞术检测细胞凋亡和细胞周期,MTT法检测细胞增殖情况。结果核-质分离法与免疫荧光法均证实DAXX主要定位在MCF-7细胞核,转染LV-DAXX-GFP慢病毒质粒主要上调了MCF-7细胞质内DAXX的表达,而转染LV-DAXX-RNAi-GFP慢病毒质粒则主要下调了DAXX在MCF-7细胞核内的表达;流式细胞术证实下调DAXX的核内表达可以使MCF-7细胞凋亡率上升,细胞增殖受抑,并将细胞阻滞在S期,而上调DAXX的细胞质内表达对MCF-7的细胞凋亡、细胞周期和细胞增殖均无显著影响。结论DAXX在乳腺癌MCF-7细胞中主要定位在细胞核,DAXX表达于细胞核或者细胞质可能对MCF-7细胞功能有着相反的作用。

Objective To explore the expression of death domain-associated protein(DAXX)and its subcellular localization in MCF-7 cells,discussing their effect on cellular function of MCF-7 cells.Methods MCF-7 cells were transfected with LV-DAXX-RNAi-GFP plasmid and LV-DAXX-GFP overexpression lentiviral plasmid,DAXX was knocked down and overexpressed.The expression and subcellular localization of DAXX in MCF-7 cells were detected by nuclear-cytoplasm separation methods and immunofluorescence,cell apoptosis and cell cycle were detected by flow cytometry,and cell proliferation was detected by MTT method.Results Both of the nuclear-cytoplasm separation methods and immunofluorescence confirmed that DAXX was mainly located in nucleus of MCF-7 cells.The transfection of LV-DAXX-GFP lentiviral plasmid mainly up-regulated the expression of DAXX in the cytoplasm of MCF-7 cells,while the transfection of LV-DAXX-RNAi-GFP lentiviral plasmid mainly down-regulated the expression of DAXX in the nucleus of MCF-7 cells.Flow cytometry confirmed that down-regulated the expression of DAXX located in nucleus could increase the apoptosis rate of MCF-7 cells,decreasing the cell proliferation,and block the cells in S phase,while up-regulated the expression of DAXX in cytoplasm had no significant effect on cell apoptosis,cell cycle and cell proliferation of MCF-7 cells.Conclusion DAXX is mainly located in the nucleus of MCF-7 cells.The expression of DAXX in the nucleus or cytoplasm may have the opposite effect on the cellular function of MCF-7 cells.