ARA55过表达激活内源性线粒体凋亡通路诱导CNE2鼻咽癌细胞凋亡

Over-expression of ARA55 promoted apoptosis in CNE2 nasopharyngeal carcinoma cells via triggering intrinsic mitochondrial apoptotic pathway

目的:研究外源性ARA55表达上调对人CNE2鼻咽癌细胞生物学特性的影响,初步探讨相关作用机制。方法:实验设过表达组、空载体组及空白对照组。采用ZLip2000将前期构建的pCMV-ARA55-EGFP重组及空白载体分别转染至人CNE2细胞系,经荧光显微镜及Western blot进一步观察和鉴定过表达组中ARA55-EGFP融合蛋白的表达;通过设置细胞增殖、侵袭迁移及凋亡等系列实验,探讨ARA55在鼻咽癌中发挥的生物学功能;Western blot检测线粒体凋亡途径中系列蛋白的表达变化。结果:转染重组过表达质粒的CNE2细胞系中可检测到ARA55-EGFP融合蛋白的表达;体外细胞功能实验结果显示,ARA55蛋白过表达后,CNE2细胞的增殖受到抑制,体外迁移和侵袭的细胞数显著减少(P<0.05)。同时,凋亡相关实验结果显示,ARA55过表达组的细胞凋亡率(早期+中晚期)较空载体组显著升高(P<0.05);Western blot进一步证实ARA55过表达组中Bcl-2蛋白的表达下调,细胞色素C(Cytochrome C)表达明显增加,下游Caspase-9和Caspase-3的剪接体表达增加。结论:过表达外源性ARA55可诱导内源性线粒体凋亡通路的激活,从而抑制CNE2鼻咽癌细胞增殖,诱导凋亡。

Objective:To evaluate the effects of exogenous ARA55 up-regulation on the biological properties of CNE2 naso-pharyngeal carcinoma cell line as well as its underlying mechanisms.Methods:Three groups including the overexpression(pCMVARA55-EGFP),mock control and blank control groups were set.The pCMV-ARA55-EGFP plasmid that we previously constructed or the mock vector were transducted into the CNE2 cells via ZLip2000 reagent.Expression of ARA55-EGFP fusion protein were observed by fluorescence microscope and detected by Western blot.A series of function experiments for cell growth,migration,invasion and apoptosis were conducted to assess the biological function of ARA55 in nasopharyngeal carcinoma;alterations of the proteins that im-plicated in the mitochondrial apoptotic pathway were finally measured through Western blot.Results:Expression of the fusion protein(ARA55-EGFP)was positive in the CNE2 cells when transfected with ARA55 recombined plasmid.In vitro function experiments showed that the cell growth was inhibited,and migrated/invaded cells were markedly decreased in the ARA55 overexpression group in response to the up-regulation of exogenous ARA55 protein(P<0.05).Besides,results of apoptosis tests showed that the total apoptosis rate(early+middle and late stage)in ARA55 overexpression group was higher than the mock control group(P<0.05).Western blot fur-ther confirmed that expressions of Bcl-2 was down-regulated,Cytochrome C,as well as the spliceosomes of downstream Caspase-9 and Caspase-3 were increased.Conclusion:ARA55 overexpression could inhibit proliferation and promote apoptosis in CEN2 cells by trig-gering the intrinsic mitochondrial apoptotic pathway.