NDV诱导HeLa细胞发生核糖体应激及对翻译起始复合体的影响

NDV induces ribosomal stress in HeLa cells and affects the translation initiation complex

摘要:

[目的]研究新城疫病毒(Newcastle disease virus,NDV) HBUN/LSRC/F3株(以下简称NDV F3)诱导宫颈癌细胞(HeLa)发生核糖体应激后对eIF2α介导的翻译起始复合体eIF4F的调控作用。[方法]流式细胞术及CCK-8检测细胞凋亡;实时荧光定量PCR (quantitative real-time polymerase chain reaction,qRT-PCR)检测c-Myc基因表达;流式细胞术分析细胞周期;Western blotting技术检测c-Myc、RPS7、Bcl-2、NP、eIF4E及eIF2α蛋白的表达;Western blotting和免疫荧光染色技术检测NP、eIF4E蛋白定位。[结果]与阴性对照组相比,NDV F3抑制HeLa细胞增殖并诱导细胞凋亡。细胞周期中G₀/G₁期出现停滞,c-Myc表达呈时间依赖性抑制,c-Myc与Bcl-2蛋白表达量在0-48 h内逐渐下降,NP蛋白在24 h时生成并逐渐增加,RPS7、eIF4E和eIF2α蛋白含量在0-48 h内呈先增加后降低趋势。Western blotting定位分析及激光共聚焦显微镜结果显示NP蛋白主要存在细胞质中,NP与eIF4E存在共定位现象。[结论]NDV F3诱导HeLa细胞凋亡并引发核糖体应激反应,NP与eIF4E相互作用而抑制eIF2α介导的翻译起始复合体eIF4F形成,阻断其与宿主mRNA之间的联系,同时促进NDV F3 mRNA的翻译,最终造成宿主蛋白翻译抑制。

Abstract:

[Objective]To study the regulation of eIF2α-mediated translation initiation complex eIF4F after Newcastle disease virus (NDV) HBUN/LSRC/F3(hereinafter referred to as NDV F3) induces ribosomal stress in cervical cancer HeLa cells.[Methods]Flow cytometry and cell counting Kit-8(CCK-8) were used to detect cell apoptosis,quantitative real-time polymerase chain reaction to examine c-Myc gene expression,flow cytometry to analyze cell cycle,Western blotting to assess the expression of c-Myc,RPS7,Bcl-2,NP,eIF4E,and eIF2α proteins,and Western blotting and immunofluorescence staining to locate NP and eIF4E proteins.[Results]Compared with the negative control group,NDV F3 inhibited the proliferation of HeLa cells and induced apoptosis.We observed G₀/G₁ arrest,inhibition of c-Myc expression in a time-dependent manner,decrease in protein expression of c-Myc and Bcl-2 in 0-48 h,generation of NP protein at 24 h followed by the increasing trend,and the increase in content of RPS7,eIF4E and eIF2α proteins followed by a decrease during 0-48 h.The results of Western blotting and laser confocal microscopy showed that NP protein was mainly in cytoplasm and that NP co-localized with eIF4E.[Conclusion] NDV F3 induces apoptosis of HeLa cells and triggers ribosomal stress.NP interacts with eIF4E to inhibit the formation of eIF2α-mediated translation initiation complex eIF4F,which blocks the connection with host mRNA and promotes the expression of NDV F3 mRNA,ultimately resulting in the inhibition of host protein translation.