Duan Jingling, Yang Yang, Wu Zhen, Lin Shiang, Zhou Chen, Sheng Guowen, Yang Fan, Bian Lihui, Zhang Xiaoling, Xiao Shengjun
Department of Pathology, The Second Affiliated Hospital, Guilin Medical University, Guilin 541199, People's Republic of China.
Graduate College, Guilin Medical University, Guilin 541199, People's Republic of China.
Cancer Manag Res. 2019 Aug 5;11:7377-7389. doi: 10.2147/CMAR.S211372. eCollection 2019.
Most Epstein-Barr virus (EBV)-positive cells lose the EBV episomes upon prolonged propagation.
The purposes of this study were to establish a simple cell model for nasopharyngeal carcinoma (NPC) research by introducing a plasmid with the EBV genome into NPC cells and then to investigate the resulting changes in malignant biological behaviour and NPC-associated signalling pathways.
HONE1 NPC cells were transfected with F-factor plasmids including the EBV genome (HONE1-EBV cells). Then cell proliferation, migration, cell cycle distribution and apoptosis were evaluated in vitro by using CCK8, transwell and flow cytometry assays respectively. EBV-encoded proteins and cell signal tranducting proteins were detected by western blot assays. EBV-encoded RNAs were detected by in situ hybridization. EBV particles were assayed by transmission electron microscope (TEM). The morphology of cells were detected by immunofluorescence assays for alpha-tubulin.
Latent membrane protein 1 (LMP1), latent membrane protein 2A (LMP2A), Epstein-Barr nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) were successfully expressed in HONE1-EBV cells. No EBV particles were founded by TEM. Introduction of the EBV genome significantly promoted proliferation, cell cycle progression and migration and inhibited apoptosis in HONE1 cells. Immunofluorescence assays showed that the morphology of HONE1-EBV cells changed into spindle. Furthermore, EBV genome introduction significantly inhibited the JAK/STAT signalling pathway, while it activated the PI3K-AKT and NF-κB signalling pathways in HONE1 cells.
These findings suggest that F-factor plasmid-mediated EBV genome introduction was successful in constructing an EBV positive cell model, which showed deteriorated biological behavior and activated NPC-associated signalling pathways. This model can serve as a good tool for studying EBV in NPC, but the subtle differences in cancer-associated pathways must be considered.
大多数爱泼斯坦-巴尔病毒（EBV）阳性细胞在长期传代培养后会丢失EBV附加体。
本研究的目的是通过将携带EBV基因组的质粒导入鼻咽癌（NPC）细胞，建立一种用于NPC研究的简单细胞模型，然后研究由此导致的恶性生物学行为和NPC相关信号通路的变化。
用包含EBV基因组的F因子质粒转染HONE1 NPC细胞（HONE1-EBV细胞）。然后分别使用CCK8、Transwell和流式细胞术检测体外细胞增殖、迁移、细胞周期分布和凋亡情况。通过蛋白质免疫印迹法检测EBV编码蛋白和细胞信号转导蛋白。通过原位杂交检测EBV编码RNA。通过透射电子显微镜（TEM）检测EBV颗粒。通过α-微管蛋白免疫荧光检测法检测细胞形态。
潜伏膜蛋白1（LMP1）、潜伏膜蛋白2A（LMP2A）、爱泼斯坦-巴尔核抗原1（EBNA1）和EBV编码的小RNA（EBERs）在HONE1-EBV细胞中成功表达。TEM未发现EBV颗粒。EBV基因组的导入显著促进了HONE1细胞的增殖、细胞周期进程和迁移，并抑制了其凋亡。免疫荧光检测显示HONE1-EBV细胞的形态变为纺锤形。此外，EBV基因组的导入显著抑制了HONE1细胞中的JAK/STAT信号通路，同时激活了PI3K-AKT和NF-κB信号通路。
这些发现表明，F因子质粒介导的EBV基因组导入成功构建了一个EBV阳性细胞模型，该模型表现出恶化的生物学行为并激活了NPC相关信号通路。该模型可作为研究NPC中EBV的良好工具，但必须考虑癌症相关通路中的细微差异。
