F factor plasmid-mediated Epstein-Barr virus genome introduction establishes an EBV positive NPC cell model

Jingling Duan; Yang Yang; Zhen Wu; Shiang Lin; Chen Zhou; Guowen Sheng; Fan Yang; Lihui Bian; Xiaoling Zhang; Shengjun Xiao

doi:10.2147/CMAR.S211372

F factor plasmid-mediated Epstein-Barr virus genome introduction establishes an EBV positive NPC cell model

Cancer Manag Res. 2019 Aug 5:11:7377-7389. doi: 10.2147/CMAR.S211372. eCollection 2019.

Authors

Jingling Duan^{1

2}, Yang Yang^{1

2}, Zhen Wu³, Shiang Lin⁴, Chen Zhou^{1

2}, Guowen Sheng^{1

2}, Fan Yang^{1

2}, Lihui Bian⁵, Xiaoling Zhang⁶, Shengjun Xiao¹

Affiliations

¹ Department of Pathology, The Second Affiliated Hospital, Guilin Medical University, Guilin 541199, People's Republic of China.
² Graduate College, Guilin Medical University, Guilin 541199, People's Republic of China.
³ Xiangya School of Medicine, Central South University, Changsha 410083, People's Republic of China.
⁴ Department of Otorhinolaryngology, The Affiliated Hospital, Guangdong Medical University, Zhanjiang 524001, People's Republic of China.
⁵ Department of Pathology, The Affiliated Hospital, Hebei University, Baoding 071000, People's Republic of China.
⁶ Department of Physiology, Faculty of Basic Medical Science, Guilin Medical University, Guilin 541199, People's Republic of China.

Abstract

Background: Most Epstein-Barr virus (EBV)-positive cells lose the EBV episomes upon prolonged propagation.

Purpose: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Epstein-Barr virus; cell cycle; oncogenesis; signal transduction.