The Study Of F Factor Plasmid-mediated Introduction With Carrying Epstein-barr Virus Genome Establishes An EBV-positive NPC Cell Model

The development and progression of nasopharyngeal carcinoma（NPC）is a complicated biological process caused by multi-factor,multi-gene and multistage.NPC exists in geographical and ethnic distribution.Undifferentiated NPC is common in Southeast Asia,with a particularly high incidence among southern Chinese.The Epstein-Barr virus（EBV）genome can be detected in practically all cancer cells in undifferentiated NPC.The association of NPC with EBV has been firmly established since 1973.Nevertheless,the role for the virus in the pathogenesis of NPC is still controversial.EBV oncogene causes the function of tumor related genes abnormal in host cells,and then causes cell transformation,based on the occurrence of cancer.Due to EB virus only infects humans and a few primates,representative EBV infected NPC animal models or cell models is a rare for the study of NPC at present.The EBV positive NPC cell lines,even if established in culture,rapidly lost their EBV episomes upon prolonged（within10–20 passages）propagation,adverse to long-term preservation and related research.The need to establish new and representative NPC cell lines is eminent for NPC and EBV research.Objective: In study of culturing NPC cells in vitro at present,most Epstein-Barr virus-positive cells lose the EBV episomes upon prolonged propagation.The purposes of this study were to establish a simple cell model for nasopharyngeal carcinoma and EBV research by introducing F factor plasmid mediated the EBV genome into NPC cells and then to investigate the effect in malignant biological behaviour including proliferation,migration,cycle,apoptosis and NPC-associated signalling pathways JAK/STAT,（PI3K）-AKT and NF-κB by introducing exogenous EBV genome.Methods: 1.HONE1 NPC cells were transfected with F-factor plasmids mediated the EBV genome by Lipofectamine? 3000 transient transfection and then was established a HONE1-EBV cell model.2.Western blot（WB）was used to assess the effect of exogenous EBV genome on LMP1 expression,and in situ hybridization（ISH）was used to detect EBER expression in NPC HONE1-EBV cells.3.CCK8 assays were used to detect the effect of exogenous EBV genome on cell proliferation in NPC cells.4.Flow cytometry was used to analyse the effect of exogenous EBV genome on cell cycle distribution and cell apoptosis in NPC cells.5.Scratch assays and transwell assays were employed to detect the effect of exogenous EBV genome on cell migration ability in NPC cells.6.Immunofluorescence assays were used to detect the effect of exogenous EBV genome on cell morphology changes in NPC cells.7.WB analysis were detected the effect of exogenous EBV genome on the proteins involved in JAK/STAT,（PI3K）-AKT and NF-κB cancer-related signalling pathways and the expression of proteins related to malignant biology E-cadherin,Bcl-2 and Cyclin D1.Results: 1.The expression of LMP1 and EBERs were successfully detected in HONE1-EBV cells by introducing EBV genome.2.EBV genome introduction on NPC enhanced the proliferation and promotes cell cycle HONE1 cells.3.EBV genome introduction on NPC promoted the migration of HONE1 cells and Immunofluorescence showed the change of cells morphology.4.EBV genome introduction on NPC inhibited of apoptosis HONE1 cells.5.EBV genome introduction significantly inhibited the JAK/STAT signalling pathway,while it activated the（PI3K）-AKT and NF-κB signalling pathways in HONE1 cells.Conclusions: The findings suggest that F-factor plasmid-mediated EBV genome introduction was successful in constructing an EBV-positive cell model;the model partially mimics the status of EBV latency type II infected NPC.This model can serve as a good tool for studying EBV in NPC,but the subtle differences in cancer-associated signalling pathways must be considered.