EBV Participates In The Occurrence And Development Of EBV-associated Gastric Carcinoma By Downregulating The Eukaryotic Translation Initiating Factor EIF4E

Background:Epstein-Barr Virus(EBV)is a member of herpes virusγherpes virus,which can infect more than 90%population of the world.EBV is a pathogen of infectious monocyte hypervisor,and it is also an important DNA tumor virus,mainly related to human lymphocytes and epithelial cytotic tumors,such as Burkitt’s lymphoma,Hodgkin lymphoma and NK/T Cell lymphoma,etc.,as well as epithelial cellular tumors such as nasopharyngeal carcinoma(NPC)and gastric carcinoma(GC).Eukaryotic translation initiation factor 4E(eIF4E)is a component of the eukaryotic cell translation complex EIF4F,which is involved in the protein translation related to m~7G cap structure in eukaryotic cells,and is regarded as the speed-limit factor of protein translation process.Studies have shown that EIF4E can translate a variety of tumor-related proteins,closely related to various human tumors such as gastric carcinoma,colorectal cancer,breast cancer,etc.,but the study of EIF4E in EBV-associated gastric carcinoma has not been reported.Objective:The objectives of the study was to clarify the expression of eIF4E in EBV-associated gastric carcinoma(EBVa GC)tissues and the regulation mechanism of EBV on eIF4E,and to explore the role of eIF4E in the occurrence and development of EBVa GC,and to further explore the role of eIF4E in the pathogenesis of EBVa GC,providing a certain theoretical basis to the study in EBVa GC.Methods:(1)Using online bioinformatics analysis website GEPIA(Gene Expression Profiling Interactive Analysis)and human tumor genome database TCGA(The cancer genome atlas)data to analyze the expression of eIF4E in a variety of human tumors,including gastric carcinoma,and to analyze the survival curves of the eIF4E high expression group and eIF4E low expression group in overall gastric carcinoma and EBVa GC.(2)37 EBV-positive(proven by EBER1 in situ hybridization experiment)and41 EBV-negative gastric carcinoma tissues were selected,and the expression of eIF4E in the tissues was detected by immunohistochemical staining technique.(3)Selected EBV-positive gastric carcinoma cell lines GT38,GT39,and SNU719,EBV-negative gastric carcinoma cell lines SGC7901,BGC823,and AGS,and used Western blot technology to detect the expression difference of eIF4E at the cell line level.(4)m TOR pathway activator and inhibitor were treated respectively to EBV-positive gastric carcinoma cell lines SNU719 and EBV-negative gastric carcinoma cell line SGC7901 to detect the expression of eIF4E.(5)Used lipo2000 transfection reagent to transfect the mimics and inhibitor of BART11-3p in EBV-negative and EBV-positive gastric carcinoma cells respectively,and detected the protein expression of eIF4E.(6)The dual-luciferase report experiment verified the targeting relationship between BART11-3p and eIF4E 3’-UTR region.(7)Interference eIF4E in EBV-negative cell lines,flow cytometry analysis of cell apoptosis and cell cycle;transwell experiment to detect cell migration;CCK8 experiment to detect cell proliferation.Results:(1)Online bioinformatics analysis showed that the expression level of eIF4E in gastric carcinoma tissue was significantly higher than that in normal gastric mucosa tissue,and the survival of the eIF4E high expression group was poor in overall gastric carcinoma and EBVa GC patients.(2)Western blot and immunohistochemical experiments showed that the expression level of eIF4E protein in EBVa GC tissue and EBV-positive gastric carcinoma cell lines was lower than that in EBVn GC and EBV-negative gastric carcinoma cell lines.(3)Dual-luciferase report experiment confirmed the direct targeting relationship of EBV-encoded miR-BART11-3p to the 3’-UTR region of eIF4E m RNA,and 48h after transfection with the mimic or inhibitor of BART11-3p,the protein of eIF4E expression was downregulated or upregulated accordingly.(4)In gastric carcinoma cell line that stably expressed the EBV latent membrane protein LMP2A,the m TOR pathway was inhibited,and the expression level of eIF4E was lower.After the m TOR pathway was activated or inhibited by drugs,the expression of eIF4E was upregulated or downregulated accordingly.(5)The downregulation of eIF4E expression may lead to increased apoptosis of gastric carcinoma cells,decreased migration ability,S phase arrest of the cell cycle,and decreased epithelial-mesenchymal transition(EMT),and downregulated the expression of apoptosis and EMT related proteins,such as Bcl2,Caspase3,Vimentin andβ-catenin etc.Conclusion:(1)In EBV-positive gastric carcinoma cell lines and EBVa GC tissues,EBV-encoded miR-BART11-3p could downregulate eIF4E protein expression by targeting the 3’-UTR region of eIF4E m RNA.(2)The EBV latent membrane protein LMP2A could reduce the expression level of eIF4E protein by inhibiting the m TOR pathway.(3)Decreased expression of eIF4E could lead to increased apoptosis of gastric carcinoma cells,cell cycle arrest in S-phase,and decreased cell migration;suggesting that the downregulation of eIF4E in EBVa GC could be the reason why it had fewer distant metastases and a relatively good prognosis,which provided new ideas for the diagnosis and treatment of EBV-associated gastric carcinoma.