The Role Of GCNT3 In Gastric Cancer And Its Relationship With EBV

Background: Epstein-Barr virus(EBV)is a ubiquitous ? herpes virus and one of the most common human herpes viruses.It can infect more than 90% of the world's adult population and establish a latent lifetime infection.This is the first virus associated with human cancer.Studies have shown that EBV causes various human malignancies,such as Hodgkin's lymphoma,nasopharyngeal carcinoma,extranodal NK / T-cell lymphoma,and nasal and lymphoplastic diseases of immunocompromised hosts.In gastric cancer cells,EBV exists as a plasmid called an episome and is not integrated into the host genome.Tumor cells often show changes in cellular glycosylation,a common cancer marker.O-glycan synthase glucosamine(N-acetyl)transferase 3(GCNT3)is a mucin type that is responsible for catalyzing core 2 and core 4 O-poly Sugars,and in which O-linked glycosylated proteins are biosynthesized.No studies have reported the relationship between EBV and GCNT3.Objective: To clarify the role of GCNT3 in the occurrence and development of EBV-associated gastric cancer(EBVaGC),and to explore the effect of EBV on GCNT3 expression in EBVaGC and the possible mechanism,and provide an experimental basis for further clarifying the mechanism of EBVaGC occurrence and development.Materials and methods:(1)The expression of GCNT3 and mTOR pathway in EBVaGC cell lines(GT38,GT39,SNU719)and EBV-negative gastric cancer(EBVnGC)cell lines(BGC823,HGC27,SGC7901,AGS)were detected by Western blot,and protein expression of related genes after treatment with different reagents were detected;the expression of GCNT3 protein in gastric cancer tissue was detected by immunohistochemistry.(2)CCK8,Transwell and flow cytometry were used to detect the effects of TGF-?1,mTOR pathway,GCNT3 and miR-BART1-5p on cell phenotype.(3)The dual luciferase experiment was used to detect whether miR-BART1-5p directly targets GCNT3.(4)qRT-PCR was used to detect the expression of GCNT3,LMP2 A and miR-BART1-5p.(5)The subcellular localization of GCNT3 in gastric cancer cell lines was detected by immunofluorescence.(6)TGF-?1,mTOR pathway activators and inhibitors and siGCNT3 treatment were used to detect changes in GCNT3 protein and EMT-related protein expression.(7)The EBV-negative gastric cancer cell line SGC7901 was transfected with the LMP2 A recombinant plasmid,and the cell clone SGC7901 stably expressing LMP2 A was selected.The si LMP2 A targeting the LMP2 A gene was transfected into the EBV-associated gastric cancer cell line GT38.The expressions of GCNT3 protein and mTOR pathway protein were detected after transfection with LMP2 A plasmid or siLMP2 A.Results:(1)The expression levels of GCNT3 and mTOR pathway proteins in EBVaGC cell lines were lower than those in EBVnGC cell lines(P <0.05);GCNT3 expression was higher in EBVnGC tissues and was positively correlated with lymph node metastasis.(2)mTORC1-GCNT3 inhibited G0/G1 cell cycle arrest and promoted cell proliferation and migration.(3)miR-BART1-5p directly targeted GCNT3 and inhibited cell proliferation and migration.(4)NF-?B signal activated mi R-BART1-5p expression and inhibited GCNT3 expression.(5)The results of immunofluorescence showed that the expression of GCNT3 in EBVaGC cell line was lower,and it was mainly localized in the nucleus.(6)LMP2A inhibited the expression of GCNT3 protein and mTOR pathway.LMP2 A down-regulated the expression of GCNT3 through the mTORC1 signaling pathway.(7)TGF-?1 activated the mTORC1-GCNT3 pathway to promote cell migration in EBVaGC and EBVnGC cells,while TGF-?1 promoted apoptosis and G0/G1 cell arrest in EBVnGC cells.Conclusion:(1)The expression of GCNT3 was down-regulated in both EBVaGC cell lines and tissues.EBV encoded mi R-BART1-5p inhibited cell proliferation and migration by targeting GCNT3.(2)The consistent activation of the NF-?B signaling pathway activated the expression of miR-BART1-5p,and down-regulated GCNT3 expression in EBVaGC.Furthermore,E-cadherin,N-cadherin,vimentin and p-ERK appeared to be downstream molecules of the miR-BART1-5p/GCNT3 pathway.(3)LMP2A inhibited the activation of mTORC1 pathway,and LMP2 A inhibited the expression of GCNT3 by down-regulating the effects of TGF-?1-mTORC1 signaling.(4)The mTORC1-GCNT3 pathway promoted cell proliferation and migration,and inhibited G0/G1 cell arrest.The TGF-?1-mTORC1-GCNT3 pathway promoted cell migration by regulating genes in its downstream EMT pathway.