Study On The Regulatory Mechanism Of Latent Membrane Protein2A On GCNT3 Expression In Nasopharyngeal Carcinoma

Background: Epstein-Barr virus(EBV)is a DNA virus which is the first to be found associated with human tumors.It is widespread in human,and the infection rate of adults can be as high as 90%.Studies have shown that EBV infection is closely related to a variety of human malignant tumors,such as gastric cancer,nasopharyngeal carcinoma(NPC),Hodgkin’s lymphoma,and Burkitt’s lymphoma.Glycosylation plays an important role in EBV infection.Glucosaminyl(N-Acetyl)Transferase3(GCNT3),an O-glycan synthase,is a mucin-type transferase that encodes a N-acetylglucosamine transferase.Its role in mucin biosynthesis is to catalyze the formation of core 2 and core 4 O-glycans on O-linked glycosylation.GCNT3 takes part in the formation and development of various tumors,but the expression mechanism and biological function of GCNT3 in nasopharyngeal carcinoma are still unclear.The role of EBV infection on GCNT3 should be further studied.Objective: This study aimed to clarify the regulatory effect of EBV latent membrane protein2A(LMP2A)on the expression of GCNT3 protein in NPC,and to explore the specific molecular mechanism of GCNT3 expression and the biological function in the occurrence and development of NPC,to provide a new experimental basis for the study of etiology of NPC.Materials and methods:(1)Western Blot was used to test the expression of GCNT3 protein level and mTORC1 pathway in EBV-negative NPC cell lines(CNE-1,HONE);The subcellular localization of GCNT3 was detected by immunofluorescence technique.(2)The recombinant plasmid of LMP2A was transfected into CNE-1 and HONE cells,and then the expression of GCNT3 protein was detected to clarify the regulatory effect of LMP2A on GCNT3 in nasopharyngeal carcinoma cells.(3)CNE-1 cells and HONE cells were treated with mTOR activator MHY1485 and mTORC1 inhibitor Rapamycin.Immunofluorescence technique and Western Blot were used to detect the protein level of GCNT3,so as to clarify the regulatory effect of mTORC1 pathway on GCNT3.(4)In CNE-1 and HONE,EBV-negative NPC cell lines,specific small interfering RNA(simTOR,siRaptor)was used to interfere with mTOR and Raptor respectively,and total protein was collected to further clarify the specific regulation of mTORC1 pathway on GCNT3.(5)The expression of p-mTOR,p-p70S6 K and p-4EBP1 in CNE-1-LMP2A and HONE-LMP2A were detected to analyze the regulatory effect of LMP2A on mTORC1.(6)Rapamycin was used in CNE-1-LMP2A and HONE-LMP2A cells to inhibit the mTORC1 pathway.Subsequently,the expression of GCNT3 protein was detected by Western Blot to elucidate the specific mechanism of LMP2A regulation to GCNT3.(7)Small interfering RNA(si GCNT3)targeting the gene encoding GCNT3 was transfected into CNE-1 and HONE cell lines,and cell apoptosis was analyzed by flow cytometry;CCK8 assay was used to detect cell proliferation;the change of EMT-related protein expression was detected.Transwell and scratch test were used to detect the cell migration ability.(8)The interaction of GCNT3 with ZEB1 and mTOR was verified by immunoprecipitation and laser confocal microscopy.Results:(1)The expression of exogenous LMP2A in EBV-negative NPC cell lines up-regulated the expression of GCNT3 protein.(2)MHY1485 activated the mTORC1 signaling pathway in NPC cells and up-regulated the expression of GCNT3;Rapamycin treatment inhibited the mTORC1 signaling pathway and could down-regulate the expression of GCNT3 in NPC cells.(3)Interference with the expression of mTOR and Raptor in NPC cells could inhibit the expression of GCNT3.(4)Compared with NC,the expression of exogenous LMP2A up-regulated p-mTOR,p-p70S6 K,p-EBP1 and activated the mTORC1 pathway in EBV-negative NPC cells.(5)Rapamycin treatment inactivated the mTORC1 pathway in CNE-1-LMP2A and HONE-LMP2A cells,partially reversing the up-regulation of GCNT3 expression caused by LMP2A.(6)Knocking down the expression of GCNT3 in CNE-1 and HONE cells inhibited cell proliferation and migration,and had no significant effect on cell apoptosis.(7)Knocking down the expression of GCNT3 in CNE-1 and HONE cells down-regulated the expression of mTORC1 pathway related proteins p-mTOR,p-p70S6 K,p-EBP1.(8)The interaction relationship between GCNT3 and mTOR and ZEB1 was determined in CNE-1.Conclusion:(1)LMP2A can activate the mTORC1 signaling pathway and promote GCNT3 protein expression by upregulating mTORC1 signaling;GCNT3 can activate the mTORC1 signaling pathway.GCNT3 regulates the mTORC1 signaling pathway as a positive feedback through its interaction with mTOR.(2)GCNT3 promotes cell proliferation and migration,but has no significant effect on cell apoptosis.GCNT3 promotes the occurrence and development of nasopharyngeal carcinoma.(3)LMP2A-mTORC1-GCNT3 pathway promotes cell migration by regulating the expression of EMT-related proteins and GCNT3 interaction with ZEB1.