Regulation Mechanism Of AQP3 And 4E-BP1 Expression In EBV-associated Gastric Cancer

Background: Epstein-Barr virus(EBV)is a ubiquitous,epithelial-and lymphocyte-derived tumour virus associated with the regulation of host cell gene expression through latencyencoded gene products,and thus involved in the development and progression of a wide range of tumours.Among epithelial cell-derived tumours are nasopharyngeal carcinoma(NPC),lymphoepithelioma-like carcinoma(LELC)and Epstein-Barr virus associated gastric cancer(EBVaGC).In the gastrointestinal tract,Aquaporin 3(AQP3)is expressed in gastric mucosal tissues,ileum and distal colon,where AQP3 is mainly involved in water and glycerol transport.AQP3 expression in gastric cancer tissues was significantly higher than that in normal gastric mucosa,and is involved in the expression of EMT-related proteins in gastric cancer tissues.How AQP3 overexpression promotes the occurrence and development of gastric cancer remains unclear.Eukaryotic translation initiation factor 4Ebindingprotein 1(4E-BP1)is a member of the translation inhibition protein family with a cap-dependent function to inhibit eukaryotic cells.High level of phosphorylated 4E-BP1 has been widely reported in human cancers and is related to the poor prognosis of some malignant tumors.However,the expression mechanism of 4E-BP1 in EBVaGC is unclear.Objective: To explore the regulatory effect of EBV on AQP3 and 4EB-P1,and to clarify the mechanism of EBV latent membrane protein 2A(LMP2A)participating in the occurrence and development of EBVaGC by affecting the expression of AQP3 and 4E-BP1.Methods and materials:(1)Western Blot was used to detect AQP3 and 4E-BP1 protein expression in EBV positive gastric cancer cell lines(SNU719,GT39,GT38)and EBV negative gastric cancer cell lines(HGC27,MGC803).(2)The cell localization of 4E-BP1 was detected by Immunofluorescence.(3)MGC803 cells were transfected with LMP2A and GT38 cells were transfected with AQP3.G418(400μg/ml)was added to screen MGC803-LMP2A cells stably expressing exogenous LMP2A and GT38-AQP3 cells stably expressing exogenous AQP3,and the transfection efficiency was observed by inverted fluorescence microscope.To detect the effect of exogenous LMP2A expression on the expression of AQP3,4E-BP1,p-4E-BP1 and p-mTOR protein,and the effect of exogenous AQP3 expression on the expression of 4E-BP1,p-4E-BP1,p-ERK,EMT-related protein and autophagy-related protein.(4)GT38 cells were treated with siLMP2A,and MGC803 cells were treated with siAQP3 and si4E-BP1.The effect of interfering LMP2A expression on the expression of AQP3,4E-BP1,p-4E-BP1 and p-mTOR protein,the effect of interfering AQP3 expression on the expression of 4E-BP1,p-4E-BP1 and p-ERK protein,and on the expression of autophagy-related protein were detected.(5)The level of LMP2A mRNA transcription in MGC803-LMP2A cells and GT38 cells interfering with LMP2A expression was detected by quantitative real-time PCR(qRT-PCR).(6)Different concentrations(1 μM and 2 μM)of mTOR pathway activators(MHY1485)were used to treat GT38 and SNU719 cells,and different concentrations(30n M,50 n M and 100 n M)of mTOR pathway inhibitors(Rapamycin)were used to act on MGC803 and HGC27 cells,respectively,to detect the expression of AQP3 and p-mTOR protein.(7)PD0325901(2 μM),an inhibitor of ERK pathway,was used to treat GT38 and GT38-AQP3 cells for 12 h and24h,respectively,and the protein expressions of 4E-BP1,p-4E-BP1 and p-ERK were detected.(8)The effect of AQP3 and 4E-BP1 on cell proliferation,cell cycle,apoptosis and cell migration in gastric cancer were analysed using CCK-8,flow cytometry and Transwell.Result:(1)The expression level of both AQP3 and 4E-BP1 protein were significantly lower in EBV-positive gastric cancer cell lines than in EBV-negative gastric cancer cell lines(P<0.05).(2)Immunofluorescence results showed that the fluorescence signal intensity of4E-BP1 in EBV positive gastric cancer cell lines was significantly lower than that in EBV negative gastric cancer cell lines,and the positive signal was located in the nucleus and cytoplasm.(3)After transfection of LMP2A and AQP3 recombinant expression plasmids and screening for 4 weeks,more than 90% of the cells showed green fluorescent protein under fluorescence inverted microscope,suggesting the successful establishment of cells MGC803-LMP2A and GT38-AQP3 that can stably express exogenous LMP2A and AQP3,respectively.(4)The transcription of LMP2A mRNA could be detected in MGC803-LMP2A cells,while no transcription of LMP2A mRNA was detected in control cells.(5)Compared with the control group,the expression of AQP3,4E-BP1,p-4E-BP1 and pmTOR was significantly down-regulated in MGC803-LMP2A cells,and the expression of AQP3,4E-BP1,p-4E-BP1 and p-mTOR was up-regulated after interfering with the expression of LMP2A in GT38 cells.(6)Compared with the control group,the expression of p-ERK,4E-BP1 and p-4E-BP1 in GT38-AQP3 cells increased significantly,and the expression of 4E-BP1,p-4E-BP1 and p-ERK protein decreased after interfering with the expression of AQP3 in MGC803 cells.(7)Activation of the mTOR signaling pathway in GT38 and SNU719 cells showed that AQP3 protein expression was up-regulated;inhibition of the mTOR signaling pathway in MGC803 and HGC27 cells showed that AQP3 protein expression was down-regulated.(8)Inhibition of the ERK signalling pathway in GT38 and GT38-AQP3 cells showed that the expression of 4E-BP1 and p-4E-BP1 protein was downregulated;the ERK pathway inhibitor effect significantly reversed the up-regulation of 4EBP1 and p-4E-BP1 protein expression by AQP3.(9)Compared with the control group,overexpression of AQP3 in GT38 cells can activate autophagy,promote cell migration,and have no significant effect on cell proliferation and apoptosis.(10)Interfering with 4E-BP1 expression in MGC803 cells inhibited cell proliferation and down-regulated autophagyrelated protein expression,with no significant effect on cell cycle or apoptosis.Conclusions:(1)EBV latent membrane protein 2A downregulates AQP3 expression by inhibiting activation of the mTOR signaling pathway,which in turn inhibits autophagy and migration of gastric cancer cells.(2)The activation of ERK signal pathway can promote the expression of 4E-BP1.AQP3 participates in the regulation of 4E-BP1 expression by activating ERK signal pathway,and then affects the expression of related protein,induces autophagy of gastric cancer cells and promotes cell proliferation.