The Role And Mechanism Of Smad2 In EBV Associated Gastric Cancer

Background:Epstein-Barr virus(EBV)is a human ?-herpes virus,infecting over 95%of adults.EBV is associated with benign and malignant diseases of both lymphoid and epithelial origin.EBV-associated GC(EBVaGC)is a unique subgroup of GC(nearly 10%of all cases of GC),but its pathogenesis is still unclear.Research has shown that latent membrane protein 2A(LMP2A)regulates the cellular survivin genes and miRNAs through several vital pathways,which plays an important role in tumor progression.LMP2A protein could affect LMP1-mediated NF-?B signaling to increase miR-155 expression.LMP2A may inhibit TGF-?1 mediated apoptosis through activation of the PI3K/AKT pathway.Meanwhile,LMP2A also has several proline-rich regions in the N-terminal tail,which can bind WW domain-containing Nedd4 family ubiquitin-protein ligases.Smad family member 2(Smad2)has been identified as a key element downstream of the TGF-? signaling pathway and acts as a repressor upstream of the beclin1(BECN1)promoter region to inhibit autophagy.Therefore,Smad2 is a transcription factor with multiple functions,such as in cell proliferation,differentiation,apoptosis,and cell motility.Objective:To investigate the effect of EBV on Smad2 transcription factor,further explore the molecular mechanisms and understand the role that low expression Smad2 plays in EBVaGC.Materials and methods:?The expression of Smad2 and miR-155-5p in EBVaGC cell lines(GT39,SNU719,GT38)and EBVnGC cell lines(BGC823,SGC7901,HGC27)was respectively detected by real-time quantitative PCR(RT-qPCR)and Western blotting?CCK-8 and apoptosis assays were used to identify the function of miR-155-5p in EBVaGC cell lines.?The expression of Smad2 and p-Smad2 after transfected miR-155-5p mimics or inhibitor into the SGC7901 and GT39 cells was detected by Western blotting.?LMP2A and control vector were transfected into SGC7901 and then the expression of Smad2 and miR-155-5p was detected by Western blotting.?The expression of Smad2 protein and miR-155-5p in SGC7901-LMP2A cell line treated with NF-?B inhibitor BAY11-7082.?The expression of Smad2 protein in EBVaGC cell line GT39 treated with PI3K inhibitor LY294002.?The expression of Smad2 protein in SGC7901-LMP2A cell line treated with proteasome inhibitor MG132.?CCK-8 and apoptosis assays identified the function of Smad2 in EBVaGC cell lines.?Western blotting was used to identify the expression of BECN1 and LC3B in cells that were knockdown of Smad2 gene.Results:? The protein expression level of Smad2 was lower in EBVaGC cell lines compared with that in the EBVnGC cell lines.The expression of miR-155-5p in the EBV-positive cell lines showed different patterns compared to the expression of Smad2.?CCK-8 and Flow cytometry assay revealed that overexpression of miR-155-5p decreases the viability of cells and promotes apoptotic rate than that of NC cells.?The protein expression of Smad2 in the miR-155-5p inhibitor-transfected GT39 cells was significantly higher compared with the NC cells(t=2.897,P<0.05).The protein level of Smad2 significantly decreased in the miR-155-5p mimic-transfected SGC7901 cells compared with the NC cells(t=3.835,P<0.05).?The expression of Smad2 in the LMP2A-transfected cells was significantly lower compared with that in the NC group cells(t=5.835,P<0.05).Expression of Smad2 shows opposite patterns with that of miR-155-5p(t=6.874,P<0.05).?LMP2A-transfected SGC7901 cells were treated with BAY 11-7982,the expression of miR-155-5p reduced significantly(2.5?M:t=5.748,P<0.05;36h:t=5.433,P<0.05;48h:t=6.175,P<0.05),but the expression of Smad2 obvious increased(2.5?M:t=3.764,P<0.05;5?M:t=5.761,P<0.05;10?M:t=4.379,P<0.05).?Upon treatment with PI3K inhibitor(LY294002)for three different concentrations in LMP2A-transfected SGC7901 cells,the expression of Smad2 and p-Smad2 significantly induced(Smad2:30?M:t=6.374 P<0.05;40?M:t=4.853,P<0.05;50?M:t=7.143,P<0.05,p-Smad2:30?M:t=3.786 P<0.05;40?M:t=6.630,P<0.05,50?M:t=3.694,P<0.05).?LMP2A-transfected SGC7901 cells were treated with MG132,the expression of Smad2 and p-Smad2 significantly induced(Smad2:6h:t=5.383,P<0.05;10h:t=3.693,P<0.05;12h:t=6.725,P<0.05,p-Smad2:6h:t=6.293,P<0.05;10h:t=5.164,P<0.05;12h:t=4.463,P<0.05).?CCK-8 and Flow cytometry assay revealed compared with NC cells,SGC7901 cells transfected with siSmad2 increases the viability of cells and decreased apoptotic rate.?Western blotting showed that SGC7901 cells transfected with siSmad2 promoted BECN1 and LC3B protein expression.Conclusion:The expression of Smad2 transcription factor in protein level was repressed by LMP2A in EBVaGC cell lines.?LMP2A up-regulated the expression of miR-155-5p through the NF-?B signaling pathway to target Smad2.?LMP2A activated PI3K/AKT to inhibit Smad2 expression and decreased the expression level of phosphorylated Smad2.?LMP2A might regulate the degradation of Smad2 protein by ubiquitination.?The the low expression of Smad2 and p-Smad2 blocked the TGF-? signaling pathway and then regulated cell proliferation,apoptosis and autophagy.