The Role And Regulatory Mechanism Of DNA Methyltransferase 3a In EBV-associated Gastric Carcinoma

Epstein-Barr virus(EBV)is a ubiquitous γ herpes virus.Approximately 90% of the world’s population has been infected with EBV and has acquired adaptive immunity.EBV infections can lead to a variety of lymphoid and epithelial tumors under certain circumstances.EBV-associated gastric carcinoma(EBVa GC)is a unique molecular subtype of gastric carcinoma.The epigenotypic status of abnormal hypermethylation in the promoter region of tumor suppressor genes plays a key role in the development of EBVa GC.Numerous studies have proved that DNA hypermethylation is more prevalent in EBVa GC than in EBV-negative gastric carcinoma(EBVn GC).DNA methylation is the clearest epigenetic mechanism that has been investigated so far,which can regulate gene expression without changing the DNA sequence,and is mainly catalyzed by the DNA methyltransferase(DNMT)family.There are at least three active DNMTs in mammalian cells: DNMT1,DNMT3 a,and DNMT3 b.Objective: EBV-positive and EBV-negative gastric carcinoma cells were selected as the research object to explore the regulatory effect of EBV on DNMT3 a,and to clarify the mechanism of EBV-encoded latent protein EBV nuclear antigen 1(EBNA1)and Latent membrane protein 2A(LMP2A)participating in the occurrence and development of EBVa GC by affecting the expression of DNMT3 a,and provide new alternative therapeutic targets for the treatment of EBVa GC.Methods:(1)GV657 vector was used to construct an overexpression plasmid(GV657-EBNA1)encoding the EBNA1 gene and transfected into EBV-negative gastric carcinoma cell lines BGC823 and SGC7901.1mg/ml puromycin was added to select cells stably expressing exogenous EBNA1,named BGC823-EBNA1 and SGC7901-EBNA1.(2)Quantitative Real-time PCR(q RT-PCR)was used to detect the m RNA expression level of EBNA1 in BGC823-EBNA1 and SGC7901-EBNA1 cells.(3)Western Blot analysis was performed to detect the expression levels of EBNA1,E2 F transcription factor 1(E2F1)and DNMT3 a protein in EBNA1 stably and transiently expressing cells.(4)Immunofluorescence technology was used to detect the location of DNMT3 a in cells.(5)Small interfering RNA targeting EBNA1,LMP2 A,E2F1 and DNMT3 a genes were transfected into EBV-positive gastric carcinoma cell line SNU719,then detected the expression level of E2F1,DNMT3 a and other related genes.(6)After treated with NF-κB inhibitor BAY11-7082,p65 and IκBα small interfering RNA,the expression level of DNMT3 a protein was detected by Western Blot in SNU719 cells.(7)CCK-8,Transwell and Flow cytometry analysis were used to analyze the effects of DNMT3 a on cell proliferation,cell migration,cell apoptosis and cell cycle in gastric carcinoma.(8)SNU719 cells were treated with DNMT inhibitor 5-Aza-Cd R,and then detected BZLF1 protein expression level and genomic DNA methylation level.Results:(1)Three weeks after transfection and screening,more than 90% of the green fluorescent protein was observed under the microscope,and the results showed that the BGC823 and SGC7901 cell lines could stably express exogenous EBNA1.(2)EBNA1 m RNA transcription can be detected in BGC823-EBNA1 and SGC7901-EBNA1 cells,but there is no EBNA1 m RNA transcription in control cells.(3)Western Blot data also confirmed that EBNA1 protein expressed in the two EBNA1 stably transfected cell lines.Compared with the control group,the expression of E2F1 and DNMT3 a was upregulated in cells stably and transiently transfected with EBNA1.(4)Immunofluorescence results showed that the fluorescence intensity of DNMT3 a was significantly increased in EBVn GC cells transiently transfected with EBNA1,and it was expressed in both nucleus and cytoplasm.(5)After interfering with EBNA1 expression in EBV-positive gastric carcinoma cell line SNU719,expressions of E2F1 and DNMT3 a were all downregulated.After interfering with LMP2 A and E2F1,respectively,the expression of DNMT3 a also showed downregulated.(6)After inhibiting the NF-κB signaling pathway with BAY11-7082 and p65 small interfering RNA,the expression of DNMT3 a was decreased.And after activating the NF-κB signaling pathway with targeted interference IκBα expression,the expression of DNMT3 a was significantly increased.(7)After inhibiting the expression of DNMT3 a in SNU719 and SGC7901 cells,compared with the control group,CCK-8 results showed that cell proliferation was inhibited,flow cytometry analysis showed that the cell cycle was blocked in G1/S phase and the number of apoptosiscellsincreased significantly,the results of transwell analysis showed that the ability of cell migration decreased significantly.(8)After treating SNU719 cells with 5-Aza-Cd R,the expression of BZLF1 protein increased significantly and the proportion of genomic DNA methylation also increased.Conclusions:(1)EBV-encoded EBNA1 and LMP2 A can promote the expression of DNMT3 a in EBVa GC cells.(2)EBNA1 upregulated the expression of DNMT3 a by activating its transcription factor E2F1.LMP2 A upregulated the expression of DNMT3 a through the activated NF-κB signaling pathway.(3)DNMT3a can promote cell proliferation,accelerate cell G1-S phase progression and cell migration,and inhibit cell apoptosis in EBVa GC and EBVn GC cells.(4)DNMT3a can facilitate the maintenance of EBV latent infection of EBV and promote genomic DNA methylation in EBVa GC cells.