Effect Of Epstein-Barr Virus On AXL Gene Expression And Its Regulatory Mechanisms In Epstein-Barr Virus-associated Gastric Cancer

Background and objective: Epstein-Barr virus(EBV)is a member of the lympho-tropic virus of the herpes virus family.It is widely infected in the human population and is closely related to the occurrence and development of various human tumors.In recent years,EBV-associated gastric carcinoma(EBVa GC)has been gradually recogn-ized as a unique molecular subtype disease.EBVa GC accounts for an average of 10% of patients with gastric cancer worldwide,but the current research on its pathogenesis is still unclear.AXL-encoded protein is a member of the receptor tyrosine kinase sub-family.Studies have shown that AXL gene is an important proto-oncogene,which can activate many pathways.Among them,AXL activation of PI3K/AKTpathway is one of the important pathways in signal transduction.Activation of this pathway plays an important role in the occurrence and development of various tumors.The aim of this study was to investigate the effect of EBV on the expression of AXL gene in EBV-a GC and the possible mechanisms affecting AXL gene expression,which provide an experimental basis for further clarifying the mechanism of EBVa GC development.Methods:(1)Quantitative Real-time PCR(q RT-PCR)was used to detect the transcrip-tional expression of AXL gene in EBV-associated gastric cancer cell lines(GT38,GT-39,SNU719)and EBV-negative gastric cancer cell lines(AGS,BGC823,SGC7901,HGC27).(2)The expression of AXL protein in EBV-associated and EBV-negative gastric cancer cell lines was detected by Western-Blotting.(3)Immunohistochemistry(IHC)was used to detect the expression of AXL protein in EBVa GC and EBV-nega-tive gastric carcinoma tissues.(4)The technique of Bisulfite genomic sequencing(B-GS)was used to detect the methylation status of the AXL gene promoter in EBV-asso-ciated gastric cancer cell lines(GT39,SNU719)and EBV-negative gastric cancer cell lines(BGC823,SGC7901,HGC27).(5)The model of latent membrane protein 2A(LMP2A)overexpression EBV-negative gastric cancer cell line SGC7901 was esta-blished by plasmid transfection to detect the effect of LMP2 A overexpression on AXL expression.(6)Bioinformatics methods were used to screen micro RNA(mi R)en-coded by EBV that may target AXL gene.q RT-PCR was used to detect the transcriptional expression of three EBV-encoded mi Rs in EBV-associated gastric cancer cell lines(GT39,GT38,SNU719)and EBV-negative gastric cancer cell lines(BGC823,SGC7901,HGC27).The synthetic mi R-BART4-3p mimics and the corresponding negative control were transfected into EBV-negative gastric cancer cell SGC7901,and the synthesized mi R-BART4-3p inhibitor and the corresponding negative control were transfected into EBV-associated gastric cancer cell GT38.The expression level of mi R-BART4-3p was detected by q RT-PCR,and the expression level of AXL gene was detected by Western-Blotting.Meanwhile,Cell Counting Kit-8(CCK-8)assay,cell scratch and flow cytometry were used to detect the proliferation,migration and apoptosis of the transfected cells and control cells.Results:(1)The transcriptional expression level of AXL gene in EBV-associated gastric cancer cell lines was lower than that in EBV-negative gastric cancer cell lines(P<0.05).(2)The results of Western-Blotting showed that AXL protein expression in EBV-associated gastric cancer cell lines was downregulated in comparision with that in EBV-negative gastric cancer cell lines(P<0.05).(3)The results of IHC suggested that there was no significant difference in AXL protein expression between EBVa GC and EBV-negative gastric carcinoma tissues(P>0.05).(4)BGS results indicated that there was no significant difference in the methylation level of AXL gene promoter between EBV-associated gastric cancer cell lines and EBV-negative gastric cancer cell lines(P>0.05).(5)After overexpression of LMP2 A in EBV-negative gastric cancer cell line SGC7901,the transcription and translation expression level of AXL gene decreased(P<0.05,P<0.05).(6)mi R-BART4-3p,mi R-BART19-5p and mi R-BART-21-3p was selected as EBV-encoded micro RNAs that may target AXL gene by using bioinformatics methods.The results of q RT-PCR showed that mi R-BART4-3p was highly expressed in three EBV positive gastric cancer cell lines(GT39,GT38,SNU-719).The expression level of mi R-BART4-3p in EBV-associated gastric cancer cell line GT38 after transfection with mi R-BART4-3p inhibitor was not significantly different from that in the corresponding negative control group(P>0.05).And the expression level of mi R-BART4-3p in EBV-negative gastric cancer cell line SGC-7901 after transfection with mi R-BART4-3p mimics was significantly higher than that in the corresponding negative control group(P<0.05).The expression level of AXL protein in EBV-associated gastric cancer cell line GT38 after transfection with mi R-BART4-3p inhibitor was significantly higher than that in the corresponding nega-tive control group(P<0.05).And the expression level of AXL protein in EBV-negative gastric cancer cell line SGC7901 after transfection with mi R-BART4-3p mimics was significantly down-regulated compared with its corresponding negative control group(P<0.05).Compared with the corresponding negative control group,the cell viability and migration ability of EBV-associated gastric cancer cell line GT38 transfected with mi R-BART4-3p inhibitor were significantly increased(P<0.05,P< 0.05),and the apoptotic rate was significantly decreased(P<0.05).While compared with the corresponding negative control group,the cell viability and migration ability of EBV-negative gastric cancer cell line SGC7901 transfected with mi R-BART4-3p mimics were significantly decreased(P<0.05,P<0.05),and the apoptotic rate was significantly increased(P<0.05).Conclusion:(1)The transcription level and translation level of AXL gene in EBV-associated gastric cancer cell lines were significantly lower than those in EBV-negative gastric cancer cell lines,suggesting that EBV can inhibit the expression of AXL.(2)There was no significant difference in the expression of AXL protein betw-een EBVa GC and EBV-negative gastric carcinoma tissues,suggesting that AXL gene may have more complex regulatory mechanisms in EBV-associated gastric cancer.(3)EBV did not induce a change in the methylation status of the promoter region of AXL gene,suggesting that the inhibition of AXL gene expression in EBV-associated gastric cancer cell lines was not caused by the change of promoter methylation status.(4)EBV latency gene LMP2 A can inhibit AXL gene transcription and protein expression,sugg-esting that LMP2 A may be involved in the regulation of AXL gene expression.(5)mi R-BART4-3p can inhibit the expression of AXL gene.Down-regulation of mi R-BA-RT4-3p expression in EBV-positive gastric cancer cell line GT38 can promote cell proliferation and migration and inhibit cell apoptosis.While up-regulation of mi R-BART4-3p expression in EBV-negative gastric cancer cell line SCG7901 can inhibit cell proliferation and migration and promote cell apoptosis.These results suggesting that mi R-BART-4-3p may be involved in the regulation of AXL gene expression.Moreover,EBV-mi R-BART4-3p may play a role in the occurrence and development of EBVa GC as a tumor suppressor.