| Background:Epstein-Barr virus(EBV)is a prevalent gamma-herpes virus,which can cause persistent infection in host cells.As an essential DNA tumor virus,EBV is associated with a variety of tumors.EBV associated gastric carcinoma(EBVaGC)is one of the most common cancers among EBV-related malignancies.The occurrence of EBVa GC is caused by multiple factors and the pathogenesis is not fully understood.FOXO1 transcription factor is a founding member of the FOXO family which plays an important role in tumor suppression.It has been proved that FOXO1 is a tumor suppressor and down-regulates in many tumors.FOXO1 is a direct target of PI3K-AKT pathway which is an important signal transduction in a variety of cell system.Recent studies have found that almost all viruses have developed the ability to modulate PI3K-AKT pathway to govern host cell functions and its proliferation.FOXO1 contain three highly conserved putative AKT phosphorylation consensus sites(Thr24,Ser256,Ser319).Phosphorylation at these sites results in the export of FOXO1 proteins from the nucleus to the cytoplasm where FOXO1 can be degraded.Objective:The aim of this study is to investigate the effect of EBV on FOXO1transcription factor in EBVaGC,and further explore the molecular mechanisms underline the effecting.Materials and Methods:(1)Expression level of FOXO1 mRNA and its upstream regulatory genes were analyzed in three EBV positive cell lines(GT38,GT39,SNU719)and three EBV negative cell lines(BGC823,HGC27,SGC7901)by qRT-PCR.(2)The methylation of FOXO1 promoter and its first exon in GC cell lines was determined by Bisulfite genome sequencing(BGS).(3)Western blotting was performed to detect the expression of FOXO1 and AKT proteins in GC cell lines.In GC tissues,the expression of FOXO1 and AKT proteins was detected using immunohistochemistry(IHC).(4)Subcellular localization of FOXO1 in GC cell lines was investigated by immunofluorescence techniques(IF).(5)EBV positive cell lines were treated by PI3K inhibitor LY294002.Then the expression changes of FOXO1 and AKT proteins were detected by Western blotting.Results:(1)The transcriptional expression level of FOXO1 gene in EBV positive GC cell lines was lower than in EBV negative GC cell lines(t=7.101,P=0.0021),while the expression level of AKT1 gene have a counter trend(t=3.650,P=0.0218).(2)BGS results showed low methylation rate of FOXO1 promoter in GC cell lines and have no significant difference between EBV positive and EBV negative cell lines(t=0.96,P>0.05).The methylation rate of FOXO1 first exon also have no significant difference between two groups(t=0.48,P>0.05).(3)Western blotting results suggested that FOXO1 protein expression in EBV positive cell lines was downregulated in comparison with the level of EBV negative cell lines(t=4.956,P=0.0077).However,the p-FOXO1(Ser256)expression of EBV positive cell lines was relatively higher than EBV negative cell lines(t=6.097,P=0.0037).AKT expression was significantly increased in EBV positive cell lines(t=3.267,P=0.0309).Nevertheless,p-AKT(Ser473)expressed in both groups.(4)IF results indicated that the expression of FOXO1 was weaker in EBV positive cell lines than in EBV negative cell lines.FOXO1 protein distributed in both nucleus and cytoplasm of EBV positive cell lines(GT38,GT39,SNU719).In the EBV negative cell lines(BGC823,SGC7901),FOXO1 located mainly in nucleus.However,in HGC27,most of FOXO1 protein was detected in cytoplasm.Moreover,expression of AKT protein in EBV positive cell lines was stronger than in EBV negative cell lines.AKT and p-AKT(Ser473)protein distributed in both nucleus and cytoplasm of six gastric carcinoma cell lines.(5)IHC results revealed that the expression of FOXO1 protein was lower in EBVaGC than in EBVnGC(?~2=4.607,P=0.032).FOXO1 proteins was rarely expressed in EBVaGC and localized mainly in the cytoplasm and nucleus.In the EBVnGC,FOXO1 proteins are almost expressed in the nucleus.In addition,expression of AKT protein in EBVaGC was stronger than in EBVnGC(?~2=5.063,P=0.024).AKT protein distributed in both nucleus and cytoplasm of EBVa GC and EBVn GC cells.(6)No p-AKT(Ser473)protein expression was detected in EBV positive cell lines after treated with LY294002,while the FOXO1 protein showed up-regulated tendency.Conclusion:EBV can represses the expression of FOXO1 transcription factor both in mRNA and protein level in EBVaGC.The effect of EBV on the methylation of FOXO1promotor in EBVaGC were not observed,indicating that the decreased FOXO1expression was not caused by hypermethylation.The FOXO1 expression was inversely correlated with the expression of AKT.EBV may down-regulate FOXO1 expression by phosphorylating FOXO1 and prompting its nuclear release via PI3K-AKT pathway in EBVaGC.This may affect the transcription of downstream apoptotic genes,inhibiting apoptosis and promoting the growth and proliferation of tumor cells. |
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