[中图分类号] R394；R735.2 [文献标志码] A

Effect and mechanism sodium butyrate on the activation Epstein-Barr virus in Epstein-Barr virus-positive gastric cancer cellsTIAN ， CHEN ，LIU （ ， ， ， ，）

[ABSTRACT]ObjectiveTo investigate theeffectand mechanism sodium butyrate（NaB）ontheactivation EpsteinBarr virus（EBV）inEBV-positivegastriccancercells.MethodsEBV-positivegastriccancercells（AGS-EBVcells）weredivided nto groups A，B，andC，and thecels in groupA were culturedroutinelyfor12 hours，while those in groups Band C were treated withNaBfor12and 24hoursrespectively.Transcriptomesequencingwas performedforthe genes AGS-EBVcelsand theEBV genes，and groups A，B，andCwerecomparedinterms therelative expressionlevelskeylatent genes and keylytic genes EBVincels.Dierentiallyexpressed genes（DEGs）were identifiedbetweengroupsA，B，andC，andthe top5upregulated DEGs and the top 5 downregulated DEGs with smaller P values were considered significant DEGs，while RT-qPCR was used to measuretherelativeexpresionlevels significantDEGs.TheGOfunctionalenrichmentanalysisandthe KEGG pathwayenrichmentanalysis were performed forallDEGs identified，andthe proteins enriched in significant pathways（the pathways with Plt;0.05 in the KEGG pathway enrichment analysis）were considered key pathway proteins，while Western blotting was used to mea-sure therelative expression levels key pathway proteinsin groups A， B，and C.TheDEGs in groupsA，B，andCwere comparedwith the transcription factors in the AnimalTFDB/PlantTFDB database，and the top5 transcription factors with smaler P values were se lectedascoretranscriptionfactors，whileRT-qPCR wasused tomeasuretherelativeexpresionlevelscoretranscriptionfactors in groups A，B，andC.ResultsCompared with group A，groups Band Chad significantincreases in therelative expresionlevels key latent genes（EBNA-1，EBNA-3A，EBNA-3B，EBNA- 3C ，EBNA .LP LMP-I ，LMP-2A，LMP-2B and RPMS1） and lytic period genes（BZLF1，BRLF1 and BHLF1）（ ：t=2.67-14.36，Plt;0.05） ，and group C had significantly higher expression levels these genes than group B （t=2.21-6.97，Plt;0.05） .Compared with group A，groupB had significant increases in the relative expression levels SCN1A，RFPL4AL1， RORB ，and CACNG7 in cells （t=2.81-13.93，Plt;0.05） and significant reductions in the relative expression levels MIR205HG ， PLAAT4 ， and OAS2 （t=4.47-623.30，Plt;0.05） ; compared with group A，group C had significant increases in the relative expression levels NLRP5 ，SCN1A，RFPL4AL1，RORB，CACNG7，and PLA2G2E Ω_t=4.32-7.43 Plt;0.05 ）and significant reductions in the relative expresson levels RS17P11，MIR205HG， PLAAT4 ，and OAS2 .Compared with group A in terms the core transcription factors，groups B and C had significant increases in the relative mRNA expression levels EGR1， PGR ，and SOX2 （ t=3.22-16.60，Plt;0.05） and significant reductions in the relative mRNA expression levels CEBPD andE2F3（ t=4.18-28.59，Plt;0.05） .Compared with group A，group B had significant reductions in the relative expression levels p65，p-p65，AKT，and p-AKT 0.05），and compared withgroupA，groupChadsignificantreductions intherelativeexpresionlevels p65，p-p65，AK，and p-AKT and a significant increase in the expression level p -ERK ⋅t=5.72-8.92，Plt;0.05） .ConclusionNaB promotes the activation EBV inEBV-positive gastric cancercels byactivating the MAPK signaling pathwayand inhibiting the NF- κB pathway， andthecoretranscriptionfactorEGRlpromotestheactivationthevirusbyfacliatingthetranscriptiontheEBVlyticgenes BZLF1 and BRLF1.

[KEY WORDs]Herpesvirus 4，human; Butyric acid; Virus activation;Gene expression regulation; Stomach neoplasms； In vitro techniques

EB病毒相关胃癌（Epstein-Barr virus-associa-tedgastriccancer，EBVaGC）是胃癌的一种独特的分子亚型[1-2]。EB 病毒（EBV）主要感染口咽部上皮细胞和B细胞，并在外周血记忆B细胞中建立长期潜伏感染状态[3]。EBV处于潜伏感染状态时复制被抑制，仅有少数潜伏期基因表达，以维持其潜伏状态[4-5]。在某些特定条件下，如机体免疫功能减弱或受到某些物理化学因素刺激后，病毒基因的启动子可能会被重新活化，启动病毒进人裂解期，并表达大量的裂解期基因[]。丁酸钠（NaB）是一种组蛋白去乙酰化酶抑制剂，属非细胞毒类抗癌化合物，可影响细胞内多种表观遗传调控机制，包括组蛋白的超乙酰化、DNA的甲基化及非组蛋白的修饰等过程]有研究显示，NaB能够诱导EBV从潜伏期进入裂解期[8-10]。但目前关于NaB诱导EBV活化的具体机制尚不明确。本研究通过对NaB处理不同时间点的EBV阳性胃癌细胞进行转录组测序，筛选出差异表达基因（DEGs）并进行生物信息学分析，同时对分析的结果进行初步验证，以全面了解NaB诱导EBV裂解复制的分子机制。

1材料与方法

1.1 细胞的分组和处理

将EBV阳性胃癌AGS-EBV细胞（中山大学第三附属医院赠予）置于含有 10% 胎牛血清、青霉素（0.1U/L） 和链霉素 （100mg/L） 的DMEM培养基（完全培养基）中，于 、含体积分数 0.05CO₂ 的湿润环境中培养，当细胞密度约达 70% 时分为 A～ C组，每组均设置3个复孔。A组细胞在完全培养基中常规培养 12h ；分别于B组和C组的完全培养基中加入 3mmol/L 的NaB后，B组细胞继续培养12h，C 组细胞继续培养 24h 。

1.2 A～C 组细胞中EBV基因的检测、转录组测序 和DEGs的富集分析