Study On Mechanism Of EBV Regulating AQP3 Expression

Epstein-barr virus(EBV)is a type 4 human herpes virus that infects more than 95%of adults.EBV infection is related to the occurrence of various tumors,but its pathogenesis is still unclear.AQP3(Aquaporin 3)is a water channel protein that facilitates transepithelial water transport through the transmembrane osmotic gradient.Some studies have shown that AQP3 plays a key role in tumor progression and metastasis,and tumor cells may promote intracellular AQP3 expression through a series of signal transduction mechanisms(tumor cells and cell membrane signal transduction),providing favorable conditions for tumor invasion and metastasis.EBV-associated gastric carcinoma(EBVaGC)accounts for about 10% of total gastric carcinoma cases,and has unique pathological and molecular characteristics.In EBVaGC,there are a variety of tumor related gene promoter regions of the whole and non-random DNA methylation,resulting in the suppression of tumor related gene expression.Viruses stimulate tumorigenesis by influencing a variety of biological pathways in human cells,among which the epigenetic mechanism of cells is one of the most characteristic pathways,and DNA methylation is involved in the occurrence process of a variety of tumors.DNA methyltransferases(DNMTs)are key effects of DNA methylation in cell genomes.The disorder of DNMTs can lead to abnormal methylation of tumor related gene promoters,including tumor suppressor genes,leading to gene silencing.Tumor viruses play an important role in the regulation of DNMTs.Objective: To investigate the effect of EBVaGC on AQP3 gene expression and its regulatory mechanism,and to clarify the mechanism of EBVaGC affecting DNA methylation of related genes,so as to provide theoretical basis for the occurrence and development of EBVaGC.Materials and methods: Quantitative real-time PCR(Quantitative real-time PCR,qRT-PCR)was used to detect EBV-positive gastric carcinoma cell line GT38 GT39SNU719 and EBV-negative gastric carcinoma.Expression levels of AQP3,methyltransferase 1(DNMT1),methyltransferase 3a(DNMT3a),methyltransferase 3b(DNMT3b)and related genes in EBVnGC cell line SGC7901,BGC823 and HGC27 were detected by Western Blot.Immunohistochemical was used to detect the protein expression of AQP3 in EBVaGC and EBVnGC tissues.Bisulfite genomic sequence(BGS,BSP)wasused to detect the methylation status of AQP3 gene promoter and first exon.Using pcDNA3.1 to construct EBV encoded small RNA(EBER)and EBV Latent membrane protein(LMP)1,LMP2 A gene expression vector,and they were used to detect the changes in the expression of AQP3,DNMTs and related genes after transfection of EBV negative gastric carcinoma cell line SGC7901.Small interfering RNA(siRNA)targeting ERK1 and ERK2 were used to SGC7901 cell line transfected with LMP2 A to detect the changes in the expression of AQP3,DNMT3 a and related genes.Results:(1)qRT-PCR results showed that the transcription level of AQP3 gene in EBV positive gastric carcinoma cell lines was significantly lower than that in EBVnGC cell lines(t=2.057,P<0.001).In contrast,the transcription level of DNMT3 a in EBV positive gastric carcinoma cell lines was higher than that in EBV negative gastric carcinoma cell lines(t=3.139,P=0.0146).There was no significant difference in transcription level of DNMT1 between EBV positive and EBV negative gastric carcinoma cell lines(t=0.6179,P=0.5594).There was no significant difference in DNMT3 b gene transcription between the two kinds of cell lines(t=0.8166,P=0.4453).(2)Western Blot showed that the expression of AQP3 protein in EBV negative gastric carcinoma cell lines was significantly higher than that in EBV positive gastric carcinoma cell lines(t=8.857,P<0.001).The expression of DNMT3 a protein in EBV positive gastric carcinoma cell lines was significantly higher than that in EBV negative gastric carcinoma cell lines(t=10.20,P<0.001).DNMT1 protein expression in EBV negative cell lines was higher than that in EBV positive cell lines(t=9.374,P<0.001).There was no significant difference in DNMT3 b protein levels between the two kinds of cell lines(t=0.1823,P=0.8576).(3)The protein expression of AQP3 in gastric carcinoma tissues was consistent with that of cell lines.Immunohistochemical results showed that the expression of AQP3 protein in EBVaGC tissues was significantly lower than that in EBVnGC.(4)The AQP3 gene promoter and the first exon showed hypermethylation in the three EBV positive gastric carcinoma cell lines,and the methylation rates were higher than 70%.In EBV negative gastric carcinoma cell lines SGC7901 and BGC823,the methylation rates were less than 3%,while in EBV negative gastric carcinoma cell line HGC27,the methylation rate of AQP3 was 58%.(5)Methyltransferase inhibitor 5-Aza-CdR treated EBV-positive cell lines GT39 and SNU719,the methylation rate of AQP3 promoter and the first exon region was reduced to less than 60%,DNMT3 a protein expression was inhibited,and the expression of AQP3 gene in transcription and protein level was restored.(6)Compared with EBVnGC cell line transfected with control plasmid,in EBVnGC cell line transfected with EBV latent membrane protein LMP2 A,DNMT3a expression increased,AQP3 gene expression decreased,p-ERK expression increased,ERK pathway was activated.However,in the cell lines transfected with EBV latent membrane protein LMP1 and EBER,the expressions of DNMT3 a and AQP3 were not significantly changed compared with the cells transfected with control plasmids.After the use of siRNA to inhibit ERK1 and ERK2 in EBVnGC cell lines that were stably transfected with EBV latent membrane protein LMP2 A,the expression of DNMT3 a was down-regulated and the expression of AQP3 was up-regulated.Conclusion: the expression inhibition of AQP3 in EBVaGC is correlated with the hypermethylation of AQP3 promoter region and the first exon region,and the hypermethylation of AQP3 may be mediated by DNMT3 a.EBV latent membrane protein LMP2 A may activate the pathway,thereby up-regulating the expression of DNMT3 a.