The Study Of Differentially Expressed Genes And Gene Polymorphisms In Ebva Gc By Transcriptome Sequencing

Objective:① The specific differentially expressed genes and the characteristic gene changes including SNP, AS, Indel of AGS cells before and after infection by EBV were studied. The expression pattern of EBV in AGS-EBV cells was also investigated.② Part of the differentially expressed genes and gene polymorphisms induced by EBV infection from transcriptome sequencing analysis were selected to be validated and compared to screen the molecular marker associated with the occurrence, infiltration, metastasis and prognosis of EBVa GC that could provide experimental basis and theoretic reference for demonstrating the carcinogenesis of EBV and finding new target for early diagnosis, prognosis and therapy of EBVa GC. Methods:① The transcriptome analysis of AGS cells before and after infection by EBV was carried out using high throughout sequencing.②The sequencing data was analysed with bioinformatic technology including preprocessing of the sequencing data, comparision with the reference genome, the enrichment analysis of Go Ontology and KEGG for differentially expressed genes and the analysis of expressed genes in EBV.③Total RNA was extracted from AGS cell line, AGS-EBV cell line, EBV positive(GT38, GT39, SNU719) and-negative gastric cancer cell lines(SGC7901, MKN-45, HGC-27). q RT-PCR was used to validate the expression of the significantly differentially expressed and closely associated with tumor genes- REG4, VEGF, LYPD3, ASNS, NDRG1 and APEX1. Immunohistochemistry(IHC) was used to detect the protein of REG4 in tumor issues of EBVa GC and EBVn GC.④The patients suffering gastric carcinoma were all from Shandong Province, Northern China. DNA was extracted from fresh tumor issues and paraffin-embedding(FFPE) gastric tumor issues to investigate the c SNPs- rs3827760, rs1008328, rs1048369, rs2065955, rs1021737, rs1131857, rs3759871, rs7177192, rs10882993 of gene EDAR, WDR62, GPC4, FLG, CTH, CPOX, SPG11, CASC5, ZFYVE27 respectively through DNA sequencing or RFLP. All the PCR products were analyzed by DNA sequencing except those of FLG, CTH and CPOX which were analyzed by PCR-RFLP with restriction endonuclease Apal I 、 Hae III 、 Apo I respectively. The gene sequences were comparatively analyzed with DNA Star software and Chromas software. Results:①Compared with AGS cells, there were 3880 gene m RNAs expressed differentially after EBV infection. Among them,1661 genes were up-regulated and the other 2259 were down-regulated.②In the enrichment analysis of GO term, more genes were involved in the function of protein binding, cytoplasm, integral to membrane, plasma membrane, cytosol, nucleus and metal ion binding. According to the analysis, 946 genes were involved in the tumor related functions of signal transduction, cell apoptosis, cell proliferation, cell adhesion, tumor suppression and immune association. There were 360 up-regulated genes and 586 down-regulated genes.③In the enrichment analysis of KEGG, more genes were involved in the metabolic pathways, pathways in cancers, endocytosis, focal adhesion.④ After stable EBV infection, characteristic changes including SNP, INDEL and AS were present in AGS-EBV cells. Some of these changes were associated with the differentially expressed genes.⑤There were 65 expressed genes of EBV in AGS-EBV cells. The top 10 expressed genes of EBV in infected AGS cell were BXLF2, BXLF1, LF3, BVRF1, BMRF2, BHLF1, LMP2, BILF1, BILF2 and BVLF1 according to the expression level.⑥The fold change of m RNA level and expression trend of differentially expressed genes(REG4, VEGF, LYPD3, ASNS, NDRG1 and APEX1) in EBV-positive and-negative gastric carcinoma cell lines was consistent with those in AGS cell line and AGS-EBV cell line.⑦As for the expression of REG4 protein, IHC analysis was done in 48 EBVn GC patients and 42 cases of EBVa GC. Only 4 cases of EBVn GC demonstrated positive reaction. There was no significant difference between EBVa GC and EBVn GC(P=0.12).⑧During the analysis of SNP, the EDAR gene was successfully amplified and sequenced in 43 EBVn GCs and 41 cases of EBVa GC, respectively. The difference between them was not significant(χ2=0.15, P=0.94). As for the distribution of rs1008328, the WDR62 gene was amplified and sequenced successfully in 50 EBVn GCs and 36 EBVa GCs respectively. There was no significant difference between the two groups(χ2=0.68, P=0.79). GPC4 gene was amplified and sequenced successfully in 47 EBVn GCs and 36 EBVa GCs. The distribution of its polymorphism(rs1048369) was significantly different between the two groups(χ2=10.17, P=0.006). Genotype AA(61.11%) and allele A(61.11%) were the risk factors for EBVa GC. In 48 cases of EBVn GC and 43 cases of EBVa GC, FLG gene was amplified and digested by restrictive enzyme. The distribution of its SNP(rs2065955) was significantly different(χ2=7.69,P=0.021). Genotype CC(54.17%) and allele C(70.83%) were the risk factors for EBVn GC. CTH gene was amplified and digested successfully in 40 EBVn GC and 31 EBVa GC. The distribution of its polymorphism(rs1021737) was not significantly different between the two groups(χ2=1.44, P=0.60). CPOX gene was amplified and digested in 46 EBVn GC and 56 EBVa GC. There was no significant difference between the two groups(χ2=0.60, P=0.74). As for the analysis of rs3759871, SPG11 gene was amplified and sequenced in 54 EBVn GCs and 40 EBVa GCs. The difference between the two groups was not significant(χ2=0.55, P=0.75). CASC5 gene was amplified and sequenced in 36 EBVn GCs and 29 EBVa GCs. The distribution of its SNP(rs7177192) was not significantly different between the two groups(χ2=0.99, P=0.61). ZFYVE27 gene was amplified and sequenced successfully in 56 EBVn GCs and 37 EBVa GCs. The distribution of its polymorphism(rs10882993) had no significant difference between the two groups(χ2=1.13, P=0.58). Conclusions: ①In AGS-EBV cells, there were specific differentially expressed genes with more down-regulated genes than up-regulated ones. The enrichment analysis of GO and KEGG showed that those genes were involved with particular functions and pathways which indicated that EBV had particular effect on the tumorigenesis of EBVa GC.②There were characteristic alterations in genes of AGS-EBV cells and some of them were associated with the differentially expressed genes which demonstrated that EBV could induce the differential expression according to change the gene structure.③The expression of many virus lytic and latent genes was detected in AGS-EBV cells. It was supposed that the expression of some virus genes induced the differential expression and characteristic alterations in host cells.④The infection of EBV could induce the differential expression of tumor related genes in host cells. EBV had universal effect on gene expression in host cells.⑤In Shandong Province, the polymorphisms of GPC4 and FLG were closely associated with EBVa GC. The genotype AA and allele A of GPC4 were the risk factors for EBVa GC. The genotype CC and allele C of FLG were the risk factors for EBVn GC.