Study On The CXCR4 Expression And Its Role In EBV-related Gastric Cancer

Epstein-Barr virus(EBV)is a double-stranded DNA-encoded virus,which is associated with the occurrence of various tumors.Approximately 90%of the healthy population had been infected with EBV in early childhood,showing a persistent latent infection status,and EBV antibodies could be detected in their bodies.EBV-associated gastric carcinoma(EBVaGC)accounts for about 3%-8%of the total number of gastric cancers,because its clinical features are different from other gastric cancers,its pathogenesis and related treatments have not yet been clear.CXCR4 is a highly conserved 7-pass transmembrane G protein-coupled receptor.Current studies indicate that CXCR4 is elevated in most tumor tissues,which can affect the development and progression of various tumors,such as promoting cell proliferation and inhibiting apoptosis,inducing cell migration and invasion and epithelial mesenchymal transformation.CXCR4 is also closely related to viral infection.For example,CXCR4 can act as a co-receptor to assist HIV infection and enter host cells.CXCL12,binding specifically to the N-terminus of CXCR4 and interacts with the second extracellular loop of CXCR4,could initiate downstream signaling pathways,thus regulating the development and progression of related tumors.Objective:To detect the CXCR4 expression and related regulation mechanism in EBVaGC,and to explore the role of CXCR4 in the occurrence and development of EBVaGC,which would provide potential theoretical basis for further clarifying the role of EBV in EBVaGC.Materials and Methods:(1)Real-time fluorescence quantitative PCR was used to detect the expression of CXCR4 and related microRNA between EBV positive and EBV negative gastric cancer cell lines.(2)The expression of CXCR4 and related proteins in EBVaGC and EBVnGC cell lines was detected by Western blotting.The expression of CXCR4 between EBVa GC and EBVnGC tissues was detected by Immunohistochemistry.(3)The methylation status of CXCR4 gene promoter and the first exon was detected by Bisulfite genomic sequencing(BGS).(4)Overexpression of LMP1 or LMP2A in EBV-associated gastric cancer cell lines and its effect on detection of CXCR4 expression levels.(5)Knock-down of LMP1 or LMP2A in EBV-related gastric cancer cell lines and detect the expression of CXCR4.(6)By transfecting mimics and inhibitors of microRNA-146a,the expression of CXCR4 proteins were detected by Western blotting at different doses and different time.(7)The expression of CXCR4 protein was detected in cells treated with different concentrations of PI3K inhibitor LY294002.(8)Small interfering RNA was used to knock down CXCR4 expression.Western blot was used to detect the expression of cell autophagy associated protein-LC3B.Transmission electron microscopy and fluorescence confocal microscopy were used to detect the formation of autophagosomes.(9)Flow cytometry was used to detect the effects of targeted silencing CXCR4 and autophagy activators on cell phenotype.(10)The interfering of CXCR4expression and autophagy activator detect the expression of EBV reactivation protein BZLF1.Results:(1)The CXCR4 mRNA and protein expressions in EBV~+gastric cancer cell lines(GT38 and GT39)were lower than those in EBV~-gastric cancer cell lines(BGC823and SGC7901)(P<0.05).The CXCR4 mRNA and protein were significantly up-regulated in AGS-EBV cell line(P<0.05).And CXCR4 was highly expressed in EBVaGC tissues(consistent with the latent type of AGS-EBV cell line).But there was no significant difference in the CXCL12 and CXCR7 expressions between EBV-positive and negative cell lines.(2)The sequence of CXCR4 promoter and first exon was hypomethylated in EBV-associated gastric carcinoma cells.(3)The expression of microRNA-146a in the EBVaGC cell line(GT38 and GT39)was higher than that of the EBVnGC cell line(BGC823,SGC7901,AGS).(4)Transfection of LMP1 plasmid could up-regulate the expression of microRNA-146a and down-regulate the CXCR4 expression,and knockdown of LMP1 expression show the opposite regulatory pattern.(5)microRNA-146a down-regulated the expression of the target gene CXCR4 by binding to the 3'UTR region of CXCR4(P<0.05).With the increase of mimics transfection concentration,the expression of CXCR4 decreases sequentially,and the inhibitory effect at 48h is significantly stronger than that at 24h(P<0.05).(6)In AGS-EBV cells,LMP2A induced phosphorylation of AKT and CXCR4 up-expression,and knockdown of LMP2A could show the opposite regulation mode.(7)PI3K inhibitor-LY294002,could inhibit CXCR4 expression in a dose-dependent manner.(8)Knockdown of CXCR4 suppressed cell autophagy.(9)CXCR4 could promote cell proliferation and inhibit cell apoptosis via cell autophagy.(10)CXCR4 could participate in the regulation of EBV persistent latent infection,but cell autophagy does not participate in this process.Conclusion:(1)LMP1 could up-regulate the expression of microRNA-146a,and then down-regulate the target gene CXCR4 expression.(2)LMP2A could induce the expression of CXCR4 through PI3K/AKT pathway.(3)LMP1 and LMP2A could regulate the CXCR4expression at different stages,and induce the occurrence and development of EBVaGC.