| Background and objective: Epstein-Barr virus(EBV)is involved in the development of tumor by changing the gene expression pattern of the host cell.CXC chemokine receptor 3(CXCR3)plays critical roles in tumorigenesis of numerous malignancies.CXCR3 has been shown to play anti-or pro-tumoral roles in tumor progression,which may depend on the different tumor tissue and cell types,as well as the expression pattern of its splice variants.Three variants of CXCR3 are identified so far(CXCR3-A,CXCR3-B and CXCR3-alt),and the aberrant expression of CXCR3 A and CXCR3 B could affect tumor progression.The role of CXCR3 A and CXCR3 B in gastric cancer remains unclear.EBV-associated gastric cancer(EBVaGC)is a special subtype of gastric cancer.Our previous studies found that EBV may up-regulate the expression of CXCR3 B in gastric cancer cells,while the underlying mechanism is unknown.This study aims to clarify the effect of EBV on CXCR3 B expression in EBVaGC and to investigate the mechanism of EBV regulating CXCR3 B expression and the role of CXCR3 B in the progression of gastric cancer.It would provide evidence for the carcinogenic mechanisms of EBV.Methods:(1)Western blot,q RT-PCR and immunofluorescence were used to detect the expression and localization of CXCR3 B in EBV-positive and-negative cell lines,AGS-EBV and AGS.Immunohistochemistry was used to detect the expression of CXCR3 B in EBVaGC and EBV-negative gastric cancer(EBVn GC)tissues and para-cancerous tissues.(2)EBV-encoded genes,latent membrane protein 2A(LMP2A),nuclear antigen 1(EBNA1)and EBV-encoded small RNA 1(EBER1),were transfected into AGS cells,respectively,and the specific si RNAs of LMP2 A and EBNA1 were transfected into AGS-EBV,respectively.Then the expression of CXCR3 B was detected.(3)The LMP2 A gene was transfected into AGS and si RNAs of LMP2 A were transfected into AGS-EBV.Then the expression of proteins related to ERK and NF-κB pathways were detected.The expression of CXCR3 B was detected in cells which were transfected with si RNAs of key proteins in ERK or NF-κB pathways,and also in cells which were treated with inhibitors of ERK or NF-κB pathway.(4)AGS was transfected with CXCR3 B gene and AGS-EBV was transfected with si CXCR3 B.AGS-EBV and AGS-CXCR3 B cells were treated with CXCL4.Then,these cells were used to detect cell proliferation,apoptosis and migration by CCK-8,colony formation assay,flow cytometry,transwell and wound healing assay.The expression of proteins related to apoptosis and epithelial-mesenchymal transition(EMT)was also detected in these cells.Results:(1)CXCR3B protein and m RNA levels in AGS-EBV were significantly higher than those in AGS cells(P<0.05).The fluorescence intensity of CXCR3 B in AGS-EBV was also stronger than in AGS.The localization of CXCR3 B was mainly in cytoplasm and cell membrane.The positive expression rate(97.7%,42/43)and high expression rate(23.3%,10/43)of CXCR3 B in EBVaGC tissues were higher than those in EBVn GC tissues(positive rate,82.2%,37/45;high expression rate,6.7%,3/45).The high expression rate in para-cancerous tissues(42.9%,12/28)was higher than that in cancer tissues(10.7%,3/28).All the differences were significant(P<0.05).(2)LMP2A and EBNA1 up-regulated CXCR3 B expression(P<0.05),while EBER1 had no effect on CXCR3 B expression.(3)LMP2A up-regulated the expression of phosphorylated ERK(p-ERK)and p-p65(P<0.01),and the expression of CXCR3 B was down-regulated by using inhibitors of ERK or NF-κB pathway or using si RNAs of key proteins in the two pathways(P<0.01).(4)Overexpression of CXCR3 B or activation of CXCR3 B by CXCL4 decreased cell proliferation rate,colony formation rate,cell migration and the ability of anti-apoptosis against cisplatin,but increased cell apoptosis rate(P<0.05).The apoptosis-related protein Bcl-2 was down-regulated,and Bax was up-regulated(P<0.05).EMT-related proteins N-cadherin,β-catenin and Vimentin were down-regulated,while E-cadherin was up-regulated(P<0.05).(5)Inhibiting CXCR3 B expression increased cell proliferation rate,colony formation rate,cell migration and the ability of anti-apoptosis against cisplatin,but decreased cell apoptosis rate(P<0.05).The apoptosis-related protein Bcl-2 was up-regulated,while Bax was down-regulated(P<0.05).EMT-related proteins N-cadherin,β-catenin and Vimentin were up-regulated,while E-cadherin was down-regulated(P<0.05).Conclusions:(1)EBV up-regulates CXCR3 B expression in tumor cells of EBVaGC.(2)EBV-encoded LMP2 A up-regulates CXCR3 B expression through activating ERK and NF-κB pathways in gastric cancer cells.(3)CXCR3B inhibits proliferation and migration and promotes apoptosis of EBVaGC cells. |
PDF Full Text Request