Epstein-barr Virus (ebv) Infection And The Molecular Mechanism Of Gastric Cancer

Objective To understand the expression of Epstein-Barr virus(EBV) latent and lytic genes in gastric carcinomas and explore the role of EBV and EBV-encoded genes in the tumorigenesis of gastric carcinoma at the molecular level.Methods 185 gastric carcinoma and corresponding para-carcinoma tissues were tested for EBV genome by polymerase chain reaction(PCR)-Southern analysis. EBV-encoded small RNA 1(EBER1) of the PCR positive specimens was detected by in situ hybridization(ISH).Gastric carcinoma with positive EBER1 signals was confirmed EBV-associated gastric carcinoma(EBVaGC).RT-PCR and Southern hybridization were used to detect the expression of nuclear antigen(EBNA) promoters(Qp,Wp and Cp), EBNA 1 and 2,latent membrane protein(LMP) 1,2A and 2B and lytic genes (immediately early genes BZLF1 and BRLF1,early genes BARF1 and BHRF1,late genes BcLF1 and BLLF1) in EBVaGCs.Results①There were 13 EBV positive samples in gastric carcinomas(7.03%),but no EBV positive sample in corresponding para-carcinomas.The difference of EBV positive rate between gastric carcinoma and corresponding para-carcinoma tissues was significant(χ~2=11.0769,P=0.0009).In our series,age,pathological differentiation, clinical stages,lymph node metastasis and location of cancer were not different between EBV-negative gastric carcinomas(EBVnGC) and EBVaGC(P=0.669,0.141,0.259,0.818, 0.064,respectively),while sex was significantly different between the two groups (χ~2=3.9404,P=0.0471).②The transcripts of Qp were detected in all of the 13 EBVaGCs tissues,while both Wp and Cp were silent.③In the detection of latent genes expression, all of the 13 cases expressed EBNA1 mRNA,but no EBNA2,LMP1 and LMP2B mRNA. LMP2A mRNA was detected in 5 of the 13 cases.④In the detection of lytic genes expression,immediate-early gene BZLF1 mRNA was detected in 6 of the 13 EBVaGCs, but no BRLF1 mRNA.6 cases exhibited early gene BARF1 transcript and 2 exhibited BHRF1 transcript.The transcripts of late gene BcLF1 were detected in 7 cases,and no BLLF1 mRNA was detected in the 13 EBVaGCs.Conclusion①EBV infection is related to the tumorigenesis of gastric carcinoma and is male predominance,but not related to age,pathological differentiation,clinical stages,lymph node metastasis and location of cancer.②The latent pattern of EBV in EBVaGCs corresponds to the latencyⅠor unique latencyⅠ/Ⅱ,intermediate between the latencyⅠandⅡ.③The lytic infection genes are partly expressed in EBVaGCs tissues. BARF1 and BHRF1 genes may play an important role in the tumorigenesis of gastric carcinoma.However,the mechanism by which EBV lytic infection regulates the pathogenesis and development of gastric carcinoma remains to be determined. Objective To study the signifcance and the mechanism of Epstein-Barr virus(EBV) infection on gastric carcinoma cells.Methods A signet ring cell line HSC-39 derived from human gastric carcinoma was infected with Akata and P3HR-1 EBV strains.Akata and P3HR-1 EBV infected cell clones were isolated by a limiting dilution method.Characterization of the parent EBV-infected HSC-39 and their clones were detected at cell and molecular level, including EBV nuclear antigen(EBNA) immunofluorescence and EBV-encoded small RNA(EBER) in situ hybridization for EBV infection,cell morphology,colorimetric (MTT) assay and colony formation in soft agar for cell growth,Western blotting and RT-PCR analysis for EBV gene expression,and flow cytometric analysis and RT-PCR for CD21 expression.Results①HSC-39cells were highly susceptible to cell-free EBV infection by Akata and P3HR1 EBV strains.EBNA and EBER1 were detected in either infected parental cells or their clones.EBV DNA was detected in all EBNA-positive clones and part of EBNA-negative clones.②The Akata and P3HR-1 infected clones differed from each other in morphology and growth pattern.Akata EBV-infected clones had lower growth rates than did P3HR-1 EBV-infected clones in both liquid and soft agar mediums.③The EBV infected parental cells and most clones expressed EBNA1,but not expressed EBNA2,latent membrane protein(LMP)1 and LMP2A.The Q promoter(p) but not Cp/Wp was active in the infected parental cells and all clones.No lytic infection was observed in the infected parental cells and all clones(EA-D,ZEBRA and MA protein were all negative).④Uninfected HSC-39 cells did not express a principal EBV receptor CD21;however,Akata but not PSHR-1 EBV-infected clones expressed low levels of CD21 mRNA.Conclusion①HSC-39 cells were susceptible to the both EBV strains.Cellular phenotypes of HSC-39 cells are altered by EBV infection in strain-specific manner, suggesting a signet ring cell line can be used as a new target for EBV infection.②The results demonstrate that EBV infects HSC-39 by CD21-independent pathway. Objective To investigate the influence of LMP1 stable silence on Ap-1 signal transduction pathway and factors involved with cell transformation,proliferation, differentiation and apoptosis.Methods The chemically synthetic siRNA649 targeting LMP1 was transfected into GT38 cell line by lipofectamine 2000 at 50nM final concentration,and then LMP1 expressions were tested by Western blotting.The expression of CDK4,c-Jun and JunB was tested by Western blotting.MMP9,c-Jun,CDK4 and survivin mRNA were tested by RT-PCR.Hoechst 33258 staining and transmission electron microscope were used to detect apoptosis.Results①Western blotting results showed obviously discrepancy 48h,72h and 96h post-transfection with siRNA649 compared with cell control group②Compared with GT38 cell control,apoptosis was observed in target cells transfected by siRNA649 with Hochest33258 staining.Chondriosome vacuolization and chromatin pyknosis appeared in target cells transfected by siRNA649 with transmission electron microscope.③Compared with the GT38 cell control,c-Jun,survivin,JunB and MMP9 were downregulated,CDK4 was upregulated.Conclusion Chemically synthetic siRNA targeted LMP1 could effectively silence the expression of LMP1 in EBV positive cell GT38.LMP1 specific silence could influence AP-1 signal transduction pathway and its related factors,inhibited cell proliferation and promoted cell apoptosis.