Meta-analysis of the relationship between Epstein-Barr virus infection and clinicopathological features of patients with gastric carcinoma

Epstein-Barr virus (EBV) infection has been causally associated with occurrence of many malignant neoplasms. EBV-encoded small RNAs (EBERs) have been detected from about 10% of gastric carcinoma tissue cells, suggesting that EBV infection is associated with the development of gastric carcinoma. The present study pooled the data from the papers concerning EBV-related gastric cancers and performed a meta-analysis of 22 research papers. Among these papers, a total of 5475 cases with gastric cancer were enrolled, of whom 411 cases were found EBV-positive, with the EBV-positive rate being 7.5%. Among the EBV-positive gastric cancer cases, the detection rate was 11.1% in males and 3.0% in females. Compared with EBV-negative gastric cancer, EBV-positive gastric cancer had less lymph node metastasis. Based on the histological typing, of the EBV-positive gastric cancers, the diffuse type was 8.1%, and intestinal type was 8.0%. The examined specimen types included stored paraffin blocks and fresh surgically removed specimens, their EBV positive rates were 7.9% and 6.5% respectively. In terms of geographical distribution, the detection rate of EBV-positive gastric cancer was 9.4% in America, 6.1% in Asia and 9.1% in Europe. Meta-analysis showed that EBV infection occurred only in gastric cancer tissue cells and was significantly associated with the patients’ gender, lymph node metastases, and the location where tumor tissue generated and geographical distribution (P0.05), but was not significantly associated with the patients’ histological types of tumor and the types of specimens (P0.05). These results suggested that EBV-positive gastric cancer has distinct clinicopathological features.     

Early Pattern of Epstein-Barr Virus Infection in Gastric Epithelial Cells by “Cell-in-cell”

Epstein-Barr virus(EBV)is an important human dsDNA virus,which has been shown to be associated with several malignancies including about 10%of gastric carcinomas.How EBV enters an epithelial cell has been an interesting project for investigation."Cell-in-cell"infection was recently reported an efficient way for the entry of EBV into nasopharynx epithelial cells.The present approach was to explore the feasibility of this mode for EBV infection in gastric epithelial cells and the dynamic change of host inflammatory reaction.The EBV-positive lymphoblastic cells of Akata containing a GFP tag in the viral genome were co-cultured with the gastric epithelial cells(GES-1).The infection situation was observed under fluorescence and electron microscopies.Real-time quantitative PCR and Western-blotting assay were employed to detect the expression of a few specific cytokines and inflammatory factors.The results demonstrated that EBV could get into gastric epithelial cells by"cell-in-cell"infection but not fully successful due to the host fighting.IL-1β,IL-6 and IL-8 played prominent roles in the cellular response to the infection.The activation of NF-κB and HSP70 was also required for the host antiviral response.The results imply that the gastric epithelial cells could powerfully resist the virus invader via cell-in-cell at the early stage through inflammatory and innate immune responses.     

Identification of aberrant cell cycle regulation in Epstein-Barr virus-associated nasopharyngeal carcinoma by cDNA microarray and gene set enrichment analysis

Previous studies have revealed that Epstein-Barr virus （EBV） was closely associated with nasopharyngeal carcinoma （NPC）. This study aimed to characterize the global pathways affected in the EBV-associated NPC. Combined with microdissection, gene expression profries in 22 NPCs and 10 non-tumor nasopharyngeal epithelial （NPE） tissue samples were analyzed. All NPC specimens served in the microarray analysis were positive for EBV, as judged by identification of the expression of EBV nuclear antigen 1 （EBNA1）. Through gene set enrichment analysis （GSEA）, we found that cell cycle pathway was the most disregulated pathway in NPC （P = 0.000, false discovery rate q-value = 0.007）, which included some aberrant expressed components. We first found that overexpression of CDK4, cyclin D1, and Rb proteins, and loss of expression of proteins p16, p27, and p19 were statistically significant in NPC tissues compared with non-cancerous NPE （P〈 0.05） by real-time RT- PCR and tissue microarray. EBV-encoded small RNA-1 （EBER-1） hybridization signals in the NPC showed significant associations with the overexpression of Rb （P = 0.000）, cyclin D1 （P = 0.000）, CDK4 （P = 0.000）, and the loss of expression of p16 proteins （P = 0.039）. In the final logistic regression analysis model, EBER-1 and abnormal expression of p16, Rb, cyclin D1, and E2F6 were inde- pendent contributions to nasopharyngeal carcinogenesis. Through survival analysis, only cyclin D1 could predict the prognosis of NPC patients. These results suggested that cell cycle pathway was the most disregulated pathway in the EBV-associated NPC, and EBER-1 was closely associated with p16, CDK4, cyclin D1, and Rb. cyclin D1 could be the prognosis biomarker for NPC.     

Association of single nucleotide polymorphism rs2065955 of the filaggrin gene with susceptibility to Epstein-Barr virus-associated gastric carcinoma and EBV-negative gastric carcinoma

The relationship between the Filaggrin gene(FLG) rs2065955 polymorphism and susceptibility to Epstein-Barr virus(EBV)-associated gastric carcinoma(EBVa GC) and EBV-negative gastric carcinoma(EBVn GC) was investigated in Shandong Province,China.We detected the FLG rs2065955 genotype and allele distribution by using PCR and restriction fragment length polymorphism(RFLP) in 64 EBVa GC,82 EBVn GC,and 111 normal control samples.Immunohistochemistry was used to detect the level of FLG protein in 35 EBVa GC and 51 EBVn GC tumor tissues.Compared with normal controls,the genotype CC and allele C of FLG rs2065955 showed higher frequency in EBVa GC and EBVn GC.There was no significant difference between EBVa GC and EBVn GC in allele distribution of FLG rs2065955,but the genotype CC was found more frequently in EBVa GC than in EBVn GC.The risk of developing either EBVa GC or EBVn GC in genotype CC was higher than in other genotypes.Furthermore,genotype CC of FLG rs2065955 may contribute more to the risk of developing EBVa GC than EBVn GC.There was no significant difference in the expression level of FLG protein between EBVa GC and EBVn GC.In conclusion,the FLG rs2065955 polymorphism was significantly related to gastric carcinoma.Allele C of FLG rs2065955 could be a risk factor for EBVa GC or EBVn GC,while genotype CC of FLG rs2065955 was especially associated with EBVa GC.     

Induction of Epstein-Barr virus lytic replication by recombinant adenoviruses expressing the zebra gene with EBV specific promoters

The latent Epstein-Barr virus (EBV) is found in the cells of many tumors. For example, EBV is detectable in almost all cases, and in almost all tumor cells, of non-keratinizing nasopharyngeal carcinoma.Activating the latent virus, which will result in its lytic replication and the death of tumor cells, is a potential approach for the treatment of EBV-associated cancers. In this study, three recombinant adenoviruses were constructed to express the Zebra gene, an EBV gene responsible for switching from the latent state to lytic replication. EBV-specific promoters were used in order to limit Zebra expression in EBV-positive cells, and reduce the potential side effects. The EBV promoters used were Cp, Zp and a dual promoter combining both promoters, CpZp. The Zebra protein was detected in HEK293 cells as well as the EBV-positive D98-HR1 cells infected with recombinant viruses. An EBV lytic replication early antigen, EA-D, was also detected in infected D98-HR1, implying the initiation of lytic replication. In the cell viability assay, Zebra-expressing adenoviruses had little effect on EBV-negative HeLa cells, while significantly reducing the cell viability and proliferation of D98-HR1 cells. The results indicate that EBV virus promoters can be used in adenovirus vectors to express the Zebra gene and induce EBV lytic replication in D98-HR1 cells.     

Regulation of survivin and CDK4 by Epstein-Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cell lines

Latent membrane protein 1 （LMP1）, an important protein encoded by Epstein Barr virus （EBV）, has been implied to link with the pathogenesis of nasopharyngeal carcinoma （NPC）. Its dual effects of increasing cell proliferation and inhibiting cell apoptosis have been confirmed. In this study, we showed that the expression of Survivin and CDK4 protein in CNE-LMP1, a LMP1 positive NPC epithelial cell line, is higher than in LMP1 negative NPC epithelial cell line- CNE1, and the expression is LMP1 dosage-dependent. Although it was reported that Survivin specifically expressed in cell cycle G2/M phase, our studies suggested that LMP1 could promote the expression of Survivin in G0/G1, S and G2/ M phase. It also showed that Survivin and CDK4 could be accumulated more in the nuclei triggered by LMP1. More interestingly, Survivin and CDK4 could form a protein complex in the nuclei of CNE-LMP1 rather than in that of CNE1, which demonstrated that the interaction between these two proteins could be promoted by LMPI. These results strongly suggested that the role of LMP1 in the regulation of Survivin and CDK4 may also shed some light on the mechanism research of LMP1 in NPC.     

Relationship of HLA-A, -Cw Polymorphisms with HIV/AIDS in Chinese Yi Ethnic Group of Sichuan Province1

The relationship of HLA-A, -Cw alleles on HIV infection and AIDS disease progression in the Chinese Yi ethnic group of Sichuan province were investigated. The genetic polymorphisms of HLA-A, -Cw alleles of 102 unrelated healthy Chinese Yi ethnic individuals, 68 HIV-1 infected and 21 HIV positive long-time survivors were typed by PCR-SSP assay. Statistic signifiance was determined by the χ^2 test with the SPSS software. No significant differences were observed between the HLA-A, -Cw alleles of the 68 HIV-1 infected and 102 non-infected Chinese Yi control individuals. Whereas the prevalence of A＊3601,Cw＊14（01-03）and Cw＊0304 was significantly higher in 21 long time survivors compared with 102 healthy controls with P values of 0.016, 0.016 and 0.000 by χ^2 or the Fisher exact test respectively. The result implies that A＊3601,Cw＊14（01-03） and Cw＊0304 may be associated with slow AIDS disease progression in the Chinese Yi ethnic group, further studies on this association may yield insight on the pathogenesis of HIV-1 infection.     

A preliminary analysis of antineoplastic activity of parvovirus MVMp NS—1 proteins

Human gastric cancer MKN-45 cells were transfected with pULB 3238,a plasmid carrying MVMp MS-1 gene with its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR.After the integration and expression of NS-1 gene,some of the transfectants died,while others remained alive,but the growth features of survived cells were changed.For further study on the antineoplastic function of parvoviral NS-1 protein in vivo,transgenic mice carrying NS-1 genes were established by conventional method.Among 4 founders,one of them was found to be able to transmit the transgene to around 50% of their offsprings.RT-PCR was performed to indicate the expression of NS-1 gene in transgenic mice and its mRNA appeared in a variety of tissues.The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.     

Active expression of Gγ globin gene on chromosome 11 with Yunnanese (Ayγδβ)~0-thalasseinia deletion in MEL cells

A permanent lymphocyte cell line of a heterozygote with Yunnanese (Aγδβ)0-thalassemia deletion, associated with an increased production of Cry globin in adult, was founded using Epstein-Barr virus transformation. The hybrids of the lymphocyte cell and mouse erythroleukemia cell (MEL) were achieved and the hybrids containing human chromosome 11 were selected with the monoclonal antibody 53/6. The subclones containing only either the normal or the abnormal human chromosome 11 were separated and the expression of the human globin genes was studied. Expression of the β-globin gene, but not the Cγ and Aγ, was observed in the hybrids containing only the normal human chromosome 11, while active expression of the Cγ globin gene was observed in the hybrids containing only the abnormal human chromosome 11. These results have confirmed that the DNA deletion in the β-globin gene cluster is the cause of persistent active expression of the Cγ globin gene in the Yunnanese mutant.     

The complete genome of Enterovirus 71 China strain

Five overlapping clones covering the full genome of Enterovirus 71 China strain SHZH98 were obtained and then the sequences were determined by the chain termination method. It showed that the full length of EV71 SHZH98 genome (not including Poly A tail) is 7408 bp. There are some diversities on the lengths and sequences of 5' UTR and 3' UTR between SHZH98 and the other EV71 strains. In P1 capsid region, which is closely associated with viral immunogenicity, EV71 strain SHZH98 shares the highest homology with Taiwan strains; but in P2 and P3 non-structural gene regions there are higher identities with Coxsakievirus A16 and EV71 strains MS, BrCr than with Taiwan strains. Phylogenetic tree constructed by structural gene region indicates that China strain SHZH98 has a closer relationship with Taiwan strains, however, in the non-coding region it has a closer relationship with Coxsakievirus A16, EV71 strains MS and BrCr. EV71 China strain was analyzed at the molecular level. The results will contribute to the     

Selective reversal of drug resistance in drug-resistant lung adenocarcinoma cells by tumor-specific expression of MDR1 ribozyme gene mediated by retrovirus

According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDR1 ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzed in vitro and in vivo ; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDR1 ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effe     

Cotton Leaf Curl Virus： Ionic Status of Leaves and Symptom Development

In the present study, the relationship between the nutritional status of leaves and the development of symptoms of cotton leaf curl virus （CLCuV） in two cotton （Gossypium hirsutum L.） cuItlvars （I.e. CIM-240 and S-12） was Investigated. The incidence of disease attack was found to be 100% In the S-12 cuItlvar and 16% in the CIM-240 cuItivar. Geminivirus particles in infected leaves were confirmed by transmission electron microscope examination of highly specific geminivirus coat protein antlsera-treated cell sap. The CLCuV Impaired the accumulation of different nutrients in both cuItivars. A marked decrease in the accumulation of Ca^2＋ and K^＋ was observed in infected leaves. However, the disease had no effect on leaf concentrations of Na^＋, N, and P. It was observed that the curling of leaf margins in CLCuV-Infected plants was associated with the leaf Ca^2＋ content; leaf curling was severe in plants with a significant reduction In Ca^2＋ content. Moreover, leaf K＆＋ content was found to be associated with resistance/susceptibility to CLCuV infection.     

Effects of garlic oil on tumoragenecity and intercellular communication in human gastric cancer cell line

Previous studies have demonstrated that garlic oil (GO) and its anti-tumor compound could inhibit DNA and RNA synthesis in human cancer cells. In order to explore the effects of garlic oil on carcinoma cells, a gastric carcinoma cell line, BGC-823 was studied at cellular and molecular levels after garlic oil treatment. Data showed that the cell differentiation and suppression of tumorigenicity were significantly induced in tumor cells after garlic oil treatment. There was a correlation between the cell-cell communication recovery and the increase of p53 and waf1/p21 gene expression in garlic oil-treated cells. This result suggested that tumor suppressor gene waf1/p21 and wt p53 might play an important role in this effect.     

In Vitro transformation of LW13 Rat liver epithelial Cells

A rat liver epithelial cell line designated LW 13 was established using a sequential sedimentation method.The cell line retained many normal proerties of liver epithelial cells and showed some structural and functional features resembling those of liver parenchymal cells,LW13 cells became malignant after the intrduction of exogenous transforming EJ Ha ras gene,Tumors produced by inoculation of the transformed cells into baby rats contained areas of poorly differentialted hepatocellular carcinoma,In situ hybridization analysis confirmed the random rather than specific integration of exogenous ras gene into host chromosomes.Furthermore,an at least tenfold increase in the expression of the endogenous c mys gene was detected among transformed cell lines,suggesting the involvement of the c myc proto oncogene in the in vitro transformation of rat liver epithelial cells by EJ Ha ras oncogene.     

Enhanced antitumor effects of tumor antigen-pulsed dendritic cells by their transfection with GM-CSF gene

To investigate the biological characterization and antitumor activitites of GM-CSF gene-transfected dendritic cells, the splenic dendritic cells were infected with GM-CSF recombinant replication-deficient adenoviruses in vitro . Their enhanced expression of B7 was demonstrated by FACS analysis, and more potent stimulatory activity was confirmed by allogeneic MLR. Immunization of dendritic cells pulsed with irradiated B16 melanoma cells induced sig-nificant CTL and enabled host to resist the challenge of wild-type B16 cells. When they were transfected with GM-CSF gene subsequently, the induced CTL activity was higher, and the produced protection against B16 cell challenge and therapeutic effect on the mice with preestablished pulmonary melastases more effective. These data suggest that the dendritic cells pulsed with tumor antigen then transfected with GM-CSF gene can be used as an effective vaccine in tumor immunotherapy.     

Regulated genes in mesenchymal stem cells and gastric cancer

AIM: To investigate the genes regulated in mesenchymal stem cells(MSCs) and diffuse-type gastric cancer(GC),gene expression was analyzed. METHODS: Gene expression of MSCs and diffuse-type GC cells were analyzed by microarray. Genes related to stem cells, cancer and the epithelial-mesenchymal transition(EMT) were extracted from human gene lists using Gene Ontology and reference information. Gene panels were generated, and messenger RNA gene expression in MSCs and diffuse-type GC cells was analyzed. Cluster analysis was performed using the NCSS software.RESULTS: The gene expression of regulator of G-protein signaling 1(RGS1) was up-regulated in diffuse-type GC cells compared with MSCs. A panel of stem-cell related genes and genes involved in cancer or the EMT were examined. Stem-cell related genes, such as growth arrest-specific 6, musashi RNA-binding protein 2 and hairy and enhancer of split 1(Drosophila), NOTCH family genes and Notch ligands, such as delta-like 1(Drosophila) and Jagged 2, were regulated.CONCLUSION: Expression of RGS1 is up-regulated, and genes related to stem cells and NOTCH signaling are altered in diffuse-type GC compared with MSCs.