Identification Of Differentially Expressed Genes In EBV- Transformed Lymphoblastes

Objective: To detect and analyse differentially expressed genes of the host cell genome between normal human lymphocytes and EBV-transformed lymphoblastes in vitro using cDNA microarray techniques, to screen important candidate genes related to the lymphocytic transformation of EBV, and to investigate the mechanism of EBV induced lymphoma.Method: Human lymphocytes were separated from fresh peripheral blood of seven health donors and each example of lymphocytes were divided into two groups for storing as normal samples (Normal) and transforming lymphoblastes (Transform) by EBV in vitro respectively. Genes of two groups of samples were detected by 4×44K Agilent whole Genome Microarray. Then obviously different genes were analyzed by comparing the host cell genome bwteen normal lymphocytes and EBV-transformed lymphoblastes respectively at transcriptional level of whole human genome, with fold change≥2 as obvious up-regulated genes and fold change≤0.5 as obvious down-regulated genes. Real-time PCR was used to confirm the results of gene chip.Result:1. EBV-transformed lymphoblastes could been immortalized. In experiment, EBV-transformed lymphoblastes in vitro were confirmed by microscope and serial subcultivation forever. Using LIMMA analysis for high-thoroughly microarray data, differential gene expression profiles of the host cell genome were obtained between seven homologous normal lymphocytes (Norms) and EBV-transformed lymphoblastes (Trans ). A test (p<0.0001) estimated that 2916 probesets measured noticeably changed expression between Norms and Trans, including 1328 up-regulated genes and 1007 down-regulated genes. Moreover, there are 581 probes can't map to genes in HUGO, which include 65 EST. By unsupervised hierarchical clustering and analysis, differential gene expression profiles clearly distinguished normal lymphocytes from EBV-transformed lymphoblastes.2. The result of systematically bioinformatic analysis of the differential expresssed genes files.Gene Ontology classfication analysis had shown, there were 958 up-regulated genes participated in 272 BP categories, in which"M phase","cell cycle process","cell cycle phase","cell cycle","mitotic cell cycle","M phase of mitotic cell","mitosis","nuclear division"and"organelle fission"had the lowest EASE score, all < 5.1E-39. 745 down-regulated genes participated in 403 GO BP categories, in which"immune response","defense response","cell activation","response to wounding","leukocyte activation","positive regulation of biological process","inflammatory response","regulation of cell activation","regulation of cell proliferatio"and"regulation of leukocyte activation"had the lowest EASE Score, all <1.7E-9.There were 870 up-regulated genes participated in 82 GO MF categories, which were mainly related to gene and protein binding activity, such as"adenyl nucleotide binding","ATP binding","nucleoside binding","purine nucleoside binding"and"nucleotide binding", had the lowest EASE Score, all <2.6E-12. There were 707 up-regulated genes participated in 70 GO MF categories, including 20 categories had EASE Score <0.001.The pathway analysis had shown, through the database of"KEGG Pathway","BioCarta"and"Reactome", in up-regulated genes, there were 404 genes participated in 25"KEGG Pathway", 119 genes participated in 7"BioCarta-pathway", and 345 genes participated in 12"Reactome-pathway". In down-regulated genes, there were 333 genes participated in 12"KEGG-pathway", 151 genes participated in 15"BioCarta-pathway"and 190 genes participated in 4"Reactome-pathway". All these pathways have the EASE Score <0.05.The result of DAVID"Functional Annotation Clustering"analysis had shown, there were 73 pathway and GO classes enriched for up-regulated genes, which had the highest Enrichment Score classes described aspects of cell cycle and cell division; there were 71 pathway and GO classes enriched for down-regulated genes, which had the highest Enrichment Score classes described aspects of cell apoptosis.There were 100 important candidated genes had been selected, by combinating STRING,GENERANK,PATHWAY,GO categorization, as well as ranking the 2916 differentially expressed probes. There were 50 up-regulated genes and 50 down-regulated genes respectively. Comprehensively analysised and forecasted the biological fuction of these 100 genes, the result indicated that: up-regulated genes , scuh as IGF1, GOT2, GOT1, AURKB, CDK2, AURKA, BRCA1, PLK1, KIF20A, KIF18B, maily participated in"cell cycly","mitosis","protein","protein biosynthesis","chromosome segregation"and so on; up-regulated genes E2F1, PTPN11, PIK3R3, PLCG2, down-regulated genes FOS, PXN, PRKCQ, FYN, ZAP70, PROK2, FCN1, HCK and so on, were related to tumor associated pathways, such as"P53 pathway","Angiogenesis","VEGF signaling pathway","FGF signaling pathway","IGF pathway-protein kinase B signaling cascade"and so on; down-regulated genes CD3G, CD3D, CD247, S100A8, CD4, GZMA, ITK, CLU, GZMH, GAMKand so on participated in cell apoptosis, immunological regulation, stress and so on, such as"apotosis","T/B cell activation","immune system process","response to stimulus"and so on . In which, there were some genes, like E2F1, PTPN11, FOS, IGF1, CDK2, had took participated in multiple biological behaviour.3. The result of systematical analysis and prediction of signal pathway and gene biological categorization had shown, EBV can up-regulate E2F1, PLK1, BIRC5 and so on to regulate cell cycle and cell apoptosis; EBV can up-regulate PTPN11 to promote tumor angiogenesis and differentiation; EBV can down-regulate FYN to degrade host immune function.4. Real time PCR revealed that when compared with normal lymphocytes and corrected by internal control gene, the transcriptional expression of E2F1, PTPN11, PLK1, BIRC5 incresed in EBV-transformed lymphoblastes, while transcriptional expression of FYN decreased. Conclusion:1. We have fist construct differential gene expression profiles of the host cell genome between normal lymphocytes and EBV-transformed lymphoblastes, and we have first indicated there are obviously difference in genes expressed between normal lymphocytes and EBV-transformed lymphoblastes.2. We indicated that the process of EBV induced lymphocytes transformed refer to multiple genes and pathway participated, and including the interacitve of virus genome and host genome. We presume that EBV mainly through promote cell cycle and inhibit host immune function to induced lymphocytes transforemed.3. We suppose that E2F1, PLK1, PTPN11, BIRC5, FYN are the target molecule in EBV induced lymphocytes transformation, in which E2F1 may have a central action in the regulation of cell cycel, cell apoptosis and humal telomerase activity.