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      Screening and functional analysis of differentially expressed genes in EBV-transformed lymphoblasts

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          Abstract

          Background

          Epstain-Barr virus (EBV) can transform human B lymphocytes making them immortalized and inducing tumorigenic ability in vitro, but the molecular mechanisms remain unclear. The aim of the present study is to detect and analyze differentially expressed genes in two types of host cells, normal human lymphocytes and coupled EBV-transformed lymphoblasts in vitro using gene chips, and to screen the key regulatory genes of lymphocyte transformation induced by EB virus.

          Methods

          Fresh peripheral blood samples from seven healthy donors were collected. EBV was used to transform lymphocytes in vitro. Total RNA was extracted from 7 cases of the normal lymphocytes and transformed lymphoblasts respectively, marked with dihydroxyfluorane after reverse transcription, then hybridized with 4 × 44 K Agilent human whole genome microarray. LIMMA, String, Cytoscape and other softwares were used to screen and analyze differentially expressed genes. Real-time PCR was applied to verify the result of gene expression microarrays.

          Results

          There were 1745 differentially expressed genes that had been screened, including 917 up-regulated genes and 828 down-regulated genes. According to the results of Generank, String and Cytoscape analyses, 38 genes may be key controlled genes related to EBV-transformed lymphocytes, including 22 up-regulated genes(PLK1, E2F1, AURKB, CDK2, PLCG2, CD80, PIK3R3, CDC20, CDC6, AURKA, CENPA, BUB1B, NUP37, MAD2L1, BIRC5, CDC25A, CCNB1, RPA3, HJURP, KIF2C, CDK1, CDCA8) and 16 down-regulated genes(FYN, CD3D, CD4, CD3G, ZAP70, FOS, HCK, CD247, PRKCQ, ITK, LCP2, CXCL1, CD8A, ITGB5, VAV3, CXCR4), which primarily control biological processes such as cell cycle, mitosis, cytokine-cytokine pathway, immunity response and so on.

          Conclusions

          Human lymphocyte transformation induced by EB virus is a complicated process, involving multiple-genes and –pathways in virus-host interactions. Global gene expression profile analysis showed that EBV may transform human B lymphocytes by promoting cell cycle and mitosis, inhibiting cell apoptosis, hindering host immune function and secretion of cytokines.

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          Most cited references27

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          Genomic analysis of regulatory network dynamics reveals large topological changes.

          Network analysis has been applied widely, providing a unifying language to describe disparate systems ranging from social interactions to power grids. It has recently been used in molecular biology, but so far the resulting networks have only been analysed statically. Here we present the dynamics of a biological network on a genomic scale, by integrating transcriptional regulatory information and gene-expression data for multiple conditions in Saccharomyces cerevisiae. We develop an approach for the statistical analysis of network dynamics, called SANDY, combining well-known global topological measures, local motifs and newly derived statistics. We uncover large changes in underlying network architecture that are unexpected given current viewpoints and random simulations. In response to diverse stimuli, transcription factors alter their interactions to varying degrees, thereby rewiring the network. A few transcription factors serve as permanent hubs, but most act transiently only during certain conditions. By studying sub-network structures, we show that environmental responses facilitate fast signal propagation (for example, with short regulatory cascades), whereas the cell cycle and sporulation direct temporal progression through multiple stages (for example, with highly inter-connected transcription factors). Indeed, to drive the latter processes forward, phase-specific transcription factors inter-regulate serially, and ubiquitously active transcription factors layer above them in a two-tiered hierarchy. We anticipate that many of the concepts presented here--particularly the large-scale topological changes and hub transience--will apply to other biological networks, including complex sub-systems in higher eukaryotes.
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            Genome-wide expression profiling reveals EBV-associated inhibition of MHC class I expression in nasopharyngeal carcinoma.

            To identify the molecular mechanisms by which EBV-associated epithelial cancers are maintained, we measured the expression of essentially all human genes and all latent EBV genes in a collection of 31 laser-captured, microdissected nasopharyngeal carcinoma (NPC) tissue samples and 10 normal nasopharyngeal tissues. Global gene expression profiles clearly distinguished tumors from normal healthy epithelium. Expression levels of six viral genes (EBNA1, EBNA2, EBNA3A, EBNA3B, LMP1, and LMP2A) were correlated among themselves and strongly inversely correlated with the expression of a large subset of host genes. Among the human genes whose inhibition was most strongly correlated with increased EBV gene expression were multiple MHC class I HLA genes involved in regulating immune response via antigen presentation. The association between EBV gene expression and inhibition of MHC class I HLA expression implies that antigen display is either directly inhibited by EBV, facilitating immune evasion by tumor cells, and/or that tumor cells with inhibited presentation are selected for their ability to sustain higher levels of EBV to take maximum advantage of EBV oncogene-mediated tumor-promoting actions. Our data clearly reflect such tumor promotion, showing that deregulation of key proteins involved in apoptosis (BCL2-related protein A1 and Fas apoptotic inhibitory molecule), cell cycle checkpoints (AKIP, SCYL1, and NIN), and metastasis (matrix metalloproteinase 1) is closely correlated with the levels of EBV gene expression in NPC.
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              Polo-like kinase 1 (Plk1) inhibits p53 function by physical interaction and phosphorylation.

              Polo-like kinase 1 (Plk1) has an important role in the regulation of M phase of the cell cycle. In addition to its cell cycle-regulatory function, Plk1 has a potential role in tumorigenesis. Here we found for the first time that Plk1 physically binds to the tumor suppressor p53 in mammalian cultured cells, and inhibits its transactivation activity as well as its pro-apoptotic function. During the cisplatin-induced apoptosis in human neuroblastoma SH-SY5Y cells, the expression level of Plk1 was significantly decreased both at mRNA and protein levels, whereas cisplatin treatment caused a remarkable stabilization of p53. Systematic immunoprecipitation analyses using a series of deletion mutants of p53 revealed that a sequence-specific DNA-binding region of p53 is required and sufficient for the physical interaction with Plk1. The ectopically overexpressed Plk1 was co-localized with the endogenous p53 in mammalian cell nucleus, as shown by confocal laser microscopy. Expression of exogenous Plk1 and p53 in p53-deficient lung carcinoma H1299 cells greatly decreased the p53-mediated transcription from the p53-responsive p21(WAF1), MDM2, and BAX promoters, whereas the kinase-deficient mutant form of Plk1 failed to reduce the transcriptional activity of p53. Consistent with the luciferase reporter analysis, Plk1 had an ability to block the p53-dependent induction of the endogenous p21(WAF1). In addition, Plk1 inhibited the pro-apoptotic function of p53 in H1299 cells. Intriguingly, Plk1-mediated repression of p53 was attenuated with ATM. Thus, our present findings strongly suggest that p53 is a critical target of Plk1, and its function is abrogated through the physical interaction with Plk1.
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                Author and article information

                Journal
                Virol J
                Virol. J
                Virology Journal
                BioMed Central
                1743-422X
                2012
                30 March 2012
                : 9
                : 77
                Affiliations
                [1 ]Cancer Research Institute, University of South China, Hunan, 421001, People’s Republic of China
                [2 ]Clinical Laboratory, The Third People Hospital of Kunshan City, Jiangsu, 215300, People’s Republic of China
                [3 ]Shanghai Center for Bioinformation Technology (SCBIT), Shanghai, 200235, People’s Republic of China
                [4 ]Pathogenic Biology Institute, University of South China, Hunan, 421001, People’s Republic of China
                [5 ]Cancer Research Institute, University of South China, School of Medicine, Chang Sheng Xi Avenue 28, Hengyang City, Hunan, 421001, People’s Republic of China
                [6 ]Pathogenic Biology Institute, University of South China, Hengyang, 421001, China
                Article
                1743-422X-9-77
                10.1186/1743-422X-9-77
                3433351
                22458412
                7ae79d42-fc86-45b8-8b0b-dec4c35325be
                Copyright ©2012 Dai et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 21 November 2011
                : 20 March 2012
                Categories
                Research

                Microbiology & Virology
                gene expression,gene chip,lymphocyte transformation,lymphoblastoid cell line (lcl),epstein-barr virus (ebv)

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