The accumulation of high-throughput gene and protein expression profile data has greatly promoted the application of computer science and technology in functional gene studies. However, complex biological functions are not simply controlled by a single gene, as certain biological processes are realized by the cooperation of a series of genes. In this study, normal peripheral blood lymphocytes (PBLs) and coupled EBV-transformed lymphoblastoid cell lines (LCLs) were analyzed using Agilent human whole genome microarrays, in which 1,745 differentially expressed genes were screened. Our results indicated that EBV-induced human lymphocyte transformation was a process involving multiple genes and pathways in virus-host interactions. Hierarchical clustering analysis of the 1,745 differentially expressed genes showed that they were well distinguished between normal PBLs and LCLs, as there were significant differences between the expression patterns of the two types of host cells. There were 88 differentially expressed genes with ≥ 10 joint-edges, accounted for 81% of the total jonit-edges, which suggested that these genes may play an important role in the process of EBV transformation of B lymphocytes.
Cell cycle and mitosis promoted differentially expressed genes
Viral genes can induce changes in the cell cycle of the host cell during the immortalization of B lymphocytes by EBV . EBV latent proteins can up-regulate cyclin D1 expression in host cell, initiate the cell cycle and promote the G1-S phase transition through several signaling pathways [9, 10]. EBV can also induce the up-regulation of CDK2 and CDK1 in the host cell . CDK2 not only acted on the boundary of G1/S phase, but also had a key regulatory effect on S phase progression and DNA synthesis. CDK1 could promote the end of S phase and start G2 phase. The cyclin B1/CDK1 complex triggered G2/M phase transition and promoted mitosis to enter into anaphase. The activation of CDK2 was dependent on the regulation of transcription factor E2F. Research has shown that EBNA-6 and EBV transcription activation factor Zta and Rta could induce up-regulation of E2F1 in the host cell [12, 13]. Annotation of differentially expressed genes and signal pathway analyses showed that up-regulated genes (CDC6, RPA3, etc.) participated in the initiation of DNA replication and DNA recombination. Therefore, we presumed that EBV might promote the progression of the cell cycle, induce B lymphocyte transformation and obtain infinite proliferation ability through up-regulating of certain cell cycle related genes.
Lacoste et al (2010) found that chromosome rearrangement might occur in subcultured early phase EBV-LCLs, mainly including chromosome deletion, chromosome fragments, dicentric chromosomes and unbalanced chromosome translocation, and aneuploidy variation may occur during the 12th week post-infection , suggesting that EBV can induce unstable variation of the host cell genome. Gruhne et al (2009) confirmed that EBNA-1 induced DNA damage, LMP-1 inhibited DNA repair and apoptosis regulation, and EBNA-3 C down-regulated BubR1 transcription resulting host cell released CDC20 and then passived spindle checkpiont . Pan et al (2009) showed that the cell spindle checkpoint could be disturbed by EBNA2 through down-regulation of MAD2 and up-regulation of PLK1, resulting in unstable chromosome variation . Up-regulation of several spindle checkpoint related genes were observed in this study, including PLK1, AURKA, AURKB, NUP37, CENPA, BUB1B, CDCA8 and others. Considering that cell aneuploidy variation was mostly induced by disruption in the spindle checkpoint , we presumed that EBV might up-regulate the expression of a series of spindle checkpoint related genes, thus inducing change in the stability of the genome, as well as inducing B lymphocyte cell transformation and abnormal proliferation.
Cell apoptosis inhibited differentially expressed genes
Abnormal cell proliferation has a close relation with disordered apoptosis regulation. Ng Siok-Bian et al  reported that LMP1 could activate NF-κB and MYC to mediate the high expression of the BIRC5 (Survivin), an inhibitor of apoptosis protein, in extranodal nasal-type NK/T cell lymphoma cells. However, LU et al  proposed that EBNA1 formed a complex with Sp1 or Sp1-like protein, and the cis-element of the complex combined with the BIRC5 promoter induced the up-regulation of BIRC5. In addition, Ando et al  reported that there existed a sequence-specific PLK1 binding region in P53, where PLK1 inhibited the transcription activity and apoptosis regulation ability of p53 by binding to this region. The high expression of BIRC5 and PLK1 in LCLs in this study suggested that EBV can inhibit B cell apoptosis and impel cell immortalization through multiple ways.
Host immune function hindered differentially expressed genes
Among the EBV-induced down-regulated genes, several were involved in T/B cell activation of the host, suggesting that EBV regulated changes in immune function of the host during transformation of B cells. A microarray experiment conducted by Sengupta et al  confirmed that EBV could limit the expression of MHC-I chain-related genes in nasopharyngeal carcinoma cells. Rovedo et al  showed that LMP2A can bind to the SH-2 functional domain of FYN and block normal BCR signal transduction. The binding of FYN specific sequences and CD3 is required for T cell activation . Gene function annotation showed that down-regulated genes, such as CD3D, CD4, CD3G, ZAP70, LCP2, ITK, etc., were involved in immunocytes activation and immune-related signal pathways, suggesting that EBV may inhibit the expression of these genes to hinder the normal immune surveillance and clearance of the host, which may allow virus-infected cells to evade immune surveillance, and ultimately induce the transformation or even malignancy of EBV-infected cells. Our previous experiments have confirmed that human-derived B-cell lymphoma could be induced in SCID mice transplanted with EBV seropositive donor’s lymphocytes [24, 25], which supported the hypothesis that EBV-induced lymphomas were related to immune suppression or deficiency.
EBV BZLF1 is the switch for EBV latent state into lytic state. The expression of the BZLF1 gene is initiated from the promoter Zp, which is normally suppressed in EBV-transformed B cell. Liang et al  demonstrated BZLF1 gene can be activated by TGF-β through the cooperation of Smad3/Smad4 and c-Jun/c-Fos that formed a complex. The proto-oncogene product c-Fos is a component of the transcription factor AP-1. Mao et al  demonstrated fos protein was rarely expressed in the primary cutaneous B-cell lymphoma (PCBCL), and the result of KEGG pathway analysis showed that fos participate in B/T cell receptor signalling pathway. In this study, we hypothesized EBV may down-regulated fos to maintain its latent infection and to evade host immune surveillance and ultimately lead to the transformation of B lymphocytes.
It is noteworthy that other studies had showed that EBV could change cytokine secretion of the host cell, thereby inhibiting normal immune function, and was also an important regulatory factor for the transformation or malignant change of the B lymphocytes induced by EBV. Ehlin-Henriksson et al  indicated that EBNA2 and LMP1 can down-regulate the expression of CXCR4 in B-cell lymphoma cells. Chen et al  confirmed that EBNA-3B inhibited the expression of CXCR4 in EBV-infected B cells, thus affecting the B-cell homing and disturbing the normal immune barrier of the host. Down-regulation of cytokine-related genes-CXCR4 and CXCL1 in LCLs were also observed in this study. Therefore, we presumed that the change in expression of cytokine-related genes induced by EBV was also a regulatory factor in hindering host immune function and transformation of B lymphocytes.
In summary, EBV-induced B lymphocyte transformation was a complicated process, which involved many genes and pathways changes between virus and host interaction. Global gene expression profile analysis showed that there existed significant differences in the gene expression patterns between LCLs and normal host lymphocytes, and EBV may induce the transformation of human B lymphocytes by promoting cell cycle and mitosis, inhibiting cell apoptosis, regulating cytokine secretion, and hindering normal immune function. Our present results filtered out 38 key regulatory genes. The function and specific effect in EBV-transformed lymphocytes of the these key regulatory genes screened from EBV-transformed lymphoblastoid cell lines (LCLs) need to be further studied. This study provided important information on the molecular mechanism of lymphocyte transformation by EBV, and laid a foundation for subsequent gene validation and functional studies.