Functions And Mechanisms Of Epstein-Barr Nuclear Antigen 1 In Human Nasopharyngeal Carcinoma Metastasis

EB virus (EBV) was paid more attention to its association with the pathogenesis of nasopharyngeal carcinoma (NPC). Especially, the undifferentiated type of NPC shows the most consistent worldwide association with EBV. Therefore, exploring the functions of EBV not only help to clarify the molecular mechanism of NPC, but also provide new markers for clinical diagnosis and treatment.In NPC, EBV adopts a type II latency gene expression program EB nuclear antigen 1 (EBNA1), latent membrane protein-1 (LMP-1), latent membrane protein-2 (LMP-2) and EBV encoded RNAs (EBERs) are the only genes detected. EBNA1 is expressed in all virus-infected cells, in which its role in the maintenance and replication of the episomal EBV genome is achieved through sequence-specific binding to the plasmid origin of viral replication, OriP. EBNA1 can also interact with certain viral promoters, thereby contributing to the transcriptional regulation of the EBNAs (including EBNA1 itself) and of LMP1. In our another study, we unexpectedly found that after EBNA1 was overexpressed, the NPC cells underwent morphological changes, from typical cobblestone-like shape to the spindle or similar to the morphology of fibroblasts. We hypothesized that this morphological change may be caused by epithelial-mesenchymal transformation (EMT). However, existing studies have not reported a direct effect of EBNA1 on NPC cells yet.The initial phase of tumor cell evasion from well-structured assemblies requires a phenotypical conversion which is referred to as epithelial to mesenchymal (fibroblastoid) transition (EMT). At this stage cells must be able to detach from the junctions that connect them to the neighboring ones, change their shape and polarity, delaminate and migrate. This process is reminiscent of early stages of embryonic development but has also been described to be crucial in transient pathological situations such as wound healing, inflammation and neural fibrosis. Accumulating evidence suggests a critical role in cancer progression, through which tissue epithelial cancers invade and metastasis.Base on our surprised findings, we want to know whether EBNA1 is correlated with EMT in NPC or not? What is its mechanism and role of EBNA1 in the invasion and metastasis of NPC? We carried out a series of studies to solve these questions.MATERIALS & METHODS1. The expression characteristics of EBNA1 in NPC tissues:Forty-eight NPC and 12 chornic nasopharyngitis paraffin-embedded specimens from Department of Otolaryngology of Nanfang Hospital of Southern Medical University (Guangdong, China) were used for immunohistochemical analysis of EBNA1.2. The effects of EBNA1 on the biological behaviors of NPC cells2.1 The establishments of NPC cell lines with stable EBNA1 over-expression or knock-downStable EBNA1-expressing CNE1, CNE2 and 5-8F cell lines were established by transfection with the pCEP4 vector (Invitrogen, Carlsbad, CA) which contained EBNA1 coding sequence and a Hygromicine B selection marker. Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Forty-eight hours post transfection, hygromycin B was added at a final concentration of 150μg/ml. After 2-week selection, positive clones CNE1/EBNA1, CNE2/EBNA1 and 5-8F/EBNA1 were picked. The expression level of EBNA1 was detected by quantitive real-time PCR and western blotting.A 68-bp SalⅠ-XbaⅠfragment containing shRNA EBNA1 (shEBNAl) sequence was cloned into the pAVU6+27 shuttle plasmid to create pAVU6+27/shEBNA1. Then U6 promoter and shEBNA1 sequence were excised from pAVU6+27/shEBNA1 plasmid and cloned into the EcoRV/Xho I sites of the pBluescript SK+(BSSK, generous gift of Dr. He) shuttle plasmid to create BSSK/shEBNA1. U6 promoter and shEBNAl fragment was amplified by polymerase chain reaction (PCR) and inserted into the ClaⅠ/MluⅠsites of pLVTHM plasmid (generous gift of Dr. Xiao) to create pLVTHM/shEBNAl.Replication incompetent lentivirus was produced by cotransfection of the pLVTHM/GFP expression vector or pLVTHM/shEBNA1 recombinant plasmid and an optimized mixture of two packaging plasmids:psPAX2 and pMD2.G (generous gift of Dr. Xiao) into 293T cells using Lipofectamine 2000. The efficiency of transfection was monitored by fluorescent microscope. Viral supernatant and polybrene were added to CNE1/EBNA1 and CNE2/EBNA1 cells. After 72h transduction, GFP positive cells were observed by fluorescent microscope. They were named as CNE1/EBNA1/Control, CNE1/EBNA1/shEBNA1, CNE2/EBNA1/Control and CNE2/EBNA1/shEBNAl cell. The expression of EBNA1 in these cells were detected by quantitive real-time PCR and western blotting.2.2 The effects of EBNA1 over-expression and knock-down on the biological behaviors of NPC cellsThe effects of EBNA1 over-expression and knock-down on cell migration and invasion were assessed by wound-healing assays and matrigel invasion assays. The effects of EBNA1 over-expression and knock-down on cell proliferation in a lower concentration were assessed by plate colony formation assays.3. EBNA1 overexpression induced EMT in NPC cells3.1 The expression characteristics of CK18 and Vimentin in NPC tissuesForty-eight NPC and 12 nasopharyngitis paraffin-embedded specimens were used for immunohistochemical analysis of CK18 and Vimentin. The relationship between EBNA1, CK18 and Vimentin were analyzed.3.2 The expression of EMT-related markersThe expression of epithelial markers (E-cadherin and CK18) and mesenchymal markers (N-cadherin, Vimentin andβ-catenin) in CNE1, CNE2,5-8F, CNE1/EBNA1, CNE2/EBNA1 and 5-8F/EBNA1 cells were detected by quantitive real-time PCR, western blotting and immunocytofluorescence assay.4. The signaling pathways involved in EMT induced by EBNAlThe quantitative real-time PCR was applied to detect the expression of EMT-related regulators, including TGF-β1/Smad, Wnt/β-catenin signaling pathway and ZEB1, ZEB2 in stably EBNAl overexpressed NPC cell lines.RESULTS1. EBNA1 was over-expressed in metastatic NPC tissues.The expression of EBNA1 in NPC tissues (42/48) was significantly higher than in chronic nasopharyngitis tissues (0/12) (Z=-4.836, P=0.000). Their expression in metastatic lymph nodes were higher than non-metastatic ones (Z=14.174, P=0.003). Furthermore, the expression of EBNA1 was correlated with N phase (χ2=14.174, P=0.003), T phase (χ2=10.047, P=0.018) and clinical stage (χ2=20.142, P=0.000).2. The effects of EBNAl overexpression or silence on the biological behaviors of NPC cells2.1 The establishment of NPC cell lines with stable EBNA1 overexpression.NPC cell lines CNE1, CNE2 and 5-8F were used to produce EBNA1 clones. The results of quantitive real-time PCR and western blot confirmed the mRNA and protein expression of EBNA1. We found that the expression level of EBNA1 in 5-8F/EBNA1 was higher than that of CNE1/EBNA1 and CNE2/EBNA1 cells.2.2 EBNA1 stimulates the migration, invasion ability of NPC cells and proliferative response in a low concentration in vitroThe results of wound healing assays showed that there was significant difference among three groups after 12h and 24h (CNE1 group:F=68.295, P=0.000; CNE2 group:F=24.242, P=0.000; 5-8F group:F=80.977, P=0.000). The migration of CNE1/EBNA1 cells was enhanced by 3.75-fold and 2.65-fold compared with CNE1 cells after 12h and 24h respectively.The Results of invasion assays showed that after cultivation for 22h, the number of cells attached to the lower surface of the membrane was increased induced by EBNA1 overexpression (CNE1 group:t=-6.506, P=0.000; CNE2 group:t=-7.834, P=0.000; 5-8F group:t=-12.498, P=0.000).The Results of plate colony formation assays showed that CNE1/EBNA1, CNE2/EBNA1 and 5-8F/EBNA1 cells with EBNA1 overexpression exhibited enhanced colony formation abilities by 2.44-fold,2.00-fold and 2.10-fold respectively compared to CNE1, CNE2 and 5-8F cells (CNE1 group:t=-2.848, P=0.046; CNE2 group:^-2.961, P=0.042; 5-8F group:t=-11.015, P=0.000).2.3 Construction of lentiviral vector of RNA interference of EBNA1 gene and its stable expression in CNE1/EBNA1, CNE2/EBNA1 cellsA recombinant lentiviral vector expressing short hairpin RNAs to target EBNA1 gene was successfully established and confirmed by DNA sequencing. Recombinant lentivirus was harvested from 293T cells. Sub-clones of CNE1/EBNA1, CNE2/EBNA1 cells infected with recombinant lentivirus were selected. We found that EBNA1 mRNA and protein expression were dramatically knocked down. 2.4 Knock-down of EBNA1 supresses the migration, invasion ability of NPC cells and proliferative response in a low concentration in vitroThe Results of wound healing assays displayed that CNE1/EBNA1/shEBNAl and CNE2/EBNA1/shEBNAl cells both showed a significantly reduced migration ability compared with CNE1/EBNA1/Control and CNE2/EBNA1/Control cells (CNE1/EBNA1 group:F=8.816, P=0.000; CNE2/EBNA1 group:F=7.258, P=0.002) after 12h and 24h.The Results of invasion assays showed that the number of cells attached to the lower surface of the membrane was decreased after EBNA1 silencing (CNE1/EBNA1 group:t=7.174,P=0.000; CNE2/EBNA1 group:t=9.111, P=0.000).The Results of plate colony formation assays showed that CNE1/EBNA1/shEBNA1 and CNE2/EBNA1/shEBNA1 cells both had a significant reduction in their colony formation abilities as compared with CNE1/EBNA1/Control and CNE2/EBNA1/Control cells (CNE1/EBNA1 group:t=3.656, P=0.022; CNE2/EBNA1 group:t=5.357, P=0.006).3. EBNA1 induced EMT in NPC cells3.1 Morphological changes after over-expression or knock-down of EBNA1After EBNA1 was overexpressed, three NPC cell lines underwent morphological changes, exhibiting a fibroblastoid shape and scattered pattern. On the contrary, knock-down of EBNA1 reversed the morphological changes.3.2. The relationship between EBNA1 and EMT markers.Although the expression of epithelial markers CK18 showed no significant difference between NPC and chronic nasopharyngitis tissues (Z=-0.720, P=0.471), expression of Vimentin was correlated with T phase (χ2=13.985, P=0.003), N phase (χ2=10.349, P=0.016) and clinical stage (χ2=21.080, P=0.000).The expression of Vimentin in NPC tissues (38/48) was significantly higher than in chronic nasopharyngitis tissues (8/12) (Z=-2.428, P=0.015). The expression of Vimentin in metastatic lymph nodes was also higher than non-metastatic ones (Z=-2.228, P=0.026). Furthermore, the expression of Vimentin was correlated with N phase (χ2=13.064, P=0.005) but not T phase (χ2=3.756, P=0.289).There was a negative correlation between EBNA1 and epithelial marker CK18 expression, and a positive correlation was found between EBNA1 and mesenchymal marker Vimentin expression.In vitro, the Results of quantitive real-time PCR, western blotting and immunocytofluorescence assay all displayed that the expression of mesenchymal markers (Vimentin, N-cadherin andβ-catenin) was increased after EBNA1 overexpressed, but EBNA1 overexpression downregulated the expression of epithelial markers (E-cadherin, and CK18), with an opposite trend to the mesenchymal markers.4. The pathways involved in EMT induced by EBNA1:Quantitative PCR assay showed that after EBNA1 was overexpressed, relative mRNA levels of TGF-β1 in three cell lines were increased by 3.75-fold,1.57-fold and 25.49-fold in CNE1, CNE2 and 5-8F cells respectively. Smad4 exprssion was increased by an average of 2.43-fold, and 1.64-fold in CNE1 and 5-8F cells respectively. The expression of Smad4 did not change significantly in CNE2 cells. These data indicated that EBNA1 induced EMT through TGF-β1/Smads pathways.After EBNA1 was overexpressed, relative mRNA level of GSK-3βincreased by an average of 1.58-fold,1.47-fold and 8.70-fold in CNE1, CNE2 and 5-8F cells respectively. C-myc expression was increased by 1.21-fold and 61.98-fold in CNE1 and 5-8F cells respectively. Combined with the results ofβ-catenin and E-cadherin, these data suggested that EBNA1 induced EMT through Wnt/β-catenin pathways. We also found that after EBNA1 was overexpressed, relative mRNA level of transcriptional repressors factor ZEB1 was increased by 2.92-fold,2.83-fold and 381.01-fold in CNE1, CNE2 and 5-8F cells respectively, and ZEB2 expression increased by an average of 3.78-fold,1.83-fold and 65.39-fold respectively. These data showed that EBNA1 was associated with transcription factors regulation.CONCLUSIONS1. The Expression of EBNA1 is associated with T phase, N phase and clinical stage of NPC. A positive correlation was found between EBNA1 and vimentin expression, and a negative correlation was found between EBNA1 and CK18 expression.2. EBNA1 overexpression stimulates the migration, invasion ability of NPC cells as well as cell proliferation in a lower concentration. On the contrary, EBNA1 silencing suppresses migration and metastasis of NPC cells.3. EBNA1 may promote EMT through TGFβ1/Smad and Wnt/β-catenin signaling pathways and transcriptional repressor ZEB1/ZEB2 in NPC cells.