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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2016, Vol. 10 Issue (1) : 61-75     DOI: 10.1007/s11684-016-0436-0
Comprehensive profiling of EBV gene expression in nasopharyngeal carcinoma through paired-end transcriptome sequencing
Lijuan Hu1,2,7,Zhirui Lin1,2,Yanheng Wu3,Juqin Dong1,2,Bo Zhao5,Yanbing Cheng6,Peiyu Huang1,4,Lihua Xu1,8,Tianliang Xia1,2,Dan Xiong1,2,Hongbo Wang1,2,Manzhi Li1,2,Ling Guo1,4,Elliott Kieff5,Yixin Zeng1,2,Qian Zhong1,2,*(),Musheng Zeng1,2,*()
1. State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine and
2. Department of Experimental Research, Sun Yat-sen University Cancer Center, Guangzhou 510060, China;
3. First Affiliated Hospital of Jinan University, Guangzhou 510630, China;
4. Department of Nasopharyngeal Carcinoma, Sun Yat-sen University Cancer Center, Guangzhou 510060, China;
5. Division of Infectious Disease, Brigham and Women’s Hospital and Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA;
6. Beijing Genomics Institute Co., Ltd., Shenzhen 518083, China;
7. Department of Medical Oncology, Affiliated Tumor Hospital of Guangzhou Medical University, Guangzhou 510095, China;
8. Department of Oncology and Hematology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, China
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The latent expression pattern of Epstein-Barr Virus (EBV) genes in nasopharyngeal carcinoma (NPC) has been extensively investigated, and the expression of several lytic genes in NPC has been reported. However, comprehensive information through EBV transcriptome analysis in NPC is limited. We performed paired-end RNA-seq to systematically and comprehensively characterize the expression of EBV genes in NPC tissue and C666-1 NPC cell line, which consistently carries EBV. In addition to the transcripts restricted to type II latency infection, the type III latency EBNA3s genes and a substantial number of lytic genes, such as BZLF1, BRLF1, and BMRF1, were detected through RNA-seq and were further verified in C666-1 cells and NPC tissue through real-time PCR. We also performed clustering analysis to classify NPC patient groups in terms of EBV gene expression, which presented two subtypes of NPC samples. Results revealed interesting patterns of EBV gene expression in NPC patients. This clustering was correlated with many signaling pathways, such as those related to heterotrimeric G-protein signaling, inflammation mediated by chemokine and cytokine signaling, ribosomes, protein metabolism, influenza infection, and ECM-receptor interaction. Our combined findings suggested that the expression of EBV genes in NPC is restricted not only to type II latency genes but also to type III latency and lytic genes. This study provided further insights into the potential role of EBV in the development of NPC.

Keywords Epstein-Barr virus      paired-end transcriptome sequencing      latency genes      lytic genes      nasopharyngeal carcinoma     
Corresponding Authors: Qian Zhong,Musheng Zeng   
Just Accepted Date: 18 February 2016   Online First Date: 16 March 2016    Issue Date: 31 March 2016
URL:     OR
Fig.1  Distribution of reads across the wild-type EBV genome in C666-1 and 12 NPC tissue samples. (A) Explanation of the EBV genome coverage. (B) Distribution of reads across the wild-type EBV genome in C666-1. (C) Distribution of reads across the wild-type EBV genome in NPC49, NPC52, and NPC66. (D) Distribution of reads across the wild-type EBV genome in the rest 9 NPC tissue samples. Coverage refers to the ratio of the area covered by reads to the length of each window, and log2 (reads number) refers to the average sequence depth in each window. 343?nt /window, 500 windows.
Fig.2  RPKM values for EBV genes in C666-1 cells and NPC tissue. (A) Summary of EBV genes in C666-1 cells and NPC tissue. (B) RPKM values for EBV type III latency genes in C666-1 cells and NPC tissue. (C) Comparison of the number of lytic genes in two subgroups. (D) Upper: RPKM values for BZLF1 and BRLF1 in C666-1 cells and NPC tissue. Lower: Venn diagram for lytic genes correlated with BZLF1 and BRLF1. (E) RPKM values for EBV lytic genes (BILF1, BNRF1, BALF3, BALF4, BALF5, LF1, and LF2) in C666-1 cells and NPC tissue. Y axis: RPKM; X axis: EBV transcripts.
Fig.3  Relative expression of latency genes in different cell lines by real-time PCR. EBV latency genes: EBER1, EBER2, EBNA1, EBNA2, LMP1, LMP2A, EBNA3A, EBNA3B, and EBNA3C.
Fig.4  Representative EBV lytic genes with normalized expression levels measured by real-time PCR in cell lines. Representative EBV lytic genes: BGLF2, BCLF1, BDLF2, BHRF1, BGLF5, BGLF4, BLLF3, BBLF4, BcRF1, and BILF2. X axis, cell lines; Y axis, relative expression normalized to GAPDH.
Fig.5  Validation of EBV latency genes in NPC tissue. EBV latency genes: EBER1, EBER2, EBNA1, EBNA2, LMP1, LMP2A, EBNA3A, EBNA3B, and EBNA3C.
Fig.6  Validation of EBV lytic genes by real-time PCR in NPC tissue. Representative EBV lytic genes: BBLF4, BLLF3, BDLF2, BCLF1, BGLF2, BGLF4, BHRF1, BGLF5, BILF2, and BcRF1. N, normal; T, tumor.
Fig.7  Overview of unsupervised hierarchical clustering of all NPC samples. Hierarchically clustered gene expression profiles of 12 NPC samples based on the EBV genes. Clustering of gene expression is shown on the top of the figure, where each column represents one gene. The NPC samples are divided into two subtypes based on differences in gene expression.
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