Please wait a minute...
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
RESEARCH ARTICLE |
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
Download: PDF(2413 KB)   HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
Abstract  

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:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-016-0436-0     OR     http://academic.hep.com.cn/fmd/EN/Y2016/V10/I1/61
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.
1 Young LS, Rickinson AB. Epstein-Barr virus: 40 years on. Nat Rev Cancer 2004; 4(10): 757–768
doi: 10.1038/nrc1452 pmid: 15510157
2 Decaussin G, Sbih-Lammali F, de Turenne-Tessier M, Bouguermouh A, Ooka T. Expression of BARF1 gene encoded by Epstein-Barr virus in nasopharyngeal carcinoma biopsies. Cancer Res 2000; 60(19): 5584–5588
pmid: 11034107
3 Pattle SB, Farrell PJ. The role of Epstein-Barr virus in cancer. Expert Opin Biol Ther 2006; 6(11): 1193–1205
doi: 10.1517/14712598.6.11.1193 pmid: 17049016
4 Xu ZJ, Zheng RS, Zhang SW, Zou XN, Chen WQ. Nasopharyngeal carcinoma incidence and mortality in China in 2009. Chin J Cancer 2013; 32(8): 453–460
doi: 10.5732/cjc.013.10118 pmid: 23863562
5 Wei KR, Zheng RS, Zhang SW, Liang ZH, Ou ZX, Chen WQ. Nasopharyngeal carcinoma incidence and mortality in China in 2010. Chin J Cancer 2014; 33(8): 381–387
pmid: 25096544
6 Dong JQ, Li MZ, Liu ZG, Zhong Q, Xiong D, Xu LH, Du Y, Xia YF, Zeng MS. Establishment and characterization of a novel nasopharyngeal carcinoma cell line (SUNE2) from a Cantonese patient. Chin J Cancer 2012; 31(1): 36–44
pmid: 22176775
7 Busson P, Keryer C, Ooka T, Corbex M. EBV-associated nasopharyngeal carcinomas: from epidemiology to virus-targeting strategies. Trends Microbiol 2004; 12(8): 356–360
doi: 10.1016/j.tim.2004.06.005 pmid: 15276610
8 Deyrup AT. Epstein-Barr virus-associated epithelial and mesenchymal neoplasms. Hum Pathol 2008; 39(4): 473–483
doi: 10.1016/j.humpath.2007.10.030 pmid: 18342658
9 Tao Q, Chan AT. Nasopharyngeal carcinoma: molecular pathogenesis and therapeutic developments. Expert Rev Mol Med 2007; 9(12): 1–24
doi: 10.1017/S1462399407000312 pmid: 17477889
10 Cabras G, Decaussin G, Zeng Y, Djennaoui D, Melouli H, Broully P, Bouguermouh AM, Ooka T. Epstein-Barr virus encoded BALF1 gene is transcribed in Burkitt’s lymphoma cell lines and in nasopharyngeal carcinoma’s biopsies. J Clin Virol 2005; 34(1): 26–34
doi: 10.1016/j.jcv.2004.12.016 pmid: 16087121
11 Martel-Renoir D, Grunewald V, Touitou R, Schwaab G, Joab I. Qualitative analysis of the expression of Epstein-Barr virus lytic genes in nasopharyngeal carcinoma biopsies. J Gen Virol 1995; 76(Pt 6): 1401–1408
doi: 10.1099/0022-1317-76-6-1401 pmid: 7782768
12 Sbih-Lammali F, Berger F, Busson P, Ooka T. Expression of the DNase encoded by the BGLF5 gene of Epstein-Barr virus in nasopharyngeal carcinoma epithelial cells. Virology 1996; 222(1): 64–74
doi: 10.1006/viro.1996.0398 pmid: 8806488
13 Lin Z, Xu G, Deng N, Taylor C, Zhu D, Flemington EK. Quantitative and qualitative RNA-Seq-based evaluation of Epstein-Barr virus transcription in type I latency Burkitt’s lymphoma cells. J Virol 2010; 84(24): 13053–13058
doi: 10.1128/JVI.01521-10 pmid: 20943983
14 Li C, Chen RS, Hung SK, Lee YT, Yen CY, Lai YW, Teng RH, Huang JY, Tang YC, Tung CP, Wei TT, Shieh B, Liu ST. Detection of Epstein-Barr virus infection and gene expression in human tumors by microarray analysis. J Virol Methods 2006; 133(2): 158–166
doi: 10.1016/j.jviromet.2005.10.032 pmid: 16384612
15 Feng L, Liu H, Liu Y, Lu Z, Guo G, Guo S, Zheng H, Gao Y, Cheng S, Wang J, Zhang K, Zhang Y. Power of deep sequencing and agilent microarray for gene expression profiling study. Mol Biotechnol 2010; 45(2): 101–110
doi: 10.1007/s12033-010-9249-6 pmid: 20432071
16 Haas BJ, Zody MC. Advancing RNA-Seq analysis. Nat Biotechnol 2010; 28(5): 421–423
doi: 10.1038/nbt0510-421 pmid: 20458303
17 Zhu JY, Pfuhl T, Motsch N, Barth S, Nicholls J, Grässer F, Meister G. Identification of novel Epstein-Barr virus microRNA genes from nasopharyngeal carcinomas. J Virol 2009; 83(7): 3333–3341
doi: 10.1128/JVI.01689-08 pmid: 19144710
18 Chen SJ, Chen GH, Chen YH, Liu CY, Chang KP, Chang YS, Chen HC. Characterization of Epstein-Barr virus miRNAome in nasopharyngeal carcinoma by deep sequencing. PLoS ONE 2010; 5(9): e12745
doi: 10.1371/journal.pone.0012745 pmid: 20862214
19 Strong MJ, Xu G, Coco J, Baribault C, Vinay DS, Lacey MR, Strong AL, Lehman TA, Seddon MB, Lin Z, Concha M, Baddoo M, Ferris M, Swan KF, Sullivan DE, Burow ME, Taylor CM, Flemington EK. Differences in gastric carcinoma microenvironment stratify according to EBV infection intensity: implications for possible immune adjuvant therapy. PLoS Pathog 2013; 9(5): e1003341
doi: 10.1371/journal.ppat.1003341 pmid: 23671415
20 Hui AB, Cheung ST, Fong Y, Lo KW, Huang DP. Characterization of a new EBV-associated nasopharyngeal carcinoma cell line. Cancer Genet Cytogenet 1998; 101(2): 83–88
doi: 10.1016/S0165-4608(97)00231-8 pmid: 9494607
21 Cheung ST, Huang DP, Hui AB, Lo KW, Ko CW, Tsang YS, Wong N, Whitney BM, Lee JC. Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus. Int J Cancer 1999; 83(1): 121–126
doi: 10.1002/(SICI)1097-0215(19990924)83:1<121::AID-IJC21>3.0.CO;2-F pmid: 10449618
22 Bernasconi M, Berger C, Sigrist JA, Bonanomi A, Sobek J, Niggli FK, Nadal D. Quantitative profiling of housekeeping and Epstein-Barr virus gene transcription in Burkitt lymphoma cell lines using an oligonucleotide microarray. Virol J 2006; 3(1): 43
doi: 10.1186/1743-422X-3-43 pmid: 16756670
23 Huang W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009; 4(1): 44–57
doi: 10.1038/nprot.2008.211 pmid: 19131956
24 Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. Gene ontology: tool for the unification of biology. Nat Genet 2000; 25(1): 25–29
doi: 10.1038/75556 pmid: 10802651
25 Al-Mozaini M, Bodelon G, Karstegl CE, Jin B, Al-Ahdal M, Farrell PJ. Epstein-Barr virus BART gene expression. J Gen Virol 2009; 90(Pt 2): 307–316
doi: 10.1099/vir.0.006551-0 pmid: 19141439
26 Miller G, El-Guindy A, Countryman J, Ye J, Gradoville L. Lytic cycle switches of oncogenic human γ herpesviruses. Adv Cancer Res 2007; 97: 81–109
doi: 10.1016/S0065-230X(06)97004-3 pmid: 17419942
27 Zhang Q, Hong Y, Dorsky D, Holley-Guthrie E, Zalani S, Elshiekh NA, Kiehl A, Le T, Kenney S. Functional and physical interactions between the Epstein-Barr virus (EBV) proteins BZLF1 and BMRF1: effects on EBV transcription and lytic replication. J Virol 1996; 70(8): 5131–5142
pmid: 8764021
28 Jung YJ, Choi H, Kim H, Lee SK. MicroRNA miR-BART20-5p stabilizes Epstein-Barr virus latency by directly targeting BZLF1 and BRLF1. J Virol 2014; 88(16): 9027–9037
doi: 10.1128/JVI.00721-14 pmid: 24899173
29 Wille CK, Nawandar DM, Panfil AR, Ko MM, Hagemeier SR, Kenney SC. Viral genome methylation differentially affects the ability of BZLF1 versus BRLF1 to activate Epstein-Barr virus lytic gene expression and viral replication. J Virol 2013; 87(2): 935–950
doi: 10.1128/JVI.01790-12 pmid: 23135711
30 Fung LF, Lo AK, Yuen PW, Liu Y, Wang XH, Tsao SW. Differential gene expression in nasopharyngeal carcinoma cells. Life Sci 2000; 67(8): 923–936
doi: 10.1016/S0024-3205(00)00684-6 pmid: 10946852
31 Bell AI, Groves K, Kelly GL, Croom-Carter D, Hui E, Chan AT, Rickinson AB. Analysis of Epstein-Barr virus latency gene expression in endemic Burkitt’s lymphoma and nasopharyngeal carcinoma tumour cells by using quantitative real-time PCR assays. J Gen Virol 2006; 87(Pt 10): 2885–2890
doi: 10.1099/vir.0.81906-0 pmid: 16963746
32 Clarke PA, Sharp NA, Clemens MJ. Expression of genes for the Epstein-Barr virus small RNAs EBER-1 and EBER-2 in Daudi Burkitt’s lymphoma cells: effects of interferon treatment. J Gen Virol 1992; 73(Pt 12): 3169–3175
doi: 10.1099/0022-1317-73-12-3169 pmid: 1335024
33 Houmani JL, Davis CI, Ruf IK. Growth-promoting properties of Epstein-Barr virus EBER-1 RNA correlate with ribosomal protein L22 binding. J Virol 2009; 83(19): 9844–9853
doi: 10.1128/JVI.01014-09 pmid: 19640998
34 Wu Y, Maruo S, Yajima M, Kanda T, Takada K. Epstein-Barr virus (EBV)-encoded RNA 2 (EBER2) but not EBER1 plays a critical role in EBV-induced B-cell growth transformation. J Virol 2007; 81(20): 11236–11245
doi: 10.1128/JVI.00579-07 pmid: 17686859
35 Cheung ST, Huang DP, Hui AB,Lo KW , Ko CW, Tsang YS, Wong N, Whitney BM, Lee JC. Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus. Int J Cancer 1999; 83(1): 121–126
pmid: 10449618
36 Sengupta S, den Boon JA, Chen IH, Newton MA, Dahl DB, Chen M, Cheng YJ, Westra WH, Chen CJ, Hildesheim A, Sugden B, Ahlquist P. Genome-wide expression profiling reveals EBV-associated inhibition of MHC class I expression in nasopharyngeal carcinoma. Cancer Res 2006; 66(16): 7999–8006
doi: 10.1158/0008-5472.CAN-05-4399 pmid: 16912175
37 Nikitin PA, Yan CM, Forte E, Bocedi A, Tourigny JP, White RE, Allday MJ, Patel A, Dave SS, Kim W, Hu K, Guo J, Tainter D, Rusyn E, Luftig MA. An ATM/Chk2-mediated DNA damage-responsive signaling pathway suppresses Epstein-Barr virus transformation of primary human B cells. Cell Host Microbe 2010; 8(6): 510–522
doi: 10.1016/j.chom.2010.11.004 pmid: 21147465
38 Gao Y, Smith PR, Karran L, Lu QL, Griffin BE. Induction of an exceptionally high-level, nontranslated, Epstein-Barr virus-encoded polyadenylated transcript in the Burkitt’s lymphoma line Daudi. J Virol 1997; 71(1): 84–94
pmid: 8985326
39 Xue SA, Griffin BE. Complexities associated with expression of Epstein-Barr virus (EBV) lytic origins of DNA replication. Nucleic Acids Res 2007; 35(10): 3391–3406
doi: 10.1093/nar/gkm170 pmid: 17478522
40 Feng P, Ren EC, Liu D, Chan SH, Hu H. Expression of Epstein-Barr virus lytic gene BRLF1 in nasopharyngeal carcinoma: potential use in diagnosis. J Gen Virol 2000; 81(Pt 10): 2417–2423
doi: 10.1099/0022-1317-81-10-2417 pmid: 10993929
41 Tanner JE, Wei MX, Alfieri C, Ahmad A, Taylor P, Ooka T, Menezes J. Antibody and antibody-dependent cellular cytotoxicity responses against the BamHI A rightward open-reading frame-1 protein of Epstein-Barr virus (EBV) in EBV-associated disorders. J Infect Dis 1997; 175(1): 38–46
doi: 10.1093/infdis/175.1.38 pmid: 8985194
42 Hitt MM, Allday MJ, Hara T, Karran L, Jones MD, Busson P, Tursz T, Ernberg I, Griffin BE. EBV gene expression in an NPC-related tumour. EMBO J 1989; 8(9): 2639–2651
pmid: 2479554
[1] FMD-16202-OF-ZMS_suppl_1 Download
[2] FMD-16202-OF-ZMS_suppl_2 Download
[3] FMD-16202-OF-ZMS_suppl_3 Download
[4] FMD-16202-OF-ZMS_suppl_4 Download
[5] FMD-16202-OF-ZMS_suppl_5 Download
[1] Hong-Lian RUAN, Feng-Hua XU, Wen-Sheng LIU, Qi-Sheng FENG, Li-Zhen CHEN, Yi-Xin ZENG, Wei-Hua JIA, . Alcohol and tea consumption in relation to the risk of nasopharyngeal carcinoma in Guangdong, China[J]. Front. Med., 2010, 4(4): 448-456.
[2] Jing CHEN MD, Guang-Yuan HU MD, Guo-Qing HU MD, Hua WU PhD, . Technetium-99m-sestamibi SPECT for the diagnosis and follow-up of nasopharyngeal carcinoma[J]. Front. Med., 2010, 4(1): 96-100.
[3] Jianqing PAN PhD, Qin ZHANG MD, Daowen WANG MD, PhD, . Construction and humoral immune response of Epstein-Barr virus latent membrane protein 2 DNA vaccine in mice[J]. Front. Med., 2009, 3(4): 390-395.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed