Expression of Epstein-Barr virus genes in EBV-associated gastric carcinomas

AIM: To understand the expression of latent and lytic genes of Epstein-Barr virus (EBV) in EBV-associated gastric carcinoma (EBVaGC) and to explore the relationship between EBV-encoded genes and development of EBVaGC at molecular level, METHODS: One hundred and seventy-two gastric carcinoma tissues and 172 corresponding para-carcinoma tissues were tested for EBV genome by polymerase chain reaction (PCR)-Southern blotting. EBV-encoded small RNA (EBER) 1 of the PCR positive specimens was detected by in situ hybridization (ISH). Gastric carcinomas with positive EBER1 signals were classified as EBVaGCs. RT-PCR and Southern hybridization were applied to the detection of expression of nuclear antigen (EBNA) promoters (Qp, Wp and Cp), EBNA 1 and EBNA 2, latent membrane proteins (LMP) 1, 2A and 2B and lytic genes (immediate early genes BZLF1 and BRLF1, early genes BARF1 and BHRF1, late genes BcLF1 and BLLF1) in EBVaGCs. RESULTS: Eleven EBV positive samples existed in gastric carcinoma tissues (6.39%). No EBV positive sample was found in corresponding para-carcinoma tissues. The difference between EBV positivity in carcinoma tissues and corresponding para-carcinoma tissues was significant (x2 = 9.0909, P = 0.0026). Transcripts of Qp and EBNA1 were detected in all the 11 EBVaGCs, while both Wp and Cp were silent. EBNA2, LMP1 and LMP2B mRNA were absent in all the cases, while LMP2A mRNA was detected in 4 of the 11 cases. Of the 11 EBVaGCs, 7 exhibited BcLFl transcripts and 2 exhibited BHRF1 transcripts. The transcripts of BZLF1 and BARF1 were detected in 5 cases, respectively. No BLLF1 and BRLF mRNA were detected. CONCLUSION: The latent pattern of EBV in gastric carcinoma corresponds to the latency I/II. Some lytic infection genes are expressed in EBVaGCs tissues. BARF1 and BHRF1 genes may play an important role in tumorigenesis of gastric carcinoma.

STRUCTURE AND EXPRESSION OF EPSTEIN-BARR VIRUS MEMBRANE ANTIGEN IN RECOMBINANT VACCINIA VIRUS

The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen was expressed under the control of a 7.5 K promoter of vaccinia virus. The antibody against the membrane antigen of EB virus was produced on rabbits vaccinated with recombinant vaccinia virus.

Regulation network and expression profiles of Epstein-Barr virus-encoded microRNAs and their potential target host genes in nasopharyngeal carcinomas

Epstein-Barr virus(EBV)is associated with nasopharyngeal carcinoma(NPC)tumorigenesis.However,the mechanism(s)connecting EBV infection and NPC remain unclear.Recently,a new class of EBV microRNAs(miRNAs)has been described.To determine how EBV miRNAs control the expression of host genes,and to understand their potential role in NPC tumorigenesis,we profiled the expression of 44 mature EBV miRNAs and potential host genes in NPC and non-tumor nasopharyngeal epithelial tissues.We found that 40 EBV miRNAs from the BART transcript were highly expressed in NPC.Analysis of potential BART miRNA target genes revealed that 3140 genes and several important pathways might be involved in the carcinogenesis of NPC.A total of 105 genes with potential EBV miRNA binding sites were significantly downregulated,suggesting that EBV miRNAs may regulate these genes and contribute to NPC carcinogenesis.An EBV miRNA and host gene regulation network was generated to provide useful clues for validating of EBV miRNA functions in NPC tumorigenesis.

Comprehensive profiling of EBV gene expression in nasopharyn- geal carcinoma through paired-end transcriptome sequencing

The latent expression pattern of Epstein-Barr Virus （EBV）igenes 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 lyric genes. This study provided further insights into the potential role of EBV in the development of NPC.

Carcinogenesis of nasopharyngeal carcinoma:an alternate hypothetical mechanism

Current proposed mechanisms implicate both early and latent Epstein-Barr virus(EBV)infection in the carcinogenic cascade,whereas epidemiological studies have always associated nasopharyngeal carcinoma(NPC)with early childhood EBV infection and with chronic ear,nose,and sinus conditions.Moreover,most patients with NPC present with IgA antibody titers to EBV capsid antigen(VCA-lgA),which can precede actual tumor presentation by several years.If early childhood EBV infection indeed constitutes a key event in NPC carcinogenesis,one would have to explain the inability to detect the virus in normal nasopharyngeal epithelium of patients at a high risk for EBV infection.It is perhaps possible that EBV resides within the salivary glands,instead of the epithelium,during latency.This claim is indirectly supported by observations that the East Asian phenotype shares the characteristics of an increased susceptibility to NPC and immature salivary gland morphogenesis,the latter of which is influenced by the association of salivary gland morphogenesis with an evolutionary variant of the human ectodysplasin receptor gene(EDAR),EDARV370A.Whether the immature salivary gland represents a more favorable nidus for EBV is uncertain,but in patients with infectious mononucleosis,EBV has been isolated in this anatomical organ.The presence of EBV-induced lymphoepitheliomas in the salivary glands and lungs further addresses the possibility of submucosal spread of the virus.Adding to the fact that the fossa of Rosen Muller contains a transformative zone active only in the first decade of life,one might be tempted to speculate the possibility of an alternative carcinogenic cascade for NPC that is perhaps not dissimilar to the model of human papillomavirus and cervical cancer.

EB病毒活化对鼻咽癌基因表达模式的影响

背景与目的:EB病毒(Epstein-Barr virus,EBV)感染在鼻咽癌发生中发挥重要作用。本研究的目的在于探讨潜伏的EBV活化后形成的复发感染对鼻咽癌发生过程中全基因组表达的影响。方法:收集EBV阳性(EBV+)/EBV阴性(EBV-)的鼻咽癌靶标基因表达谱和EBV复发感染次数不同的鼻咽癌细胞株基因表达谱,通过生物信息学手段对其进行交叉比较,统计学分析筛选出25个差异基因。运用DAVID(database for annotation,visualization and integrated discovery)、pSTIING(protein,signaling,transcriptional interactions and inflammation networks gateway)、GATHER(gene annotation tool to help explain relationships)和TELiS(transcriptionelement listening system)在线分析工具对25个共同差异基因进行表达模式的预测。结果:与EBV初次感染时的基因表达谱比较,仅有DUSP1、TOP1、HOXA9、DEK、IMPDH2、PABPC1等25个基因在EBV复发感染时仍然呈明显差异表达;这25个差异基因及其相关转录因子主要通过2种模式相互作用:一条主要涉及TOP1、DUSP1、DUSP6和RPS28等,另一条是由PITX1、CD9、HOXA9和IMPDH2组成的转录相关环。结论:EBV潜伏后的复发感染,可能通过筛选到的差异基因的2种表达模式发挥功能,最终导致鼻咽癌的发生。

人鼻咽癌细胞裸鼠体内演进中bcl-2、p53及c-erbB-2表达与EB病毒LMP1的关系

目的 研究 EB病毒 L MP1在人高分化鼻咽癌细胞裸鼠体内演进中的作用及与 bcl- 2、p5 3及 c- erb B- 2蛋白表达的关系。方法 将表达 EB病毒 L MP1的人高分化鼻咽癌细胞及对照阴性细胞移植于裸鼠体内 ,然后将移植瘤细胞再移入裸鼠建立第二代移植瘤 ,如此传至第三代 ,分别观察移植瘤的成瘤和生长的变化 ,以免疫组化和图像分析技术检测移植瘤细胞 bcl- 2、p5 3及 c- erb B- 2蛋白表达。结果 表达 L MP1的鼻咽癌细胞原代移植成瘤率达 75 .0 %,比对照组显著增高 (P<0 .0 1)。平均潜伏期及体内倍增时间分别为 2 9.6± 7.7天和 2 .81± 0 .13天 ,比对照组明显缩短 (P<0 .0 1)。表达 L MP1的鼻咽癌细胞 (C1L)各代移植瘤的 bcl- 2蛋白表达阳性率显著低于对照组对应的各代细胞的表达 (P<0 .0 1) ;bcl- 2蛋白表达阳性率有显著下降趋势 ,对照组细胞 bcl- 2蛋白表达则呈峰值变化 ;各细胞系体内移植瘤细胞的 p5 3及 c- erb B- 2蛋白表达的变化趋势较为相似 ,即各细胞系不同代间 p5 3表达的阳性率有显著下降趋势 ,c- erb B- 2蛋白表达阳性率有显著上升趋势。表达 L MP1的鼻咽癌各代移植瘤细胞的 p5 3及 c- erb B- 2蛋白表达阳性率显著高于对照组对应的各代 (P<0 .0 1)。结论 L MP1使鼻咽癌细胞的生长能力增强 。

Epstein—Barr病毒基因组在鼻咽癌组织中转录的特征

对ＥＢ病毒基因在鼻咽癌活检组织细胞内的转录进行了较系统的探测。实验结果表明，ＥＢ病毒基因组在鼻咽癌活检组织中以附加体（Ｅｐｉｓｏｍｅ）形式存在，而其基因转录有如下特征：（１）ＥＢ病毒在所有鼻咽癌组织细胞中都表达ＥＢＮＡ－１，并且此基因转录产物由一个在ＢａｍＨＩ－Ｆ区的启动子（Ｆｐ）驱动；（２）；潜伏感染膜蛋白（Ｌａｔｅｎｔｍｅｍｂｒａｎｅｐｒｏｔｅｉｎ，ＬＭＰ和末端蛋白（Ｔｅｒｍｉｎａｌｐｒｏｔｅｉｎ，ＴＰ）的转录只在约５０％的活检组织中检测到，并且显示利用不同启动子的现象；（３）少部分的病例表达个别裂解基因；（４）在所有活检组织中，ＥＢ病毒基因组的ＢａｍＨＩ－Ａ区，表达一组功能未明的ｍＲＮＡ。通过对ＥＢ病毒基因在鼻咽癌细胞和其它受ＥＢ病毒感染的Ｂ淋巴细胞中表达情况的比较，本文对ＥＢ病毒基因在鼻咽癌组织细胞中的表达调控及其在癌变过程中的可能作用进行了讨论。

鼻咽癌中抗凋亡基因bcl-2的表达及其与EB病毒的关系

为了探讨鼻咽癌（ＮＰＣ）中抗凋亡基因ｂｃｌ－２的表达及其与ＥＢ病毒的关系。用免疫组化方法对曾用ＥＢ病毒探针进行原位杂交的ＮＰＣ标本再进行ｂｃｌ－２检测。结果：非角化型鳞状细胞癌５４例，阳性５１例，阴性３例；角化型鳞状细胞癌１２例，阳性３例，阴性９例；腺癌３例，阳性１例，阴性２例；ＮＰＣ淋巴结转移性鳞状细胞癌１１例，阳性９例，阴性２例；非ＮＰＣ淋巴结转移性鳞状细胞癌１０例，阳性２例，阴性８例。ＮＰＣ各组织学类型中ｂｃｌ－２的表达与ＥＢ病毒原位杂交表达的结果相似。提示ｂｃｌ－２基因表达与ＮＰＣ组织学类型关系密切，非角化型大部分为阳性表达，角化型大部分为阴性表达，并提示ＥＢ病毒在ＮＰＣ的发生机制上可能与抑制细胞凋亡的基因有协同作用。

EB病毒BHRF1基因表达对鼻咽癌细胞增殖能力的影响

为了解ＥＢ病毒编码基因ＢＨＲＦ１的表达对鼻咽癌细胞增殖能力的影响，通过构建携有ＢＨＲＦ１的高表达载体，并转染低分化鼻咽癌细胞株ＣＮＥ２细胞，观察转染后的癌细胞在缺乏营养条件下增殖能力的改变。结果发现：ＢＨＲＦ１的表达能抑制增殖核抗原的表达，并促进其在缺乏营养条件下生存能力。提示ＥＢ病毒编码的ＢＨＲＦ１基因可能与鼻咽癌的发生和发展过程有关。

转染人pIgR的小鼠鼻咽上皮细胞在EBV感染前后基因表达谱差异

目的应用cDNA阵列技术研究转染人多聚免疫球蛋白受体(hpIgR)的二亚硝基哌嗪(n, n'-dinitrosoperazine, DNP)转化的小鼠鼻咽上皮(TMNE)细胞系在EBV感染前后基因表达谱差异,探索EBV在鼻咽癌发病过程中的作用及鼻咽癌的发病机制。方法提取转染hpIgR 的TMNE细胞在EBV感染前后的总RNA,逆转录成α-32P-dATP标记的cDNA探针,与AtlasTM mouse cancer array 1.2 阵列膜进行杂交。利用AtlasImageTM 软件分析基因表达差异。结果共有25个基因表达水平发生了改变,有23个基因表达上调,2个基因表达下调。结论EBV感染可使转染细胞的多个基因表达发生改变,这些基因可能与鼻咽癌的发生、发展有关。

BHRF1表达对鼻咽癌细胞抗凋亡能力的影响

目的了解ＥＢ病毒（ＥｐｓｔｅｉｎＢａｒｖｉｒｕｓ）基因ＢＨＲＦ１（ＢａｍＨＩＨｒｉｇｈｔｗａｒｄｒｅａｄｉｎｇｆｒａｍｅＩ）表达对鼻咽癌细胞抗凋亡能力的影响。方法构建表达ＢＨＲＦ１重组载体并转入鼻咽癌细胞株ＣＮＥ２细胞中，以检测癌细胞在６０Ｃｏ照射后生物学行为的改变。结果ＢＨＲＦ１表达可抑制增殖细胞核抗原的表达，促使癌细胞的细胞周期重新分布，癌细胞对辐射的敏感性下降，生存能力增强。结论ＢＨＲＦ１的表达可增强ＣＮＥ２细胞抵抗放射线诱发细胞凋亡的能力。

鼻咽癌相关肿瘤基因及基因治疗

鼻咽癌是一种特殊的头颈肿瘤,它是一种非淋巴瘤性的非角质化的鳞状上皮恶性肿瘤,发生与病毒慢性感染、环境致癌因素以及基因突变等多因素相关.本文从鼻咽癌肿瘤基因的角度,探讨鼻咽癌的发病因素以及基因治疗方向研究.