EB病毒miRNAs在鼻咽癌细胞株C666-1和CNE-2Z中的差异性表达分析

目的:探讨ebv-miRNA在低分化NPC细胞株C666-1(EBV+)和CNE-2Z(EBV表达水平随传代次数增加而逐渐降低)中的差异性表达进而分析其功能.方法:常规培养NPC细胞株C666-1和CNE-2Z,分别提取总RNA并进行质检;RT-PCR检测LMP-1和LMP-2A mRNA的表达以判断EBV的感染情况;miRNA芯片技术检测EBV miRNAs的表达并对其表达谱进行差异性分析;荧光定量RT-PCR进行验证;复习文献了解ebv-miRNA调控的靶基因以了解其功能.结果:从C666-1和CNE-2Z两株细胞抽提的总RNA纯度高,A260/A280>2.0,A260/A230>2.1,RNA完整性好.C666-1具有比CNE-2Z更高的LMP-1和LMP-2A mRNA表达.ebv-miRNA表达谱的差异分析表明39个ebv-miRNAs中19个在两株细胞显著差异性表达;荧光定量RT-PCR结果证实ebv-miR-BHRF1-1和ebv-miR-BART14*在C666-1中表达升高而ebv-miR-BART8*表达降低,与芯片结果趋势一致;复习文献,EBV miRNAs功能远不清楚,少数已知的靶基因涉及信号转导、转录调控、细胞凋亡、增殖、免疫反应和病毒DNA复制等方面.结论:ebv-miRNA在低分化NPC中具有差异性表达并广泛参与基因调控.不同NPC细胞中EBV miRNA表达差异预示着基于ebv-miRNA的NPC个性化治疗的可能性.     

EB病毒miRNAs在鼻咽癌细胞株C666-1和CNE-2Z中的差异性表达分析

目的：探讨ebv-miRNA在低分化NPC细胞株C666-1（EBV＋）和CNE-2Z（EBV表达水平随传代次数增加而逐渐降低）中的差异性表达进而分析其功能.方法：常规培养NPC细胞株C666-1和CNE-2Z,分别提取总RNA并进行质检;RT-PCR检测LMP-1和LMP-2A mRNA的表达以判断EBV的感染情况;miRNA芯片技术检测EBV miRNAs的表达并对其表达谱进行差异性分析;荧光定量RT-PCR进行验证;复习文献了解ebv-miRNA调控的靶基因以了解其功能.结果：从C666-1和CNE-2Z两株细胞抽提的总RNA纯度高,A260/A280〉2.0,A260/A230〉2.1,RNA完整性好.C666-1具有比CNE-2Z更高的LMP-1和LMP-2A mRNA表达.ebv-miRNA表达谱的差异分析表明39个ebv-miRNAs中19个在两株细胞显著差异性表达;荧光定量RT-PCR结果证实ebv-miR-BHRF1-1和ebv-miR-BART14＊在C666-1中表达升高而ebv-miR-BART8＊表达降低,与芯片结果趋势一致;复习文献,EBV miRNAs功能远不清楚,少数已知的靶基因涉及信号转导、转录调控、细胞凋亡、增殖、免疫反应和病毒DNA复制等方面.结论：ebv-miRNA在低分化NPC中具有差异性表达并广泛参与基因调控.不同NPC细胞中EBV miRNA表达差异预示着基于ebv-miRNA的NPC个性化治疗的可能性.     

EB病毒感染与鼻咽癌诊治的研究进展

鼻咽癌（Nasopharyngeal Carcinoma,NPC）是指原发于鼻咽腔部的恶性肿瘤。EB病毒（Epstein-Barr Virus,EBV）感染与NPC有着密切关系。EBV感染人体后,可通过多种特异性病毒抗原介导NPC的发生发展。目前的研究显示,针对NPC高危人群进行的多种EBV抗体标志物的联合筛查,在NPC的预防、诊断及随访中有着重要意义。EBV相关的最新NPC免疫治疗方式,如病毒相关疫苗,过继性免疫治疗等都展示出了较好的疗效及应用潜力。对NPC患者血清中病毒抗原滴度及EBV DNA的定量检测,为其预后和生存率的判断有着一定的帮助。本文就EBV感染与NPC诊治及预后判断的最新研究进展作一综述。     

HHV-6感染在鼻咽癌发病中的意义

目的：探计鼻咽癌组织中人疱疹病毒6型(HHV—6)在鼻咽癌(Nasopharyngeal carcinoma，NPC)发病中的意义。方法：PCR检测27例正常鼻咽组织、21例异型增生及42例鼻咽癌组织中HHV一6DNA和EB病毒(Epstein—barr vims，EBV)DNA存在状况，用免疫组化检测36例NPC组织中EBVLMPl蛋白的表达及40例NPC组织中p53基因的表达。结果：异型增生和NPC组织中HHV一6 DNA的阳性率分别为4．7％和26．2％，EBV DNA分别为4．6％和95．2％；正常鼻咽组织不存在HHV一6DNA，EBVDNA为25．9％。NPC组织中EBV—LMPl蛋白阳性率为47．2％，HHV一6阳性NPC中LMN表达阳性率(81．8％)显著高于HHV一6阴性者(32．0％)。HHV一6阳性与阴性的NPC中p53表达的阳性率无显著性差异，但HHV—6阳性的NPC中p53表达强度显著性增高。结论：NPC组织中存在HHV—6的感染，与EBV—LMP1蛋白表达上调呈正相关。提示在NPC的发生中HHV—6可能直接参与和(或)通过上调EBV—LMP1的表达而起作用，并可能参与鼻咽癌发病中p53过表达的调控。     

EBV BART microRNAs 在鼻咽癌中的作用研究进展

摘 要：MicroRNAs(miRNAs)是一类长度大约22nt的内源性非编码单链RNA,在转录后水平调控基因的表达。EBV BART microRNAs是由EBV BART基因编码,其与鼻咽癌(nasopharyngeal carcinoma,NPC)的发生及发展有着重要的关系。目前研究发现BART miRNAs在NPC中的作用主要与免疫逃逸、侵袭转移及抗凋亡作用等相关。文章主要对EBV BART microRNAs 在NPC中的作用进行综述。     

EBV阳性鼻咽癌相关性中性粒细胞抑制肿瘤微环境中的CD8+ T细胞活化

目的：分析鼻咽癌（NPC）肿瘤微环境（TME）中的肿瘤相关中性粒细胞（TAN）浸润与患者的预后和临床病理特征之间的相关性,初步探讨EBV阳性NPC的TAN对CD8+ T细胞活化的作用。方法：收集2008年至2012年间中山大学肿瘤防治中心收治的118例初治且无转移的EBV阳性NPC患者的肿瘤组织标本,通过免疫组织化学染色法观察并分析NPC组织中CD8+T细胞、TAN浸润和EBV感染之间的关系,检测NPC组织标本中的TAN浸润程度并分析其与患者预后和临床病理特征之间的关系;流式细胞术检测HK1-EBV细胞培养上清液对TAN极化的N2型标志物（CD182和CD206）表达水平的影响,并进一步检测极化后的TAN对CD8+ T细胞活化标志物（CD69和PD-1）表达水平的影响。结果：EBV阳性NPC组织中CD8+T细胞和TAN浸润增多且两者在数量上呈负相关关系（P=0.005 2）;EBV阳性NPC组织中高水平浸润的TAN与患者的不良预后密切相关（OS：P=0.025,PFS：P=0.027）,TAN浸润水平是EBV阳性NPC患者总生存时间（P=0.035）的独立预后因子;与对照组相比,HK1-EBV细胞培养上清液可诱导TAN极化并高表达N2型标志物（CD182：P<0.001;CD206：P<0.01）;极化后的TAN可抑制CD8+T细胞CD69的表达并促进PD-1的表达（P<0.001）。结 论：EBV阳性NPC能够促进TME中的TAN浸润增多并使其极化成N2型,从而抑制CD8+ T细胞的活化,发挥免疫抑制作用,与NPC患者的不良预后密切相关。     

EB病毒感染与鼻咽癌发病机制的关系

鼻咽癌（nasopharyngeal carcinoma,NPC）是一种在中国南方、台湾、新加坡及特定地域的其它国家或地区很常见的肿瘤,其发病因素尚不明确。目前,还未发现与NPC发生过程的遗传因素有关的主要基因;而一些环境因素可能与诱发NPC有关。如食用腌鱼和长期暴露于硫酸蒸汽,研究指出EB病毒（Epstein—Barr virus,EBV）与NPC发病机理密切相关。为了探索NPC与EBV之间的关系,我们建立了10个NPC细胞株。经过广泛研究,我们认为EBV通过IgA受体（分泌组分蛋白）介导的细胞内吞作用,可能仅感染鼻咽肿瘤细胞,而不感染化生上皮细胞。我们发现EBV对于促进NPC进展起着重要作用,但并不参与启动及促进NPC的发生。     

EB病毒与鼻咽癌相关性的研究进展

Epstein-Barr病毒（EBV）的潜伏感染与鼻咽癌（Nasopharyngeal carcinoma,NPC）密切相关,在感染人体后长期潜伏,可表达多种基因,其潜伏膜蛋白（LMP）编码基因、EB病毒核抗原（EBNA）基因及EBV编码的小RNA（EBER）可以通过不同的机制致鼻咽癌。本文就EBV与NPC的关系,与NPC相关的EBV的生物学特性和进展作一综述。     

鼻咽癌病人外周血和骨髓中的EB病毒与预后的临床相关性

目的通过检测NPC病人治疗前外周血和骨髓中EBV,研究EBV与NPC放疗后失败及远期生存的临床相关性.方法1998年2月至同年8月,选择诊断明确的27例晚期NPC病人,其中24例为初治、3例为局部复发,疗前用常规PCR分别检测外周静脉血和骨髓中EB病毒.同时检测13例非NPC肿瘤病人静脉血中的EBV,作为对比.结果13例非NPC病人静脉血中EB病毒均为阴性.27例NPC中,15例EBV阳性(外周血和骨髓中EBV均阳性者13例),阳性病例中有10例治疗失败,失败率66.7%,其中6例为远处转移,远处转移率40.0%.12例阴性病人,3例疗后失败,失败率25.0%:其中仅1例为远处转移,远处转移率8.3%.两组间失败率差异明显.结论部分NPC病人外周血和骨髓中疗前即存在着EB病毒.EBV阳性组放疗后失败的发生率明显高于EBV阴性组,而且外周血与骨髓EBV有很好的相关性,提示外周血EBV的检测在临床上具有判定预后的应用价值.     

鼻咽癌初诊患者EB病毒与2008分期相关性的临床研究

[目的]探讨EB病毒(EBV)与鼻咽癌(NPC)2008分期的相关性;比较EBV IgA抗体滴度与EBV DNA拷贝数在NPC诊断与疗效评价中的差异.[方法]将初诊为NPC的患者98例,在放化疗前予以TNM分期;在放化疗前、后分别监测血清EBV抗体滴度与全血EBV DNA拷贝数.在放化疗后2个月,复查鼻咽部磁共振及全身检查后评价疗效.[结果]治疗前,血清EBV IgA抗体滴度阳性患者28例,全血EBV DNA拷贝数阳性42例.EBV IgA抗体滴度及全血EBV DNA拷贝数在不同TNM分期上差异均无统计学意义(P=-0.189,P=-0.074).全血EBV DNA拷贝数在Ⅲ期与Ⅱ期、Ⅲ期与Ⅳa期间比较,差异均具有统计学意义(P=0.024,P=0.022).放疗后,全血EBV DNA拷贝数转阴率高;但在提示转移或复发方面,EBV IgA抗体与EBV DNA比较二者差异无统计学意义(P=-0.095).[结论]血清EBV IgA抗体与NPC 2008分期无相关性.全血EBV DNA拷贝数与NPC 2008分期具有一定相关性.全血EBV DNA拷贝数转阴率高,优于EBV IgA抗体,在一定程度上能有效评价治疗疗效及监测病情变化.     

Vesicle-bound EBV-BART13-3p miRNA in circulation distinguishes nasopharyngeal from other head and neck cancer and asymptomatic EBV-infections

Cell-free microRNA (miRNA) in biofluids released by tumors in either protein or vesicle-bound form, represent promising minimally-invasive cancer biomarkers. However, a highly abundant non-tumor background in human plasma and serum complicates the discovery and detection of tumor-selective circulating miRNAs. We performed small RNA sequencing on serum and plasma RNA from Nasopharyngeal Carcinoma (NPC) patients. Collectively, Epstein Barr virus-encoded miRNAs, more so than endogenous miRNAs, signify presence of NPC. However, RNAseq-based EBV miRNA profiles differ between NPC patients, suggesting inter-tumor heterogeneity or divergent secretory characteristics. We determined with sensitive qRT-PCR assays that EBV miRNAs BART7-3p, BART9-3p and BART13-3p are actively secreted by C666.1 NPC cells bound to extracellular vesicles (EVs) and soluble ribonucleoprotein complexes. Importantly, these miRNAs are expressed in all primary NPC tumor biopsies and readily detected in nasopharyngeal brushings from both early and late-stage NPC patients. Increased levels of BART7-3p, BART9-3p and particularly BART13-3p, distinguish NPC patient sera from healthy controls. Receiver operating characteristic curve analysis using sera from endemic NPC patients, other head and neck cancers and individuals with asymptomatic EBV-infections reveals a superior diagnostic performance of EBV miRNAs over anti-EBNA1 IgA serology and EBV-DNA load (AUC 0.87–0.96 vs 0.86 and 0.66 respectively). The high specificity of circulating EBV-BART13-3p (97%) for NPC detection is in agreement with active secretion from NPC tumor cells. We conclude EV-bound BART13-3p in circulation is a promising, NPC-selective, biomarker that should be considered as part of a screening strategy to identify NPC in endemic regions.     

Profiling of Epstein-Barr virus-encoded microRNAs in nasopharyngeal carcinoma reveals potential biomarkers and oncomirs

BACKGROUND:

Epstein‐Barr virus (EBV) microRNAs are abundant in nasopharyngeal carcinoma (NPC) tumors. With recent advances in serum microRNA detection, the distinct presence of EBV microRNAs in serum could aid in screening endemic regions for NPC. A proposed network of genes targeted by these microRNAs could also shed light on EBV‐associated tumorigenesis.

METHODS:

MicroRNA microarray profiling of 5 paired NPC biopsies was followed by validation of 12 up‐regulated EBV microRNAs (BART1‐3p, 2‐5p, 5, 6‐5p, 6‐3p, 7, 8, 9, 14, 17‐5p, 18‐5p, 19‐3p) in 15 additional cases by real‐time polymerase chain reaction. Tumor (cellular) and serum microRNA copy numbers from the same 15 patients were correlated. Expression of the same microRNAs were also examined in EBV‐positive cell lines C666 and NP460hTERT+EBV. Bioinformatic tools helped predict cellular target genes, which were later confirmed by gene expression analysis.

RESULTS:

The authors' high‐throughput approach shows that EBV microRNAs are generally more up‐regulated than microRNAs of human origin. Twenty‐nine of 39 EBV microRNAs were significantly up‐regulated in tumor versus their nontumor biopsies (P < .05). Upon successfully validating 12 selected EBV microRNAs in 15 additional paired NPC cases, the authors found that their distinct presence in the serum of NPC patients positively correlated with cellular copy numbers of EBV microRNAs. Further investigation of potential EBV microRNA target genes revealed inhibition of tumor suppressor genes (eg, PTEN) and extensive deregulation of several pathways frequently involved in NPC (eg, Wnt signaling).

CONCLUSIONS:

Increasing knowledge of host‐virus interaction via microRNAs may provide feasible explanations underlying NPC tumorigenesis along with the development of biomarkers for screening high‐risk populations. Cancer 2012;. © 2011 American Cancer Society.     

Gold nano-particles (AuNPs) carrying anti-EBV-miR-BART7-3p inhibit growth of EBV-positive nasopharyngeal carcinoma

Epstein-Barr virus (EBV) infection is a major etiological factor for nasopharyngeal carcinoma (NPC). Several EBV-encoded BART miRNAs have been associated with viral latency, immune escape, cell survival, cell proliferation and apoptosis. Here, we report that EBV-miR-BART7-3p, an EBV-encoded BART miRNA highly expressed in NPC, was correlated with cell-cycle progression in vitro and increased tumor formation in vivo. This viral miRNA stimulated the PTEN/PI3K/Akt pathway and induced c-Myc and c-Jun. Knockdown of PTEN mimicked EBV-miR-BART7-3p-induced tumorigenic phenotype. Based on these results, we conducted a therapeutic experiment by using gold nano-particles (AuNPs) carrying anti-EBV-miR-BART7-3p. Silencing of EBV-miR-BART7-3p reduced tumor growth in animal model. We conclude that EBV-miR-BART7-3p favors carcinogenesis, representing a potential target for miRNA-based therapy.     

Distribution of Epstein-Barr viral load in serum of individuals from nasopharyngeal carcinoma high-risk families in Taiwan

The utility of EBV load as a tumor marker in nasopharyngeal carcinoma (NPC) patients suggests that it might also serve as a screening test for individuals who are at high risk for developing NPC. We previously demonstrated that unaffected individuals from high-risk families had elevated anti-EBV antibody levels compared to community controls. In this study, we measured EBV load using 2 different real-time PCR assays (targeting BamH1W and polymerase gene sequences, respectively) carried out in 2 independent research labs in serum samples from 19 untreated NPC cases, 11 healthy community controls and 100 unaffected individuals from families in which 2 or more individuals were affected with NPC. EBV genomes were detectable in 68% of NPC cases by the EBV BamH1W assay and in 74% by the EBV polymerase assay (kappa = 0.64). Patients with stage III or IV disease had significantly higher EBV load compared to those with stage I or II disease (p = 0.008). EBV DNA was detected in a single community control sample by the EBV BamH1W assay and in none of the samples by the EBV polymerase assay. Only one of 100 unaffected family members tested positive by both assays. An additional 14 were positive by only one of the 2 EBV load assays used and usually in only one of the duplicate wells tested, all with very low viral loads (3-50 copies/ml). In addition, EBV load did not correlate with EBV serology results (anti-VCA, anti-DNase, anti-EBNA-1) among these unaffected family members. In conclusion, our study suggests limited clinical utility of the EBV load test, in its current configuration, to screen individuals from high-risk families. Should a more sensitive or specific molecular assay be developed that is capable of detecting and distinguishing tumor-derived EBV genomes or gene products from true negatives, it could be evaluated as a possible screening tool for asymptomatic and early-stage NPC.     

EBV⁃miR⁃BART17⁃5p对鼻咽癌细胞增殖及迁移功能的调控及其作用机制

目的 探讨EB病毒（epstein⁃barr virus,EBV）编码的EBV⁃miR⁃BART17⁃5p对鼻咽癌细胞增殖及迁移的调控及其机制。方法 将慢病毒Vector control和 EBV⁃miR⁃BART17⁃5p mimic分别感染至HK1⁃EBV细胞,记为Vector control组和Mimic组,采用RT⁃qPCR验证慢病毒感染效果;EdU和CCK⁃8实验分别检测细胞的增殖能力;平板克隆形成实验检测细胞的集落形成能力;细胞划痕实验及Transwell 细胞迁移实验检测细胞的迁移能力;Western blot检测EBV⁃miR⁃BART17⁃5p靶基因PTEN的蛋白表达水平。结果 与Vector control组比较,Mimic组HK1⁃EBV细胞中EBV⁃miR⁃BART17⁃5p表达量显著升高（P<0.001）,细胞增殖、集落形成和迁移能力均明显增强（均P<0.05）,PTEN蛋白表达水平显著降低（P<0.001）。结论 EBV⁃miR⁃BART17⁃5p能调控鼻咽癌细胞HK1⁃EBV增殖和迁移,其作用机制可能与EBV⁃miR⁃BART17⁃5p反向调控肿瘤抑制因子PTEN表达有关。