Yoshizaki Tomokazu, Endo Kazuhira, Ren Qingchun, Wakisaka Naohiro, Murono Shigeyuki, Kondo Satoru, Sato Hiroshi, Furukawa Mitsuru
Division of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan.
Auris Nasus Larynx. 2007 Mar;34(1):73-8. doi: 10.1016/j.anl.2006.09.025. Epub 2006 Nov 28.
Epstein-Barr virus (EBV) is a causative agent of nasopharyngeal carcinoma (NPC), and EBV gene expression is considered to be closely associated with the pathogenesis of NPC. Among EBV genes expressed in NPC, EBV-encoded non-polyadenylated RNAs, termed EBERs, are the most abundant transcripts of EBV in NPC. However, the role of EBERs still remains unclear. This study was designed to investigate the relevance of EBERs to the oncogenesis of NPC.
Two types of EBERs expression vectors (EBERs-high-expression vector and EBERs-low-expression vector) were constructed and transfected into EBV-negative cells, MDCK or EBV-negative clones of NPC-KT cells. Then, malignant transformation, represented by anchor independent growth, was evaluated between the EBERs-transfected cells and EBERs-negative cells using a soft agar colony formation assay. Apoptosis was induced by serum deprivation (0.1% concentration of fetal bovine serum) and interferon-alpha (IFN-alpha) (500 U/ml) treatment. Cell viability was evaluated with a trypan blue exclusion test. The activation of cellular transcriptional factor NF-kappaB was studied with the IL-8 promoter sequence using a luciferase reporter assay.
EBERs-high-expression vector-transfected MDCK cells showed enhanced growth ability in soft agar compared with either EBERs-low-expression vector-transfected MDCK cells or EBERs-untransfected MDCK cells. However, they did not show the acquisition of any anti-apoptotic potential against either IFN-alpha or serum deprivation. Introduction of EBERs-low-expression vector into MDCK cells did not show anchor independent growth characteristics. Neither EBV-negative NPC-KT cells nor MDCK cells transfected with EBERs-high-expression vector showed any difference from EBERs-untransfected EBV-negative NPC-KT cells. Introduction of EBERs into MDCK cells did not transactivate the IL-8 promoter, indicating that neither NF-kappaB nor AP-1 was activated by EBERs.
EBERs are believed to induce the initial transformation of epithelial cells, thus contributing to the oncogenesis of NPC. Expression of abundant EBERs is considered to be critical for this transforming property of EBERs.
爱泼斯坦-巴尔病毒（EBV）是鼻咽癌（NPC）的致病因子，EBV基因表达被认为与NPC的发病机制密切相关。在NPC中表达的EBV基因中，EBV编码的非多聚腺苷酸化RNA（称为EBERs）是NPC中EBV最丰富的转录本。然而，EBERs的作用仍不清楚。本研究旨在探讨EBERs与NPC肿瘤发生的相关性。
构建两种类型的EBERs表达载体（EBERs高表达载体和EBERs低表达载体），并将其转染到EBV阴性细胞、MDCK或NPC-KT细胞的EBV阴性克隆中。然后，使用软琼脂集落形成试验评估EBERs转染细胞和EBERs阴性细胞之间以锚定非依赖性生长为代表的恶性转化。通过血清剥夺（0.1%浓度的胎牛血清）和α干扰素（IFN-α）（500 U/ml）处理诱导细胞凋亡。用台盼蓝排斥试验评估细胞活力。使用荧光素酶报告基因试验通过IL-8启动子序列研究细胞转录因子NF-κB的激活。
与EBERs低表达载体转染的MDCK细胞或未转染EBERs的MDCK细胞相比，EBERs高表达载体转染的MDCK细胞在软琼脂中显示出增强的生长能力。然而，它们对IFN-α或血清剥夺均未显示出任何抗凋亡潜能。将EBERs低表达载体导入MDCK细胞未显示出锚定非依赖性生长特征。用EBERs高表达载体转染的EBV阴性NPC-KT细胞和MDCK细胞与未转染EBERs的EBV阴性NPC-KT细胞均未显示出任何差异。将EBERs导入MDCK细胞未激活IL-8启动子，表明EBERs未激活NF-κB或AP-1。
EBERs被认为可诱导上皮细胞的初始转化，从而促进NPC的肿瘤发生。丰富的EBERs表达被认为对EBERs的这种转化特性至关重要。
