Kanda Teru, Miyata Mamiko, Kano Makoto, Kondo Satoru, Yoshizaki Tomokazu, Iizasa Hisashi
Division of Microbiology and Oncology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Japan
Division of Microbiology and Oncology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Japan.
J Virol. 2015 Mar;89(5):2684-97. doi: 10.1128/JVI.03189-14. Epub 2014 Dec 17.
The Epstein-Barr virus (EBV) encodes its own microRNAs (miRNAs); however, their biological roles remain elusive. The commonly used EBV B95-8 strain lacks a 12-kb genomic region, known as BamHI A rightward transcripts (BART) locus, where a number of BART miRNAs are encoded. Here, bacterial artificial chromosome (BAC) technology was used to generate an EBV B95-8 strain in which the 12-kb region was fully restored at its native locus [BART(+) virus]. Epithelial cells were stably infected with either the parental B95-8 virus or the BART(+) virus, and BART miRNA expression was successfully reconstituted in the BART(+) virus-infected cells. Microarray analyses of cellular gene expression identified N-myc downstream regulated gene 1 (NDRG1) as a putative target of BART miRNAs. The NDRG1 protein was barely expressed in B cells, highly expressed in epithelial cells, including primary epithelial cells, and strongly downregulated in the BART(+) virus-infected epithelial cells of various origins. Although in vitro reporter assays identified BART22 as being responsible for the NDRG1 downregulation, EBV genetic analyses revealed that BART22 was not solely responsible; rather, the entire BART miRNA cluster 2 was responsible for the downregulation. Immunohistochemical analyses revealed that the expression level of the NDRG1 protein was downregulated significantly in EBV-positive nasopharyngeal carcinoma specimens. Considering that NDRG1 encodes an epithelial differentiation marker and a suppressor of metastasis, these data implicate a causative relationship between BART miRNA expression and epithelial carcinogenesis in vivo.
EBV-related epithelial cancers, such as nasopharyngeal carcinomas and EBV-positive gastric cancers, encompass more than 80% of EBV-related malignancies. Although it is known that they express high levels of virally encoded BART miRNAs, how these miRNAs contribute to EBV-mediated epithelial carcinogenesis remains unknown. Although a number of screenings have been performed to identify targets of viral miRNAs, many targets likely have not been identified, especially in case of epithelial cell infection. This is the first study to use EBV genetics to perform unbiased screens of cellular genes that are differentially expressed in viral miRNA-positive and -negative epithelial cells. The result indicates that multiple EBV-encoded miRNAs cooperatively downregulate NDRG1, an epithelial differentiation marker and suppressor of metastasis. The experimental system described in this study should be useful for further clarifying the mechanism of EBV-mediated epithelial carcinogenesis.
爱泼斯坦-巴尔病毒（EBV）编码自身的微小RNA（miRNA）；然而，它们的生物学作用仍不清楚。常用的EBV B95-8毒株缺少一个12kb的基因组区域，即BamHI A向右转录本（BART）基因座，该基因座编码多个BART miRNA。在此，利用细菌人工染色体（BAC）技术构建了一个EBV B95-8毒株，其中12kb区域在其天然基因座处完全恢复[BART（+）病毒]。上皮细胞用亲本B95-8病毒或BART（+）病毒稳定感染，并且在BART（+）病毒感染的细胞中成功重建了BART miRNA表达。细胞基因表达的微阵列分析确定N- myc下游调控基因1（NDRG1）为BART miRNA的推定靶标。NDRG1蛋白在B细胞中几乎不表达，在包括原代上皮细胞在内的上皮细胞中高表达，并且在各种来源的BART（+）病毒感染的上皮细胞中强烈下调。虽然体外报告基因分析确定BART22负责NDRG1的下调，但EBV遗传学分析表明BART22并非唯一负责；相反，整个BART miRNA簇2负责下调。免疫组织化学分析显示，NDRG1蛋白的表达水平在EBV阳性鼻咽癌标本中显著下调。鉴于NDRG1编码一种上皮分化标志物和转移抑制因子，这些数据暗示了BART miRNA表达与体内上皮癌发生之间的因果关系。
EBV相关的上皮癌，如鼻咽癌和EBV阳性胃癌，占EBV相关恶性肿瘤的80%以上。虽然已知它们表达高水平的病毒编码的BART miRNA，但这些miRNA如何促进EBV介导的上皮癌发生仍不清楚。尽管已经进行了许多筛选以鉴定病毒miRNA的靶标，但许多靶标可能尚未被鉴定，特别是在上皮细胞感染的情况下。这是第一项利用EBV遗传学对病毒miRNA阳性和阴性上皮细胞中差异表达的细胞基因进行无偏筛选的研究。结果表明，多种EBV编码的miRNA协同下调NDRG1，NDRG1是一种上皮分化标志物和转移抑制因子。本研究中描述的实验系统应有助于进一步阐明EBV介导的上皮癌发生机制。
