Oncogenic role of Epstein-Barr virus-encoded small RNAs (EBERs) in nasopharyngeal carcinoma.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Yoshizaki
Tomokazu
T
Division of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan. tomoy@med.kanazawa-u.ac.jp
Endo
Kazuhira
K
Ren
Qingchun
Q
Wakisaka
Naohiro
N
Murono
Shigeyuki
S
Kondo
Satoru
S
Sato
Hiroshi
H
Furukawa
Mitsuru
M
D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D014408:Biomarkers, Tumor; D002277:Carcinoma / Q000188:drug therapy / Q000821:virology*; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization; D016898:Interferon-alpha / Q000494:pharmacology / Q000627:therapeutic use; D016328:NF-kappa B; D009303:Nasopharyngeal Neoplasms / Q000188:drug therapy / Q000821:virology*; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000187:drug effects / Q000235:genetics*; D018808:Transcription Factor AP-1; D014162:Transfection; D014412:Tumor Virus Infections / Q000188:drug therapy / Q000235:genetics / Q000821:virology
D000998:Antiviral Agents; D014408:Biomarkers, Tumor; C099537:Epstein-Barr virus encoded RNA 1; C106732:Epstein-Barr virus encoded RNA 2; D016898:Interferon-alpha; D016328:NF-kappa B; D012367:RNA, Viral; D018808:Transcription Factor AP-1