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

Abstract

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 (chi(2) = 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 BcLF1 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.

Figure 1

In situ hybridization for EBER1 in gastric carcinoma tissue. A: In situ EBER1 hybridization with antisense probes. Strong signals were observed in the nuclei of all tumor cells; B: In situ EBER1 hybridization with sense probes; C: Hematoxylin/eosin (H&E) staining in the adjacent pair of EBER1 in situ hybridization. original magnification ×200.

Figure 2

RT-PCR and Southern hybridization analysis of EBNA gene transcription using promoters. M: DIG-labeled DNA molecular weight marker VIII (Roche). Lane 1: EBV-positive LCL(positive control); lane 2: EBV-negative Ramos cells(negative control); lanes 3-13: EBV-positive gastric carcinoma samples. GAPDH mRNA was amplified to check pertinent RNA extraction and results were shown by EB staining.

Figure 3

Detection of EBV latent gene expression (A) and EBV lytic gene expression (B) by RT-PCR and Southern hybridization. M: DIG-labeled DNA molecular weight marker VIII (Roche). Lane 1: EBV-positive LCL (positive control); lane 2: EBV-negative Ramos cells (negative control); lanes 3-13: EBV-positive gastric carcinoma samples. GAPDH mRNA was amplified to check pertinent RNA extraction and results were shown by EB staining.