Background & objective: Epstein-Barr virus (EBV) A73 gene encodes for a main mRNA of BamHI A rightward transcripts (BARTs) family which is widely transcribed in EBV-carrying cells and tumors. This study was designed to investigate the expression of A73 mRNA in nasopharyngeal carcinoma(NPC) from Guangdong region,from which A73 coding sequence(CDS) was cloned and introduced into epithelial cell line to observe the subcellular fraction of its encoding protein.
Methods: DNA and total RNA were isolated from NPC and noncarcinoma nasopharyngeal tissues,peripheral blood lymphocyte of NPC, SUNE1, B95.8, Raji, and BJAB cells; Nested PCR was designed to identify the EBV infection by amplifying the W sequence of EBV DNA in above- mentioned samples, and RT-PCR was used to analyze the transcription of A73 gene in the same samples. A73 CDS amplifying from a NPC tissue was cloned into pEGFP-C2 on special direction. After being transfected with this recombinant plasmid by lipofectin mediation, human embryo kidney(HEK293) cells were analyzed by RT-PCR and laser confocal microscope to observe the expression of A73 gene and its subcellular fraction.
Results: Except for BJAB cells, EBV W sequence existed in all samples. A73 transcribed in 79.63%(43/54)of NPC and 4%(1/25)of noncarcinoma nasopharynx biopsies between which the difference was significant, and no A73 mRNA was detected in all peripheral blood lymphocyte of NPC.A73 expressed at lower levels in B95.8 and Raji than in SUNE1. The constructed recombinant plasmid expressed stably in HEK293 cells, in which the fusion protein was found to be mainly cytoplasmic.
Conclusion: EBV-A73 gene expresses widely in NPC tissue from Guangdong region and its encoding protein can stably express in cytoplasm of epithelial cell in vitro,suggesting A73 is highly correlated with the occurrence and development of NPC, and A73 gene may take part in the signal conduction related to epithelial oncogenesis as a signal molecule.