Objective: To study the role of Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) in nasopharyngeal carcinoma (NPC) cell growth and metastasis in vivo by recombinant adeno-associated virus (rAAV)-mediated RNA interference (RNAi).
Methods: Specific small hairpin RNA (shRNA) targeting EBV-LMP-1 gene was designed and synthesized to construct two rAAV vectors rAAV-shRNA-LMP-1 and rAAV-EGFP. The multiplicity of infection (MOI) was confirmed using different titers of rAAV-EGFP to transfect the NPC cell line C666-1. The C666-1 cells were transfected by rAAV-shRNA-LMP-1 at the optimal MOI titer and the inhibition efficiency of the target gene expression was determined with RT-PCR. The C666-1 cells with RNAi of LMP-1 gene were injected into nude mouse liver via laparotomy to establish the animal model of hepatic and lung metastases of NPC cells. The metastases of the C666-1 cells in the liver and lungs were observed to assess the effect of LMP-1 gene silencing on the tumorigenic and metastatic potentials of the cells in vivo.
Results: The transfection efficiency of 5 x 10(4) virus genome/cell rAAV-EGFP exceeded 95%. The expression of the target gene was suppressed by over 90% as shown by RT-PCR after transfection with rAAV-shRNA-LMP-1 at 5 x 10(4) virus genome/cell. Animal experiments showed that compared with rAAV-EGFP, rAAV-shRNA-LMP-1 transfection did not reduce the primary tumor volume implanted into the liver, but significantly inhibited the intrahepatic and lung metastases of the NPC cells.
Conclusion: LMP-1 expression can be suppressed effectively by rAAV-mediated RNAi, and LMP-1 suppression does not obviously affect the tumor cell growth but can inhibit their metastasis in vivo.