Suppression Of EBV-encoded Latent Membrane Protein-1 By RNA Interference Inhibits Metastasis Potential Of NPC Cells

Objective: To evaluate the application of RNA interference for silencing latent membrane protein-l(LMP-l) expression in EBV-positive NPC cell line C666-1. Analysis the inhibiting effects of LMP-1 silencing on NPC cell metastasis. Methods: Four synthesized siRNAs were transfected into C666-1 cell using OligofectamineTMreagent, and LMP-1 inhibition effect was determined by semi-quantitative RT-PCR. DNA vectors were constructed as shRNA vector according to the critical target region of LMP-1. A LMP-1 silenced sub-clone named C611L was isolated by standard calcium phosphate transfection procedure and G418 selection. Then, effect of LMP-1 inhibition on its metastatic properties was evaluated by eelL adhesion, invasion and wound healing assays. Results: The the critical target sequence was identified from the 4 candidates. Single dose of this siRNA caused nearly 90% loss of LMP-1 mRNA in C666-1 cell. The specific inhibition could last for 96 hours following a re-transfectinn. Moreover, shRNA vector also mediated stable RN.Ai against LMP L in C666-1. Compared with control groups, loss of LMP-1 significantly altered cell motility, substratum adhesion and transmembrane invasion ability. Conclusions: These results provided solid testimony thai EBV-encoded latent membrane protein-1 is vulnerable to RNA interference. Stable suppression of LMP-1 by shRNA vector can inhibit metastatic properties of- 4 -NPC cell, which indicated that DNA vector-based RNA interference , as a powerful tool, is benefit to further investigating LMP-1 functions in NPC. It also suggested a novel RNAi-mediated therapeutic strategy for EBV-reLated NPCs.