The Effect Of LMP1Over Expression On The Metastasis Of Human Nasopharyngeal Carcinoma CNE2Cells And Its Mechanism

Objective Nasopharyngeal carcinoma (NPC) is one of the most commoncancers in South China. Epstein-Barr Virus (EBV) is the first human virusidentified to be associated with NPC. The EBV-encoded Latent membraneprotein1(LMP1) displays potent oncogenic properties in some epithelial cells.LMP1expression has an effect on the cell proliferation, apoptosis and migration.LMP1functions as a constitutively active tumor necrosis factor receptor (TNFR)engaging a multitude of signaling pathways which is very complex and themechanism is not very clear. The eukaryotic expression plasmidpEGFP-C1-LMP1carried with the enhanced green fluorescent protein (EGFP)reporter gene was constructed and transiently transfected into CNE2cells toinvestigate the effect of LMP1over expression on the metastasis of humannasopharyngeal carcinoma CNE2cells. The possible mechanism involved inwill be discussed and the theory and experiment will be enriched.Methods Acquire the cDNA segment encoding LMP1from human nasopharyngeal carcinoma cell line C15and cloned into a eukaryote expressionvector pEGFP-C1after double digestion. The recombinant vector was identifiedby restriction enzyme analysis and nucleotide sequence determination. Theeukaryotic expression plasmid pEGFP-C1-LMP1was transiently transfectedinto CNE2cells by lipofectamine2000. The location and expression of LMP1was detected by laser scanning confocal microscope. Cell viability and cell cyclewere determined by CCK-8and flow cytometry. Migration and invasion assaywere utilized to analyze the metastasis of CNE2in vitro. The expression ofLMP1and Rac1were examined by real-time PCR and Western blot,respectively.Results The LMP1was correctly inserted into pEGFP-C1, the sequencingresults were100%identical with those reported in GenBank. GFP-LMP1couldbe detected mainly in cell membrane and partly in cytoplasm of CNE2. Theviability of the LMP1groups was higher than that of the GFP groups on thesixth day(p＜0.05). There was a difference between the control groups and theLMP1groups on the G0/G1and S phase of the cell cycle. The capacity of cellmigration and invasion were weaken after transfection. The optical density ofGFP control groups and pEGFP-C1-LMP1groups in the invasion assay were0.1160±0.009and0.066±0.0097, which had statistics significant (p＜0.001).The number of migration cells were99.3±13.05and63.0±8.54(p＜0.05) incontrol groups and pEGFP-C1-LMP1groups, respectively. Compared with the control groups, the LMP1mRNA in pEGFP-C1-LMP1groups increaseddramatically while the Rac1mRNA decreased sharply in real-time PCR(p<0.05). The result of western blot indicated that LMP1protein could not beexamined in GFP control groups but could be checked inpEGFP-C1-LMP1groups, in which the protein of LMP1rised gently from24hafter the transiently transfection, and reached an apex at48h, after that it beganto fall after72h. A decrease level of Rac1protein was observed in GFP-LMP1group during these three periods (p<0.05).Conclusions Recombinant plasmid pEGFP-C1-LMP1was constructedsuccessfully. Transiently transfected of pEGFP-C1-LMP1in vitro inhibited themetastasis of human nasopharyngeal carcinoma cells and down-regulated Rac1.