The Mechanism Of Cellular Proliferation Induced By Epstein-barr Virus Encoded Latent Membrane Protein1in Nasopharyngeal Cancer Stem Cell

Objective: The pathogenesy of nasopharyngeal carcinoma is closely related withEpstein Barr-virus (EBV) infection. The latent membrane protein1(LMP1) wasknown for important oncogenic protein encoded by EBV. This study aimed toinvestigate action and mechanism of Nasopharyngeal Cancer Stem Cell SP18proliferation affected by TNF receptor-associated death domains (TRADD) of latentmembrane protein1and provided the experiment datas to reveal tumorigenesismechanism of EBV.Methods: The PA317cell packaging retrovirus RV-LMP1and RV-LMP1TRADDwascultivated. Then, Retrovirus RV-LMP1and RV-LMP1TRADDprepared was respectivelycollected to infect the nasopharyngeal cancer stem cell SP18. The infected cells wereselected by geneticin, and then the cellular clones were mixed together. Theexpression of LMP1and LMP1TRADDprotein was detected by immunocytochemicalstain. The SP18-LMP1cell and SP18-LMP1TRADDcell lines, expressing LMP1andLMP1TRADDprotein, were established. The proliferation of SP18cells affected byLMP1TRADDwas detected by cellular growth curve, colony formation, soft agarformation, and flow cytometry (FCM). Moreover, the expression of differential genesbetween SP18-LMP1cell and SP18-LMP1TRADDcell were detected by gene chips.Then, the expression of part gene was verified by RT-PCR.Results:1.The result of immunocytochemical stain showed that the LMP1andLMP1TRADDprotein expressed in membrane and plasma of SP18-LMP1cell andSP18-LMP1TRADDcell.2. The result of cellular growth curve displayed that thegrowth velocity of SP18-LMP1cell were faster than that of SP18-LMP1TRADD Compared with SP18-LMP1TRADDcell (n=3， p<0.01).3.The result of colonyformation and soft agar formation showed that, Compared with SP18-LMP1TRADDcell,the number of colonies of SP18-LMP1cell were bigger and more than that ofSP18-LMP1TRADD(n=3，p<0.01).4. The result of flow cytometry (FCM) indicatedthat cellular proliferation index of SP18-LMP1cell was increased than ofSP18-LMP1TRADDcell (n=3，p<0.01).5. The genic chip all detected428differentialexpression genes, which they included that the differential expression genes relatedwith cellular proliferation (39genes up-regulation expression and36genesdown-regualtion expression).6. The results of RT-PCR was verified as same as theresults of genic chip detection.Conclusion:1. The TNF receptor-associated death domain (TRADD) of LMP1was importantactivity site to affect SP18cellular proliferation.2. TRADD activing region probably enhanced the ratio of S and G2periods andincreased the cellular proliferation index to affect the SP18cell proliferation.3. TRADD active domain of LMP1carboxy terminal activating region probablyaffected the expression of CYP1A1and NGFR genes to regulate the proliferation ofSP18cell.