Transcriptional Activation Of EIF4E Is Induced By Lmp1and The Activation Regulates The Proliferation, Migration And Invasion Of Human Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a unique head and neck cancerbecause of its close relationship with the infection of Epstein-Barr virus (EBV).NPC features early metastasis (about60%of the firstly diagnosed cases occurmetastasis), easy recurrence and low5-year survival rate (under50%). The earlymetastasis is one of the important factors that harm the health and life of theNPC patients. However, the molecular mechanism of early metastasis of NPC isstill unclear. It has been one of the hot topics of research around the world.The novel oncogene eIF4E (eukaryotic translation initiation factor4E)functions as a key regulator of cap-dependent protein translation, and plays arate-limiting role in protein translation of multiple oncogenes and growth factors.The abnormal activation of eIF4E initiates the mRNA translation of selectedoncogenes and growth factors. Clinical studies showed that eIF4Eover-expresses in carcinomas such as lung carcinoma, breast carcinoma andesophageal carcinoma, and ect., and this over-expression closely relates to highinvasion and metastasis of these cancers. Our preliminary study demonstratedthat eIF4E over-expresses in100%of metastatic NPC, indicating an importantrole of eIF4E in invasion and metastasis of NPC. Zhang J et al showed that eIF4E immunohistochemically over-expresses in NPC tissues and the eIF4Eexpression is positively associated with LMP1expression and5-year survivalrates of the patients. Recently we performed a meta-analysis of718cases ofNPC reported worldwide and confirmed that LMP1induces NPC metastasis.Based on the previous studies, we transfected LMP1expression plasmid intoCNE2cells and found that eIF4E transcription is strongly increased. Therefore,we suggest,“the transcriptional activation of eIF4E induced by LMP1promotescell proliferation, migration, invasion and metastasis in NPC.”In comparison with the transcriptional level, the translational level is muchmore closed to and effective on the oncogenesis and development of cancer. Sofar, the study on regulation of oncogenes in translational level in NPC is stillvery rare. Latent membrane protein1(LMP1), a transmembrane protein,initiates a lot of oncogenesis signals through induction of transcription. ForLMP1how to transmit the oncogenesis signals to the effector’s protein, it needsurgent study. This paper shall study the mechanism of transcriptional activationof eIF4E and the activation that regulates the proliferation, migration andinvasion of NPC, in order to elucidate the mechanism of NPC metastasis and tosupply a new parameter for the prognosis of NPC and a useful target point forNPC treatment.Objectives1. To investigate the mechanism of eIF4E overexpression in NPC cells2. To study the mechanism of that LMP1activates the transcription of eIF4E inNPC cell3. To study the transcriptionally activated eIF4E that effects on the proliferation,migration and invasion of NPC cellsMethods1. Cell linesCNE1(well differentiated nasopharyngeal carcinoma cell line, EBV-)CNE2(poor differentiated nasopharyngeal carcinoma cell line, EBV-) B95-8(marmoset leukocyte line immortalized by EBV extracted from ahuman leukemia, EBV+)CNE1and CNE2were for the object of study, B958was for the control.2. Experimental methods①The pGL3Basic-eIF4E recombinant vector (Luciferase reporter gene vectorcarrying1500bp sequence of eIF4E promoter), eIF4E expressionrecombinant vector carrying full-length eIF4E, eIF4E-shRNA recombinantvector, LMP1-shRNA recombinant vector and c-myc-shRNA recombinantvector were constructed by the sub-clone technique.②The mRNA expression levels of eIF4E, LMP1and c-myc in NPC cells andB95-8cell were determined by qPCR or qReal-time PCR.③The protein expression levels of LMP1and eIF4E in NPC cells weredetermined by western blotting.④The promoter activity of eIF4E was evaluated by the dual luciferase reporterassays.⑤The stable eIF4E interference cell line (eIF4E-CNE2siEp) was establishedby transfection of eIF4E-shRNA.⑥Proliferation, cell cycle progression, migration and invasion of NPC cellswere tested by CCK8method, flow cytometry, migration assay and invasionassay, respectively.Results1. eIF4E is transcriptionally activated by LMP1in NPC cell lines①After the transient transfection of LMP1, eIF4E mRNA level in CNE1cellsexpressing LMP1was1.6times as many as that in the control (P <0.01).②After the transient transfection of LMP1, eIF4E mRNA level in CNE2cellsexpressing LMP1was3.0times as many as that in the control (P <0.01),which was significantly higher than in CNE1cells.③After the stable transfection of LMP1, eIF4E mRNA level in CNE1cells expressing LMP1was1.7times as many as that in the control (P <0.01),which was consistent with the change of the transient transfection.④In the dosage gradient transfection of LMP1expression vector for CNE2cells, LMP1and eIF4E mRNA levels were significantly and coordinatelyincreasing as the transfected dose of LMP1expression vector was raising.⑤The shRNA-LMP1vectors were successfully constructed. The rate of LMP1knockdown in NPC cells was above65.0%.⑥After the transient transfection of shRNA-LMP1vectors into B958cells, themRNA levels significantly decreased by70.0%for LMP1and72.0%foreIF4E.⑦After the transient transfection of LMP1, eIF4E protein level in CNE1cellsexpressing LMP1was2.8times as many as that in the control (P <0.01),which was consistent with the change in mRNA level.⑧After the transient transfection of LMP1, eIF4E protein level in CNE2cellsexpressing LMP1was4.7times as many as that in the control (P <0.01),which was consistent with the change in mRNA level, and significantlyhigher than in CNE1cells.⑨eIF4E protein level in LMP1stably transfected CNE1cells (CNE1/LMP1)was3.0times as many as that in the control (P <0.01), which was consistentwith the change in mRNA level.2. The mechanism of transcriptional activation of eIF4E induced by LMP1inNPC cells①The pGL3Basic-eIF4E (Luciferase reporter gene vector containing sequenceof eIF4E promoter) was successfully constructed.②After the transient transfection of LMP1, the activity of eIF4E promoter inthe CNE1cells expressing LMP1was20.0times as many as that in thecontrol (P <0.01).③After the transient transfection of LMP1, the activity of eIF4E promoter in the CNE2cells expressing LMP1was47.0times as many as that in thecontrol (P <0.01), which was significantly higher than in CNE1cells.④After the stable transfection of LMP1, the mRNA expression of c-myc andeIF4E in CNE1/LMP1cells were8.2and1.8times as many as that in thecontrol (P <0.01).⑤After the knock-down of LMP1, the mRNA levels were decreased by55.0%for LMP1and60.0%for eIF4E in the B958cells, compared with the control(P <0.01).⑥Under the over-expression of LMP1, the knock-down of c-myc led themRNA levels to decrease by35.0%for eIF4E and30.0%for LMP1,compared with the control (P <0.05).⑦The eIF4E expression vector (pEGFPN1-eIF4E) was successfullyconstructed.⑧After the transient transfection of pEGFPN1-eIF4E, the mRNA levels inCNE1cells were increased by6.0times for LMP1and18.0times for eIF4E,compared with the control (P <0.01).3. Effect of the transcriptional activation of eIF4E induced by LMP1onproliferation, migration and invasion of NPC cells①The shRNA-eIF4E vector was successfully constructed, with an inhibition ofeIF4E by70.0%in CNE2cells.②CNE2siEp cell line was successfully established, with eIF4E proteinknock-down by70.0%.③After the transient transfection of LMP1, the proliferation, migration andinvasion of CNE1cells increased by1.1,2.4and1.4times of those in thecontrol, respectively (P <0.05).④After the transient transfection of LMP1, the proliferation, migration andinvasion of CNE2cells increased by1.1,3.0and1.6times of those in thecontrol, respectively (P <0.05), among which, the changes in migration and invasion were more obviously in CNE2cells than CNE1cells.⑤After the stable transfection of LMP1in CNE1cells, the cell cycle appearedabnormal with proportion declined in G0/G1phase and raised in S phase,compared with the control (P <0.05). The migration and invasion of thetransfected cells increased by2.0and1.5times of those in the control,respectively (P <0.01).⑥After the stable knock-down of eIF4E, the CNE2siEp cells appearedproliferation inhibition, prolonged mass doubling time, G1phase blockage,and S phase decline. The migration and invasion of the eIF4E knockdowncells decreased by41.0%and27.0%, respectively, compared with thecontrol (P <0.05).⑦Under the over-expression of LMP1, the proliferation, migration andinvasion in the eIF4E knock down CNE1cells decreased by10.0%(P <0.05),40.0%(P <0.01) and50.0%(P <0.01), respectively, compared withthe control.⑧Under the over-expression of LMP1, the proliferation, migration andinvasion in the eIF4E knock down CNE2cells decreased by10.0%(P <0.05),63.0%(P <0.01) and37.0%(P <0.01), respectively, compared withthe control.Conclusion①To our best knowledge, this is the first time for us to discover that thetranscriptional activation of eIF4E is induced by LMP1in NPC. TheLMP1induction could be the direct factor that causes eIF4Eover-expression in NPC. The transcriptional activation of eIF4E results indirect rise of eIF4E protein, indicating that the transcriptional activation ofeIF4E plays an important role in the initiation and development of NPC.The transcriptional activation of eIF4E is significantly higher in the poordifferentiated NPC (accounting for over90.0%of the clinical NPCs thatare associated with EBV infection) than in the well differentiated NPC (accounting for only5.0%of the clinical NPCs that are not associated withEBV infection), indicating that the transcriptional activation of eIF4Eparticipates in the oncogenesis of EBV. In the B95-8(marmoset leukemiacell line), eIF4E is also induced by LMP1, indicating that thetranscriptional activation of eIF4E induced by LMP1is a commonphenomenon in the EBV related cancer.②The mechanism of the transcriptional activation of eIF4E induced by LMP1in NPC is that LMP1induces c-myc over-expression and theover-expressed c-Myc in turn activates the transcription of eIF4E bydirectly binding to the promoter sequence of eIF4E. An unsuspectedfinding is that the over-expression of eIF4E promotes LMP1mRNA inNPC, indication that eIF4E could induce LMP1transcription, and both smight form an LMP1/eIF4E positive feedback loop. However, the detailmechanism of the feedback loop is unclear and needs further study.③The transcriptional activation of eIF4E induced by LMP1significantlyenhances proliferation, migration and invasion of NPC cells. Theenhancement is much stronger in the poor differentiated NPC cells than inthe well-differentiated NPC cells and to the migration and invasion than tothe proliferation and cell cycle of NPC cells.④eIF4E might be a critical point in the pathway for transmitting theoncogenesis signal of LMP1. Therefore, eIF4E is a potential target pointthat definitely values further study in order to control NPC metastasis.