Study On The Mechanism About The LMP1 Regulation On Capn4 Promoting The Metastasis Of Nasopharyngeal Carcinoma Cells

Nasopharyngeal carcinoma(NPC)is a typical as well as wicked tumor that occurs on the head and neck,especially in Southeast Asia,Mediterranean countries,and Northern Africa.An vital pathogenic factor that results in NPC is the infection of EB virus(epsteinbarr virus,EBV).LMPl of EBV gene is considered as the most important oncogene and it can promote the proliferation,metastasis and anti-apoptosis of NPC cells.It can phosphorylate NF-kappa B p65 and nuclear translocation.It can also activate AP-1 by inducing ERK1 and JNK cascade reactions to mediate the occurrence and metastasis of NPC.AP-1 can activate Capn4 by combining with Capn4 initiator.Capn4,which belongs to the Calpain family,participates in a lot of physiological courses inclouding the cell proliferation,differentiation and apoptosis.It is also closely related to tumorigenesis,metastasis and cytoskeleton remodeling.The previous study of our group found that Capn4 in NPC was higher than that in normal nasopharyngeal tissue significantly.It was also closely relevant to the infection of EBV as well as the stage of patients with NPC.It could promote the phosphorylation of NF-κB p65 and upregulate the expression of MMP2,which leaded to the metastasis of NPC.In order to explore the key molecules of LMP1 regulating Capn4 promoting NPC metastasis and its mechanism,we further clarify the role of Capn4 in LMP1 signaling pathway and its important significance in promoting the occurrence and metastasis of NPC.Objective:(1)To explore the expression as well as the clinical meaning of Capn4 and LMP1 in NPC.(2)To investigate whether Capn4 took part in the signaling pathway of LMP1 for promoting metastasis of NPC and its underlying molecular mechanism.Methods:(1)The expression of Capn4 and LMP1 in patients with NPC was detected by immunohistochemistry(IHC)and their correlation was analyzed.(2)Kaplan-Meier was used for analysing that whether the expressions of Capn4 and LMP1 had any relevance to disease-free survival(DFS)and distant metastasis-free survival(DMFS)in patients with NPC.(3)The eukaryotic expression vectors of Capn4 and LMP1 were established,and the stably transfected NPC cell line was operated.Real-time PCR and Western blot were adopted to learn the effects of LMP1 on Capn4 expression.(4)The binding site,where transcription factor was acted on Capn4 promoter was predicted on TFSEARCH and its role was explore.(5)Through the construction of LMP1 series deletion mutants and the principle of site-directed mutagenesis,we search for the functional domain of Capn4 expression induced by LMP1.(6)The luciferase report experiment explored the effect of over-expressing Capn4 and LMP1 on the activity expression of NF-kappa B;Western blot was used for detecting the downstream proteins of NF-kappa B.(7)The Transwell migration test was adopted to learn the migration capacity of NPC cells which were treated with Capn4 and LMP1 later;Immunofluorescence was used for studying the rearrangement about actin of NPC cells after Capn4 and LMP1.Results:(1)IHC showed that a high expression of LMP1 Capn4 in NPC tissues and a positive correlation between them.(2)Kaplan-Meier showed that the DFS phase and DMFS phase of NPC patients with positive expression of LMP1 and Capn4 were significantly lower than those NPC patients of negative expression.(3)In NPC cell lines that overexpress LMP1,Capn4 significantly increased protein levels and m RNA levels;LMP1 transcription enhanced Capn4 promoter activity in a dose-dependent manner.(4)AP-1 participates in LMP1 to regulate Capn4 promoter activity.(5)LMP1C-terminal cytoplasmic tail CTAR1 / CTAR2 domain induces Capn4 expression by AP-1 activation.(6)LMP1 can regulate the activity of NF-kappa B through Capn4.(7)After Capn4 was upregulated by LMP1,NPC cells caused actin rearrangement migration increasing.Conclusion:(1)The expression of Capn4 and LMP1 in NPC tissues is improved than that in the normal nasopharyngeal tissue significantly,and the DFS phase with DMFS phase are significantly shortened in the patients expressing positively.(2)LMP1 can induce Capn4 expression by activating AP-1 through the CTAR1 and CTAR2 domains.(3)LMP1 can regulate the activity of NF-kappa B through Capn4.(4)LMP1 promotes the migration of NPC cells by actin rearrangement mediated by Capn4.