The Action And Mechanism Of Migration And Invasion By Carboxy Terminal Activating Region3of EBV-LMP1in Nasopharyngeal Carcinoma Stem Cell

Objective:This study selected nasopharyngeal carcinoma stem cells SP18cells as object, observed the effects of migration and invasion by carboxy terminal activating region3of EBV-LMP1in SP18cells; and investigated the molecular mechanism of migration and invasion by LMP1-CTAR3in SP18cell.Methods:The SP18cell which expressed LMP1and the SP18cell which expressed deletion mutant type LMP1(LMP1Δ232-351), were established by liposome transfection techniques; detected the proliferation of SP18cells by MTT assay and Plate clone formation assay; observed the invasion and migration of SP18cells by transwell experiments and the scratch test; and then used cDNA microarray techniques testing the differential gene expression between SP18-LMP1and SP18-LMP1Δ252-351, preliminary analyzed the function of those differentially expressed genes accorded to the bioinformatics.Result:1. Effect of LMP1-CTAR3on proliferation, migration and invasion in nasopharyngeal carcinoma stem cell SP18.The growth curve of MTT assay showed that, cell growth rate of transfection of LMP1group and transfection of LMP1Δ232-351group were higer than untransfected group and negative transfection group (P<0.05), cell growth rate of transfection of LMP1group was higer than transfection of LMP1Δ232-351group(P<0.05). Plate clone formation test showed that, The number of cell clones of transfection of LMP1group and transfection of LMP1Δ232-351group were more than untransfected group and negative transfection group (P<0.05), the number of cell clones of transfection of LMP1group was more than transfection of LMP1Δ232-351group(P<0.05). Demonstrated that LMP1-CTAR3may play a positive regulatory role in the process of LMP1promote the proliferation of SP18cells. Scratch test showed that, scratch healing of transfection of LMP1group and transfection of LMP1Δ232-351group were faster than untransfected group and negative transfection group after24h (P<0.05), scratch healing of transfection of LMP1group was faster than transfection of LMP1Δ232-351group(P<0.05). And transwell experiments showed that, migration and invasion capacity of transfection of LMP1group and transfection of LMP1Δ232-351group were better than untransfected group and negative transfection group(P<0.05), migration and invasion capacity of transfection of LMP1group was better than transfection of LMP1Δ232-351group(P<0.05). It indicated that LMP1can promote migration and invasion of the SP cells, and LMP1-CTAR3is the important active area in the process.2.Screening the differential expressed genes of LMP1-CTAR3affects cell migration and invasion.Detected by gene chip, we filtered out314differentially expressed genes, including180up-regulated genes and134downregulated genes. Using Functional Annotation Clustering analysis the biological function of314differentially expressed genes showed that those differentially expressed genes were classified into24categories, the three highest Enrichment Score function categories, mainly involved in cell apoptosis, migration and other related biological processes. These indicated that CTAR3may be closely related in migration of SP cells. Gene Ontology classification of314differentially expressed genes by DAVID online software; the result revealed that there were18differentially expressed genes relative with tumor migration and invasion, including13up-regulated genes and5down-regulated genes.Analyzed the signaling pathways, gene biological classification and gene interaction analysis, we had screened several differentially expressed genes, including EFNB2, THBS1, FN1, MMP14, which were promoted the migration and invasion of nasopharyngeal carcinoma, which also may promote tumor angiogenesis by promoting cell adhesion and cell motility, degrading extracellular matrix and basement membrane. Thus, these differentially expressed genes play a role in invasion and migration of nasopharyngeal carcinoma.Conclusion: 1.CTAR3is the important active area of LMP1to promote the invasion and migration of nasopharyngeal carcinoma stem cells SP18cells.2.LMP1may promote the migration and invasion of SP18cells by regulated EFNB2, THBS1, FN1, MMP14and so on.