Effects Of EphA2and Its Phosphorylation At Serine897Site On The Growth And Migration Of Nasopharyngeal Carcinoma Cells

Objective:EphA2is a member of the Eph (Erythropoietin-producing hepatoma cell line, Eph) receptor tyrosine kinase family, which mediates growth factor to promote tumorigenesis and metastasis. In our previous quantitative proteomics study, we found EphA2expression level and phosphorylation level of EphA2S897site were up-regulated in nasopharyngeal carcinoma cell line5-8F with highly metastatic potential. The main purpose of this research is to investigate the effect of EphA2expression and S897site phosphorylation on NPC cell proliferation, cycle distribution and migration, to explore whether the EGFR phosphorylates EphA2S897sites via activating PI3K-AKT, and eventually lay the foundation for revealing the effect and mechanism of EphA2in NPC development and metastasis.Methods:(1) Western boltting was used to detect the expressional levels of EphA2and EGFR, and phosphorylation levels of EphA2S897site and EGFR in NPC cell lines5-8F and6-1OB. In addition, the mRNA expression level of EphA2ligand (EphrinAl) in these two cell lines with differential metastatic potential was determined by real-time PCR;(2) Western blotting combined with small molecule kinase inhibitor was used to detect whether EGFR phosphorylates EhpA2S897site by means of activating PI3K-AKT;(3) Liposome transfection method was used to establish the stable transfected5-8F cell line with mutant EphA2(S897A) overexpression,6-1OB cell line with the wild-type EphA2overexpression and their corresponding control cell lines with empty vector transfection, respectively;(4) MTT assay, flow cytometry and wound healing assay were performed to compare the differences of cell proliferation, cell cycle distribution and cell migration in the stable transfected NPC cell lines.Results:(1) The expressional levels of EphA2and EGFR, and phosphorylation levels of EphA2S897site and EGFR in5-8F cells were significantly higher than in6-1OB cells, whereas the expressional level of EphA2ligand Ephrin Al was significantly lower in5-8F cells than in6-10B cells;(2) The mRNA expression level of EphrinAl in5-8F cells was significantly lower than in6-10B cells;(3) EGFR induced the phosphorylation of EphA2S897site in the both NPC cell lines through activating PI3K-AKT;(4) The stable transfected5-8F cell line with mutant EphA2(S897A) overexpression,6-10B cell line with the wild-type EphA2overexpression, and their corresponding control cell lines were established;(5) compared with empty vector transfected and untransfected5-8F cells, the5-8F cells with overexpressed mutant EphA2showed the decreased growth, increased percent of G0/G1phase cells, reduced percent of S phase cells, and decreased migration ability. Compared with empty vector transfected and untransfected6-10B cells, the6-10B cells with overexpressed wild-type EphA2showed increased growth, increased percent of S phase cells, reduced percent of G2/M phase cells, and increased migration ability.Conclusions:1. The expression level of EphA2and the phosporylation level of EphA2S897site in5-8F cells were significantly higher than that in6-10B cells, whereas the expressional level of EphA2ligand Ephrin A1was significantly lower in5-8F cells than that in6-10B cells;2. EGFR induces EphA2S897site phosphorylation through activating PI3K-AKT signaling pathway;3.5-8F cell line with overexpressed mutant EphA2and6-10B cell line with overexpressed wild-type EphA2were successfully established;4. Overexpression of EphA2and phosphorylation of EphA2S897site play an important role in promoting NPC cell growth and migration.