EphA2及其S897位点磷酸化在鼻咽癌细胞生长和迁移中的作用

发布时间：2018-03-24 13:36

  本文选题：鼻咽癌　切入点：EphA2　出处：《中南大学》2013年硕士论文

【摘要】：目的：EphA2是Eph (Erythropoietin-producing hepatoma cell line, Eph)受体酪氨酸激酶家族的成员,具有介导生长因子促进肿瘤发生和转移的作用。我们前期的定量蛋白质组学研究发现,EphA2表达水平及其EphA2S897位点的磷酸化水平在高转移5-8F鼻咽癌(Nasopharyngeal carcinoma, NPC)细胞中上调。本课题研究EphA2表达水平及其S897位点的磷酸化水平对鼻咽癌细胞增殖、细胞周期分布和细胞迁移能力的影响,并探讨EGFR是否通过激活PI3K-AKT使EphA2S897位点磷酸化,为揭示EphA2在NPC发生和转移中的作用及其机制奠定基础。 方法：(1)Western blotting检测高转移的NPC细胞株5-8F细胞和低转移的NPC细胞株6-10B细胞中EphA2表达水平及其EphA2S897位点的磷酸化水平,以及EGFR表达水平和磷酸化EGFR的水平,实时荧光定量PCR检测两株细胞中EphA2配体EphrinA1的mRNA表达水平；(2)Western blotting结合小分子激酶抑制剂检测EGFR是否通过激活PI3K-AKT使EphA2S897位点磷酸化；(3)脂质体转染法将突变型EphA2(S897A)表达载体和空载体转染5-8F细胞,将野生型EphA2表达载体和空载体分别转染6-10B细胞,建立稳定转染细胞系；(4)以突变型EphA2高表达的5-8F细胞系、野生型EphA2高表达的6-10B细胞系及其空白载体转染细胞系为样本,采用MTT法检测细胞增殖,采用流式细胞仪检测细胞周期分布,划痕实验观察细胞的迁移。 结果：(1)5-8F细胞EphA2的表达水平和EphA2S897位点的磷酸化水平以及EGFR的表达水平和磷酸化EGFR的水平明显高于6-10B细胞,证实了我们前期的定量蛋白质组学结果；(2)EphA2配体Ephrin A1mRNA在5-8F细胞的表达水平低于6-10B细胞；(3)在5-8F细胞和6-10B细胞中,EGFR通过激活PI3K-AKT使EphA2S897位点磷酸化；(4)建立了突变型EphA2高表达的5。8F细胞系,野生型EphA2高表达的6-10B细胞系及其空白载体转染细胞系；(5)与空载体转染的5-8F细胞和未转染5-8F细胞比较,突变型EphA2高表达的5-8F细胞的增殖能力降低,G0/G1细胞增加,而S期细胞减少,细胞迁移能力降低；与空载体转染的6-10B细胞和未转染6-10B细胞比较,野生型EphA2高表达的6-10B细胞增殖能力增加,S期细胞增加,而G2/M期细胞减少,细胞迁移能力增强。 结论：1.高转移5-8F NPC细胞的EphA2表达水平及其S897位点的磷酸化水平显著高于低转移的6-1OB NPC细胞,而Ephrin A1的表达水平显著低于6-1OB NPC细胞；2. EGFR通过激活PI3K-AKT信号通路使NPC细胞EphA2S897位点磷酸化；3.建立了突变型EphA2高表达的5-8F细胞系和野生型EphA2高表达的6-10B细胞系；4.EphA2高表达及其S897位点的磷酸化促进NPC细胞的生长和迁移。
[Abstract]:Objective: EphA2 is a member of the tyrosine kinase family of Eph Erythropoietin-producing hepatoma cell line. Our previous quantitative proteomics studies showed that the expression of EphA2 and the phosphorylation level of EphA2S897 site were up-regulated in high metastatic 5-8F nasopharyngeal carcinoma (NPCs) cells. The aim of this study was to investigate the effect of EphA2 expression and phosphorylation at S897 on the proliferation of nasopharyngeal carcinoma cells. The effects of cell cycle distribution and cell migration ability, and whether EGFR phosphorylates EphA2S897 sites by activating PI3K-AKT are discussed, which lays a foundation for revealing the role and mechanism of EphA2 in the pathogenesis and metastasis of NPC. Methods the expression of EphA2 and the phosphorylation of EphA2S897 site, EGFR expression and phosphorylated EGFR in 5-8F cells with high metastasis and 6-10B cells with low metastasis were detected by Western blotting. Real-time fluorescence quantitative PCR was used to detect the mRNA expression level of EphA2 ligand EphrinA1 in the two cell lines. Western blotting combined with small molecular kinase inhibitor was used to detect whether EGFR phosphorylated EphA2S897 site by activating PI3K-AKT. The mutant EphA2S897A) expression vector was transfected by liposome. The empty vector was transfected into 5-8F cells. The wild-type EphA2 expression vector and empty vector were transfected into 6-10B cell line respectively. A stable transfection cell line was established. The mutant EphA2 overexpression 5-8F cell line, the wild-type EphA2 overexpression 6-10B cell line and its blank vector transfected cell line were used as samples. Cell proliferation was detected by MTT assay, cell cycle distribution was detected by flow cytometry, cell migration was observed by scratch test. Results the levels of EphA2 expression, EphA2S897 site phosphorylation, EGFR expression and phosphorylated EGFR in the 5-8F cells were significantly higher than those in 6-10B cells. It was confirmed that the expression level of EphA2 ligand Ephrin A1mRNA in 5-8F cells was lower than that in 6-10B cells (5-8F cells and 6-10B cells). In 5-8F cells and 6-10B cells, EphA2S897 sites were phosphorylated in 5-8F cells and 6-10B cells by activating PI3K-AKT. Compared with 5-8F cells transfected with empty vector and 5-8F cells transfected with empty vector, the proliferative ability of 5-8F cells with high expression of mutant EphA2 decreased, while that of G-0 / G1 cells increased, and that of S-phase cells decreased, compared with 5-8F cells transfected with wild type EphA2 and its blank vector. Compared with 6-10B cells transfected with empty vector and non-transfected 6-10B cells, the proliferative ability of wild-type EphA2 overexpression 6-10B cells increased in S phase, while that in G _ 2 / M phase decreased and cell migration increased. Conclusion the level of EphA2 expression and phosphorylation at S897 site in high metastatic 5-8F NPC cells were significantly higher than those in 6-1OB NPC cells with low metastasis. The expression level of Ephrin A1 was significantly lower than that of 6-1OB NPC cells. EGFR phosphorylated EphA2S897 sites in NPC cells by activating PI3K-AKT signaling pathway. 5 8F cell lines with high expression of mutant EphA2 and 6-10B cell lines with high expression of wild type EphA2 were established. Phosphorylation of S897 and S897 promoted the growth and migration of NPC cells.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R739.63

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