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目的 阐明EB病毒潜伏膜蛋白 1(LMP1)在鼻咽癌 (NPC)中调节表皮生长因子受体(EGFR)磷酸化水平。方法 采用已建立的、受四环素调控的、LMP1表达的NPC细胞系 (pTet on LMP1HNE2 ) ,定量诱导pTet on LMP1HNE2细胞LMP1动态表达水平 ,并观察EGFR表达和磷酸化水平。采用瞬间转染方法 ,将EGFR显性负性突变体和LMP1antisense表达质粒分别导入pTet on LMP1HNE2细胞中 ,采用免疫共沉淀和Westernblot检测EGFR磷酸化 ;同时观察EGF刺激后对EGFR磷酸化前后的影响。结果 在pTet on LMP1HNE2细胞中 ,LMP1能诱导EGFR的表达和磷酸化 ,并随LMP1表达增加而增加。pTet on LMP1HNE2细胞在导入EGFR显性负性突变体后 ,不但完全阻断EGFR磷酸化 ,而且完全抑制EGF所引起的EGFR磷酸化 ;而导入LMP1antisense后 ,可显著地抑制EGFR磷酸化水平。结论 LMP1能同时诱导EGFR表达和上调EGFR磷酸化 ,并呈剂量相关效应 ,提示EGFR磷酸化可能在NPC发生发展过程中起着重要作用。
Objective To elucidate the role of Epstein-Barr virus latent membrane protein 1 (LMP1) in the regulation of epidermal growth factor receptor (EGFR) phosphorylation in nasopharyngeal carcinoma (NPC). Methods pTet on LMP1HNE2 was used to detect the expression of LMP1 in pTet on LMP1HNE2 cells. The expression of EGFR and the phosphorylation of EGFP were observed. Transient transfected EGFR dominant negative mutant and LMP1antisense expression plasmid were respectively introduced into pTet on LMP1HNE2 cells. EGFP phosphorylation was detected by co-immunoprecipitation and Western blotting. Meanwhile, the effect of EGF stimulation on EGFR phosphorylation was observed. Results In pTet on LMP1HNE2 cells, LMP1 induced EGFR expression and phosphorylation, and increased with the increase of LMP1 expression. pTet on LMP1HNE2 cells not only completely blocked the phosphorylation of EGFR, but also completely inhibited the phosphorylation of EGFR induced by EGF after introducing dominant negative mutant of EGFR. However, the phosphorylation of EGFR was significantly inhibited by LMP1antisense. Conclusion LMP1 can induce both EGFR expression and up-regulation of EGFR phosphorylation in a dose-dependent manner, suggesting that EGFR phosphorylation may play an important role in the development and progression of NPC.