Ezrin在食管癌细胞的生长和侵袭过程中的作用及其机制的初步研究

　　汕头大学医学院生物化学与分子生物学教研室  谢剑君

　　【摘要】目的: Ezrin从属于ERM(ezrin–radixin–moesin)蛋白家族，其基本功能是将肌动蛋白维系到细胞膜和锚定到膜蛋白的特定部位上，从而参与细胞的移动和粘附。研究表明Ezrin在多种肿瘤中异常表达并可能参与肿瘤的生长和转移。然而，到目前为止，Ezrin参与肿瘤发生发展的直接证据仍然缺乏。食管癌中Ezrin的表达最近才得到初步地阐明，但其功能和相关机制还不清楚。本研究旨在运用RNA干扰等方法探讨Ezrin在食管癌中的功能。方法:我们首先利用免疫蛋白印迹和免疫荧光染色的方法检测了Ezrin在多种食管癌细胞系中的表达情况。然后以其中一种细胞(EC109)为模型，用RNA干扰的方法成功降低了该细胞中Ezrin的表达。然后用克隆形成、MTT生长曲线、裸鼠体内成瘤、Chamber移动实验和细胞侵袭以及细胞粘附等实验检测了Ezrin表达对细胞生物学行为的影响。最后，联合运用cDNA芯片和免疫印迹等方法，初步探讨了Ezrin发挥功能的机制。结果:免疫蛋白印迹的结果提示Ezrin在多种食管癌细胞系中高表达。免疫荧光染色发现，Ezrin不但定位于胞膜下和胞浆中，同时还表达于细胞核内，核内Ezrin主要分布于核浆中。克隆形成和MTT实验表明，Ezrin表达减少后，细胞生长减慢。裸鼠体内成瘤实验也直接证明了Ezrin对细胞生长的影响。移动、侵袭和粘附实验揭示，Ezrin表达的减少，可以导致细胞移动、侵袭以及粘附能力的减弱。接着我们进一步用cDNA芯片的方法揭示，Ezrin表达减少后一系列细胞生长和凋亡相关基因、细胞移动和侵袭相关基因以及细胞粘附相关基因均发生了差异表达，比如TGM2, CTGF和CYR61等。最后，信号通路分析发现，Ezrin表达的减少可以抑制MAPK和TGF-β信号通路。结论:以上结果表明Ezrin参与了食管癌细胞的生长和侵袭过程。其可能的机制是Ezrin通过调节MAPK和TGF-β信号途径影响某些细胞生长与侵袭相关基因的表达，或者是直接参与这些基因的转录调控(国家高技术研究发展计划(2006AA02A403)、国家自然科学基金(30672376和30772485)和广东省自然科学基金(5104541，7118419)资助项目)。

　　Roles of ezrin in the growth and invasiveness of the esophageal squamous cell carcinoma cells

　　【Abstract】Objective: Ezrin belongs to the ezrin–radixin–moesin (ERM) family proteins, which cross-link actin cytoskeleton and plasma membrane, and is actively involved in regulating the growth and metastatic capacity of certain cancer cells, although little direct evidence has been collected in this regard. The expression of ezrin in esophageal squamous cell carcinoma (ESCC) has been described only recently, but its roles and mechanism still remained unclear. The purpose of this study is to further explore the expression and roles of ezrin in ESCC. Methods: We first detected the expression of ezrin in several ESCC cell line. Then, by using RNA interference (RNAi), we stably silenced the expression of ezrin in EC109 cells, an ESCC cell line. Effects of ezrin down-expression on cell growth and invasiveness were demonstrated. Finally, cDNA microarray and pathways analysis were performed on the ezrin down-expression cells to explore some mechanisms possibly involved. Results: Western blotting and fluorescence staining showed that ezrin in ESCC cell is expressed in the nucleus as well as in the cytoplasm and plasma membrane. Down-regulation of ezrin resulted in a suppression of cell growth, cell adhesion and as well as cell invasiveness. Tumorigenesis experiments in nude mice also revealed that ezrin might have direct functions in regulating tumor growth in vivo. Data of cDNA microarrays on ezrin knockdown cells and the control cells revealed that some proliferation- and invasiveness-related genes such as TGM2, CTGF and CYR61, were differentially expressed upon ezrin knockdown. Finally, pathway analysis showed that ezrin knockdown led to decreased activation of the TGF- and MAPK pathways. Conclusions: Taken together, we propose that ezrin might function in growth and invasiveness of ESCC by affecting the expression of certain genes that function in the MAPK and TGF-β pathways, or by influencing the transcription of the MAPK and TGF-β genes directly.