Ezrin基因在人涎腺腺样囊性癌中的表达及对转移细胞增殖侵袭性的影响

Effects of Ezrin gene on the proliferation and invasion activity of human salivary gland adenoid cystic carcinoma

目的检测Ezrin基因在人涎腺腺样囊性癌（salivary gland adenoid cystic carcinoma，SACC）中的表达，探讨Ezrin基因对SACC肺高转移细胞（adenoid cystic carcinoma，ACC）-M增殖、凋亡及侵袭活性的影响和作为SACC基因治疗靶点的可行性。方法采用免疫组织化学链霉素抗生物素蛋白-过氧化物酶连结法（streptavidin-perosidase，SP）检测石蜡包埋的43例SACC、40例涎腺多形性腺瘤（pleomorphic adenoma，PA）及15例正常涎腺组织中Ezrin的表达。设计并合成Ezrin特异性经化学修饰的双链siRNA和双链SiRNA阴性对照组，经脂质体介导转染人ACC—M，将细胞分为实验组、阴性对照组、空白对照组。反转录聚合酶链反应（RT—PCR）及免疫细胞化学分析各组细胞中Ezrin基因的mRNA及蛋白表达变化；甲基噻唑基四唑（MTT）法绘制细胞生长曲线，检测各组细胞体外增殖能力；流式细胞术测定细胞周期及凋亡情况；Transwell实验检测各组细胞侵袭能力差异。结果15例正常涎腺组织Ezrin表达阳性4例，40例PA中23例、43例SACC中37例，Ezrin表达阳性依次升高，差异有统计学意义（P〈0．05）。Ezrin特异性siRNA转染ACC—M后，Ezrin基因mRNA及蛋白表达水平均降低，细胞增殖活性受抑制，细胞凋亡增加，细胞侵袭能力下降（P〈0．05）。结论Ezrin的高表达可能在SACC的发生、发展及转移中发挥一定作用，Ezrin特异性siRNA能有效沉默ACC—M细胞Ezrin基因，使体外培养的ACC—M细胞在一定程度上增殖受抑制并诱导细胞凋亡及降低侵袭能力。

Objective To examine the expression of Ezrin in human salivary gland adenoid cystic carcinoma and investigate the effects of Ezrin gene silence on cell proliferation, apoptosis and invasion of adenoid cystic carcinoma （ ACC ）-M. Methods The expression of Ezrin was detected by immunohistochemistry in normal salivary gland tissue（ n = 15 ） , pleomorphic adenoma（ n = 40） and salivary gland adenoid cystic carcinoma （n = 43 ）. The Ezrin StealthTM RNAi Duplex, containing StealthTM RNAi Negtive Control Duplex were contructed and transfected into ACC-M cells by Lipofectamine TM 2000. The expression levels of Ezrin were detected by RT-PCR and immunohistochemistry. The cell cycle and apoptosis rate were analyzed by flow cytomtery （FCM）. The cell proliferation was detected by methyl thiazolyl tetrazolium（MTT） and cell invasion by Transwell test. Results The positive rate of Ezrin expression in ACC was significantly higher than that in normal salivary gland tissue and pleomorphic adenoma （P 〈 0. 05 ）. After transfection of Ezrin SteahhTM RNAi Duplex, the mRNA and protein expression of Ezrin were down-regulated, the cell proliferation activity was inhibited, the G0-G1 Phase cells were increased, and the apoptosis rate of Ezrin StealthTM RNAi Duplex group was higher than that in control groups and cell invasion ability was decressed. Conclusions Over expression of Ezrin in human salivary gland adenoid cystic carcinoma may promote genesis, development and metastasis of tumors. Ezrin StealthTM RNAi Duplex could efficiently down-regulate the expression of Ezrin gene, and partly inhibite proliferation of ACC-M cells, induce apoptosis and degrease invasion ability of these cells in vitro.