amiRNA-Snai1逆转人胃癌细胞株SGC7901/DDP对顺铂的耐药性及其机制研究  

Reversion of Resistance of Human Gastric Cancer Cell Line SGC7901/DDP to Cisplatin by amiRNA-Snai1 and its Possible Mechanism

作　　者：许春红[1] 郭慧敏[2] 王军[2] 李建琦[2] 陈敏[2] 邹晓平[1] 

机构地区：[1]南京医科大学附属鼓楼临床医学院消化科,210008 [2]南京大学医学院附属鼓楼医院消化科

出　　处：《胃肠病学》2012年第3期146-150,共5页Chinese Journal of Gastroenterology

基　　金：国家自然科学基金资助项目(81071816)

摘　　要：背景:多药耐药是恶性肿瘤化疗失败的主要原因。研究显示锌指转录因子Snai1介导肿瘤细胞发生上皮间质转化(EMT)可促成肿瘤对化疗耐药。目的:探讨人工合成microRNA-Snai1(amiRNA-Snai1)逆转人胃癌细胞株SGC7901/DDP对顺铂(DDP)耐药性的作用及其可能机制。方法:构建稳定转染amiRNA-Snai1质粒或阴性对照质粒的SGC7901/DDP细胞株(SGC7901/DDP-amiRNA组和SGC7901/DDP-Mock组),以蛋白质印迹法、免疫荧光法检测各组细胞中的Snai1、耐药基因ERCC1、Snai1下游靶基因E-cadherin蛋白表达,以CCK-8法检测各组细胞经不同浓度DDP(0.01、0.1、1.0、10.0 mg/L)作用后的存活率,计算DDP对细胞的半数抑制浓度(IC50)。结果:DDP耐药SGC7901/DDP细胞中的Snai1蛋白相对表达量显著高于DDP敏感亲本SGC7901细胞(P<0.05)。SGC7901/DDP-amiRNA组细胞Snai1、ERCC1蛋白相对表达量显著低于SGC7901/DDP-Mock组细胞(P<0.05),荧光强度明显减弱;E-cadherin蛋白相对表达量显著高于SGC7901/DDP-Mock组细胞(P<0.05),荧光强度明显增强。DDP对SGC7901/DDP-amiRNA组细胞的IC50值显著低于SGC7901/DDP-Mock组细胞(P<0.05)。结论:以amiRNA-Snai1沉默Snai1基因表达可能通过下调ERCC1表达、上调E-cadherin表达而逆转人胃癌细胞株SGC7901/DDP对DDP的耐药性。Development of multidrug resistance is a major deterrent in the effective treatment of cancers by chemotherapy. Emerging evidences suggest that epithelial-mesenchymal transition （EMT） mediated by zinc finger transcription factor Snail might contribute to chemoresistance in cancer cells. Aims： To investigate the effect of artificial microRNA-Snail （amiRNA-Snail） in reversing resistance of human gastric cancer cell line SGC7901/DDP to eisplatin （DDP） and its possible mechanism. Methods： SGC7901/DDP cell lines stably transfected with amiRNA-Snail plasmid （SGC7901/ DDP-amiRNA group） or negative control plasmid （SGC7901/DDP-Mock group） were constructed, respectively. Western blotting and immunofluorescence were used to determine the protein expressions of Snail, drug resistance gene ERCC 1, and E-cadherin, the downstream target of Snail in transfected and untransfected cells. Survival rates of cells treated with DDP at different concentrations （0.01, 0.1, 1.0, and 10.0 mg/L） were assessed by CCK-8 assay and the 50% inhibiting concentration （IC50） of DDP was calculated. Results： Expression level of Snail protein in DDP-resistant SGC7901/I）DP cells was significantly higher than that in DDP-sensitive parent SGC7901 cells （P〈0.05）. Expression levels of Snail and ERCC1 proteins were significantly decreased, and that of E-cadherin protein was significantly increased in SGC7901/DDP-amiRNA group than in SGC7901/DDP-Mock group （P〈0.05）, and the fluorescence intensities of these proteins were concordant with their expression levels. IC5o of DDP was significantly lower in SGC7901/DDP-amiRNA group than in SGC7901/DDP-Mock group （P〈0.05）. Conclusions： Silencing Snail gene by amiRNA-Snail may reverse resistance of human gastric cancer cell line SGC7901/DDP to DDP through down-reulatin ERCC1 exnression and un-reulatinz E-cadherin exoression.

关 键 词：胃肿瘤 细胞系 肿瘤 抗药性 上皮间质转化 Snai1 RNA干扰 钙黏着糖蛋白类 

分 类 号：R735.2[医药卫生—肿瘤]