富含亮氨酸重复和免疫球蛋白样结构域1基因过表达增强膀胱癌T24细胞对顺铂敏感性的实验研究  

Overexpression of LRIG1 gene enhances chemosensitvity of human bladder cancer T24 cell line to cisplatin

作　　者：严泽军[1] 夏术阶[2] 蒋军辉[1] 谢国海[1] 钱君海[1] 蒋照辉[1] 周成[1] 程跃[1] 

机构地区：[1]宁波大学医学院附属宁波市第一医院泌尿外科,315010 [2]上海交通大学泌尿外科研究所博士后流动站

出　　处：《中华泌尿外科杂志》2015年第7期505-510,共6页Chinese Journal of Urology

基　　金：中国博士后科学基金资助项目（2013M541524）;浙江省自然科学基金项目（LY12H05002）;浙江省医药卫生科技计划项目（2013KYA180）;宁波市自然科学基金项目（2013A610208）

摘　　要：目的 探讨富含亮氨酸重复和免疫球蛋白样结构域1 （leucine-rich repeats and immunoglobulin-like domains-1,LRIG1）基因过表达对膀胱癌T24细胞顺铂（cis-diaminodichloroplatinum,CDDP）敏感性的影响及其机制.方法 2012年12月至2014年2月选取膀胱癌T24细胞株,将建立并鉴定的过表达LRIG1基因的T24细胞分为3组：实验组,转染Ad-Surp-LRIGl;对照组,转染Adeno-SP-EGFP;空白组,未转染.将3组细胞分别加入CDDP,构建为CDDP/Ad-Surp-LRIG1组、CDDP/Adeno-SP-EGFP组、CDDP组,与未加入CDDP的空白组共4组进行诱导凋亡实验.采用RT-PCR和蛋白质印迹法检测转染前后T24细胞中LRIG1和表皮生长因子受体（epidermal growth factor receptor,EGFR）的表达水平.蛋白质印迹法检测CDDP对T24细胞的总EGFR和核磷酸化EGFR表达的影响.采用CCK-8法检测各组细胞对CDDP的敏感性.流式细胞学检测各组细胞的凋亡率.结果 实验组、对照组和空白组的LRIG1 mRNA和EGFR mRNA的表达量分别为2.79±0.15和0.65±0.05、0.76±0.09和1.98±0.07、0.66±0.05和2.05±0.04,LRIG1蛋白和EGFR蛋白分别为1.98±0.12和0.92±0.05、0.88 ±0.07和2.51 ±0.07、1.00 ±0.08和2.42±0.09,实验组与对照组和空白组比较差异均有统计学意义（P＜0.05）,对照组与空白组比较差异无统计学意义（P＞0.05）.CDDP组、CDDP/Adeno-SP-EGFP组和CDDP/Ad-Surp-LRIGl组的半数抑制浓度IC50值分别为（30.96±0.57）、（31.55 ±0.48）和（14.09±0.31） μg/ml,CDDP组与CDDP/Adeno-SP-EGFP组和CDDP/Ad-Surp-LRIGl组比较差异均有统计学意义（P＜0.05）,CDDP/Adeno-SP-EGFP组和CDDP/Ad-Surp-LRIGl组比较差异无统计学意义（P＞0.05）.CDDP/Ad-Surp-LRIGl组的细胞凋亡率[（69.77±2.14）％]高于CDDP/Adeno-SP-EGFP组[（48.15±1.53）％]、CDDP组[（46.82±1.23）％]、空白组[（3.17±0.21）％],差异均有统计学意义（P＜0.05）,CDDP组和CDDP/Adeno-SP-EGFP组比较差异无统计学意义（P＞0.05）.空白组、CDDP组、CDDP/Adeno-SP-EGFPObjective To investigate the effects and mechanisms of LRIG1 overexpression on chemosensitivity of human bladder cancer T24 cell line to cisplatin.Methods Human bladder cancer T24 cell line was used in the experiment since December 2012.Firstly,human bladder cancer T24 cells which were capable of highly expressing exogenous LRIG1 gene were established and identified.The T24 cells were divided into 3 groups：experimental group （transfected with Ad-Surp-LRIGl）,control group （transfected with Adeno-SP-EGFP）,blank control group （untransfected）.Secondly,experiments of CDDP-induced apoptosis were divided into 4 groups：control group （untreated）,CDDP group （treated with CDDP）,CDDP/Adeno-SP-EGFP group （treated with CDDP/Adeno-SP-EGFP and CDDP）,CDDP/Ad-Surp-LRIG1 group （treated with CDDP/Ad-Surp-LRIG1 and CDDP）.The expression of mRNA and protein of LRIG1 and EGFR were detected by RT-PCR and Western blot.The effect of LRIG1 and/or CDDP on the expression of EGFR and pEGFR of T24 cells were measured by Western blot.The cellular sensitivity to CDDP was evaluated by cell counting Kit-8 （CCK-8）.The cells apoptosis rates were examined by flow cytometry.Results The mRNA expression of LRIG1 and EGFR in experimental group,control group and blank control group were （2.79±0.15） and （0.65±0.05）,（0.76±0.09） and （1.98 ±0.07）,（0.66±0.05） and （2.05 ± 0.04）;the protein expression of LRIG1 and EGFR in experimental group,control group and blank control group were （1.98 ± 0.12） and （0.92 ± 0.05）,（0.88 ± 0.07） and （2.51 ± 0.07）,（1.00 ± 0.08） and （2.42 ± 0.09）.The differences were significant between experimental group and the other groups （P 〈 0.05）,the difference was not significant between control group and blank control group （P 〉 0.05）.The IC50 values in CDDP group,CDDP/Adeno-SP-EGFP group and CDDP/Ad-Surp-LRIG1 group were （30.96 ± 0.57） μg/ml,（31.55 ± 0.48） μg/ml and （14.09 ± 0.31） μg/ml.The difference were significa

关 键 词：富含亮氨酸重复和免疫球蛋白样结构域1基因 顺铂 膀胱肿瘤 细胞凋亡 

分 类 号：R737.14[医药卫生—肿瘤]