磷脂酰肌醇-3激酶调节亚基3在非小细胞肺癌上皮-间充质转化及迁移中的作用  

The effect of Phosphoinositide-3-kinase, regulatory subunit 3 in epithelial-mesenchymal transition and migration of non-small cell lung cancer

作　　者：杨熹[1] 胡福清 李海杰[1] 兰静芩 罗学来[1] 龚建平[1] 胡俊波[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院肿瘤研究所/胃肠外科,武汉430030

出　　处：《中华实验外科杂志》2015年第4期787-791,共5页Chinese Journal of Experimental Surgery

基　　金：国家973计划资助项目（2009CB521802）;国家自然科学基金资助项目（81272278、81372662）

摘　　要：目的 观察磷脂酰肌醇-3激酶调节亚基3（PIK3R3）在非小细胞肺癌（NSCLC）组织中的表达,及PIK3R3对NSCLC细胞株A549和PC-9上皮-间充质转化（EMT）及迁移的影响.方法 分别提取22对NSCLC患者的肿瘤组织和癌旁组织中的总RNA及和7对标本中总蛋白,分别检测PIK3R3mRNA水平和蛋白表达;在A549和PC-9中通过感染慢病毒高表达或抑制PIK3R3后,通过Transwell迁移实验检测其对细胞迁移的影响,通过Western blot法检测上皮细胞钙黏蛋白（CDH1）、波形蛋白（VIM）、蜗牛族锌指1（SNAI1）及蜗牛族锌指2（SNAI2）等EMT相关蛋白的变化,并通过染色质免疫共沉淀法检测SNAI1及SNAI2结合于CDH1启动子的能力,通过双荧光素酶报告系统检测CDH1的转录活性变化.结果 在NSCLC临床标本中,PIK3R3在肿瘤组织中的表达水平高于癌旁组织;在A549和PC-9中高表达PIK3R3后,穿膜细胞数增加[A549 Control：（46 ±3）个,PIK3R3：（92±5）个;PC-9 Control：（25±2）个,PIK3R3：（53±3）个],细胞的迁移能力增强,同时EMT相关的上皮标志蛋白CDH1表达降低,而间质标志蛋白VIM、SNAI1及SNAI2的表达升高,SNAI1及SNAI2结合于CDH1启动子的能力分别增强9.0倍及3.8倍,同时CDH1的转录活性降低70％;而抑制HK3R3的表达后,穿膜细胞数[A549 sh-Control：（58±5）个,sh-HK3R3：（13±3）个;PC-9 sh-Control：（28±5）个,sh-PIK3R3：（10±3）个],细胞的迁移能力减弱,伴随CDH1表达增高,而VIM、SNAI1及SNAI2的表达降低,同时SNAI1及SNAI2结合于CDH1启动子的能力分别降低70％,相应CDH1的转录活性增高3.6倍.结论 在NSCLC标本中PIK3R3的表达增高,在NSCLC细胞株中高表达PIK3R3可诱使其发生EMT,并促进其迁移能力,而抑制PIK3R3的表达可逆转上述过程.Objective To test the expression of phosphoinositide-3-kinase,regulatory subunit 3 （PIK3R3） in the human non-small cell lung cancer （NSCLC） tissues,and to observe the effect of PIK3R3 on the epithelial-mesenchymal transition （EMT） and migration of NSCLC cell lines A549 and PC-9.Methods Total RNA and total protein lysate from the NSCLC tissues and paratumor tissues of 22 or 7 patients with NSCLC were prepared respectively,and the expression of PIK3R3 was tested by real-time quantitative polymerase chain reaction（Real-time PCR） and Western blotting; PIK3R3 was overexpressed or down-regulated by lentivirus infection,then the effect of PIK3R3 on the migration was detected by Transwell assay,and the expression of EMT related proteins were detected by Western blotting,and the binding of snail family zinc finger （SNAI） 1 and SNAI2 on the promoter of cadherin 1 （CDH1） were measured by chromatin immunoprecipitation assay （ChIP）,and the transcription activity of CDH1 was detected by luciferase reporter assay.Results The expression of PIK3R3 was elevated in NSCLC patients＆#39; tumor tissues compared with the paratumor tissues; The migration of A549 and PC-9 cells was enhanced after PIK3R3 overexpression （A549 Control：46 ±3,PIK3R3：92 ±5; PC-9 Control：25 ±2,PIK3R3：53 ± 3）,with the expression of CDH1 depressed and elevation of VIM,SNAI1 and SNAI2,which were related to the progress of EMT; The binding activity of SNAI1 and SNAI2 on the CDH1 promoter was elevated 9 times and 3.8 times,respectively,and the transcriptive activity of CDH1 was depressed to 30%.To the opposite,the migration of A.549 and PC-9 cells were inhibited after PIK3R3 down-regulated （A549 sh-Control：58 ± 5,sh-PIK3 R3：13-± 3 ; PC-9 sh-Control：28 ± 5,sh-PIK3 R3：10 ± 3）,with the expression of CDH1 elevation and depressed of VIM,SNAI1 and SNAI2.The binding activity of SNAI1 and SNAI2 on the CDH1 promoter was reduced,and the transcriptive activity of CDH1 was increased 3.6 times.Conclusion The expression

关 键 词：磷脂酰肌醇-3激酶调节亚基3 非小细胞肺癌 迁移 上皮-间充质转化 

分 类 号：R734.2[医药卫生—肿瘤]