CAJ | 학술논문

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.
목적 관찰린지선기순-3격매조절아기3(PIK3R3)재비소세포폐암(NSCLC)조직중적표체,급PIK3R3대NSCLC세포주A549화PC-9상피-간충질전화(EMT)급천이적영향.방법 분별제취22대NSCLC환자적종류조직화암방조직중적총RNA급화7대표본중총단백,분별검측PIK3R3mRNA수평화단백표체;재A549화PC-9중통과감염만병독고표체혹억제PIK3R3후,통과Transwell천이실험검측기대세포천이적영향,통과Western blot법검측상피세포개점단백(CDH1)、파형단백(VIM)、와우족자지1(SNAI1)급와우족자지2(SNAI2)등EMT상관단백적변화,병통과염색질면역공침정법검측SNAI1급SNAI2결합우CDH1계동자적능력,통과쌍형광소매보고계통검측CDH1적전록활성변화.결과 재NSCLC림상표본중,PIK3R3재종류조직중적표체수평고우암방조직;재A549화PC-9중고표체PIK3R3후,천막세포수증가[A549 Control:(46 ±3)개,PIK3R3:(92±5)개;PC-9 Control:(25±2)개,PIK3R3:(53±3)개],세포적천이능력증강,동시EMT상관적상피표지단백CDH1표체강저,이간질표지단백VIM、SNAI1급SNAI2적표체승고,SNAI1급SNAI2결합우CDH1계동자적능력분별증강9.0배급3.8배,동시CDH1적전록활성강저70％;이억제HK3R3적표체후,천막세포수[A549 sh-Control:(58±5)개,sh-HK3R3:(13±3)개;PC-9 sh-Control:(28±5)개,sh-PIK3R3:(10±3)개],세포적천이능력감약,반수CDH1표체증고,이VIM、SNAI1급SNAI2적표체강저,동시SNAI1급SNAI2결합우CDH1계동자적능력분별강저70％,상응CDH1적전록활성증고3.6배.결론 재NSCLC표본중PIK3R3적표체증고,재NSCLC세포주중고표체PIK3R3가유사기발생EMT,병촉진기천이능력,이억제PIK3R3적표체가역전상술과정.
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' 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 of PIK3R3 was elevated in NSCLC,and overexpression of PIK3R3 could induce the EMT and migration of A549 and PC-9 cells; on the contrary,down-regulation of PIK3R3 could inhibit the EMT and migration of A549 and PC-9 cells.