Wang Bin, Qin Hao, Wang Yuejian, Chen Weixiong, Luo Jie, Zhu Xiaolin, Wen Weiping, Lei Wenbin
Otorhinolaryngology Hospital, Otorhinolaryngology Institute, The First Affiliated Hospital, Sun Yat-sen University, National Key Department of Otorhinolaryngology of People's Republic of China, Guangzhou, Guangdong 510080, P.R. China.
Department of Otolaryngology, The First People's Hospital of Foshan, Foshan, Guangdong 528000, P.R. China.
Oncol Rep. 2014 Sep;32(3):1108-16. doi: 10.3892/or.2014.3286. Epub 2014 Jun 24.
The aim of the present study was to explore the effect of DJ-1-mediated PI3K/AKT/mTOR pathway on the proliferation, apoptosis, invasion, migration and other tumor biological characteristics of laryngeal squamous cell SNU-46, through stable transfection and overexpression of the DJ-1 gene. Retrovirus carrying DJ-1 gene was used to stabilize transfected human laryngeal squamous carcinoma SNU-46 cell line, and monoclonal cell line of stably overexpressed DJ-1 protein was screened out by G418. DJ-1 protein expression was determined by western blotting, and changes of p-AKT, p-mTOR and PTEN protein content were detected, followed by the detection of changes in proliferation, apoptosis, invasion, migration and other tumor biological characteristics of laryngeal squamous carcinoma cell line with stably transfected DJ-1 protein overexpression by flow cytometry, CCK-8 method and Transwell. We successfully constructed a laryngeal squamous carcinoma cell line of stably overexpressed DJ-1 protein and termed it SNU-46-DJ-1. After overexpression of DJ-1 protein, the levels of PTEN expression in laryngeal squamous cell SNU-46 decreased and p-AKT and p-mTOR protein expression levels increased. Compared to the untreated SNU-46 cells, the proliferation rate of SNU-46-DJ-1 cells increased (0.834±0.336 vs. 0.676±0.112; p<0.001); invasiveness was enhanced (165.7±13.6 vs. 100.0±17.4; p=0.001), the migration ability was enhanced (207.3±13.1 vs. 175.3±13.3; p=0.036), and the apoptosis rate decreased (3.533±5.167 vs. 16.397±5.447%; p=0.019). The overexpression of DJ-1 protein in laryngeal squamous carcinoma SNU-46 cells can accelerate proliferation rate, increase the invasion and migration capacity, and reduce apoptosis, by activating the PI3K/AKT/mTOR pathway.
本研究旨在通过稳定转染和过表达DJ-1基因,探讨DJ-1介导的PI3K/AKT/mTOR通路对喉鳞状细胞SNU-46增殖、凋亡、侵袭、迁移及其他肿瘤生物学特性的影响。采用携带DJ-1基因的逆转录病毒对人喉鳞状癌细胞系SNU-46进行稳定转染,并用G418筛选出稳定过表达DJ-1蛋白的单克隆细胞系。通过蛋白质免疫印迹法检测DJ-1蛋白表达,并检测p-AKT、p-mTOR和PTEN蛋白含量的变化,随后采用流式细胞术、CCK-8法和Transwell检测稳定转染DJ-1蛋白过表达的喉鳞状癌细胞系增殖、凋亡、侵袭、迁移及其他肿瘤生物学特性的变化。我们成功构建了稳定过表达DJ-1蛋白的喉鳞状癌细胞系,并将其命名为SNU-46-DJ-1。DJ-1蛋白过表达后,喉鳞状细胞SNU-46中PTEN表达水平降低,p-AKT和p-mTOR蛋白表达水平升高。与未处理的SNU-46细胞相比,SNU-46-DJ-1细胞的增殖率增加(0.834±0.336对0.676±0.112;p<0.001);侵袭性增强(165.7±13.6对100.0±17.4;p=0.001),迁移能力增强(207.3±13.1对175.3±13.3;p=0.036),凋亡率降低(3.533±5.167对16.397±5.447%;p=0.019)。喉鳞状癌细胞SNU-46中DJ-1蛋白的过表达可通过激活PI3K/AKT/mTOR通路加速增殖率,增加侵袭和迁移能力,并减少凋亡。