【作者】 赵力； 辛彦； 肖玉平；

【Author】 Li Zhao;Yan Xin;Yuping Xiao;Department of Gastrointestinal Tumor Pathology of Cancer Institute,the First Hospital of China Medical University;

【机构】 中国医科大学附属第一医院肿瘤研究所胃肠肿瘤病理研究室；

【摘要】 研究目的:AEG-1稳定沉默后对BGC-823胃癌细胞生物学特性的影响及其机制研究。研究方法:筛选出高表达AEG-1的BGC-823胃癌细胞系,将成功构建稳定沉默AEG-1基因的胃癌细胞,通过MTT法、克隆形成实验、caspase-3活性检测、Hoechst 33258荧光染色检测、划痕愈合实验和Transwell小室侵袭实验检测BGC823细胞体外生物学特性的变化。采用Western blot方法检测PI3k/Akt/mTOR信号传导通路、自噬相关因子、凋亡相关因子和转移相关因子的表达变化,建立裸鼠皮下移植瘤模型,检测了AEG-1稳定沉默后对BGC-823胃癌细胞体内生长的影响,免疫组化方法检测瘤体组织中AEG-1、VEGF、CD31蛋白表达的变化。研究结果:成功构建沉默效率最高的载体质粒建立了稳定沉默AEG-1基因的BGC823细胞系。沉默AEG-1基因后,减弱了细胞的增殖能力和克隆形成能力,促进了细胞凋亡,降低了BGC-823细胞的迁移和侵袭能力。Western-blot实验检测发现,与增殖、凋亡相关的P-Akt、p-mTOR、NF-κB蛋白的表达量降低,caspase-3蛋白的表达量增加。自噬相关蛋白P62的表达量减少,Beclinl和LC3Ⅱ型蛋白的表达量增多,并且LC3Ⅱ型与LC3Ⅰ型的比值增加,与侵袭转移相关的MMP9、MMP2,VEGF蛋白的表达量减少。裸鼠皮下移植瘤生长速度减慢,瘤重低于对照组。与对照组相比,沉默AEG-1后的BGC823细胞瘤体组织中VEGF表达显著降低,CD31标记的微血管数量减少。研究结论:沉默胃癌细胞系BGC823中的AEG-1基因,可能通过抑制PI3k/Akt/mTOR通路的活性,增强caspase-3的活性,抑制NF-κB的作用、促进细胞凋亡。增强Beclin1和LCⅡ的活性,减弱P62的作用,诱导细胞自噬,增加细胞死亡。抑制基质金属蛋白酶MMP9,2和血管内皮生长因子VEGF的作用降低细胞侵袭转移能力。沉默AEG-1基因能减慢BGC823细胞裸鼠皮下移植瘤的生长,降低皮下移植瘤组织中AEG-1蛋白的表达,抑制移植瘤中血管的形成,减少微血管的数量。更多还原

【Abstract】 Objective:To observe the changes of gastric cancer cell biological behavior by silencing AEG-1 gene expression and analyze its possible mechanism.Method:After stable silencing AEG-1 gene expression in gastric cancer cell line BGC-823by RNAi,MTT method was used to detect cell proliferation and clone forming experiment test cell clone forming ability,caspase 3 activity detection and Hoechst 33258 fluorescent staining detected apoptosis,and scratches healing experiment and Transwell little room invasion experiment detected cell migration and invasion ability..Using Western Blot method to detect the expression of PI3K/AKT/mTOR signaling pathway,autophagy related molecular,apoptosis related molecular.Establishing a nude mice subcutaneous transplantation tumor model,we observed the growth of tumor and used immunohistochemical method to detect the changes of protein expression of AEG-1,VEGF and CD31 in tumor tissues.Results:Stable AEG-1-silencing vectors were successfully constructed,and BGC-823 gastric cancer cell clone with stable silenced AEG-1 was obtained.After silencing AEG-1 gene in BGC-823 gastric cancer cells,the cell proliferation and clone formation ability were decreased significantly.It promoted the apoptosis of cells and reduced the cell migration and invasion ability of BGC-823cell.Western blot method detected the expressions of p-Akt,p-mTOR and NF-kappa B with proliferation and apoptosis were decreased,caspase 3 were increased.The expression of autophagy related protein Beclinl and LC3 Ⅱ were increased,P62 were decreased,and LC3 Ⅱ/LC3 Ⅰ was increased,the expression of MMP9, MMP2 and VEGF protein associated with invasion and metastasis were decreased.After silencing AEG-1 gene in BGC-823 gastric cancer cells,subcutaneous transplantation tumor in nude mice grew slow,tumor weight was lower than the control group.The AEG-1 protein expression in tumors tissue was significantly lower than control group.Conclusions:AEG-1 gene stable silencing in gastric cancer cell line BGC823 silence may promote cell apoptosis by inhibiting PI3k/Akt/mTOR pathway,strengthen NF-kappa B and caspase 3 activity.It can Induce cell autophagy and promote cell death by increasing the activity of Beclinl and LC3Ⅱ,decreasing p62.It can reduce cell invasion and metastasis ability by inhibiting matrix metalloproteinases MMP9,2,and vascular endothelial growth factor VEGF.AEG-1 gene silencing can increase the growth of BGC823 cells in nude mice subcutaneous transplantation tumour,reduce subcutaneous transplantation AEG-1 protein expression in tumor tissue.更多还原