背景:CHIP是一种E3泛素连接酶,在多种人类恶性肿瘤中起着对比作用,取决于目标。迄今为止,CHIP在胃癌中的作用机制尚不清楚.这里,我们旨在进一步阐明CHIP在胃癌发生发展中的作用,并探讨其潜在的作用靶点.
方法:使用Lipofectamine2000建立稳定转染的CHIP-shRNA和TRAF2-shRNAAGS胃癌细胞系。通过xCelligence实时监测系统和集落形成测定来测量细胞生长。使用CCK-8、Ki-67或CFSE测定法检测细胞增殖。通过TUNEL测定或膜联蛋白V/PI-染色随后通过流式细胞术分析来检测细胞凋亡。通过PI染色然后通过流式细胞术分析检测细胞周期分布。通过实时xelligence系统测量细胞迁移和侵袭能力,Transwell插入,和划痕试验。细胞周期相关蛋白的表达,凋亡相关蛋白,AKT,ERK,NF-κB信号亚基,通过蛋白质印迹分析检测MMP2、MMP9和整合素β-1。使用基于ELISA的NF-κB活性测定定量NF-κBDNA结合能力。分析胃癌组织芯片以研究CHIP和TRAF2的表达及其临床意义。
结果:AGS细胞中的CHIP沉默是致癌的,表现为能够锚定非依赖性生长。CHIP沉默显着增强了AGS细胞的增殖能力,这可能是由于ERK的诱导磷酸化所致。由于Bcl-2的表达增加,CHIP沉默显著抑制细胞凋亡。CHIP沉默促进AGS细胞迁移和侵袭能力,可能通过调节整合素β-1的表达。TRAF2表达在CHIP过表达细胞的蛋白质水平上显著降低,但不是在mRNA水平。TRAF2沉默显著抑制AGS细胞的增殖能力,参与了细胞增殖缺陷和凋亡增强。TRAF2沉默也减弱了AGS细胞的细胞迁移和侵袭能力。此外,胃癌组织中CHIP的表达下调,而TRAF2的表达上调。TRAF2表达是胃癌的独立预后因素。CHIP和TRAF2在胃癌组织中的表达呈负相关。较低的CHIP或较高的TRAF2与胃癌患者的总生存期缩短显著相关。
结论:TRAF2影响胃癌细胞的细胞行为的各个方面,包括细胞生长,迁移,和入侵,这与CHIP的功能相反。TRAF2可作为胃癌患者的独立预后因子。TRAF2可能是CHIP的底物,CHIP调节TRAF2/NF-κB轴,在AGS胃癌细胞中调节多种细胞行为。
BACKGROUND: CHIP is an E3 ubiquitin ligase that plays contrast roles in diverse human malignancies, depending on its targets. To date, the mechanisms underlying the function of CHIP in gastric cancer remains unclear. Here, we aim to further clarify the effects of CHIP on the development and progression of gastric cancer and explore its potential target.
METHODS: Stably transfected CHIP-shRNA and TRAF2-shRNA AGS gastric cancer cell lines were established using Lipofectamine 2000. Cell growth was measured by an xCelligence real-time monitoring system and colony formation assay. Cell proliferation was detected using CCK-8, Ki-67, or CFSE assays. Apoptosis was detected by TUNEL assay or Annexin V/PI-staining followed by flow cytometric analysis. Cell cycle distribution was detected by PI-staining followed by flow cytometric analysis. Cell migration and invasion abilities were measured by a real-time xCelligence system, Transwell insert, and scratch assays. The expression of cell cycle-related proteins, apoptosis-related proteins, AKT, ERK, NF-κB signaling subunits, MMP2, MMP9, and Integrin β-1 were detected by Western blotting analysis. NF-κB DNA-binding capability was quantified using an ELISA-based NF-κB activity assay. Gastric cancer tissue microarray was analyzed to investigate the expression of both CHIP and TRAF2, and their clinical significance.
RESULTS: The CHIP-silencing in the AGS cells was oncogenic evidenced by the appearance of capable of anchorage-independent growth. The CHIP-silencing significantly enhanced the AGS cell proliferation capability likely due to the induced phosphorylation of ERK. The CHIP-silencing significantly inhibited apoptosis due to increased expression of Bcl-2. The CHIP-silencing promoted the AGS cell migration and invasion abilities, likely by regulating the expression of Integrin β-1. TRAF2 expression was markedly decreased in the CHIP-overexpressing cells at protein level, but not at mRNA level. The TRAF2-silencing markedly inhibited the proliferation ability of the AGS cells, the defected cell proliferation and enhanced apoptosis were involved in. The TRAF2-silencing also attenuated the cell migration and invasion capacities of the AGS cells. Furthermore, the expression of CHIP was downregulated while the expression of TRAF2 was upregulated in gastric cancer tissues. TRAF2 expression is independent prognostic factors of gastric cancer. The expression of CHIP and TRAF2 was negatively correlated in the gastric cancer tissue. Lower CHIP or higher TRAF2 was significantly linked to shorter overall survival in gastric cancer patients.
CONCLUSIONS: TRAF2 influenced diverse aspects of cellular behavior of gastric cancer cells, including cell growth, migration, and invasion, which was in contrast to the functions of CHIP. TRAF2 could be considered as an independent prognostic factor in gastric cancer patients. It is possible that TRAF2 was a substrate of CHIP and CHIP regulated the TRAF2/NF-κB axis, which modulated diverse cellular behaviors in the AGS gastric cancer cells.