Yang Ya, Li Min, Yan Yan, Zhang Jia, Sun Kai, Qu Jing-Kun, Wang Jian-Sheng, Duan Xiao-Yi
The Second Department of Thoracic Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
Department of Radiology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
Oncol Rep. 2015 Nov;34(5):2385-94. doi: 10.3892/or.2015.4234. Epub 2015 Sep 1.
Metastasis is the major cause of death among gastric cancer (GC) patients, and altered expression of Ras-related protein RAP1B is associated with cancer development. The present study assessed RAP1B expression ex vivo and the effect of hypoxia‑induced RAP1B overexpression on the promotion of the metastatic potential of GC cells in vitro. Immunohistochemistry was used to detect the expression of RAP1B and hypoxia‑inducible factor-1α (HIF-1α) in 178 GC tissue specimens. GC cell lines were used to assess the effects of hypoxia and RAP1B knockdown with RAP1B small interfering RNA (siRNA). Tumor cell viability was detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, invasion capacity was evaluated by a Transwell assay, and gene expression was determined by qRT-PCR and western blotting. The data showed that expression levels of RAP1B and HIF-1α proteins were high in the GC tissue specimens, and RAP1B expression was significantly associated with tumor-node-metastasis (TNM) stage and tumor size, while HIF-1α expression was significantly associated with TNM stage, Borrmann type and tumor differentiation. Moreover, RAP1B expression was associated with HIF-1α expression (r=0.547, P<0.001). The expression of RAP1B and HIF-1α proteins was associated with a shorter overall survival of patients according to the univariate analysis (log-rank test, P<0.01), and RAP1B expression and TNM stage were independent prognostic predictors in patients using a multivariate analysis (P<0.001). In vitro, hypoxia induced the invasion of GC cells and the expression of RAP1B and HIF-1α (P<0.05); whereas knockdown of RAP1B expression using siRNA inhibited the tumor cell invasion capacity even under hypoxic culture conditions (P<0.05). In conclusion, the protein expression of RAP1B and HIF-1α contributed to GC malignant behavior and poor prognosis. Future studies will evaluate whether targeting RAP1B expression can be used as a novel strategy to control GC or as a biomarker for prognosis prediction.
转移是胃癌（GC）患者死亡的主要原因，而Ras相关蛋白RAP1B表达的改变与癌症发展相关。本研究评估了RAP1B的体外表达以及缺氧诱导的RAP1B过表达对体外促进GC细胞转移潜能的影响。采用免疫组织化学法检测178例GC组织标本中RAP1B和缺氧诱导因子-1α（HIF-1α）的表达。利用GC细胞系评估缺氧和用RAP1B小干扰RNA（siRNA）敲低RAP1B的影响。通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）法检测肿瘤细胞活力，通过Transwell法评估侵袭能力，通过qRT-PCR和蛋白质印迹法测定基因表达。数据显示，GC组织标本中RAP1B和HIF-1α蛋白表达水平较高，RAP1B表达与肿瘤-淋巴结-转移（TNM）分期和肿瘤大小显著相关，而HIF-1α表达与TNM分期、Borrmann分型和肿瘤分化显著相关。此外，RAP1B表达与HIF-1α表达相关（r = 0.547，P < 0.001）。单因素分析（对数秩检验，P < 0.01）显示，RAP1B和HIF-1α蛋白表达与患者较短的总生存期相关，多因素分析显示，RAP1B表达和TNM分期是患者独立的预后预测指标（P < 0.001）。在体外，缺氧诱导GC细胞侵袭以及RAP1B和HIF-1α表达（P < 0.05）；而使用siRNA敲低RAP1B表达即使在缺氧培养条件下也能抑制肿瘤细胞侵袭能力（P < 0.05）。总之，RAP1B和HIF-1α的蛋白表达促成了GC的恶性行为和不良预后。未来研究将评估靶向RAP1B表达是否可作为控制GC的新策略或作为预后预测的生物标志物。
