Chen Yan, Ouyang Guo-Liang, Yi Hong, Li Mao-Yu, Zhang Peng-Fei, Li Cui, Li Jian-Ling, Liu Ying-Fu, Chen Zhu-Chu, Xiao Zhi-Qiang
Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, China.
J Proteome Res. 2008 Dec;7(12):5254-62. doi: 10.1021/pr800602c.
To identify novel proteins associated with the pathogenesis of nasopharyngeal carcinoma (NPC), a proteomic approach was used to screen for differential proteins between NPC and adjacent noncancerous nasopharyngeal epithelial tissue (ANNET). As a result, 21 differential proteins were identified by two-dimensional electrophoresis and mass spectrometer. Raf kinase inhibitor protein (RKIP), one of the downregulated proteins in NPC compared to ANNET, was investigated for its role in the metastasis of NPC. Western blot analysis and immunohistochemistry were used to detect RKIP expression in 5-8F and 6-10B NPC cell lines with the different metastatic potentials, and in NNET, primary NPC and NPC metastasis. Furthermore, high metastatic 5-8F with low RKIP expression and nonmetastatic 6-10B with high RKIP expression were stably transfected with plasmids that expressed sense and antisense RKIP cDNA, respectively, or with empty vector. The effects of RKIP expression on in vitro cell invasion, and the activity of Raf-1/MEK/ERK signaling pathway were analyzed in the transfected cells. The results showed that RKIP was significantly downregulated in 5-8F compared with 6-10B, in NPC compared with NNET, and not detectable in NPC metastasis. Overexpressed RKIP in 5-8F could decrease its in vitro cell invasion, whereas downregulated RKIP in 6-10B could increase its in vitro cell invasion. RKIP negatively regulated Raf-1/MEK/ERK signaling pathway in NPC cells, and activation of this signaling pathway by RKIP downregulation increased in vitro invasion of NPC cells. Taken together, our results suggest that RKIP may be a NPC metastasis suppressor, and decreased RKIP expression is associated with the increased invasive capability of NPC cells possibly through the activation of Raf-1/MEK/ERK pathway.
为了鉴定与鼻咽癌（NPC）发病机制相关的新蛋白，采用蛋白质组学方法筛选NPC与相邻鼻咽非癌上皮组织（ANNET）之间的差异蛋白。结果，通过二维电泳和质谱仪鉴定出21种差异蛋白。Raf激酶抑制蛋白（RKIP）是NPC中与ANNET相比下调的蛋白之一，研究了其在NPC转移中的作用。采用蛋白质印迹分析和免疫组织化学检测具有不同转移潜能的5-8F和6-10B NPC细胞系、ANNET、NPC原发灶和NPC转移灶中RKIP的表达。此外，分别用表达有义及反义RKIP cDNA的质粒或空载体稳定转染RKIP表达低的高转移5-8F细胞和RKIP表达高的非转移6-10B细胞。分析转染细胞中RKIP表达对体外细胞侵袭及Raf-1/MEK/ERK信号通路活性的影响。结果显示，与6-10B相比，5-8F中RKIP明显下调；与ANNET相比，NPC中RKIP下调；在NPC转移灶中未检测到RKIP。5-8F中过表达RKIP可降低其体外细胞侵袭能力，而6-10B中下调RKIP可增加其体外细胞侵袭能力。RKIP在NPC细胞中负向调节Raf-1/MEK/ERK信号通路，RKIP下调激活该信号通路可增加NPC细胞的体外侵袭能力。综上所述，我们的结果表明RKIP可能是一种NPC转移抑制因子，RKIP表达降低可能通过激活Raf-1/MEK/ERK通路与NPC细胞侵袭能力增加有关。
