胃癌相关新蛋白Raf激酶抑制蛋白相互作用蛋白质的鉴定

Characterization of Interaction Proteins for Gastric Cancer Related Novel Protein RKIP

Raf激酶抑制蛋白(RKIP)的异常表达在胃癌的发生发展中起重要的作用,为了阐明其作用机制,应用脂质体将RKIP-3xFLAG-pcDNA3.1质粒转染至SGC7901细胞,建立RKIP-3xFLAG高表达的SGC7901细胞;并利用3xFLAG标签的亲和层析技术联合质谱分析,分离、鉴定与RKIP相互作用的蛋白质,并应用免疫共沉淀联合Western-blot进一步验证质谱结果.共鉴定出66个RKIP相互作用蛋白质,功能分类包括蛋白质代谢酶类、生物氧化相关酶类,细胞骨架蛋白、分子伴侣、信号转导相关蛋白、酶解相关蛋白等.并首次证实14-3-3蛋白与RKIP存在相互作用.为阐明RKIP在胃癌发生发展中的作用机制提供了重要的线索,为胃癌的早期诊治及预后监测提供了新的靶点.

Gastric cancer （GC） seriously impacts on human health with high incidence and mortality rate in the world. Raf kinase inhibitor protein （RKIP） that was discovered in our previous studies is down-regulated in GC, which is associated with the occurrence, differentiation, invasion, and metastasis of GC. In order to investigate the molecular mechanisms and biological functions of RKIP in the occurrence and metastasis of GC, the fusion expression plasmid pcDNA3.1-RK/P-3xFLAG was transiently transfected into SGC7901 cells with liposome-mediated approach, the RKIP fusion proteins were purified with 3xFLAG tag affinity chromatography, and the RKIP-interacted proteins were identified with electrospray ionization-Quadrupole-time of flight （ESI-Q-TOF） tandem mass spectrometry （MS/MS）. A total of 66 RKIP-interacted proteins were MS/MS-identified. The MS/MS-characterized components of the existing interaction complex （RKIP and 14-3-3） were confirmed with Western blotting in combination with co-immunoprecipitation. It is the first time to discover the interaction of RK/P with 14-3-3. This preliminary study on the molecular mechanisms that how RKIP inhibits the occurrence and development of GC provides novel clues for the early-stage diagnosis, prognosis monitoring, and targeted gene therapy of GC.