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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.
In order to elucidate its mechanism of action, the abnormal expression of Raf kinase inhibitor protein (RKIP) plays an important role in the development and progression of gastric cancer. The RKIP-3xFLAG-pcDNA3.1 plasmid was transfected into SGC7901 cells by lipofectamine to establish RKIP-3xFLAG High expression of SGC7901 cells; 3xFLAG tag affinity chromatography and mass spectrometry analysis, separation and identification of proteins interacting with RKIP, and the application of co-immunoprecipitation and Western-blot to further verify the mass spectrometry results were identified 66 RKIP The functional classifications include protein metabolism enzymes, biooxidation related enzymes, cytoskeleton proteins, chaperones, signal transduction related proteins, proteolysis related proteins, etc. For the first time, it is confirmed that there is mutual interaction between 14-3-3 protein and RKIP Which provided important clues for elucidating the mechanism of RKIP in the development of gastric cancer and provided a new target for the early diagnosis and prognosis of gastric cancer.