Identification Of RKIP As A Metastasis Suppressor Protein In Nasopharyngeal Carcinoma By Proteomic Approach

Nasopharyngeal carcinoma(NPC) is one of the most common malignant tumors in southern China,with an incidence rate ranging from 20 to 50/100,000,and it poses one of the most serious public health problems in southern China.Early metastasis is one of distinctive characteristics of NPC and the main cause of death for NPC patients. Although numerous efforts have been made to reveal the molecular mechanism of NPC metastasis,it remains poorly understood. Identification of NPC metastasis-related molecules will be helpful for diagnosis and treatment of NPC,and may provide new insights into its metastasis.High throughput omics technologies such as microarrays and proteomics offer the potential ability to find alterations previously unidentified in cancer.Analyses for gene expression profiles of NPC have been reported using a cDNA array,found the genes with aberrant expressions possibly contributed to pathogenesis of NPC.Because the functional molecules in cells are proteins,proteome analysis is believed to have an advantage over cDNA microarray for clinical use.Proteomics has introduced a new approach to cancer research which aims at identifying differential expression proteins associated with the development and progression of cancer,providing new opportunities to uncover biomarkers and therapeutic targets for cancer.To screen for NPC associated proteins,we carried out the following 3 parts of research in this study.1.Differential proteome study of NPC and adjacent non-cancerous nasopharyngeal epithelial tissue(ANNET).2-DE was performed to separate the total proteins from 10 paired NPC and ANNET,image analysis was used to fine the differential protein spots between the two types of tissues,and both MALDI-TOF-MS and ESI-Q-TOF MS/MS were performed to identify the differential proteins.As a result,21 differential proteins were identified.Among them,nine proteins including Raf kinase inhibitor protein(RKIP) were significantly downregulated in NPC as compared ANNET.2.Differential phosphoproteome study of NPC and non-cancerous nasopharyngeal epithelial tissue(NNET).2-DE was used to separate the total proteins from 10 NPC and 10 NNET.After transferring the proteins to PVDF membranes,2-D Western blotting was did to find the differential tyrosine-phosphorylated proteins between the two types of tissues by using monoclonal anti-phosphotyrosine antibody,and ESI-Q-TOF MS/MS was performed to identify the differential tyrosine-phosphorylated proteins.And then NetPhos software was used to predict the tyrosin-phosphorylation sites of the identified proteins,and 1-D Western blotting was did to verify the differential tyrosine-phosphorylated protein RKIP.As a result,13 differential tyrosine-phosphorylated proteins were identified,and tyrosine-phosphorylated levels of six proteins including RKIP were significantly downregulated in NPC as compared NNET.3.Study on the roles and mechanisms of RKIP in NPC metastasis.To explore the roles and mechanisms of RKIP in NPC metastasis,Western blotting analysis and immunohistochemistry was respectively 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 non-metastatic 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 and NF-κB 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 increased its in vitro cell invasion.RKIP negatively regulated Raf-1/MEK/ERK and NF-κB signaling pathway in NPC cells,and activation of the two signaling pathways 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 and NF-κB signaling pathway.