The Role Of Raf Kinase Inhibitor Protein In Nasopharyngeal Carcinoma Metastasis

Background: Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China and Southeast Asia. Early Metastasis is one of the most important characteristics of NPC, and the main cause of death for most patients with NPC. However, the mechanism underlying the metastasis of NPC remains poorly understood. Raf kinase inhibitor protein (RKIP), a conserved, small, cytosolic protein, has multiple physiological and pathophysiological functions. Recently, a lot of studies demonstrated RKIP could suppress the metastasis of prostate cancer and breast cancer, and RKIP has been recognized as a strong candidate for a metastasis suppressor gene.Methods: Proteomic analysis was performed to identify the differential expression proteins between NPC and normal nasopharyngeal mucosal tissues. Role of RKIP, one of the differential expression proteins identified by proteomic analysis, in the metastasis of NPC was further investigated. Western blot analysis was used to measure the expression of RKIP in the human NPC cell line 5-8F with high metastatic potential and 6-10B without metastatic potential. Immunohistochemistry was performed to detect RKIP expression in paraffin-embedded human normal nasopharyngeal mucosal tissues, primary NPC and their lymph node metastases. 5-8F and 6-10B cells were stably transfected with plasmids that constitutively expressed sense and antisense RKIP cDNA, respectively, or with empty vector. And then In vitro transwell migration and invasion assays were used to examine the effect of RKIP expression on migration and invasion of NPC cells using stably transfected cell lines. MTT assay, flow cytometry (FCM) analysis and soft-agar colony formation assay were used to examine the effect of RKIP expression on proliferation, cell cycle distribution and anchorage independence growth of NPC cells using stably transfected cell lines. Results: (1) 2-DE reference patterns of human NPC and normal nasopharyngeal mucosal tissues were established. 19 differential expression proteins between the two types of tissues were identified by mass spectrometer, of which RKIP was upregulated in NPC tissue as compared with normal nasopharyngeal mucosal tissues. (2) The expression level of RKIP was significantly lower in 5-8F cells than 6-10B cells, and in NPC lymph node metastases than in primary NPC tissues. (3) 5-8F cell line stably transfected with sense RKIP expression vector, and 6-10B cell line stably transfected with antisense RKIP expression vector were successfully established. (4) Up-regulation of RKIP expression in 5-8F cells was associated with decreased in vitro cell invasion and migration, and down-regulation of RKIP expression in 6-10B cells was associated with increased in vitro cell invasion and migration. (5) Up-regulation of RKIP expression in 5-8F cells could inhibit cell proliferation and anchorage independence growth, and cause an increase in the number of G₀/G₁ phase cells. Down-regulation of RKIP expression in 6-10B cells could promote cell proliferation and anchorage independence growth, and cause a decrease in the number of G₀/G₁ phase cells.Conclusion: RKIP can inhibit cell invasion, migration, proliferation and anchorage independence growth of human NPC cells, suggesting that RKIP may be a suppressor gene of NPC metastasis. To our knowledge, this is the first time to find that RKIP may function as a NPC metastasis suppressor gene, which not only is helpful to elucidating the mechanism underlying the metastasis of NPC, but also provides a potential therapeutic target for the metastasis of NPC.