RKIP在鼻咽癌侵袭转移中的作用及机制研究
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摘要
目的:研究Raf激酶抑制蛋白(Raf kinase inhibitory protein,RKIP)在鼻咽癌(nasopharyngeal c a r c i n o m a,N P C)侵袭转移中的作用及其机制。方法:采用免疫组织化学染色检测R K I P在转移潜能不同的NPC组织中的表达,分析其表达水平与NPC临床病理特征和患者预后的关系;采用脂质体转染技术建立RKIP表达改变的稳定转染NPC细胞系,采用刮痕愈合实验和Transwell小室侵袭实验检测细胞的体外运动和侵袭能力,采用Western blot检测NF-κB信号分子磷酸化水平。结果:RKIP在NPC组织中的表达显著低于正常鼻咽粘膜组织,在有转移NPC组织中的表达明显低于无转移NPC组织,在颈淋巴结转移癌组织中的表达明显低于原发癌;RKIP表达水平与NPC的淋巴结转移、远处转移、临床分期以及NPC患者的总生存期负相关;RKIP表达上调降低5-8F NPC细胞的体外运动和侵袭能力,而RKIP表达下调增强6-10B NPC细胞的体外运动和侵袭能力;RKIP表达水平与NPC细胞NF-κB信号通路的活性负相关,NF-κB抑制剂Bay11-7082能抑制RKIP下调的6-10B细胞的体外运动和侵袭。结论:RKIP可能是NPC的一个转移抑制蛋白,RKIP低表达的NPC患者预后差,RKIP表达下调通过活化NF-κB通路促进NPC的侵袭和转移。
Objective: To investigate the roles and mechanisms of Raf kinase inhibitory protein(RKIP) in the invasion and metastasis of nasopharyngeal carcinoma(NPC). Methods: Immunohistochemistry was performed to detect the expression levels of RKIP in the NPC tissue specimens with different metastasis; Establishment of stable transfected NPC cell lines with RKIP expression changes using lipofectamine 2000; Scratch wound-wealing assay and traswell invasion assay were performed to determine the ability of cell migration and invasion; Western blot was used to detect the phosphorylated levels of NF-κB signaling molecules in NPC cells. Results: RKIP expression was signii cantly reduced in the NPCs relative to normal nasopharyngeal mucosa, and in the NPCs with metastasis relative to NPCs without metastasis, and there was not PKIP expression in the almost all lymphonode metastases. RKIP expression was found to be significantly associated with lymph node and distant metastasis, advanced clinical stage and reduced overall survival. Stable overexpression of RKIP in 5-8F NPC cells dramatically decreased tumor cell migration and invasion, whereas stable knockdown of RKIP in 6-10 B NPC cells signii cantly increased tumor cell migration and invasion. RKIP expression levels were negatively associated with the activity of NF-κB signaling pathway, and Bay11-7082 could inhibit the migration and invasion of 6-10 B cells with RKIP knockdown. Conclusion: RKIP may be a metastatic suppressor protein of NPC, NPC patients with low RKIP level had a poor prognosis, and RKIP downregulation promoted NPC invasion and metastasis by activating NF-κB signaling pathway.
Objective: To investigate the roles and mechanisms of Raf kinase inhibitory protein(RKIP) in the invasion and metastasis of nasopharyngeal carcinoma(NPC). Methods: Immunohistochemistry was performed to detect the expression levels of RKIP in the NPC tissue specimens with different metastasis; Establishment of stable transfected NPC cell lines with RKIP expression changes using lipofectamine 2000; Scratch wound-wealing assay and traswell invasion assay were performed to determine the ability of cell migration and invasion; Western blot was used to detect the phosphorylated levels of NF-κB signaling molecules in NPC cells. Results: RKIP expression was signii cantly reduced in the NPCs relative to normal nasopharyngeal mucosa, and in the NPCs with metastasis relative to NPCs without metastasis, and there was not PKIP expression in the almost all lymphonode metastases. RKIP expression was found to be significantly associated with lymph node and distant metastasis, advanced clinical stage and reduced overall survival. Stable overexpression of RKIP in 5-8F NPC cells dramatically decreased tumor cell migration and invasion, whereas stable knockdown of RKIP in 6-10 B NPC cells signii cantly increased tumor cell migration and invasion. RKIP expression levels were negatively associated with the activity of NF-κB signaling pathway, and Bay11-7082 could inhibit the migration and invasion of 6-10 B cells with RKIP knockdown. Conclusion: RKIP may be a metastatic suppressor protein of NPC, NPC patients with low RKIP level had a poor prognosis, and RKIP downregulation promoted NPC invasion and metastasis by activating NF-κB signaling pathway.
引文
1 .Yu MC,Yuan JM.Epidemiology of nasopharyngeal carcinoma[J].Semin Cancer Biol,2002,12(6):421-429.
2 .King AD,Ahuja AT,Leung SF,et al.Neck node metastases from nasopharyngeal carcinoma:MR imaging of pat erns of disease[J].Head Neck,2000,22(3):275-281.
3 .Huang CJ,Leung SW,Lian SL,et al.Patterns of distant metastases in nasopharyngeal carcinoma[J].Kaohsiung J Med Sci,1996,12(4):229-234.
4 .Chen Y,Ouyang GL,Yi H,et al.Identii cation of RKIP as an invasion suppressor protein in nasophar yngeal carcinoma by proteomic analysis[J].J Proteome Res,2008,7(12):5254-5262.
5 .Keller ET,Fu Z,Brennan M.h e role of Raf kinase inhibitor protein(RKIP)in health and disease[J].Biochem Pharmacol,2004,68(6):1049-1053.
6 .Yeung KC,Rose DW,Dhillon AS,et al.Raf kinase inhibitor protein interacts with NF-kappa B-inducing kinase and TAK1 and inhibits NFkappa B activation[J].Mol Cell Biol,2001,21(21):7207-7217.
7 .Fu Z,Smith PC,Zhang L,et al.Ef ects of raf kinase inhibitor protein expression on suppression of prostate cancer metastasis[J].J Natl Cancer Inst,2003,95(12):839-841.
8 .Hagan S,Al-Mulla F,Mallon E,et al.Reduction of R af-1 kinase inhibitor protein expression correlates with breast cancer metastasis[J].Clin Cancer Res,2005,11(20):7392-7397.
2 .King AD,Ahuja AT,Leung SF,et al.Neck node metastases from nasopharyngeal carcinoma:MR imaging of pat erns of disease[J].Head Neck,2000,22(3):275-281.
3 .Huang CJ,Leung SW,Lian SL,et al.Patterns of distant metastases in nasopharyngeal carcinoma[J].Kaohsiung J Med Sci,1996,12(4):229-234.
4 .Chen Y,Ouyang GL,Yi H,et al.Identii cation of RKIP as an invasion suppressor protein in nasophar yngeal carcinoma by proteomic analysis[J].J Proteome Res,2008,7(12):5254-5262.
5 .Keller ET,Fu Z,Brennan M.h e role of Raf kinase inhibitor protein(RKIP)in health and disease[J].Biochem Pharmacol,2004,68(6):1049-1053.
6 .Yeung KC,Rose DW,Dhillon AS,et al.Raf kinase inhibitor protein interacts with NF-kappa B-inducing kinase and TAK1 and inhibits NFkappa B activation[J].Mol Cell Biol,2001,21(21):7207-7217.
7 .Fu Z,Smith PC,Zhang L,et al.Ef ects of raf kinase inhibitor protein expression on suppression of prostate cancer metastasis[J].J Natl Cancer Inst,2003,95(12):839-841.
8 .Hagan S,Al-Mulla F,Mallon E,et al.Reduction of R af-1 kinase inhibitor protein expression correlates with breast cancer metastasis[J].Clin Cancer Res,2005,11(20):7392-7397.