摘要
背景:鼻咽癌是我国南方及东南亚地区最常见的恶性肿瘤之一,早期转移是鼻咽癌最重要的特征之一,也是患者治疗失败的主要原因,但鼻咽癌转移机制尚不清楚。Raf激酶抑制蛋白(RKIP)是一种高度保守、广泛表达、具有多种生理与病理功能的蛋白质,近年来的研究表明,RKIP具有抑制前列腺癌和乳腺癌转移的作用,可能是一种新的肿瘤转移抑制基因。
方法:采用蛋白质组学技术分离、鉴定鼻咽癌组织与鼻咽正常粘膜组织的差异表达蛋白质。以差异蛋白质RKIP为研究对象,采用Western blot检测RKIP在不同转移潜能的鼻咽癌细胞5-8F(高转移)和6-10B(不转移)细胞中的表达水平,采用免疫组织化学检测RKIP在鼻咽正常粘膜组织、鼻咽癌组织和颈淋巴结转移癌组织中的表达水平;采用脂质体转染方法将正义、反义RKIP核酸表达质粒pcDNA3.1(+)-ssRKIP和pcDNA3.1(-)-asRKIP及其相应空白载体pcDNA3.1(+)和pcDNA3.1(-)分别转染5-8F和6-10B细胞,建立相应的稳定转染细胞系。以稳定转染细胞系为样本,Transwell小室侵袭迁移实验检测RKIP表达水平改变对鼻咽癌细胞的侵袭迁移能力的影响;MTT、流式细胞仪和软琼脂集落形成实验检测RKIP表达水平改变对鼻咽癌细胞增殖、细胞周期分布和停泊非依赖性生长的影响。
结果:(1)建立了鼻咽癌组织和鼻咽正常粘膜组织的2-DE图谱,识别了31个差异表达的蛋白质点,质谱鉴定了19个差异表达蛋白质,其中RKIP等蛋白质在鼻咽癌组织中下调;(2)RKIP在高转移5-8F细胞系中的表达水平低于无转移潜能的6-10B细胞,在淋巴结转移鼻咽癌组织中的表达水平低于原发鼻咽癌组织;(3)建立了RKIP表达上调的5-8F细胞系和RKIP表达下调的6-10B细胞系;(4)上调RKIP在5-8F细胞中的表达水平能明显降低其侵袭迁移能力,下调RKIP在6-10B细胞中的表达水平能明显增强其侵袭迁移能力;(5)上调RKIP在5-8F细胞中的表达水平能抑制其增殖和停泊非依赖性生长、并导致其发生G_0/G_1期阻滞,下调RKIP在6-10B细胞中的表达水平能促进其增殖和停泊非依赖性生长、并加速6-10B细胞越过G_0/G_1期。
结论:RKIP具有抑制鼻咽癌细胞侵袭迁移、增殖和停泊非依赖性生长的作用,RKIP可能是鼻咽癌的转移抑制基因。本研究首次发现RKIP可能是鼻咽癌的转移抑制基因,不仅有助于揭示鼻咽癌的转移机制,而且为靶向抑制鼻咽癌的转移提供了实验依据。
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 G_0/G_1 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 G_0/G_1 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.
引文
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