应用比较蛋白质组学技术筛选肾透明细胞癌细胞差异表达蛋白的初步研究

Preliminary analysis of differentially expressed proteins of clear-cell renal cell carcinoma by comparative proteomic technologies

目的探讨人肾透明细胞癌细胞株RLC-310和人正常肾近曲小管上皮细胞株HK-2蛋白表达的差异，寻找新的肾癌差异表达蛋白。方法体外培养RLC-310和HK-2细胞，采用Proteome Lab PF2D蛋白分离系统分离细胞总蛋白，采用毛细管反相色谱-电喷雾-线性离子阱串联质谱（CapillaryLC—ESI-MS／MS）分析和鉴定差异蛋白组分。采用逆转录聚合酶链反应（RT—PCR）和Westernblot法对代表性差异表达蛋白进行mRNA和蛋白质表达水平的验证。结果RLC-310和HK-2细胞株共鉴定出196个差异蛋白，涉及肾透明细胞癌细胞增殖和抗凋亡、能量代谢、线粒体还原氧化、氧化应激和耐药、细胞信号传导、侵袭和黏附、细胞骨架和运动、肿瘤血管形成等。RT—PCR和Westernblot检测显示，Septin-9mRNA和蛋白在RLC-310细胞中高表达，Septin-9mRNA在HK-2细胞中不表达，Septin-9蛋白在RLC-310细胞中的表达水平明显高于HK-2细胞（P〈0．05）。结论肾透明细胞癌和正常。肾细胞在蛋白质组的表达存在显著差异，鉴定出的差异表达蛋白涉及肾透明细胞癌发生、发展的各方面，并有助于深入研究和阐明。肾透明细胞癌发生、发展的分子机制。Septin-9作为重要的差异表达蛋白，在肾透明细胞癌肿瘤血管形成中的作用值得深入研究。

Objective To explore the different expression of proteins between human clear-cell renal cell carcinoma （ccRCC） cell line RLC-310 and normal renal proximal tubule epithelial cell line HK-2, and to search new differentially expressed proteins of RCC. Methods RLC-310 and HK-2 cells were cultured in vitro. The total proteins were separated by ProteomeLab PF 2D protein fractionation system and the differential expression protein fractions of the two cell lines were analyzed and identified by capillary liquid chromatography-electrospray ionization-mass spectrometry （LC-ESI-MS/MS）. RT-PCR and Western blot were used to confirm the representative differential expression at mRNA and protein levels. Results One hundred and ninty-six differentially expressed proteins were identified. These differentially expressed proteins involved in many aspects, including cell proliferation and anti-apoptosis, energy metabolism, mitochondria reduction and oxidation, oxidative stress and resistance, cell signaling, invasion and adhesion, cytoskeleton and motion, neovascularization, etc. Except for previously reported RCC associated proteins： annexin A2, fatty acid-binding protein, vimentin, fibronectin, and so on, Septin-9 was firstly found highly expressed in RLC-310 cells when compared with that in the HK-2 cells. Moreover, the overexpression of Septin-9 was confirmed by RT-PCR and Western blot analysis at both mRNA and protein levels（ P 〈 0.05 ）. Conclusions The human ecRCC cell line RLC-310 cells display differential protein profiles compared with those of the normal renal cell line HK-2 cells. The identified differential expression proteins are involved in many aspects of RCC development. It is worth further study and elucidate the molecular mechanisms of RCC. The representative differential protein Septin-9 deserves further study its role in the angiogenesis of ccRCC.