Zhang Wei, Li Yanting, Yang Shuanying, Li Wei, Ming Zongjuan, Zhang Yuping, Hou Yanli, Niu Zequn, Rong Biaoxue, Zhang Xuede, Liu Xiaoli
Respiratory Medicine, Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China.
Ultrasound Department, People's Hospital of Tongchuan City, Tongchuan, Shaanxi, China.
Thorac Cancer. 2013 Nov;4(4):373-379. doi: 10.1111/1759-7714.12031.
Lung cancer is one of the higher incidences of malignant tumors around the world. At present, tumor markers CEA, CA19-9, and CA-125 in serum are used for the diagnosis of lung cancer, however, fewer studies have shown tumor markers for early diagnosis. Therefore, using quantitative mass spectrometry, differential mitochondrial proteome analysis was performed, comparing human lung adenocarcinoma and normal bronchial epithelium cells.
A human lung adenocarcinoma cell line A549 and a normal human bronchial epithelial cell line 16HBE were cultured in vitro. The cell mitochondria of the two cell lines were extracted and purified by differential centrifugation and percoll density gradient centrifugation. The integrity and purity of mitochondria were validated by electron microscopy and Western-blot. The proteins/peptides from lung cancer cells and normal cells were marked by the same amount of relative and absolute quantification of ectopic tags (iTRAQ). The mixed samples were analyzed and identified by two-dimensional liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS). The proteome was analyzed with different bioinformatic tools.
One hundred and sixty-one mitochondrial proteins were identified. One hundred and fifty-three mitochondrial proteins, which were expressed differently between 16HBE cells and A549 cells, were identified. Sixty-seven proteins were high expression, while 86 proteins were lower expression. Expression of three proteins: ornithine aminotransferase (OAT), heat shock protein beta90 (HSP90), and vimentin (VIM), was increased more than twice. Our results, in combination with the literature review, suggest that HSP90 and Vimentin may be the new tumor markers of lung cancer.
肺癌是全球发病率较高的恶性肿瘤之一。目前，血清中的肿瘤标志物癌胚抗原（CEA）、糖类抗原19-9（CA19-9）和糖类抗原125（CA-125）用于肺癌诊断，然而，较少有研究表明这些肿瘤标志物可用于早期诊断。因此，采用定量质谱法，对人肺腺癌和正常支气管上皮细胞进行差异线粒体蛋白质组分析。
体外培养人肺腺癌细胞系A549和正常人支气管上皮细胞系16HBE。通过差速离心和Percoll密度梯度离心法提取并纯化两细胞系的线粒体。通过电子显微镜和蛋白质免疫印迹法验证线粒体的完整性和纯度。用等量的异位标签相对和绝对定量（iTRAQ）标记肺癌细胞和正常细胞中的蛋白质/肽段。混合样本经二维液相色谱-串联质谱（2D-LC-MS/MS）分析鉴定。使用不同的生物信息学工具分析蛋白质组。
共鉴定出161种线粒体蛋白。鉴定出153种在16HBE细胞和A549细胞中表达差异的线粒体蛋白。其中67种蛋白高表达，86种蛋白低表达。鸟氨酸氨基转移酶（OAT）、热休克蛋白β90（HSP90）和波形蛋白（VIM）这三种蛋白的表达增加了两倍以上。我们的研究结果结合文献综述表明，HSP90和波形蛋白可能是肺癌新的肿瘤标志物。
