Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan.
BMC Cancer. 2012 Jun 29;12:273. doi: 10.1186/1471-2407-12-273.
Lung cancer is one of the most lethal malignancies worldwide, but useful biomarkers of lung cancer are still insufficient. The aim of this study is to identify some membrane-bound protein(s) associated with migration and invasion in human non-small cell lung cancer (NSCLC) cells.
We classified four NSCLC cell lines into high and low migration/invasion groups by Transwell and Matrigel assays. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), we identified 10 membrane-associated proteins being significantly overexpressed in the high migration/invasion group. The expression of the target protein in the four NSCLC cell lines was then confirmed by reverse transcription polymerase chain reaction (RT-PCR), western blot and immunostaining. RNA interference technique was applied to observe the influence of the target protein on migration and invasion. Gelatin zymography was also performed to evaluate the activities of matrix metalloproteinase (MMP)-2 and MMP-9. Expression condition of the target protein on surgical specimens was further examined by immunohistochemical staining and the clinicopathologic data were analyzed.
We identified a mitochondria-bound protein cytochrome c oxidase subunit Va (COX Va) because of its abundant presence found exclusively in tumorous areas. We also demonstrated that migration and invasion of NSCLC cells decreased substantially after knocking down COX Va by siRNA. Meanwhile, we found a positive correlation between COX Va expression, Bcl-2 expression and activities of MMP-2 and MMP-9 in NSCLC cells. Immunohistochemical staining of surgically resected lung adenocarcinomas in 250 consecutive patients revealed that strong COX Va expression was found in 54.8% (137/250) of patients and correlated positively with the status of lymph node metastasis (P = 0.032). Furthermore, strong COX Va expression was associated with the presence of distant metastasis (P = 0.033).
Our current study showed that COX Va may play a role in migration and invasion of NSCLC cells and can be used as a biomarker to predict aggressiveness of NSCLC.
肺癌是全球最致命的恶性肿瘤之一，但仍缺乏有用的肺癌生物标志物。本研究旨在鉴定与人类非小细胞肺癌（NSCLC）细胞迁移和侵袭相关的一些膜结合蛋白。
我们通过 Transwell 和 Matrigel 分析将四种 NSCLC 细胞系分为高迁移/侵袭组和低迁移/侵袭组。使用二维凝胶电泳和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS），我们鉴定了在高迁移/侵袭组中显著过表达的 10 种膜相关蛋白。然后通过逆转录聚合酶链反应（RT-PCR）、western blot 和免疫染色法确认了四种 NSCLC 细胞系中目标蛋白的表达。应用 RNA 干扰技术观察靶蛋白对迁移和侵袭的影响。还进行了明胶酶谱分析以评估基质金属蛋白酶（MMP）-2 和 MMP-9 的活性。通过免疫组织化学染色进一步检查了目标蛋白在手术标本上的表达情况，并分析了临床病理数据。
我们发现一种线粒体结合蛋白细胞色素 c 氧化酶亚基 Va（COX Va），因为它在肿瘤区域中大量存在。我们还证明，通过 siRNA 敲低 COX Va 后，NSCLC 细胞的迁移和侵袭明显减少。同时，我们发现 COX Va 表达与 NSCLC 细胞中 Bcl-2 表达以及 MMP-2 和 MMP-9 的活性呈正相关。对 250 例连续肺腺癌患者的手术切除标本进行免疫组织化学染色显示，54.8%（137/250）的患者 COX Va 表达强烈，与淋巴结转移状态呈正相关（P=0.032）。此外，COX Va 表达强烈与远处转移存在相关（P=0.033）。
本研究表明，COX Va 可能在 NSCLC 细胞的迁移和侵袭中发挥作用，并可用作预测 NSCLC 侵袭性的生物标志物。
