Hua Yingqi, Jia Xiaofang, Sun Mengxiong, Zheng Longpo, Yin Lin, Zhang Lijun, Cai Zhengdong
Musculoskeletal Oncology Center, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai, 200072, China.
Tumour Biol. 2011 Oct;32(5):1013-21. doi: 10.1007/s13277-011-0203-4. Epub 2011 Jun 25.
Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. To identify new biomarkers for early diagnosis of OS and novel therapeutic candidates, we carried out a plasma membrane proteomic study based on two-dimensional electrophoresis (2DE). The OS cell line MG-63 and the human osteoblastic cell line hFOB1.19 were adopted as the comparison model. We extracted plasma membrane by aqueous two-phase partition extraction. The proteins were separated through 2DE. We analyzed the differentially expressed proteins by Imagemaster software and then identified them by liquid chromatography-tandem mass spectrometry, and the location and function of differential proteins were searched through the Gene Ontology database. In total, 220 protein spots were separated by 2DE. Seven proteins with more than 2.0-folds of difference were successfully identified from 13 gel spots, with 6 up-regulated and 1 down-regulated. Gene Ontology analysis of the differentially expressed proteins indicated that these proteins were involved in seven kinds of functions including binding, structural, cell motility, receptor activity, electron carrier activity, NADH dehydrogenase (ubiquinone) activity, and transcription repressor activity. The up-regulation of NDRG1 was verified in osteosarcoma through Western blotting and by immunohistochemistry in paraffin-embedded tissues. The plasma membrane proteins identified in this study may provide new insights into osteosarcoma cancer biology and potential diagnostic and therapeutic biomarkers.
骨肉瘤（OS）是儿童和青少年中最常见的原发性恶性骨肿瘤。为了鉴定用于骨肉瘤早期诊断的新生物标志物和新的治疗候选物，我们基于二维电泳（2DE）进行了质膜蛋白质组学研究。采用骨肉瘤细胞系MG-63和人成骨细胞系hFOB1.19作为比较模型。我们通过双水相分配萃取法提取质膜。蛋白质通过2DE进行分离。我们使用图像分析软件分析差异表达的蛋白质，然后通过液相色谱-串联质谱对其进行鉴定，并通过基因本体数据库搜索差异蛋白的定位和功能。总共通过2DE分离出220个蛋白点。从13个凝胶点中成功鉴定出7种差异倍数超过2.0倍的蛋白质，其中6种上调，1种下调。对差异表达蛋白质的基因本体分析表明，这些蛋白质参与了包括结合、结构、细胞运动、受体活性、电子载体活性、NADH脱氢酶（泛醌）活性和转录抑制因子活性在内的七种功能。通过蛋白质免疫印迹法以及石蜡包埋组织的免疫组织化学方法验证了骨肉瘤中NDRG1的上调。本研究中鉴定出的质膜蛋白可能为骨肉瘤癌症生物学以及潜在的诊断和治疗生物标志物提供新的见解。
