采用定量蛋白质组学技术筛选小鼠肝癌淋巴道转移相关蛋白

Identification Lymphatic Metastasis-associated Proteins in Mouse Hepatocarcinoma Cell Lines Using Quantitative Proteomics Technique

淋巴道转移是上皮来源恶性肿瘤转移的早期阶段,其发生机制不清,一直是肿瘤学研究面临的难题,为寻找淋巴道转移相关蛋白,以一对来源于同一亲本细胞,且淋巴道转移潜能显著不同的小鼠肝癌腹水型细胞株为研究对象,其中Hca-F为高淋巴道转移力细胞株,Hca-P为低淋巴道转移力细胞株,采用定量蛋白质组学技术——荧光差异双向凝胶电泳,建立了高低淋巴道转移力小鼠肝癌细胞荧光差异蛋白表达图谱,高通量筛选与肿瘤淋巴道转移相关的蛋白质.经DeCyde软件分析,共得到163个有统计学差异的蛋白质点,选择2倍以上的差异性蛋白质点23个,经质谱鉴定得到17个蛋白质,在Hca-F中高表达的蛋白质有7个:转羟乙醛酶、波形蛋白、肌酸激酶(脑)、膜联蛋白7、膜联蛋白5、烯酰辅酶A水合酶1(过氧化物酶体)、核内异质核糖核蛋白A2/B1异构体1.而在Hca-F中低表达的蛋白质有10个:真核翻译延长因子2、Ero1样蛋白、乙醛脱氢酶2(线粒体)、苹果酸盐脱氢酶2(NAD)、β-内酰胺酶2、谷胱甘肽S转移酶"1、泛素C末端水解酶同工酶L3、内质网蛋白29(前体)、溶血磷脂酶1、微管不稳定蛋白.这些差异性蛋白质的功能涉及到代谢、蛋白质分泌、蛋白质结合、核苷酸结合,钙离子结合、凋亡和调节生长等过程.对这些蛋白质功能的进一步验证,将有助于解析肿瘤淋巴道转移的分子机制.

Lymphatic metastasis is the first step in the metastatic process of magligant tumors deriving from epithelia, and its uncertain mechanisms are always the major problem in the field of oncology. In order to obtain lymphatic metastasis-associated proteins, the protein expressed profiles of mouse hepatocarcinoma ascites syngeneic cell lines Hca-F with highly lymphatic metastatic potentiality and Hca-P with low lymphatic metastatic potentiality were compared using fluorescent two-dimensional difference gel electrophoresis（2D DIGE）. DeCyde software was applied to analyze 2D DIGE images,and 163 differential protein spots between Hca-F and Hca-P were detected, including 86 up-regulated protein spots in Hca-F and 77 down-regulated protein spots in Hca-F. 23 protein sports representing differential ratio more than 2-fold were chosen to be analyzed by MS. 17 metastasis-associated proteins were identified, and the partial identified proteins were further validated by Western blotting analysis. Of the identified proteins, the expression oftransketolase, vimentin, creatine kinase（brain）, anxa7 protein, anxa5 protein,enoyl coenzyme A hydratase 1 （peroxisomal）, heterogeneous nuclear ribonucleoprotein A2/B1 isoform 1 were up-regulated in Hca-F. Whereas eukaryotic translation elongation factor 2, Eroll, Aldh2 protein, malate dehydrogenase 2 （NAD）, lactamase beta 2, glutathione S-transferase omegal, ubiquitin carboxyl-terminal hydrolase isozyme L3, endoplasmic reticulum protein ERp29（precursor）, lyplal, stathmin were down-regulated in Hca-F. On the basis of the Gene Ontology （GO）classification, the differential expression proteins were found to be involved in many of biological process, such as metabolism, protein secretion, protein binding, nucleic acid binding, calcium ion binding,apoptosis and regulation of growth etc. Validating the function of these proteins is helpful to elucidate the mechanisms of lymphatic metastasis.