结直肠癌原发灶和正常肠黏膜组织相关差异表达蛋白研究

Proteomic analysis of differentially expressed proteins involved in primary focus of human colorectal carcinomas and normal colonic mucosa

目的探讨结直肠癌原发灶和正常肠黏膜组织中蛋白质组的表达差异。方法采用双向荧光差异凝胶电泳法(2-DDIGE),对比分析结直肠癌原发灶、癌旁正常肠黏膜组织中蛋白质组的表达差异,经质谱分析鉴定差异蛋白点;采用细胞转染方法,将差异蛋白基因导入结直肠癌细胞,观察细胞生物学行为的改变。结果结直肠癌原发灶与正常肠黏膜组织中蛋白质组分有明显差别。结直肠癌原发灶组织与正常肠黏膜组织比较,差异倍数为1.5倍,差异点数目为60个。共分析了8个差异蛋白点,其中2个蛋白点在原发灶组织中表达下调,6个蛋白点在原发灶组织中表达上调。经质谱鉴定,碳酸酐酶Ⅱ(CAⅡ)、蛋白质二硫键异构酶(PDI)在原发性组织中表达下调,醛缩酶A等在原发灶中表达上调。将CAII cDNA克隆转染结直肠癌细胞后,癌细胞侵袭、趋化运动能力及耐药性均明显降低。结论结直肠癌原发灶和正常肠黏膜蛋白质组表达有显著差异,CAⅡ、PDI表达降低和醛缩酶A表达增强可能与结直肠癌的发生相关。

Objective To study the differentially expressed proteins and their biological behavior in colorectal carcinoma tissues and the normal colonic mucosa by proteomics and molecular biology techniques. Methods The technique of fluorescence two dimension differential gel electrophoresis （2-D DIGE） was used to analyze the expression of differential proteins in normal colorectal mucosa and primary cancer loci. Liquid chromatography with electrospray ionization tandem mass spectrometry （LC-ESI-MS/MS） was used to identify the differential proteins. Transfection experiment of colorectal cancer cells was performed with the differential protein cDNA, and the changes in cytobiological behavior were observed. Results Significant differences of protein expression levels were found by two-dimension electro phoresis. Eight differential protein spots were analyzed and identified. Human carbonic anhydrase Ⅱ and protein disulfide isomerase were detected in normal colorectal mucosa, but not in primary cancer loci. Phosphoglycerate kinase 1, fumarate hydratase and aldolase A were expressed in primary cancer. After transfection with human carbonic anhydrase Ⅱ cDNA, the abilities of Lovo cells were obviously reduced in invasiveness, chemotaxy motor and drug resistance. Conclusions Differences on protein expression levels are found between normal colorectal mucosa and primary cancer loci by 2-DE DIGE. The pathogenesis of colorectal carcinoma is related to the reduced expressions of carbonic anhydrase Ⅱ and protein disulfide isomerase and enhanced expression of aldolase A. The technique of differential proteomics is useful in reaching a indepth understanding of the pathogenesis mechanisms of human colorectal cancer.