食管鳞癌恶性表型相关蛋白的蛋白质组学研究

目的:利用蛋白质组学技术探讨与人食管鳞癌细胞(esophageal squamous cell carcinoma, ESCC)恶性表型转化相关的差异表达的蛋白质谱.方法:采用二维双向电泳(two-dimensional electrophoresis, 2-DE)和基质辅助激光解吸电离飞行时间质谱(matrix assisted laser desorption ionisation timE-of-flight mass spectrometry, MALDI-TOF-MS)法鉴定人食管上皮永生化细胞株NECA-E6E7-hTERT和ESCC细胞株EC1、EC18、EC109差异表达的蛋白质分子,采用Western印迹法和免疫细胞化学法验证annexin A2在人食管上皮永生化细胞和ESCC细胞中的差异表达,实时荧光定量PCR(real-time fluorogentic quantitative PCR,RFQ-PCR)分析annexin A2 mRNA的表达水平.结果:鉴定出5倍以上差异表达的蛋白质分子15个,其中3个蛋白质在ESCC细胞中表达下调,12个蛋白质表达上调;Western 印迹法和免疫细胞化学法验证了ESCC细胞中annexin A2蛋白的表达低于人食管上皮永生化细胞;而annexin A2 mRNA表达与其蛋白表达模式不一致.结论:本研究鉴定出的差异表达蛋白质谱为建立食管癌高发区高危人群筛查和早期诊断的分子指标和生物预防提供了重要线索.annexin A2翻译后调控可能是导致ESCC中annexin A2蛋白表达下调的主要原因.

Proteomic identification of malignant transformation-related proteins in esophageal squamous cell carcinoma

Objective:To identify differentially expressed proteins related with malignant transformation of esophageal squamous cell carcinoma (ESCC) using proteomic analysis. Methods:Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization timE-of flight mass spectrometry (MALDI-TOF-MS) in combination with protein database searching were used to determine and identify differentially expressed proteins in esophageal cancer cell lines (EC1, EC18, and EC109) and immortal cell line (NECA-E6E7-hTERT). Western blotting and immunocytochemistry were used to verify the differential expression of annexin 2 in esophageal cancer cell lines and immortal cell line (NECA-E6E7-hTERT). Real-time fluorogentic quantitative PCR(RFQ-PCR) was performed to analyze the expression level of annexin A2 mRNA.Results: A total of 15 differentially expressed proteins were identified with more than 5 folds difference. Among them three proteins were down-regulated and 12 proteins were up-regulated. Western blotting and immunocytochemical analysis verified the down-regulation of annexin A2 protein in ESCC cell lines. However, differential expression pattern of annexin A2 mRNA was not consistant with its protein expression in ESCC cell lines and immortal cell line (NECA-E6E7-hTERT). Conclusion:The findings provide important clues for identifying the candidate biomarkers for high-risk population screening and early diagnosis of ESCC. Post-translative regulation/modification contributes to the down-regulation of annexin A2 protein.