胆囊良恶性组织中蛋白质组差异表达及膜联蛋白A3的功能

Screening for the differentially expressed proteins in human gallbladder cancer by two-dimensional gel electrophoresis and mass spectrometry and functional exploration of AnnexinA3

目的 分离并鉴定胆囊癌和胆囊良性组织的差异表达蛋白质,以发现可能用于早期诊断胆囊癌的肿瘤标志物.观察RNA干扰沉默膜联蛋白A3（AnnexinA3）基因对胆囊癌细胞增殖及周期的影响.方法 提取人胆囊癌和胆囊良性组织的总蛋白质,用双向电泳分离蛋白并进行比较.选择差异表达超过2倍的蛋白点进行MALDI-TOF/TOF质谱和生物学分析.miRNA干扰胆囊癌细胞株中AnnexinA3的表达后,通过噻唑蓝（MTT）实验及流式细胞仪观察癌细胞增殖能力及细胞周期的变化.结果 筛选出在胆囊癌组织中明显差异表达的46个蛋白点,共有17个蛋白质被成功鉴定,其中在胆囊癌组织中高表达的为9个,低表达的为8个,包括AnnexinA3、TTR蛋白等.RNA干扰胆囊癌细胞株AnnexinA3蛋白的表达后,MTT显示干扰后细胞增殖明显下降效率为44.14%（P〈0.05）.流式细胞仪观察显示干扰后G1期增加（P〈0.05）,S期减少（P〈0.05）.结论 胆囊癌组织相对于胆囊良性组织蛋白存在明显差异.干扰AnnexinA3蛋白的表达可以改善胆囊癌细胞的某些恶性生物学行为.

Objective To find potential molecular targets for gallbladder cancer diagnostics by analyzing and comparing the proteomes expressed in human gallbladder carcinoma and benign gallbladder tissues, and investigate whether annexinA3 down-regulation by miRNA leads to inhibition of proliferation and arrest of cell cycle in human gallbladder cancer cell line SGC-996. Methods The proteins expressed in these tissues were analyzed by the two-dimensional gel electrophoresis. Proteins expressed differently of a 2-fold change were cut and analyzed by MALDI-TOF/TOF mass spectrometry. After miRNA expression plasmid targeting to annexinA3 gene was constructed and transfected into cancer cells, the methyl thiazolyl tetrazolium （ MTT） assay was performed for measuring cell proliferation and flow cytometic analysis for cell cycle. Results Protein extracts of individual sample in each type of tissues were separated on two-dimensional gels. There were 46 differentially expressed proteins in the tissues of gallbladder cancer. Seventeen proteins were successfully identified by MS, in which 9 proteins were overexpressed in tumors and the rest 8 proteins were underexpressed, including Annexi-nA3, TTR protein, etc. After interfering annexinA3 in gallbladder cancer SGC-996 cell line, the cell proliferation was suppressed. Flow cytometry revealed that after interfering annexinA3 in gallbladder cancer SGC-996 cell line, the cells in G1 phase were increased, while those in S phase were declined as compared with control group （P〈0.05）. Conclusion Significant discrepancies in protein expression exist between gallbladder cancer tissues and benign gallbladder tissues. Some of the differentially expressed proteins found by the proteomic approach may be potential molecular targets for early diagnosis of gallbladder cancer. miRNA interfering to annexi-nA3 effectively reverses some of the malignant biological characters of gallbladder cancer cells.