欢迎访问《解剖学报》官方网站！今天是
English
二维电泳和质谱技术鉴定肝细胞癌相关N-连接糖蛋白
Identification of hepatocellular carcinoma associated N-linked glycoproteins by two dimensional electrophoresis and mass spectrometry
目的 鉴定与肝细胞癌(HCC)发生发展相关的差异表达N-连接糖蛋白。 方法 采用刀豆凝集素A(ConA)、晶状体凝集素(LCH)、雪花凝集素(GNA)等3种植物凝集素组成的亲和层析柱，分别从肝永生化细胞系L02和HCC细胞系Huh7、PLC5、SNU449中纯化N-连接糖蛋白、二维电泳分析差异表达的蛋白质斑点，质谱、生物信息学等技术鉴定差异表达蛋白，Western blotting验证了转录调控肿瘤蛋白(TCTP)、上皮细胞黏附分子（EpCAM）和膜联蛋白A2（annexin A2）在肝永生化及HCC细胞和HCC及癌旁组织中的表达规律，体外侵袭实验检测TCTP沉默后对SNU449的侵袭力影响。 结果 质谱、生物信息学等技术共鉴定出42个差异表达的蛋白质分子/异质体（HCC细胞中高、低表达的蛋白/异质体分别为14个、28个），代表32个蛋白质分子，其中的22个蛋白含有至少1个N糖基化位点（NetNGlyc 1.0预测）。这些差异表达的蛋白主要参与氧化还原内稳态维系、碳水化合物/能量代谢、糖酵解、抗凋亡等生物学过程。Western blotting检测表明，TCTP、EpCAM和annexin A2在6个肝癌细胞系PLC5、HepG2、SNU449、Huh7、HCC7721和SNU473及肝癌组织中表达显著升高，siRNA介导的TCTP沉默明显抑制了HCC细胞系SNU449的体外侵袭能力。 结论 TCTP、EpCAM和annexin可能参与了HCC发生发展过程，TCTP可能成为HCC靶向治疗的分子靶点之一。
Objective To identify differentially expressed N-linked glycoproteins associated with hepatocellular carcinoma (HCC). Methods Multi-lectin affinity chromatography comprising concanavalin A (ConA), lentil lectin (LCH), snowdrop lectin (GNA) were utilized to isolate N-linked glycoproteins from human immortalized liver cell line L02 and HCC cell lines Huh7, PLC5 and SNU449, followed by 2 dimensional electrophoresis-based quantification and MS/MS identification. Western blotting was used to verify different expression of translationally-controlled tumor protein (TCTP), epithelial cell adhesion molecule (EpCAM) and annexin A2 between two immortalized cell lines vs six HCC cell lines, HCC vs adjacent nontumor liver tissue. Invasion potential in vitro was examined after si-RNA mediated TCTP gene scilencing. Results A total of 42 proteins/isoforms including 14 up-regulated and 28 down-regulated proteins/isoforms were identified. These proteins/isoforms represented 32 unique proteins of which 22 had more than one glycosylation site predicted by NetNGlyc 1.0. These differentially expressed proteins plaid biological functions in redox homeostasis, carbohydrate/energy metabolism, glycolysis, anti-apoptosis, etc. Western blotting validated the up-regulated expression of TCTP, EpCAM and Annexin A2 in 6 HCC cell lines and HCC tissue in comparison with two immortalized cell lines L02, chang liver and adjacent non-tumor tissue, respectively. siRNA mediated downregulation of TCTP remarkably inhibited the invasion potential of in SUN449. Conclusion TCTP, EpCAM and annexin A2 may participate the pathogenesis of HCC and TCTP may become one of molecular targets for the targeted therapy of HCC.
肝细胞癌 / N-连接糖蛋白 / 植物凝集素 / 质谱 / 二维电泳
Hepatocellular carcinoma / N-lined glycoproteins / Lectin / Mass spectrometry / Two-dimensional electrophoresis
［1］Wong CM, Ng IO. Molecular pathogenesis of hepatocellular carcinoma ［J］. Liver Int, 2008, 28(2): 160-174.
［2］Song P, Gao J, hagaki Y, et al. Biomarkers: evaluation of screening for and early diagnosis of hepatocellular carcinoma in Japan and China ［J］. Liver Cancer, 2013, 2(1): 31-39.
［3］Tang ZY, Ye SL, Liu YK, et al. A decade’s studies on metastasis of hepatocellular carcinoma ［J］. Cancer Res Clin Oncol, 2004, 130(4): 187-196.
［4］Poon RT, Fan ST. Hepatectomy for hepatocellular carcinoma: patient selection and postoperative outcome ［J］. Liver Transpl, 2004, 10(2 Suppl 1): S39-S45.
［5］Wang M, Huang ZhH, Lei P, et al. Proteomic profiling of N-linked glycoproteins involved in hepatocellular carcinoma by two-dimensional electrophoresis and mass spectrometry ［J］. Chinese Journal of Biochemistry and Molecular Biology, 2013, 29 29(5): 482-489. (in Chinese)
王明, 黄志红, 雷鹏, 等. 二维电泳和质谱技术鉴定肝癌血清差异表达的N-连接糖蛋白［J］. 中国生物化学与分子生物学报, 2013, 29(5): 482-489.
［6］Ma J, Qi YJ, Liu RM, et al. Proteomic fingerprinting of N-linked glycoproteins involved in hepatocellular carcinoma ［J］. Acta Anatomic Sinica, 2014, 45(4): 493-499. (in Chinese)
马瑾，齐义军，刘瑞敏，等. 肝细胞癌中差异表达N-连接糖蛋白的分析鉴定［J］. 解剖学报, 2014, 45(4): 493-499.
［7］Sun S, Xu MZ, Poon RT, et al. Circulating Lamin B1 (LMNB1 ) biomarker detects early stages of liver cancer in patients ［J］. J Proteome Res, 2010, 9(1): 70-78.
［8］Bruix J, Sherman M. Management of hepatocellular carcinoma: an update ［J］. Hepatology, 2011, 53(3): 1020-1022.
［9］Gatenby RA, Gillies RJ. Glycolysis in cancer: a potential target for therapy ［J］. Int J Biochem Cell Biol, 2007, 39(78): 1358-1366.
［10］Klaunig JE, Xu Y, Isenberg JS, et al. The role of oxidative stress in chemical carcinogenesis ［J］. Environ Health Perspect, 1998, 106 (Suppl 1): 289-295.
［11］Yilmaz M, Christofori G. EMT, the cytoskeleton, and cancer cell invasion ［J］. Cancer Metastasis Rev, 2009, 28(1-2): 15-33.
［12］Wu D, Guo Z, Min W, et al. Upregulation of TCTP expression in human skin squamous cell carcinoma increases tumor cell viability throughanti-apoptotic action of the protein ［J］. Exp Ther Med, 2012, 3(3): 437-442.
［13］ChenYL,Yan MY, Chien SY, et al. Sann-Joong-Kuey-Jian-Tang inhibits hepatocellular carcinoma Hep-G2 cell proliferation by increasing TNF-α, Caspase-8, Caspase-3 and Bax but by decreasing TCTP and Mcl-1 expression in vitro ［J］. Mol Med Rep, 2013, 7(5): 1487-1493.
［14］Zhang F, Liu B, Wang Z, et al. A novel regulatory mechanism of Pim-3 kinase stability and its involvement in pancreatic cancer progression ［J］. Mol Cancer Res, 2013, 11(12): 1508-1520.
［15］Kaarb ? M, Storm ML, Qu S, et al. TCTP is an androgen-regulated gene implicated in prostate cancer ［J］. PLoS One, 2013, 8(7): e69398.
［16］Tuynder M, Susini L, Prieur S, et al. Biological models and genes of tumor reversion: cellular reprogramming through tpt1/TCTP and SIAH-1 ［J］. Proc Natl Acad Sci USA, 2002, 99(23): 14976-14981.
［17］Yu J. The role of epithelial cell adhesion molecule in the expression and metastasis of hepatocellular carcinoma ［D］. Tianjin: Tianjie Medical School, 2010: 4-6. (in Chinese)
于娟.上皮细胞黏附分子在肝癌中的表达及转移中的作用［D］.天津:天津医科大学，2010: 4-6.
［18］Pai RK, West RB. MOC-31 exhibits superior reactivity compared with Ber-EP4 in invasive lobular and ductal carcinoma of the breast: a tissue microarray study ［J］. Appl Immunohistochem Mol Morphol, 2009, 17(3): 202-206.
［19］Seimetz D, Lindhofer H, Bokemeyer C. Development and approval of the trifunctional antibody catumaxomab (anti-EpCAM x anti-CD3) as a targeted cancer immunotherapy ［J］. Cancer Treat Res, 2010, 36(6): 458-467.
［20］Zhao P, Zhang W, Tang J, et al. Annexin II promotes invasion and migration of human hepatocellular carcinoma cells in vitro via its interaction with HAb18G/CD147 ［J］. Cancer Sci, 2010, 101(2): 387-395.
［21］Deng S, Wang J, Hou L, et al. Annexin A1, A2, A4 and A5 play important roles in breast cancer, pancreatic cancer and laryngeal carcinoma, alone and/or synergistically ［J］. Oncol Lett, 2013, 5(1): 107-112.
［22］Gurluler E, Guner OS, Tumay LV, et al. Serum annexin A2 levels in patients with colon cancer in comparison to healthy controls and in relation to tumor pathology ［J］. Med Sci Monit, 2014, 20: 1801-1807.
［23］Emoto K, Sawada H, Yamada Y, et al. Annexin II overexpression is correlated with poor prognosis in human gastric carcinoma ［J］. Anticancer Res, 2001, 21(2B): 1339-1345.
［24］Zhai H, Acharya S, Gravanis I, et al. Annexin A2 promotes glioma cell invasion and tumor progression ［J］. J Neurosci, 20115, 31(40): 14346-14360.
［25］Wang CY, Chen CL, Tseng YL, et al. Annexin A2 silencing induces G2 arrest of non-small cell lung cancer cells through p53-dependent and -independent mechanisms ［J］. J Biol Chem, 2012, 287(39): 32512-32524.
［26］Zhang X, Liu S, Guo C, et al. The association of annexin A2 and cancers ［J］. Clin Transl Oncol, 2012, 14(9): 634-640.
食管鳞癌糖蛋白差异表达谱研究
/
| 〈 |
|
〉 |
