O-N-乙酰葡萄糖胺糖基化水平正调控促血管生成素-2表达从而促进小鼠肝癌中肿瘤新生血管形成

doi:10.3969/j.issn.1674-5817.2020.01.007

摘要/Abstract

摘要： 目的探索在肝癌的发生发展过程中,O-N-乙酰葡萄糖胺(O-GlcNAc)糖基化水平与促血管生成素-2(Ang-2)表达的关系。方法采用二乙基硝胺(DEN)诱导C57BL/6小鼠肝癌模型,检测O-GlcNAc糖基化、Ang-2及内皮细胞黏附分子(CD31)在肝癌中表达; 采用N-乙酰氨基葡萄糖转移酶(OGT)抑制剂及N-乙酰氨基葡萄糖苷酶(OGA)抑制剂干预HepG2细胞,检测Ang-2的表达是否随O-GlcNAc糖基化水平的变化而改变;采用MTT检测O-GlcNAc糖基化的水平与肝癌细胞增殖的关系;采用Transwell法检测HUVEC细胞中O-GlcNAc糖基化水平与细胞迁移侵袭能力的关系。结果在DEN诱导的肝癌模型中,小鼠肝脏体积明显增大,肝脏上可见大小不一的肿瘤。免疫组织化学结果显示,与对照组相比,O-GlcNAc糖基化水平与Ang-2的表达在模型组小鼠肝脏中均明显上升(P<0.05),且肝癌组织中CD31染色的微血管密度明显上升(P<0.01); qRT-PCR检测结果显示,通过检测OGT、OGA、Ang-2 mRNA水平,得出O-GlcNAc糖基化水平影响Ang-2的表达水平;Western blotting结果显示,采用OGT抑制剂干预HepG2细胞后,Ang-2的表达随着O-GlcNAc糖基化水平的降低而下调;采用OGA抑制剂干预HepG2细胞后,Ang-2的表达随着O-GlcNAc糖基化水平的上升而升高。MTT结果显示,O-GlcNAc糖基化水平与肝癌细胞的增殖有密切关系,当O-GlcNAc糖基化水平高时,肝癌细胞增殖能力较强,当O-GlcNAc糖基化水平较低时,肝癌细胞的增殖能力明显受到抑制。Transwell结果显示,HUVEC细胞迁移侵袭能力与O-GlcNAc糖基化水平呈正相关。结论在肝癌的发生发展过程中,O-GlcNAc 糖基化水平逐渐上升,且Ang-2的表达水平受到O-GlcNAc 糖基化的正调控,从而促进肿瘤新生血管的生成。

关键词: 肝癌, O-N-乙酰葡萄糖胺(O-GlcNAc), 糖基化, HepG2细胞, 促血管生成素-2(Ang-2), 肿瘤新生血管

Abstract: Objective To explore the relationship between O-linked N-acetylglucosamine (O-GlcNAc) glycosylation and Angiopoietin-2(Ang-2) expression in the development of hepatocellular carcinoma. Methods C57BL/6 mice model of hepatocellular carcinoma was induced by Diethylnitroamine (DEN) to detect the changes of O-GlcNAc glycosylation, Ang-2 and cell adhesion molecule-1 (CD31) expression in hepatocellular carcinoma. HepG2 cells were intervened with O-linked Glc NAc-transferase (OGT) inhibitors and O-Glc NAcase (OGA) inhibitors to detect whether the expression of Ang-2 changed with the changes of O-GlcNAc glycosylation level. MTT was used to detect the relationship between O-GlcNAc glycosylation level and proliferation of hepatocellular carcinoma cells. Transwell method was used to detect relationship between the level of O-GlcNAc glycosylation and cell migration and invasion in HUVEC cells. Results In the DEN-induced hepatocarcinoma model, the liver volume of mice increased significantly, and tumors of different sizes could be seen in the liver. The results of immunohistochemistry showed that the glycosylation level of O-GlcNAc and the expression of Ang-2 increased significantly in the liver of the model group compared with the control group (P<0.05). The results of qRT-PCR showed that the level of O-GlcNAc glycosylation affected the expression of Ang-2 by detecting the levels of OGT, OGA and Ang-2. Western blotting showed that the expression of Ang-2 decreased with the decrease of the glycosylation level of O-GlcNAc after the intervention of OGT inhibitors on HepG2 cells. After the intervention of HepG2 cells with OGA inhibitors, the expression of Ang-2 increased with the decrease of the glycosylation level of O-GlcNAc. MTT results showed that the glycosylation level of O-GlcNAc was closely related to the proliferation of hepatocellular carcinoma cells. The proliferation ability of hepatocellular carcinoma cells was stronger when the glycosylation level of O-GlcNAc was higher,. The proliferation ability of hepatocellular carcinoma cells was significantly inhibited, when the glycosylation level of O-GlcNAc was low,. Transwell results showed that HUVEC cell migration and invasion ability was positively correlated with O-GlcNAc glycosylation level. Conclusions During the development of hepatocellular carcinoma, the glycosylation level of O-GlcNAc increases gradually, and the expression level of Ang-2 positively regulated by the glycosylation of O-GlcNAc, thus promoting neovascularization.

Key words: Hepatocellular carcinoma, O-linked N-acetylglucosamine (O-GlcNAc), Glycosylation, HepG2 cell, Angiopoietin-2(Ang-2), Neovascularization of tumors

中图分类号:

Q95-33

王爱红, 王明全, 杜娟. O-N-乙酰葡萄糖胺糖基化水平正调控促血管生成素-2表达从而促进小鼠肝癌中肿瘤新生血管形成[J]. 实验动物与比较医学, 2020, 40(1): 39-46.

WANG Aihong, WANG Mingquan, DU Juan. O-GlcNAc Glycosylation Level Positively Regulates Ang-2 Expression and Promotes Tumor Angiogenesis in Mice with Liver Cancer[J]. Laboratory Animal and Comparative Medicine, 2020, 40(1): 39-46.

参考文献

[1] Bray F, Ferlay J, Soerjomataram I, et al.Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018, 68(6):394-424.
[2] McGlynn KA, Petrick JL, London WT. Global epidemiology of hepatocellular carcinoma: an emphasis on demographic and regional variability[J].Clin Liver Dis, 2015, 19(2):223-238.
[3] Xu W, Zhang X, Wu JL, et al.O-GlcNAc transferase promotes fatty liver-associated liver cancer through inducing palmitic acid and activating endoplasmic reticulum stress[J].J Hepatol, 2017, 67(2):310-320.
[4] Zhu Q, Zhou L, Yang Z, et al.O-GlcNAcylation plays a role in tumor recurrence of hepatocellular carcinoma following liver transplantation[J]. Med Oncol, 2012, 29(2): 985-993.
[5] Ma Z, Vosseller K.Cancer metabolism and elevated O-GlcNAc in oncogenic signaling[J]. J Biol Chem, 2014, 289(50):34457-34465.
[6] 张肖冰, 师以康. 蛋白质O-GlcNAc糖基化与肿瘤[J]. 肿瘤, 2013, 33(11):1027-1032.
[7] Bupathi M, Kaseb A, Janku F.Angiopoietin 2 as a therapeutic target in hepatocellular carcinoma treatment: current perspectives[J]. Onco Targets Ther, 2014, 7:1927-1932.
[8] Fodor D, Jung I, Turdean S, et al.Angiogenesis of hepatocellular carcinoma: An immunohistochemistry study[J]. World J Hepatol, 2019, 11(3):294-304.
[9] Yoshiji H, Kuriyama S, Noguchi R, et al.Angiopoietin 2 displays a vascular endothelial growth factor dependent synergistic effect in hepatocellular carcinoma development in mice[J].Gut, 2005, 54(12):1768-1775.
[10] Scholz A, Rehm VA, Rieke S,et al.Angiopoietin-2 serum levels are elevated in patients with liver cirrhosis and hepatocellular carcinoma[J]. Am J Gastroenterol, 2007, 102(11): 2471-2481.
[11] Mitsuhashi N, Shimizu H, Ohtsuka M, et al.Angiopoietins and Tie-2 expression in angiogenesis and proliferation of human hepatocellular carcinoma[J].Hepatology, 2003, 37(5): 1105-1113.
[12] 张舒曼, 王源, 李淑莲. 间断低剂量二乙基亚硝胺诱导 C57BL /6J 小鼠肝癌模型[J]. 河南大学学报: 医学版, 2019, 38(2):110-112.
[13] Chaparro M, Sanz-Cameno P, Trapero-Marugan M, et al.Mechanisms of angiogenesis in chronic inflammatory liver disease[J].Ann Hepatol, 2007, 6(4):208-213.
[14] Verna L, Whysner J, Williams GM.N-nitrosodiethylamine mechanistic data and risk assessment: bioactivation, DNA-adduct formation, mutagenicity, and tumor initiation[J].Pharmacol Ther, 1996, 71(1-2):57-81.
[15] 李果, 朱柱, 戴小明, 等. 肝癌动物模型建立的研究进展[J]. 医学综述, 2018, 24(2):285-289.
[16] 赛文莉, 姚登福, 邰伯军. 血管生成素2对肝癌的发生和发展及治疗的研究进展[J]. 中华临床医师杂志: 电子版, 2010, 4(4):471-473.
[17] Wada H, Nagano H, Yamamoto H, et al.Combination therapy of interferon-alpha and 5-fluorouracil inhibits tumor angiogenesis in human hepatocellular carcinoma cells by regulating vascular endothelial growth factor and angiopoietins[J]. Oncol Rep, 2007, 18(4):801-809.