Yang Ganglong, Tan Zengqi, Lu Wei, Guo Jia, Yu Hanjie, Yu Jingmin, Sun Chengwen, Qi Xiaowei, Li Zheng, Guan Feng
The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education; School of Biotechnology, Jiangnan University , Wuxi, China.
J Proteome Res. 2015 Feb 6;14(2):639-53. doi: 10.1021/pr5006026. Epub 2015 Jan 8.
Diagnosis of bladder cancer, one of the most common types of human cancer, at an early (nonmuscle-invasive) stage is the best way to reduce the mortality rate. Tumor malignancy in general is closely associated with alterations of glycan expression. Glycosylation status, particularly global glycomes, in bladder cancer has not been well studied. We integrated lectin microarray and mass spectrometry (MS) methods to quantitatively analyze and compare glycan expression in four bladder cancer cell lines (KK47, YTS1, J82, T24) and one normal bladder mucosa cell line (HCV29). Glycopattern alterations were analyzed using lectin microarray analysis and confirmed by lectin staining and lectin blotting. Associations of glycopatterns with diverging stages were evaluated by lectin histochemistry on tissue microarrays. N-Glycans were derivatized by amidation of sialylated glycans with acetohydrazide and reductive amination with the stable isotope tags [(12)C6]- and [(13)C6]-aniline, and were quantitatively analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). N-Glycan biosynthesis-associated proteins were quantitatively analyzed by a stable isotope labeling by amino acids in cell culture (SILAC) proteomics method, which revealed significant differences in expression of 13 glycosyltransferases and 4 glycosidases. Our findings indicate that sialyl Lewis X (sLe(x)), terminal GalNAc and Gal, and high mannose-type N-glycans were more highly expressed in bladder cancer cells and tissues than in normal cells. Bladder cancer cells showed high expression of core-fucosylated N-glycans but low expression of terminally fucosylated N-glycans. Each of these glycome changes may be directly related to bladder cancer progression.
膀胱癌是人类最常见的癌症类型之一，早期（非肌层浸润性）诊断是降低死亡率的最佳方法。肿瘤恶性程度一般与聚糖表达改变密切相关。膀胱癌中的糖基化状态，特别是整体糖组，尚未得到充分研究。我们整合了凝集素微阵列和质谱（MS）方法，以定量分析和比较四种膀胱癌细胞系（KK47、YTS1、J82、T24）和一种正常膀胱黏膜细胞系（HCV29）中的聚糖表达。使用凝集素微阵列分析来分析糖型改变，并通过凝集素染色和凝集素印迹进行确认。通过组织微阵列上的凝集素组织化学评估糖型与不同阶段的关联。通过用乙酰肼对唾液酸化聚糖进行酰胺化并用稳定同位素标签[(12)C6]-和[(13)C6]-苯胺进行还原胺化，对N-聚糖进行衍生化，并通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF/TOF-MS）进行定量分析。通过细胞培养中氨基酸稳定同位素标记（SILAC）蛋白质组学方法对N-聚糖生物合成相关蛋白进行定量分析，结果显示13种糖基转移酶和4种糖苷酶的表达存在显著差异。我们的研究结果表明，唾液酸化路易斯X（sLe(x)）、末端GalNAc和Gal以及高甘露糖型N-聚糖在膀胱癌细胞和组织中的表达高于正常细胞。膀胱癌细胞显示核心岩藻糖基化N-聚糖高表达但末端岩藻糖基化N-聚糖低表达。这些糖组变化中的每一种都可能与膀胱癌进展直接相关。
