The Effect Of Sialylation On Proliferation And Apoptosis Of Bladder Cancer Cells

Sialic acid,an acidic sugar containing nine carbon atoms as the backbone,often exists as the form of monomers or polymers at the end of the sugar chain.The synthesis of sialic acid starts with glucose and is methodically catalyzed by a series of enzymes.Sialyltransferases are responsible for the addition of sialic acids to the sugar chain.In tumor biology,high sialylation has been proven to help tumor cells evade immune surveillane,promote tumor-related angiogenesis,promote tumor cell proliferation and migration,and increase cancer cell resistance to chemotherapy and physical therapy.The alteration in sialylation is controlled by changes of sialyltransferases and sialidases.Sialyltransferases have been shown with high expression in different tumor types,but the role of sialidases in tumors has not been well studied.Bladder cancer originates in the urothelial cell and is the most common malignant tumor in human urinary system.Bladder cancer is a serious threat to patients' quality of life due to its rapid progress and limited treatment options.Using glycomics and proteomics techniques,this study revealed the correlation and mechanism between sialylation and bladder cancer progression,and uncovered the function of sialylation on small extracellular vesicles,as well as improved the sample preparation method for detection of different sialic acid linkages.The main conclusions are as follows:(1)We improved the sample preparation method of sialic acid detection,using dimethylamine/ammonia derivatization method to distinguish ?2,3 and ?2,6 linked sialic acid.Based on the lactone formation properties of ?2,3 sialic acid to the adjacent galactose,dimethylamine and ammonia were used to derivatize ?2,3 and ?2,6 linked sialic acids for mass spectrum detection,under the catalysis of EDC and HOBt.Using standard trisaccharides(?2,3sialyl lactose and ?2,6 sialyl lactose),standard glycoproteins(fetuin and lactoferrin),we verified that the derivatization method could effectively identify two sialic acid modifications.Furthermore,the sialyl N-glycans of tumor cells under normoxia and hypoxia conditions were analyzed using above strategy.This method can identify the N-glycan structures as well as characterize the ?2,3 and ?2,6 linked sialic acid,and extends the glycan-information obtained by mass spectrum analysis.(2)Abnormal hypersialylation of bladder cancer was related to low expression of sialidase NEU1.High-throughput N-glycomic analysis revealed the abnormal sialylation expressed in malignant bladder cancer cells.An increased sialylation was related to low sialidase activity in bladder cancer cells.High-throughput mass spectrometry analysis showed the key sialidase of bladder cancer cells was NEU1,and low NEU1 expression in malignant bladder cancer was observed.Using analytical methods such as lectin staining,fluorescence staining and confocal microscopy,we concluded that the expression of NEU1 in bladder cancer cells was negatively correlated with sialylation.We also found the NEU1 expression in bladder cancer tissues was significantly lower than that in the matched adjacent tissues at protein level,and NEU1 level was negatively correlated with sialylation level.Our data further showed the low expression of NEU1 in patients with bladder cancer indicated the worse survival time.(3)Sialidase NEU1 reduced the sialylation on fibronectin(FN),and inhibited Akt phosphorylation and cell proliferation mediated by integrin ?5?1.We cloned NEU1 gene and transfected it into bladder cancer cell YTS-1 by lentivirus infection.The increasing of NEU1 in YTS-1 cells(YTS-1/NEU1)reduced cell proliferation,increased apoptosis,and arrested cell cycle at G0/G1 phase.Analysis of protein interaction and co-immunoprecipitation experiments confirmed that the elevated NEU1 was mainly located on cell membrane.NEU1 catalyzed FN desialylation and affected its affinity towards integrin ?5?1.Therefore,FN desialylation weakened cell adhesion and its related signal pathway mediated by integrin,resulting in reduced cell proliferation and increased apoptosis.Tumor formation assay confirmed that NEU1 regulated the decreased sialylation,leading to growth inhibition and cell apoptosis of bladder cancer cell in vivo.(4)Desialylation of small extracellular vesicles(sEVs)retarded the sEV intake,and can provide a reference for clinical diagnosis of bladder cancer.By performing lectin blot assay and ELISA assay,we found that sEVs secreted by bladder cancer cells were of hypersialylation.sEVs could be uptaken by normal bladder epithelial cells,and activated Akt signaling pathway to promote cell proliferation.Reducing the sialylation on sEVs significantly reduced the endocytosis rate and disactivated Akt pathway of recepient cells.Moreover,the sialylation in plasma or in plasma sEVs from patients with bladder cancer(N = 48)were significantly higher than those from healthy donors(N = 30).When using plasma sEV as the detection index of bladder cancer,AUC value of its ROC curve was 0.9583.These data indicated that sialylation level on plasma sEV may offer a reference for clinical diagnosis of bladder cancer.