| Glycosylation is one of the most common post-translational modifications on eukaryotic cell surface.The main glycan structures are N-glycans,O-glycans,GAGs and glycolipids.Oligosaccharide units of glycoconjugates are involved in cellular recognition and regulation processes,so changes in the expression and structure of carbohydrates on surface could lead to malignant transformation of cells,such as under-/ over expression of naturally-occurring glycans and some new abnormal structures of glycans.In tumor cells,alterations in cellular glycosylation may play a key role in their metastasis behaviors.The increasing expression ofβ1,6 GlcNAc-branching (N-glycans) caused by stronger activity of N-acetylglucosaminyltransferaseⅤ(GnT-Ⅴ),is often observed in tumor cells.More and more cancer-related glycosylations are revealed based on the developments in Genomics,Proteomics and application of Lectins and Mass Spectrometry.However,little literature described whether oligosaccharide expression on cell surface could influence cell migration in different phases of cell cycle and the responsible molecular mechanisms are still unclear.Thus this work aims to investigate the relationships among oligosaccharides expression,cell cycle and cell migration.1.Preparation of 4TO7 cells in different phases of cell cycle.4TO7 cells were blocked under certain cell cycle phase for next experiments,using cell synchronizing methods. 2.Migrations of synchronizing 4TO7 cells in different phases of cell cycle were observed,using Transwell method.The results showed that 4TO7 cells in all the phases of cell cycle can migrate,and that the G2/M phase cells can migrate is the firsttime reported.The migration of cells in S and G2/M phases were further confirmed by BrdU incorporating and wound healing assays.The oligosaccharide expressions on surfaces of 4TO7 cells were studied.First,oligosaccharide expressions on surfaces of synchronized cells in G0/G1 and G2/M phases were analyzed.The results indicated that there were no significant differences between the two phases.Next, double-stained flow cytometry experiments were performed to measure the oligosaccharide levels on surfaces during the cell cycle,using Lectins and PI.The data showed similar result with synchronized cells assay described above.3.Influences of GnT-Ⅴandβ1,6 branching on the cell migration in G2/M phase were determined.Previous studies had demonstrated that theβ1,6 GlcNAc branching could promote the cell migration.However,whether the oligosaccharides on cell surfaces modulate cell migration with the same efficiency in different cell cycle phases still needed to be confirmed.So the migration ability in G2/M phase of GnT-ⅤsiRNA-3 cells that have lower GnT-Ⅴandβ1,6 branching expression levels were investigated.The results provided evidences that expression suppression of the GnT-Ⅴandβ1,6 GlcNAc branching reduced the cell migration ability in G2/M phase.In summary,analysis of relations among oligosaccharides,cell cycle and migration showed that the oligosaccharides on cell surfaces can modulate the cell migration of all cell cycle.
|
PDF Full Text Request