The Study Of Effects And Mechanisms Of O-GlcNAc Modification On The Warburg Effect Of MCF-7 Cells

Metabolic reprogramming is one of the features of tumors,which intake of large amounts of glucose for glycolysis to produce lactate in the case of sufficient O₂,this phenomenon is known as aerobic glycolysis,also called the"Warburg effect".The occurrence of metabolic reprogramming can be induced by a variety of factors.It is known that the post-translational modification of the key metabolic enzymes can regulate the metabolic process by altering the enzyme activity.O-GlcNAc modification is known as a kind of protein post-translational modifications that is widely present on serine or threonine residues,which involved in the regulation of tumor cell proliferation,signal transduction and metabolism processes.However,the effect and mechanism of O-GlcNAc modification on the Warburg effect of tumor cells is unclear.In this study,we selected the human breast cancer MCF-7 cell line,firstly to detect the whether the up-regulated O-GlcNAc modification level could influence the Warburg effect,followed by the possible mechanism of the preliminary study.Detecting glucose consumption,lactate production,ATP concentration changes,we found the three indicators have increased which indicate that O-GlcNAc modification can promote the Warburg effect of MCF-7 cells.Then we explored the mechanism of this event.Western blot and qPCR showed increased O-GlcNAc modification can induce Glut1and LDHA expression.Western blot and qPCR were used to detect the expression of Glut1and LDHA upstream regulatory gene Myc which was also up-regulated.Since the activation marker of the Myc is the histone H3 T11 site phosphorylated and the K9 site acetylation at the promoter of Myc,chromatin immunoprecipitation showed increased O-GlcNAc modification can upregulate Myc gene expression which is bindg with histone H3 T11 site phosphorylation and K9 site acetylation.As it is showned that O-GlcNAc modification may promote Warburg effects by up-regulating histone H3 T11 site phosphorylation and histone H3 K9 site acetylation resulting of the expression of Myc,and then promoting the expression of Myc downstream target genes Glut1 and LDHA.In addition,since the post-translational modification of the key metabolic enzymes can regulate metabolic reprogramming,we use western blot,immunofluorescence assay finding that O-GlcNAc modification promotes PKM2 from cytoplasm into the nucleus.Further more,chromatin immunoprecipitation with PKM2 to detect the expression of Myc gene binging with PKM2,showing that increased O-GlcNAc modification could up-regulate the expression of the Myc gene regulated by PKM2 in the nuclear.This study found that O-GlcNAc modification can promote MCF-7 cells Warburg effect and described the possible mechanism:O-GlcNAc modification can promote PKM2 into the nucleus,PKM2 may promote the expression of PKM2-dependent Myc gene by modulating the phosphorylation and acetylation of histone H3,which switch on the expression of Glut1and LDHA,as a result of facilitating the Warburg effect of MCF-7 cells.This research revealing the important role of O-GlcNAc glycosylation in the tumor metabolic reprogramming.