The Mechanism Of O-GlcNac Modified STAT3 In Hypoxia Induced-EMT Of Breast Cancer Cells

Hypoxia is a common tumor microenvironment and a typical feature of most solid tumors.The response of cancer cells to hypoxia directly affects the occurrence and development of tumors,such as epithelial-mesenchymal transition(EMT)and etc.Epithelial-mesenchymal transition(EMT)is a significant phases of cancer cell development,in which hypoxia can be used as a crucial inducing factor to promote the EMT process of cancer cells.In the process of rapid proliferation of malignant tumors,in order to meet the supply of energy,the metabolic pathways need to be changed to adapt to the microenvironment such as hypoxia.Similarly,hypoxia also has effects on the metabolic reorganization of cancer cells.O-GlcNAcylation is a dynamic and reversible post-translational modification.The homeostasis of energy in cells depends on the interaction between nutrient sensing mechanisms and energy production pathways,one of which is the hexosamine synthesis pathway(HBP).O-GlcNAcylation utilizes UDP-GlcNAc,the end product of the HBP pathway,as a sugar donor to regulate the signaling pathways and metabolic processes,and affects proliferation,migration,and metabolism of cancer cells.However,the mechanism of O-GlcNAcylation level in hypoxia-induced EMT remains to be researched.STAT3 is one of the essential transcription factors in hypoxia induced-EMT.Many studies have confirmed that the abnormal activation of STAT3 protein in cancer cells will lead to promoting malignant transformation,growth and survival of cancer cells.Although many studies have shown that STAT3 protein can be modified by O-GlcNAc,the regulation of STAT3 structure and function and the underlying regulatory mechanism between its O-GlcNAcylation and phosphorylation remain unclear.The thesis explored the mechanism of O-GlcNAcylation level in hypoxia-induced EMT and the relationship between O-GlcNAcylation and phosphorylation on STAT3 protein.By means of Pug NAc and OSMI-1 to regulate intracellular O-GlcNAcylation level,it is found that elevated O-GlcNAcylation level can promote EMT process and cell migration.The O-GlcNAc modified protein STAT3 was identified and screened by mass spectrometry.Next,the ex-OGT and sh OGT cell lines were constructed,and the results showed that O-GlcNAcylation can stabilize the expression of STAT3 protein and avoid ubiquitination degradation.Taking ex-OGT and sh OGT cells as the subject,it was found that the O-GlcNAcylation level of STAT3 protein increased and decreased accompanied by p-STAT3 protein.When the p-STAT3 protein was elevated,the O-GlcNAcylation level of STAT3 decreased,whereas,the O-GlcNAc modified STAT3 increased.In brief,it shows that the O-GlcNAcylation of STAT3 protein promotes its phosphorylation,and when its phosphorylation occurs,phosphorylated STAT3 protein can no longer undergo O-GlcNAcylation.Therefore,there is a sequential regulatory relationship between O-GlcNAcylation and phosphorylation.To further explore,the O-GlcNAcylation T717 site and phosphorylation site Y705 on STAT3 protein was screened out through the database and the corresponding mutant cells were constructed.The results illustrated when the T717 site was mutated,p-STAT3 protein level reduced significantly and was prevented from entering the nucleus to regulate downstream target genes,which inhibited the EMT process and cell migration.Moreover,phosphorylation site mutation does not affect its O-GlcNAcylation modification.In summary,STAT3 protein is dynamically regulated by O-GlcNAcylation and phosphorylation,which affects the EMT process and migration ability of breast cancer cells under hypoxia.In addition,the O-GlcNAcylation T717 site of STAT3 protein may be a potential target,which may contribute to intervention or treatment of cancer in the future.