The Mechanism Of FBXO31 Maintaining The Level Of Intracellular O-GlcNAcylation

Research purpose:Protein O-GlcNAcylation is an important post-translation modification of proteins,and its abnormally increased level can affect intracellular signal transduction,transcriptional regulation and cell cycle,contributing to tumorigenesis.Due to the lack of effective detection methods for protein O-GlcNAcylation,it was difficult to study the maintenance mechanism of protein O-GlcNAcylation in vivo,and a systematic and comprehensive study was lacking in the field.In the previous work,our research group developed a novel fluorescent probe Cp OGA^D298N that can detect the protein O-GlcNAcylation.The probe was effective and specific.In this study,we combined the new fluorescent probe of protein O-GlcNAcylation with CRISPR/cas9 technology to conduct genome-wide screening to reveal the key genes and molecular mechanisms that maintain the level of protein O-GlcNAcylation in cells.We then validated this mechanism in a tumor model associated with abnormal O-GlcNAcylation homeostasis.The completion of this project not only helps to explain the maintenance mechanism of protein O-GlcNAcylation but also provides a new treatment plan for diseases related to abnormal O-GlcNAcylation levels.Method:1.Using of CRISPR/Cas9 technology and novel O-GlcNAcylation probe for genome-wide screening,sorting out cells with increased levels of O-GlcNAcylation by flow cytometry,and extracting DNA from these cells,amplifying their sg RNAs and performing next-generation sequencing;2.Analysing the sequencing results by bioinformatics methods to obtain a list of genes that can regulate the level of O-GlcNAcylation;3.Using RNAi technology to knock down the genes we were interested in,western blot and immunofluorescence are used to detect the changes of O-GlcNAcylation level and selecting the target gene;4.After understanding the function of the target gene through literature search,the possible pathway that it may affect protein O-GlcNAcylation levels is speculated.And then designing experiments to figure out the specific mechanism of the target gene regulating protein O-GlcNAcylation;5.Meta-analysis and TCGA database digging are used to select cancer types most associated with abnormally elevated O-GlcNAcylation levels,and validate it by immunohistochemistry with patient-derived tissue samples;6.Validating the previously revealed mechanism of key genes maintaining O-GlcNAcylation levels by immunohistochemistry,data analysis,etc.in selected tumor models.Results:1.Through the analysis of the screening results and the later experimental validation,FBXO31 was identified as a key gene for maintaining O-GlcNAcylation levels;2.FBXO31 acts as an E3 ubiquitin ligase to maintain and regulate the level of intracellular O-GlcNAcylation by promoting the proteasomal degradation of OGT;3.Endometrial cancer is highly associated with abnormally high levels of protein O-GlcNAcylation;4.FBXO31 expression is abnormally low in endometrial cancer patients,and in survival analysis,it was found that the group with low FBXO31expression had worse survival and prognosis than the group with high FBXO31 expression.Conclusion:FBXO31 is a key gene for maintaining intracellular protein O-GlcNAcylation level;loss of FBXO31 promotes endometrial carcinogenesis by increasing cellular O-GlcNAcylation level.