Application of O-GlcNAc modified OPG in preparation of drugs for enhancing drug sensitivity of tumor cells

Abstract

The invention relates to application of O-GlcNAc modified OPG in preparation of drugs for enhancing drug sensitivity of tumor cells. The amino acid sequence of the OPG is shown as SEQ ID NO.1, and O-GlcNAc modification exists at the site S151 of the OPG. According to the invention, the O-GlcNAc modification obviously enhances the stability and activity of the OPG protein and the correlation between the O-GlcNAc modified OPG and the malignant progression of tumors. The method focuses on an O-GlcNAc modification related protein spectrum and identifies and defines a new substrate modified by O-GlcNAc; the invention discloses that O-GlcNAc modified OPG is mediated by membrane protein EHD1 to promote malignant progression of tumors in a manner of promoting a cell cycle axis, and clarifies a novel tumor treatment means, and drug resistance of lung adenocarcinoma tumor cells to carboplatin and pemetrexed can be reversed by breaking an O-GlcNAc modified OPG axis, so that the curative effect of chemical treatment is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of O-GlcNAc modified OPG in the preparation of drugs that enhance the drug sensitivity of tumor cells. Background Technology

[0002] Glycosylation is the most common and varied type of post-translational modification in organisms. Glycan structures are rich and diverse in normal cells, mainly classified into nitrogen- or oxygen-linked glycosylation, carbon-linked mannosylation, and glycosylated phosphatidylinositol anchoring. O-linked β-N-acetylglucosamine (O-GlcNAc) modification is an important biological phenomenon discovered in 1984 by scientists Carmen-Rosa Torres and Gerald W. Hart (CR TORRES, GW Hart et al., Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc, 1984).

[0003] O-GlcNAc modification, as a special type of monosaccharide modification, is widely present on serine (S) or threonine (T) residues in nuclear, cytoplasmic, and mitochondrial proteins (CR TORRES, GW Hart et al., Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc, 1984; Junfeng Ma et al., O-GlcNAcomic Profiling Identifies Widespread O-Linked β-N-Acetylglucosamine Modification (O-GlcNAcylation) in Oxidative Phosphorylation System Regulating Cardiac Mitochondrial Function, 2015). It is mainly produced by O-GlcNAc transferase (O-GlcNAc...). O-GlcNAc transferase (OGT) and O-GlcNAc glycosidase (O-GlcNAcase, OGA) are responsible for the transfer and hydrolysis of the donor substrate uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) on amino acid residues, respectively, to achieve dynamic regulation of O-GlcNAc modification.

[0004] Within rapidly proliferating tumor cells, glucose uptake and glycolysis rates are significantly increased. Tumor cells rely heavily on glycolysis to generate high levels of ATP and metabolic intermediates for complex anabolic metabolism and to adapt to the harsh environment (Douglas Hanahan et al., Hallmarks of cancer: the next generation, 2011). The aggressive uptake of glucose and glutamine by tumor cells leads to generally elevated HBP flux, UDP-GlcNAc levels, and O-GlcNAc modification levels (John C Chatham et al., Role of O-Linked N-Acetylglucosamine Protein Modification in Cellular (Patho) Physiology, 2021).Many functional proteins, signaling molecules, and transcription factors are substrates for O-GlcNAc modification (Ying-Ying Liu et al., O-GlcNAcylation of MORC2 atthreonine 556 by OGT couples TGF-β signaling to breast cancer progression, 2022; Xiao Zhang et al., The essential role of YAP O-GlcNAcylation in high-glucose stimulated liver tumorigenesis, 2017; Xinghui Li et al., Myeloid-derivedcullin 3 promotes STAT3 phosphorylation by inhibiting OGT expression and protects against intestinal inflammation, 2017). O-GlcNAc modification achieves precise regulation of tumorigenesis and development through a combination of competitive phosphorylation, proteasome degradation, and altered protein localization (Hang Song et al., Global profiling of O-GlcNAcylated and / or phosphorylated proteins in hepatoblastoma, 2019; Yang Yang et al., O-GlcNAcylation of YTHDF2 promotes HBV-related...). Hepatocellular carcinoma progression in an N(6)-methyladenosine-dependent manner, 2023; Hao Nie et al., O-GlcNAcylation of PGK1 coordinates glycolysis and TCA cycle to promote tumor growth, 2020).

[0005] Currently, O-GlcNAc modification is a highly promising approach to cancer therapy (Xue-Fen He et al., O-GlcNAcylation in cancer development and immunotherapy, 2023). Exploring new O-GlcNAc modified proteins and their progressive effects is of great significance for advancing O-GlcNAc-based cancer therapy. Summary of the Invention

[0006] The purpose of this invention is to provide the application of O-GlcNAc modified OPG in the preparation of drugs that enhance the drug sensitivity of tumor cells.

[0007] Osteoporosis protectant (OPG) is a secreted cytokine belonging to the tumor necrosis factor receptor superfamily (TNFRSF). OPG directly binds to the receptor activator of nuclear transcription factor-κB ligand (RANKL), inhibiting the maturation and activation of osteoclast precursors by antagonizing the RANK / RANKL signaling pathway, thereby preventing osteoclast resorption (Sandrine Theoleyre et al., The molecular triad OPG / RANK / RANKL: involvement in the orchestration of pathophysiological bone remodeling, 2004), hence the name "osteoporosis protectant." Another endogenous ligand of OPG protein is called TNF-related apoptosis-inducing ligand (TRAIL). OPG blocks TRAIL-induced apoptosis by inhibiting the interaction between TRAIL and death receptors, jointly regulating vascular calcification. Given the crucial roles of the RANKL and TRAIL signaling pathways in the immune system and tumor progression, OPG is not only a bone-specific regulator but also a vital regulatory molecule in tumors, broadly influencing pathological processes such as tumorigenesis, invasion, metastasis, and angiogenesis (Emma Harper et al., Vascular calcification in type-2 diabetes and cardiovascular disease: Integrative roles for OPG, RANKL, and TRAIL, 2016). Current research has revealed a novel mechanism by which mesenchymal stem cell p53 participates in tumor-related bone remodeling through OPG, partially explaining the regulation of tumor bone metastasis by OPG (Tania Velletri et al., Loss of p53 in mesenchymal stem cells promotes alteration of bone remodeling through negative regulation of osteoprotegerin, 2021). OPG expression can be detected in various tumor cell types, and OPG extensively remodels the tumor microenvironment (TME) to exert its biological activity.Given the secretory protein characteristics of OPG, it holds promise as a biomarker for tumor diagnosis, treatment, and efficacy evaluation (Lorenz C Hofbauer et al., Clinical implications of the osteoprotegerin / RANKL / RANK system for bone and vascular diseases, 2004).

[0008] This invention clarifies that O-GlcNAc modification significantly enhances the stability and activity of OPG protein, and establishes a correlation between O-GlcNAc-modified OPG and malignant tumor progression. This invention focuses on the proteomic profile of O-GlcNAc modification and identifies novel substrates for O-GlcNAc modification; it reveals that O-GlcNAc-modified OPG, mediated by the membrane protein EHD1, promotes malignant tumor progression by driving the cell cycle axis; and it elucidates a novel tumor treatment approach that can reverse the resistance of lung adenocarcinoma cells to carboplatin and pemetrexed by disrupting the O-GlcNAc modification-OPG axis, thereby improving the efficacy of chemotherapy.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] The technical solution of the present invention is to provide the application of an inhibitor of O-GlcNAc modified OPG in the preparation of a drug that enhances the drug sensitivity of tumor cells. The amino acid sequence of the OPG is shown in SEQ ID NO.1, and the S151 site of the OPG is modified with O-GlcNAc.

[0011] In some specific embodiments, the drugs used to enhance tumor cell drug sensitivity include carboplatin and pemetrexed.

[0012] In some specific embodiments, the tumor cells include lung adenocarcinoma cells, lung adenocarcinoma cells with concurrent hyperglycemia and / or cytokine disorders, lung cancer cells, and human non-small cell lung cancer cells.

[0013] More preferably, the tumor cells include lung adenocarcinoma cells A549, primary lung adenocarcinoma cells with hyperglycemia and / or cytokine disorders, Lewis lung cancer cells, and human non-small cell lung cancer cells NCI-H1299.

[0014] In some specific embodiments, the drug is a drug that inhibits the CDK2-Rb-E2F1-WNT5A cell cycle axis.

[0015] In some specific embodiments, the drug is a drug that inhibits the expression of the membrane protein EHD1.

[0016] In some specific embodiments, the drug is a drug that inhibits the expression of RNF2 protein.

[0017] In some specific embodiments, the drug is a drug that inhibits the expression of OGT protein.

[0018] In some specific embodiments, the drug is a drug that binds to the Q839 site of the OGT protein.

[0019] In some specific embodiments, the inhibitor of O-GlcNAc modified OPG is an inhibitor that inhibits the regulation of O-GlcNAc-modified OPG.

[0020] In some specific embodiments, the inhibitors of the O-GlcNAc modified OPG include celecoxib and OSMI-1.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention focuses on the O-GlcNAc modification of cytokines secreted by tumor cells and their tumor phenotypes, and identifies a novel O-GlcNAc-modified protein, OPG. It reveals that OPG can undergo O-GlcNAc modification inside tumor cells and be secreted into the tumor stroma, driving the cell cycle in a manner dependent on the membrane protein EHD1. Furthermore, it can enhance the efficacy of carboplatin- and pemetrexed-based tumor treatments by targeting the O-GlcNAc modification-OPG axis. Attached Figure Description

[0023] Figure 1 A is a schematic diagram showing the presence of O-GlcNAc-modified cytokines secreted by tumor cells in the tumor macroenvironment; Figure 1 B is a graph verifying the efficiency of successful OGT gene knockout in A549 cells; Figure 1 C is a schematic diagram showing the results of O-GlcNAc modification-related candidate cytokines OPG, IL-4, and CCL5 in the tumor macroenvironment screened by the cytokine chip; Figure 1 D is a flowchart for the proteomics detection of extratumor O-GlcNAc modification-related cytokines; Figure 1 E is the result of proteomics analysis of extratumor O-GlcNAc modification-related cytokines confirming the abundance of OPG, IL-4, and CCL5.

[0024] Figure 2 A final confirmation diagram of candidate cytokines related to O-GlcNAc modification in the tumor macroenvironment;

[0025] Figure 3 AC is a schematic diagram of the results of multiple immunohistochemical staining of tumor cells as the dominant cells secreting OPG. Figure 3D shows the results of OPG expression levels in different cell types; Figure 3 E shows the results of OPG expression levels in tumor cells and mononuclear cells;

[0026] Figure 4 A is a graph verifying the efficiency of successful OPG gene knockout; Figure 4 Figure B shows the changes in the in vitro proliferative capacity of tumor cells (including colony formation and relative cell proliferation) after OPG knockout. Figure 4 Figure C shows the changes in the in vivo proliferative capacity of tumor cells (including expression level, tumor cell volume, and IHC value) after OPG knockout.

[0027] Figure 5 Figure A shows the changes in the proliferative capacity (including colony formation and relative cell proliferation) of OGT knockout tumor cells after exogenous OPG purification protein replenishment in vitro. Figure 5 Figure B shows the changes in the proliferative capacity (including tumor cell volume) of OGT knockout tumor cells after in vivo exogenous replenishment of purified OPG protein.

[0028] Figure 6 To verify that OPG underwent O-GlcNAc modification via co-immunoprecipitation, immunofluorescence staining and co-localization quantification, and in vivo and in vitro chemiluminescent immunoassays;

[0029] Figure 7 A represents the specific site where O-GlcNAc modification occurs in OPG, specifically serine residue at position 151. Figure 7 B represents the sequence alignment of this locus among different species (Homo sapiens, Mus musculus, Rattus norvegicus, Bostaurus, Oryctolagus cuniculus, and Sus scrofa). Figure 7 C represents the validation of this site by immunoprecipitation; Figure 7 D represents the verification of O-GlcNAc modification sites using a chemical enzyme labeling method; Figure 7 E and F are validations of the O-GlcNAc modification site using the adjacent connection method;

[0030] Figure 8 A represents the effect of OGA overexpression on the O-GlcNAc modification level of OPG protein; Figure 8 B represents the effect of OSMI-1 treatment on the O-GlcNAc modification level of OPG protein;

[0031] Figure 9 Figure A shows the changes in the in vitro proliferation ability of tumor cells caused by the O-GlcNAc modification site of OPG protein. Figure 9Figure B shows the changes in the in vivo proliferative capacity of tumor cells caused by the O-GlcNAc modification site of OPG protein. Figure 9 Figure C shows the changes in the exogenous tumor-promoting ability of the O-GlcNAc modification site of OPG protein (taking A549 and NCI-H1299 cell lines as examples, respectively). Figure 9 D shows the localization and expression of OPG in a nude mouse subcutaneous xenograft tumor (CDX) model;

[0032] Figure 10 The graph shows the changes in the half-life of wild-type OPG and O-GlcNAc-modified mutant OPG proteins by OGT (MOTS-c, a peptide that does not undergo O-GlcNAc modification, was selected as a negative control).

[0033] Figure 11 A is a flowchart of the screening process to identify E3 ubiquitin ligase zinc finger binding protein 2 (RNF2) that interacts with OPG; Figure 11 B represents the regulation of OPG protein stability and ubiquitination levels by RNF2; Figure 11 C is an in vitro ubiquitination experiment that confirmed RNF2 to be the true E3 ubiquitin ligase of OPG; Figure 11 D represents the effect of OGT overexpression on the stability and ubiquitination level of wild-type or O-GlcNAc-modified OPG proteins regulated by RNF2.

[0034] Figure 12 A represents 261 differentially expressed genes positively regulated by both OGT and OPG, identified by RNA-seq. Figure 12 B represents the pathways identified by KEGG enrichment analysis of the above genes, with the cell cycle pathway appearing prominently. Figure 12 C represents cell cycle-related genes that were upregulated after cells were treated with exogenous OPG purified protein, as screened by RNA-seq.

[0035] Figure 13 A represents the ability of gradient OPG supplementation in OGT gene knockout cell lines to gradually reverse G0 / G1 phase arrest caused by OGT gene knockout. Figure 13 B represents the gradient replenishment of OPG in OGT gene knockout cell lines, which can gradually reverse the proliferation inhibition caused by OGT gene knockout. Figure 13 C is a schematic diagram of the classic CDK2-Rb-E2F1 cell cycle axis; Figure 13 Figure D shows the changes in cell cycle-related proteins in the OGT gene knockout cell line A549 caused by the addition of exogenous OPG recombinant protein. Exogenous OPG can reverse the expression of G0 / G1 phase cell arrest-related proteins.

[0036] Figure 14A is a flowchart showing the screening of membrane proteins that interact with OPG, namely protein 1 (RNF2) containing the EH domain and protein 1 (IQGAP1) containing the IQ motif to activate GTPases. Figure 14 Figure B shows the changes in the regulation of tumor cell cycle distribution by exogenous OPG purified protein after EHD1 gene knockout. After EHD1 knockout, exogenous OPG cannot drive the cell cycle.

[0037] Figure 15 A is a schematic diagram illustrating the hypothesis that exogenous OPG regulates the endogenous cell cycle through the membrane protein EHD1. Figure 15 B is a graph verifying the efficiency of successful EHD1 gene knockout; Figure 15 Figure C shows the changes in the regulation of cell cycle-related protein expression in tumor cells by EHD1 gene knockout and the use of exogenous OPG purified protein. Figure 15 Figure D shows the changes in the regulation of CDK2 and CCNE1 / 2 interactions by EHD1 gene knockout with gradient-increased exogenous OPG purified protein. Figure 15 E represents the change in the ability of EHD1 gene knockout to inhibit tumor cells from endocytizing exogenous OPG recombinant protein;

[0038] Figure 16 A is a flowchart of the O-GlcNAc experiment for OPG-purified protein in vitro; Figure 16 B is a verification diagram of the results of the in vitro O-GlcNAc experiment of OPG purified protein; Figure 16 C represents the concentration of OPG-purified protein obtained from the in vitro O-GlcNAc experiment; Figure 16 Figure D shows the changes in cell cycle distribution of tumor cells caused by OPG purified protein as determined by in vitro O-GlcNAc experiment. Figure 16 E is a graph showing the changes in the proliferation ability of tumor cells by OPG purified protein after in vitro O-GlcNAc experiment. Figure 16 F shows the changes in the expression of cell cycle-related proteins in tumor cells by OPG purified protein as determined by the in vitro O-GlcNAc experiment.

[0039] Figure 17 A is a schematic diagram showing the binding strength between OPG purified protein and EHD1 obtained by in vitro O-GlcNAc experiment. Figure 17 B represents the endocytosis status of OPG-purified protein obtained by in vitro O-GlcNAc experiment; Figure 17 C represents the docking diagram of wild-type and O-GlcNAc-modified OPG with EHD1 molecules; Figure 17 D represents the hydrogen bonding situation revealed by the docking of wild-type and O-GlcNAc-modified OPG with EHD1 molecules; Figure 17 E represents the binding energy revealed by the docking of wild-type and O-GlcNAc-modified OPG with EHD1 molecules; Figure 17 F is a verification diagram of the results of in vitro O-GlcNAc experiments on wild-type and related hydrogen-bonded mutant OPG; Figure 17 G is a schematic diagram showing the binding strength of wild-type and related hydrogen-bonded mutant OPG to EHD1 after in vitro O-GlcNAc experiments.

[0040] Figure 18 A is a schematic diagram illustrating the hypothesis that exogenous OPG promotes the cell cycle axis through the membrane protein EHD1 and by inhibiting cyclic adenosine monophosphate (cAMP). Figure 18 Figure B shows the changes in cAMP levels in OPG-purified protein and OPG / EHD1 dual-gene knockout tumor cells after O-GlcNAc assay. Figure 18 Figure C shows the changes in cAMP levels in tumor cells after treatment with exogenous OPG-purified protein and dibutyryl cyclic adenosine monophosphate (dbcAMP). Figure 18 Figure D shows the changes in CDK2 and CCNE1 / 2 interactions after exogenous OPG-purified proteins and dbcAMP treatment of tumor cells. Figure 18 E represents the expression of cell cycle-related proteins in tumor cells after treatment with exogenous OPG-purified proteins and dbcAMP. Figure 18 F shows the expression of cAMP in xenografts in a nude mouse CDX progression model;

[0041] Figure 19 A shows the gross lungs of Control KPC and Ogt KO KPC mice after tamoxifen induction; Figure 19 B is a schematic diagram of HE staining of the lungs of Control KPC and Ogt KO KPC mice; Figure 19 C is a validation of the success rate of Ogt knockout in Control KPC and Ogt KO KPC mice; Figure 19 D is O S151 - Validation of GlcNAc-OPG antibody specificity; Figure 19 E represents the lung contents of ControlKPC and Ogt KO KPC mice: O-GlcNAc, OPG, and O. S151 -GlcNAc-OPG expression level map; Figure 19 F represents primary cells derived from lung tumors in ControlKPC and Ogt KO KPC mice, containing O-GlcNAc, OGT, OPG, and O S151 -GlcNAc-OPG expression level map; Figure 19 G represents serum OPG and O in Control KPC and Ogt KO KPC mice. S151 -GlcNAc-OPG expression level map;

[0042] Figure 20 A represents the survival curves of Control KPC and Ogt KO KPC mice; Figure 20 B shows the expression levels of pCDK2, CDK2, and Ki67 in the lungs of Control KPC and Ogt KOKPC mice.

[0043] Figure 21 A is a flowchart of the construction of a type II diabetes mouse model; Figure 21 B is a graph showing the fasting blood glucose verification in type II diabetic mice; Figure 21 C represents the gross lung view of a mouse lung in a type II diabetic mouse and a control mouse model of tail vein lung transfer. Figure 21 D represents the O-GlcNAc level in the lungs or tumor-derived primary cells of type II diabetic mice and control mice with tail vein lung metastasis model. Figure 21 E represents the lung OPG and O2 levels in type II diabetic mice and control mice with tail vein lung transfer model. S151 -GlcNAc-OPG, pCDK2, CDK2 expression level map; Figure 21 F represents serum OPG and O2 levels in type II diabetic mice and control mice in a tail vein lung metastasis model. S151 -GlcNAc-OPG expression level map;

[0044] Figure 22 A represents the survival curve of OPG expression level versus overall survival in LUAD from the KM database; Figure 22 B refers to the serum OPG and O2 levels in thoracic tumor patients with or without hyperglycemia (HbA1c > 7.0% or < 6.5%). S151 -GlcNAc-OPG expression level; Figure 22 C represents serum OPG and O2 levels in thoracic tumor patients with or without cytokine disorders (more than 4 abnormal factors in the dodecagonal cytokine system or no abnormalities). S151 -GlcNAc-OPG expression level;

[0045] Figure 23 Figure A shows the proliferation level of primary tumor cells from LUAD patients with or without hyperglycemia (HbA1c > 7.0% or < 6.5%). Figure 23 B shows the proliferation level of primary tumor cells from LUAD patients with or without cytokine disorders (more than 4 abnormal factors in the twelve-cytokine pair or no abnormality). Figure 23 C is a 3D tumorigenesis diagram of primary tumor cells from LUAD patients with or without hyperglycemia (HbA1c > 7.0% or < 6.5%). Figure 23D is a 3D tumorigenesis diagram of primary tumor cells from LUAD patients with or without cytokine disorders (more than 4 abnormal factors in the twelve-cytokine pair or no abnormality). Figure 23 E represents the OGT and O-GlcNAc modification and EHD1 expression levels in primary cells of LUAD patients with or without hyperglycemia (HbA1c > 7.0% or < 6.5%). Figure 23 F represents the OGT and O-GlcNAc modification and EHD1 expression levels in primary cells of LUAD patients with or without cytokine disorders.

[0046] Figure 24 A is for constructing primary cells from LUAD patients. S151 -GlcNAc-OPG knockout strategy; Figure 24 B represents primary cells from LUAD patients. S151 -Efficiency verification of wild-type and knockout GlcNAc-OPG strains and levels of endogenous and exogenous OPG; Figure 24 C represents primary cells from LUAD patients. S151 Cell proliferation levels and 3D tumorigenesis of wild-type and knockout GlcNAc-OPG strains under different chiral glucose treatment conditions. Figure 24 D represents primary cells from LUAD patients. S151 The proliferation of wild-type GlcNAc-OPG strains and knockout strains in type II diabetic and control mice;

[0047] Figure 25 A represents the OGT and O-GlcNAc modification, OPG, and O after celecoxib treatment of A549 cells. S151 -Graph showing decreased GlcNAc-OPG expression levels; Figure 25 B indicates that celecoxib exerts its OGT inhibitory effect by regulating the Q839 site of the OGT protein; Figure 25 C suggests that the inhibitory effect of celecoxib on OGT protein may involve the proteasome degradation pathway. Figure 25 Figure D shows that parecoxib treatment of A549 cells had no significant effect on the expression levels of OGT and O-GlcNAc modification, and OPG, OGT and O-GlcNAc modification.

[0048] Figure 26 Figure A shows the cell survival of primary cells derived from malignant pleural effusion in patients treated with celecoxib in combination with carboplatin and pemetrexed. Figure 26 B represents the tumor growth curve of a PDX model derived from LUAD patients with hyperglycemia (HbA1c>7.0%) treated with celecoxib in combination with carboplatin and pemetrexed. Figure 26C represents the survival curve of a PDX model derived from LUAD patients with hyperglycemia (HbA1c>7.0%) treated with celecoxib combined with carboplatin and pemetrexed. Figure 26 D represents the tumor growth curve of a PDX model derived from LUAD patients with celecoxib combined with carboplatin and pemetrexed and complicated by cytokine dysregulation (more than 4 abnormal factors in the 12-cytokine combination); Figure 26 E represents the tumor growth curve of a PDX model derived from LUAD patients with celecoxib combined with carboplatin and pemetrexed and complicated by cytokine dysregulation (more than 4 abnormal factors in the 12-cytokine combination); Figure 26 F represents xenografts of LUAD patients from a PDX model, treated with celecoxib in combination with carboplatin and pemetrexed, and modified with OGT and O-GlcNAc, OPG, and O2. S151 -GlcNAc-OPG expression level; Figure 26 G represents the xenografts of LUAD patients from a PDX model, derived from patients with celecoxib combined with carboplatin and pemetrexed and complicated by cytokine dyskinia (more than 4 abnormal factors in the dodecagonal cytokine pair), containing OGT and O-GlcNAc modified tumors, OPG, and O. S151 -GlcNAc-OPG expression level;

[0049] Figure 27 A represents the tumor growth rate of primary cells in malignant pleural effusion patients treated with OSMI-1 combined with carboplatin and pemetrexed. Figure 27 B represents the tumor growth rate of a CDX model constructed from primary cells of malignant pleural effusion from patients treated with OSMI-1 combined with carboplatin and pemetrexed. Figure 27 C represents OSMI-1 combined with carboplatin and pemetrexed treatment of OGT and O-GlcNAc-modified OPG and O2 in a CDX model xenograft constructed from primary cells of malignant pleural effusion from patients. S151 -GlcNAc-OPG expression level.

[0050] Figure 28 This is a schematic diagram of the principle of the present invention, namely: OPG undergoes O-GlcNAc modification inside tumor cells, and after being secreted outside the tumor cells, it promotes the cell cycle under the delivery of the membrane protein EHD1, while celecoxib has the potential to inhibit this process. Detailed Implementation

[0051] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. In the following embodiments, unless otherwise specified, the raw materials or processing techniques are all conventional commercially available raw materials or conventional processing techniques in the art.

[0052] Some of the materials involved in the following embodiments:

[0053] (1) Cell source: Human lung adenocarcinoma A549, NCI-H1299, HEK293T and mouse Lewis lung cancer LLC cell lines were all purchased from Shanghai Fuheng Biotechnology Co., Ltd.; Primary cells from patients were dissociated, isolated and cultured from fresh thoracic tumor tissue, and cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin.

[0054] (2) The reagents used in cell and animal experiments were: DMSO (D1435, Sigma-Aldrich), GlcNAc (U4375, Sigma-Aldrich), Thiamet-G (S7213, Selleck), PuGNAc (A7229, Sigma-Aldrich), Cycloheximide (CHX, HY12320, MedChemExpress), WT-rhTR11B (HY-P70805, MedChemExpress), Celecoxib (HY-14398, MedChemExpress), Parecoxib (HY-17474, MedChemExpress), Carbohydrate, and Carbohydrate. platin(HY-17393,MedChemExpress),Pemetrexed(HY-10820,MedChemExpress),OSMI-1(HY-119738,MedChemExpress),dibutyryl-cAMP(dbcAMP; HY-B0764,MedChem Express),cAMP(HY-B1511,MedChemExpress),MG132(HY-13259,MedChemExpress),L-glucose(S3131,Selleck),D-glucose(S2123,Selleck),Dextran(D1816,Thermo Fisher).To prevent TR11B degradation, cells were also treated with cOmplete TM protease inhibitor cocktail (11873580001, Roche).

[0055] (4) Plasmid constructs based on Lenti CRISPR v2 were used to knock out OGT, OPG and EHD1.

[0056] (5) OPG (SEQ ID NO.1):

[0057] MNNLLCCALVFLDISIKWTTQETFPPKYLHYDEETSHQLLCDKCPPGTYLKQHCTAKWKTVCAPCPDHYYTDSWHTSDECLYCSPVCKELQYVKQECNRTHNRVCECKEGRYLEIEFCLKHRSCPPGFGVVQAGTPERNTVCKRCPDGFFSNETSSKAPCRKHTNCSVFGLLLTQKGNATHDNICSGNSESTQKCGIDVT LCEEAFFRFAVPTKFTPNWLSVLVDNLPGTKVNAESVERIKRQHSSQEQTFQLLKLWKHQNKDQDIVKKIIQDIDLCENSVQRHIGHANLTFEQLRSLME SLPGKKVGAEDIEKTIKACKPSDQILKLLSLWRIKNGDQDTLKGLMHALKHSKTYHFPKTVTQSLKKTIRFLHSFTMYKLYQKLFLEMIGNQVQSVKISCL

[0058] (6) sgRNA primers used to construct the human OGT KO1 plasmid:

[0059] Forward primer (SEQ ID NO.2): caccgGTCACCCGCTGCTACCAAGG

[0060] Reverse primer (SEQ ID NO.3): aaacCCTTGGTAGCAGCGGGTGACc

[0061] (7) sgRNA primers used to construct the human OGT KO2 plasmid:

[0062] Forward primer (SEQ ID NO.4): caccgGGGAATGTGTACAAGGAAAG

[0063] Reverse primer (SEQ ID NO.5): aaacCTTTCCTTGTACACATTCCCc

[0064] (8) sgRNA primers used to construct the human OPG KO1 plasmid:

[0065] Forward primer (SEQ ID NO.6): caccgGCAGTATAGACACTCGTCAC

[0066] Reverse primer (SEQ ID NO.7): aaacGTGACGAGTGTCTATACTGCc

[0067] (9) sgRNA primers used to construct the human OPG KO2 plasmid:

[0068] Forward primer (SEQ ID NO.8): caccgTGGAGGAAACGTTTCCTGGG

[0069] Reverse primer (SEQ ID NO.9): aaacCCCAGGAAACGTTTCCTCCAc

[0070] (10) sgRNA primers used to construct the human EHD1 KO1 plasmid:

[0071] Forward primer (SEQ ID NO.10): caccgTGATCGAGCAGGACTTCCCG

[0072] Reverse primer (SEQ ID NO.11): aaacCGGGAAGTCCTGCTCGATCAc

[0073] (11) sgRNA primers used to construct the human EHD1 KO2 plasmid:

[0074] Forward primer (SEQ ID NO.12): caccgCAAGGCTCTGAAGAACCATG

[0075] Reverse primer (SEQ ID NO.13): aaacCATGGTTCTTCAGAGCCTTGc

[0076] (12) The ZV302-OGT overexpression plasmid was synthesized by Zorun Biotechnology.

[0077] The ZV302-OGT overexpression vector is formed by inserting an OGT CDS fragment (SEQ ID NO.15) between EcoRI and BamHI in the original ZV302 vector (SEQ ID NO.14).

[0078] ZV302 original vector (SEQ ID NO.14):

[0079] ACCGTGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTA

[0080] GCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTA

[0081] AGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGG

[0082] ACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGAT

[0083] ACAATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTA

[0084] ACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAG

[0085] TGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGT

[0086] CAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAGCGAAAGGG

[0087] AAACCAGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAG

[0088] GCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAA

[0089] GGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGA

[0090] TGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATA

[0091] TAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAA

[0092] ACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAG

[0093] GATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCAT

[0094] CAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAG

[0095] CAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCACTGATCTTCAGACCTGGA

[0096] GGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAA

[0097] AAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAG

[0098] AGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCA

[0099] GGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTAT

[0100] TGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAG

[0101] CATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGC

[0102] TGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAA

[0103] AACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGG

[0104] AACAGATTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTAC

[0105] ACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATG

[0106] AACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTTGTGGAATTGGTTTAACATA

[0107] ACAAATTGGCTGTGGTATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGG

[0108] TTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTC

[0109] ACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAG

[0110] GAATAGAAGAAGAAGGTGGAGAGAGACAGAGACAGATCCATTCGATTAGTGA

[0111] ACGGATCTCGACGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGTACAGTGC

[0112] AGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAA

[0113] AAACAAATTACAAAAATTCAAAATTTTATCGATACTAGTATTATGCCCAGTACATG

[0114] ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC

[0115] ATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACG

[0116] GGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAA

[0117] ATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGC

[0118] GGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCA

[0119] GATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGATTCTAGAGCT

[0120] AGCGAATTCGAATTTAAATCGGATCCGACTACAAAGACCATGACGGTGATTATAAA

[0121] GATCATGATATCGATTACAAGGATGACGATGACAAGTGATAAGCGGCCGCAAGGA

[0122] TCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCC

[0123] GAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCG

[0124] GGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGG

[0125] GGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTT

[0126] TGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCC

[0127] GCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCC

[0128] GCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTC

[0129] GAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCT

[0130] CCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCT

[0131] GCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGACGCCACCATGGAGA

[0132] GCGACGAGAGCGGCCTGCCCGCCATGGAGATCGAGTGCCGCATCACCGGCACCCT

[0133] GAACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGGGCACCCCCAAGCAGGGC

[0134] CGCATGACCAACAAGATGAAGAGCACCAAAGGCGCCCTGACCTTCAGCCCCTACCT

[0135] GCTGAGCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCTACCCCAGCGGCT

[0136] ACGAGAACCCCTTCCTGCACGCCATCAACAACGGCGGCTACACCAACACCCGCATC

[0137] GAGAAGTACGAGGACGGCGGCGTGCTGCACGTGAGCTTCAGCTACCGCTACGAGG

[0138] CCGGCCGCGTGATCGGCGACTTCAAGGTGGTGGGCACCGGCTTCCCCGAGGACAGC

[0139] GTGATCTTCACCGACAAGATCATCCGCAGCAACGCCACCGTGGAGCACCTGCACCC

[0140] CATGGGCGATAACGTGCTGGTGGGCAGCTTCGCCCGCACCTTCAGCCTGCGCGACG

[0141] GCGGCTACTACAGCTTCGTGGTGGACAGCCACATGCACTTCAAGAGCGCCATCCAC

[0142] CCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGCCTTCCGCCGCGTGGAGGAGCT

[0143] GCACAGCAACACCGAGCTGGGCATCGTGGAGTACCAGCACGCCTTCAAGACCCCC

[0144] ATCGCCTTCGCCAGATCCCGCGCTCAGTCGTCCAATTCTGCCGTGGACGGCACCGC

[0145] CGGACCCGGCTCCACCGGATCTCGCGAGGGCAGAGGAAGTCTTCTAACATGCGGTG

[0146] ACGTGGAGGAGAATCCCGGCCCTATGACCGAGTACAAGCCCACGGTGCGCCTCGC

[0147] CACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACT

[0148] ACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGA

[0149] GCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCG

[0150] CGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGG

[0151] GGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGG

[0152] CCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGC

[0153] GTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCA

[0154] GCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTC

[0155] CTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGT

[0156] CACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAG

[0157] CCCGGTGCCTGAAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTAT

[0158] TCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTAT

[0159] CATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGC

[0160] TGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACT

[0161] GTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTT

[0162] TCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGC

[0163] CTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTT

[0164] GTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCT

[0165] GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTC

[0166] CCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTCCGCCTTCGCCCTCAGAC

[0167] GAGTCGGATCTCCCTTTGGCCGCCTCCCCGCCTGGTACCTTTAAGACCAATGACTTA

[0168] CAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGG

[0169] CTAATTCACTCCCAACGAAAATAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGG

[0170] TTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAA

[0171] GCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGA

[0172] CTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA

[0173] GTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATAT

[0174] CAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAAT

[0175] AGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTG

[0176] TCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTC

[0177] CGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAG

[0178] AGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTT

[0179] GGAGGCCTAGACTTTTGCAGAGACGGCCCAAATTCGTAATCATGGTCATAGCTGTT

[0180] TCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCA

[0181] TAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG

[0182] CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAAT

[0183] CGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGC

[0184] TCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCA

[0185] AAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT

[0186] GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT

[0187] TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGA

[0188] GGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC

[0189] CTCGTGCGCTTCCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTC

[0190] CCTTCGGGAAGCGTGGCCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTG

[0191] TAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCG

[0192] CTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATC

[0193] GCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGT

[0194] GCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATT

[0195] TGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAGAGTTGGTAGCTCTT

[0196] GATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAG

[0197] ATTACGCGCAGAAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTCTACGGGGTC

[0198] TGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAA

[0199] AAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAA

[0200] AGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC

[0201] TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTA

[0202] GATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGC

[0203] GAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAG

[0204] GGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATT

[0205] GTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT

[0206] GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGC

[0207] TCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGC

[0208] GGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATC

[0209] ACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATG

[0210] CTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGC

[0211] GACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGA

[0212] ACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGAT

[0213] CTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTC

[0214] AGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATG

[0215] CCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTT

[0216] TTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTT

[0217] GAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGT

[0218] GCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGC

[0219] GTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGA

[0220] CACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCA

[0221] GACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAA

[0222] CTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATAC

[0223] CGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCT

[0224] GCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGG

[0225] CGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCA

[0226] GTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTG

[0227] OGT CDS fragment (SEQ ID NO.15):

[0228] CGTCTTCCGTGGGCAACGTGGCCGACAGCACAGAACCAACGAAACGTATGCTTTCCTTC

[0229] CAAGGGTTAGCTGAGTTGGCACATCGAGAATATCAGGCAGGAGATTTTGAGGCAGCTGAGA

[0230] GACACTGCATGCAGCTCTGGAGACAAGAGCCAGACAATACTGGTGTGCTTTTATTACTTTCA

[0231] TCTATACACTTCCAGTGTCGAAGGCTGGACAGATCTGCTCACTTTAGCACTCTGGCAATTAAA

[0232] CAGAACCCCCTTCTGGCAGAAGCTTATTCGAATTTGGGGAATGTGTACAAGGAAAGAGGGC

[0233] AGTTGCAGGAGGCAATTGAGCATTATCGACATGCATTGCGTCTCAAACCTGATTTCATCGATG

[0234] GTTATATTAACCTGGCAGCCGCCTTGGTAGCAGCGGGTGACATGGAAGGGGCAGTACAAGCT

[0235] TACGTCTCTGCTCTTCAGTACAATCCTGATTTGTACTGTGTTCGCAGTGACCTGGGGAACCTG

[0236] CTCAAAGCCCTGGGTCGCTTGGAAGAAGCCAAGGCATGTTATTTGAAAGCAATTGAGACGC

[0237] AACCGAACTTTGCAGTAGCTTGGAGTAATCTTGGCTGTGTTTTCAATGCACAAGGGGAAATT

[0238] TGGCTTGCAATTCATCACTTTGAAAAGGCTGTCACCCTTGACCCAAACTTTCTGGATGCTTAT

[0239] ATCAATTTAGGAAATGTCTTGAAAGAGGCACGCATTTTTGACAGAGCTGTGGCAGCTTATCTT

[0240] CGTGCCCTAAGTTTGAGTCCAAATCACGCAGTGGTGCACGGCAACCTGGCTTGTGTATACTA

[0241] TGAGCAAGGCCTGATAGATCTGGCAATAGACACCTACAGGCGGGCTATCGAACTACAACCAC

[0242] ATTTCCCTGATGCTTACTGCAACCTAGCCAATGCTCTCAAAGAGAAGGGCAGTGTTGCTGAA

[0243] GCAGAAGATTGTTATAATACAGCTCTCCGTCTGTGTCCCACCCATGCAGACTCTCTGAATAAC

[0244] CTAGCCAATATCAAACGAGAACAGGGAAACATTGAAGAGGCAGTTCGCTTGTATCGTAAAG

[0245] CATTAGAAGTCTTCCCAGAGTTTGCTGCTGCCCATTCAAATTTAGCAAGTGTACTGCAGCAG

[0246] CAGGGAAAACTGCAGGAAGCTCTGATGCATTATAAGGAGGCTATTCGAATCAGTCCTACCTT

[0247] TGCTGATGCCTACTCTAATATGGGAAACACTCTAAAGGAGATGCAGGATGTTCAGGGAGCCT

[0248] TGCAGTGTTATACGCGTGCCATCCAAATTAATCCTGCATTTGCAGATGCACATAGCAATCTGG

[0249] CTTCCATTCATAAGGATTCAGGGAATATTCCAGAAGCCATAGCTTCTTACCGCACGGCTCTGA

[0250] AACTTAAGCCTGATTTTCCTGATGCTTATTGTAACTTGGCTCATTGCCTGCAGATTGTCTGTGA

[0251] TTGGACAGACTATGATGAGCGAATGAAGAAGTTGGTCAGTATTGTGGCTGACCAGTTAGAGA

[0252] AGAATAGGTTGCCTTCTGTGCATCCCTCATCATAGTATGCTATATCCTCTTTCTCATGGCTTCAG

[0253] GAAGGCTATTGCTGAGAGGCACGGCAACCTGTGCTTAGATAAGATTAATGTTCTTCATAAACC

[0254] ACCATATGAACATCCAAAAGACTTGAAGCTCAGTGATGGTCGGCTGCGTGTAGGATATGTGA

[0255] GTTCCGACTTTGGGAATCATCCTACTTCTCACCTTATGCAGTCTATTCCAGGCATGCACAATCC

[0256] TGATAAATTTGAGGTGTTCTGTTATGCCCTGAGCCCAGACGATGGCACAAACTTCCGAGTGA

[0257] AGGTGATGGCAGAAGCCAATCATTTCATTGATCTTTCTCAGATTCCATGCAATGGAAAAGCA

[0258] GCTGATCGCATCCATCAGGATGGAATTCATATCCTTGTAAATATGAATGGCTATACTAAGGGCG

[0259] CTCGAAATGAGCTTTTTGCTCTCAGGCCAGCTCCTATTCAGGCAATGTGGCTGGGATACCCTG

[0260] GGACGAGTGGTGCGCTTTTCATGGATTATATTATCACTGATCAGGAAACTTCGCCAGCTGAAG

[0261] TTGCTGAGCAGTATTCCGAGAAATTGGCTTATATGCCCCACACTTTTTTTATTGGTGATCATGC

[0262] TAATATGTTCCCTCACCTGAAGAAAAAAGCAGTCATCGATTTTAAGTCCAATGGGCACATTTA

[0263] TGACAATCGGATAGTTCTGAATGGCATCGACCTCAAAGCATTTCTTGATAGTCTACCAGATGT

[0264] GAAAATTGTCAAGATGAAGTGTCCTGATGGAGGAGACAATGCAGATAGCAGTAACACAGCT

[0265] CTTAATATGCCTGTTATTCCTATGAATACTATTGCAGAAGCAGTTATTGAAATGATTAACCGAG

[0266] GACAGATTCAAATAACAATTAATGGATTCAGTATTAGCAATGGACTGGCAACTACTCAGATCA

[0267] ACAATAAGGCTGCAACTGGAGAGGAGGTTCCCCGTACCATTATTGTAACCACCCGTTCTCAG

[0268] TACGGGTTACCAGAAGATGCCATCGTATACTGTAACTTTAATCAGTTGTATAAAATTGACCCT

[0269] TCTACTTTGCAGATGTGGGCAAACATTCTGAAGCGTGTTCCCAATAGTGTACTCTGGCTGTTG

[0270] CGTTTTCCAGCAGTAGGAGAACCTAATATTCAACAGTATGCACAAAACATGGGCCTGCCCCA

[0271] GAACCGTATCATTTTTTCACCTGTTGCTCCTAAAGAGGAACACGTCAGGAGAGGCCAGCTGG

[0272] CTGATGTCTGCTTGGACACTCCACTCTGTAATGGGCACACCACAGGGATGGATGTCCTCTGG

[0273] GCAGGGACCCCCCATGGTGACTATGCCAGGAGAGACTCTTGCTTCTCGAGTTGCAGCATCCCA

[0274] GCTCACTTGCTTAGGTTGTCTTGAGCTTATTGCTAAAAACAGACAAGAATATGAAGACATAG

[0275] CTGTGAAGCTGGGAACTGATCTAGAATAACCTGAAGAAAGTTCGTGGCAAAGTCTGGAAGCA

[0276] AAGAATATCTAGCCCTCTGTTCAACACCAAACAATACACAATGGAACTAGAGCGGCTCTATC

[0277] TACAGATGTGGGAGCATTATGCAGCTGGCAACAAACCTGACCACATGATTAAGCCTGTTGAA

[0278] GTCACTGAGTCAGCA

[0279] (13) ZV302-Q839A-OGT overexpression plasmid (catalog number G294259-3) was mutated by Junji Biotechnology based on ZV302-OGT by site-directed mutation, which is to mutate “CAG” in the bold underline of SEQ ID NO.15 to “GCG”.

[0280] (14) RNF2 overexpression plasmid, synthesized by Junji Biotechnology, catalog number G25325S-1

[0281] The pLVX-RNF2 overexpression vector inserts the RNF2 CDS fragment (SEQ ID NO.16) between XhoI and KpnI on the pLVX original vector. The RNF2 CDS fragment (SEQ ID NO.16) is as follows:

[0282] ATGTACCCATACGACGTCCCAGACTACGCTTCTCAGGCTGTGCAGACAAACGGAACTCA

[0283] ACCATTAAGCAAAACATGGGAACTCAGTTTATATGAGTTACAACGAACACCTCAGGAGGCAA

[0284] TAACAGATGGCTTAGAAATTGTGGTTTCACCTCGAAGTCTACACAGTGAATTAATGTGCCCA

[0285] ATTTGTTTGGATATGTTGAAGAACACCATGACTACAAAGGAGTGTTTACATCGTTTTTGTGCA

[0286] GACTGCATCATCACAGCCCTTAGAAGTGGCAACAAAGAATGTCCTACCTGTCGGAAAAAAC

[0287] TAGTTTCCAAAAGATCACTAAGGCCAGACCCAAACTTTGATGCACTCATCAGCAAAATTTAT

[0288] CCAAGTCGTGATGAGTATGAAGCTCATCAAGAGAGAGTATTAGCCAGGATCAACAAGCACA

[0289] ATAATCAGCAAGCACTCAGTCACAGCATTGAGGAAGGACTGAAGATACAGGCCATGAACAG

[0290] ACTGCAGCGAGGCAAGAAACAACAGATTGAAAATGGTAGTGGAGCAGAAGATAATGGTGA

[0291] CAGTTCACACTGCAGTAATGCATCCACACATAGCAATCAGGAAGCAGGCCCTAGTAACAAAC

[0292] GGACCAAAACATCTGATGATTCTGGGCTAGAGCTTGATAATAACAATGCAGCAATGGCAATT

[0293] GATCCAGTAATGGATGGTGCTAGTGAAATTGAATTAGTATTCAGGCCTCATCCCACACTTATG

[0294] GAAAAAGATGACAGTGCACAGACGAGATACATAAAGACTTCTGGTAACGCCACTGTTGATC

[0295] ACTTATCCAAGTATCTGGCTGTGAGGTTAGCTTTAGAAGAACTTCGAAGCAAAGGTGAATCA

[0296] AACCAGATGAACCTTGATACAGCCAGTGAGAAGCAGTATACCATTTATATAGCAACAGCCAG

[0297] TGGCCAGTTCACTGTATTAAATGGCTCTTTTTCTTTGGAATTGGTCAGTGAGAAATACTGGAA

[0298] AGTGAACAAACCCATGGAACTTTATTACGCACCTACAAAGGAGCAAAATGA

[0299] (15) WT-OPG overexpression plasmid, synthesized by Junji Biotechnology, catalog number G253678-1

[0300] The pLVX-WT-OPG overexpression vector is formed by inserting an OPG CDS fragment (SEQ ID NO. 17) between XhoI and KpnI in the original pLVX vector. The OPG CDS fragment (SEQ ID NO. 17) is as follows:

[0301] ATGAACAACTTGCTGTGCTGCGCGCTCGTGTTTCTGGACATCTCCATTAAGTGGACCACCCAAGAGACATTTCCACCTAAGTACCTTCATTATGACGAAGAAACCTCTCATCAGCTGTTGTGTGACAAATGTCCTCCTGGTACCTACCTAAAACAACACTGTACAGCAAAGTGGAAGACCGTGTGCGCCCCTTGCCCTGACCACTACTACACAGACAGCTGGCACACCAGTGACGAATGCCTGTACTGCAGCCCCGTGTGCAAGGAGCTGCAGTACGTCAAGCAGGAGTGCAATCGCACCCACAACCGCGTGTGCGAATGCAAGGAAGGGCGCTACCTTGAGATAGAGTTCTGCTTGAAACATAGGAGCTGCCCTCCTGGATTTGGAGTGGTGCAAGCTGGAACCCCAGAGCGAAATACAGTTTGCAAAAGATGTCCAGATGGGTTCTTCTCAAATGAGACGTCATCTAAAGCACCCTGTAGAAAACACACAAATTGCAGTGTCTTTGGTCTCCTGCTAACTCAGAAAGGAAATGCAACACACGACAACATATGTTCCGGAAACAGTGAATCAACTCAAAAATGTGGAATAGATGTTACCCTGTGTGAGGAGGCATTCTTCAGGTTTGCTGTTCCTACAAAGTTTACGCCTAACTGGCTTAGTGTCTTGGTAGACAATTTGCCTGGCACCAAAGTAAACGCAGAGAGTGTAGAGAGGATAAAACGGCAACACAGCTCACAAGAACAGACTTTCCAGCTGCTGAAGTTATGGAAACATCAAAACAAAGACCAAGATATAGTCAAGAAGATCATCCAAGATATTGACCTCTGTGAAAACAGCGTGCAGCGGCACATTGGACATGCTAACCTCACCTTCGAGCAGCTTCGT AGCTTGATGGAAAGCTTACCGGGAAAGAAAGTGGGAGCAGAAGACATTGAAAAAACAATAAAGGCATGCAAACCCAGTGACCAGATCCTGAAGCTGCTCAGTTTGTGGCGAATAAAAAATGGCGACCAAGACACCTTGAAGGGCCTAATGCACGCACTAAAGCACTCAAAGACGT ACCACTTTCCCAAAACTGTCACTCAGAGTCTAAAGAAGACCATCAGGTTTCCTTCACAGCTTCACAATGTACAAATTGTATCAGAAGTTATTTTTAGAAATGATAGGTAACCAGGTCCAATCAGTAAAAATAAGCTGCTTAGAGCAGAAACTCATCTCTGAAGAGGATCTGTAA

[0302] (16) S151A-OPG overexpression plasmid (catalog number G253678-2) was synthesized by Junji Biotechnology. Based on the pLVX-WT-OPG overexpression vector, the "AGC" was mutated to "GCA" at the underlined part of the OPG CDS fragment (SEQ ID NO.17).

[0303] The experimental methods involved in the following embodiments are as follows:

[0304] (1) Animal experiments (All mouse experiments were approved by the Ethics Committee of Shanghai Chest Hospital.)

[0305] (1-1) To investigate the effects of different O-GlcNAc modifications and OPG levels on cell proliferation in vivo, we constructed a subcutaneous xenograft model. The human LUAD cell line A549, in logarithmic growth phase, with few passages and in good condition, was selected for the construction of the subcutaneous xenograft model (CDX). Serum-free cell pellets were collected and diluted to the appropriate volume using 1×PBS. Cell number injected subcutaneously into nude mice: 4×10⁶ cells / mL. 6 Tumors were inoculated into 200 μL of 1×PBS. Tumor length and width were recorded at regular time points after inoculation. The tumor volume was calculated using the following formula: Tumor volume (mm²) 3 ) = 0.5 × length × width 2Once small, firm tumors are observed growing under the skin of the mice, the next step of drug administration can be performed. Recombinant human OPG (rhOPG) is reconstituted in 1×PBS at a dose of 2.5 μg / mouse / time, administered every five days. After anesthetizing the mice, the eyeballs are removed, and blood is collected from the orbital cavity into EP tubes. The mice are then euthanized, and normal tissue samples are collected.

[0306] (1-2) To investigate the roles of Ogt and O-GlcNAc modification in spontaneous tumor formation, we constructed a mouse model of spontaneous lung adenocarcinoma. Ogt floxed transgenic mice (customized from Shanghai Southern Model Biotechnology Co., Ltd., referring to mice containing paired LoxP sites in the OGT gene) and Kras / p53 gene conditional point mutation knock-in mice (Kras / p53 gene knock-in mice) were used. LSL-G12D / + p53 LSL-R172H / + (Purchased from Jackson Laboratories, USA, catalog numbers #008179 and #008652), and tamoxifen (Tam)-inducible Cre recombinase (Sftpc) carrying a specific promoter for pulmonary surfactant protein C (Sftpc). CreERT2 / + Mice (purchased from Cyagen Biosciences, catalog number #C001459) were used to construct Ogt KO Kras LSL-G12D / + p53 LSL-R172H / + Sftpc CreERT2 / + The mouse was named Ogt KO KPC mouse model. Ogtfloxed transgenic mice produced during the breeding process of Ogtfloxed transgenic mice. NC Mice, used to build Ogt NC Kras LSL-G12D / + p53 LSL -R172H / + Sftpc CreERT2 / + A control mouse model, named the Control KPC mouse model, was used. At 6 weeks of age, target mice were intraperitoneally injected with 10 mg / mL Tam (75 mg / kg) every other day for a total of 5 times. This conditionally induced activation of the mouse oncogene Kras and inactivation of the tumor suppressor gene p53 in type II alveolar epithelial cells, with or without Ogt gene knockout, leading to spontaneous LUAD. Mice were euthanized when they reached the humane endpoint (e.g., 20% weight loss, cachexia, loss of appetite, inability to eat or drink). The time of active or passive death was recorded, and survival curves were plotted for different groups. At the experimental endpoint, the mice underwent enucleation to collect blood and serum, and the lungs were dissected to observe differences in the size and number of lung tumor lesions between different groups.

[0307] (1-3) To investigate the roles of hyperglycemia and O-GlcNAc modification in tumor progression and metastasis, we constructed a mouse model of type II diabetes with tail vein lung metastasis. C57BL / 6J mice were fed a high-fat / high-sucrose diet (HFSD, 41.4 kJ / kg) or a normal chow diet (NCD, 25 kJ / kg) for 5 weeks. Mice in the high-fat / high-sucrose diet group received intraperitoneal injections of 10 mg / mL streptozotocin (STZ, 40 mg / kg, fasted before injection) for three consecutive days, while mice in the normal chow diet group received an equal volume of citrate-sodium citrate solution as a control. After the last STZ injection, mice were fasted for 16 hours, and blood glucose levels were measured. A fasting blood glucose level exceeding 11.1 mM was considered a successful establishment of the type II diabetes model. LLC cells in logarithmic growth phase, with few passages and in good condition were selected for tail vein injection. The number of cells injected was 5 × 10⁶. 5 150μL per cell.

[0308] (1-4) To more realistically explore drug efficacy and sensitivity at the in vivo level, we constructed a patient-derived tumor xenograft model (PDX model). Mice were anesthetized with isoflurane. Hair was removed and the skin was prepared near the transplantation site (near the rib-side axilla). A 2-3 mm incision was made ophthalmologically, and then the subcutaneous channel was dissected with forceps. A trocar aspirated the pre-cut tissue and matrix adhesive, removed air bubbles, and inserted subcutaneously through the incision. After reaching the center of the transplantation area along the subcutaneous channel, the entire tissue block was pushed into the subcutaneous tissue, and the trocar was carefully withdrawn to avoid tissue leakage. The PDX mice constructed in the above manner were fed and observed until the tumor volume reached 1,500 mm. 3 Around 10:00 AM, the current generation of PDX mice were marked as F0, sacrificed, and the xenografts were isolated. The F0 generation xenografts were minced, mixed with a high concentration of matrix gel, and then passaged into a new batch of NSG mice to construct the F1 generation of PDX mice. This process was repeated until the F3 generation. F3 generation PDX mice were periodically administered intraperitoneal injections of carboplatin (60 mg / kg), pemetrexed (80 mg / kg), or celecoxib (10 mg / kg) to evaluate the efficacy of combined drug therapy. The size of the xenografts was measured periodically; when the xenograft volume exceeded 1,500 mm², the mice were considered Xenografts. 3 Mice were euthanized under certain circumstances. The time of active or passive death of mice was recorded, and survival curves were plotted for different groups.

[0309] (2) Human cytokine antibody chip G5

[0310] Purchased from RayBiotech, USA. This chip, combining a dual-antibody sandwich method with a biotin-streptavidin amplification system, converts cytokines in the sample into relative fluorescence signal intensities, enabling high-throughput relative quantification of cytokines. The corresponding experiments and detection procedures were performed by RayBiotech (Guangzhou) Biotechnology Co., Ltd.

[0311] (3) Mass spectrometry identification of O-GlcNAc-modified secreted proteins in tumor tissue

[0312] Tumor-associated secretory protein supernatant was used to digest tumor tissue with type I collagenase (yeasen). After centrifugation, the lower cell precipitate was tumor-associated cells, while the supernatant contained secretory proteins, including corresponding cytokines from the tumor microenvironment. The supernatant was separated and filtered through a 0.22 μm cell filter to obtain a supernatant containing secretory proteins containing cytokines secreted by the tumor tissue. Immunoprecipitation was performed using O-GlcNAc antibody or isotype control antibody, followed by capture of the magnetic bead-antibody-antigen complex using Protein A / G magnetic beads. The magnetic beads were eluted with acid and immediately neutralized; the eluent was adjusted to neutral using pH test strips. Data-independent mass spectrometry was used to acquire mass spectrometry data of the protein solution obtained from the immunoprecipitation. Differential proteins (FC>2) were screened by comparing the O-GlcNAc precipitation group matched with the isotype control group from the same patient.

[0313] (4) Immunoblotting (IB)

[0314] All immunoblotting procedures in this invention were performed according to standard protocols. Proteins were extracted using cell lysis buffer (Beyotime, P0013), and the supernatant was obtained by centrifugation at 4°C. Protein samples were prepared after BCA quantification. Proteins were separated by SDS-PAGE and transferred to a membrane, creating a "transfer sandwich" in the following order: negative electrode - sponge - three-layer filter paper - protein gel - PVDF membrane - three-layer filter paper - sponge - positive electrode. Transfer time was positively correlated with molecular weight. After transfer, non-specific sites were blocked. The PVDF membrane was placed in a primary antibody solution of appropriate dilution and incubated overnight at 4°C on a shaker, followed by washing. A secondary antibody labeled with horseradish peroxidase (HRP) corresponding to the species of the primary antibody was selected and diluted. The PVDF membrane was placed in the secondary antibody and incubated at room temperature for 60 min. After secondary antibody incubation, the membrane was washed three times with 1×PBST for 10 min each time.The main antibodies include anti-ogt (Abcam, ab96718 or Santa Cruz Biotechnology, sc74546), anti-o-glcnac (Cell Signaling Technology, 82332 or Abcam, ab2739), anti-oga (ACTIVE MOTIF, 61425), anti-OPG (Abcam, ab73400 or ab183910 or Santa Cruz Biotechnology, sc390518 or Novus Biologicals, NB100-56505), anti-myc (Abcam, ab9106 or Cell Signaling Technology, 2276), anti-MOTS-c (Fabgennix, MOTSC-101AP), anti-ubiquitin (Cell Signaling Technology, 3936), anti-rnf2 (Cell Signaling Technology, 5694), and anti-phospho-cdk2-T160 (Abclonal). Technology, AP0325), anti-CDK2 (Santa Cruz Biotechnology, sc6248 or Proteintech, 10122-1-AP), anti-RB (Santa Cruz Biotechnology, sc102), anti-phospho-RB-Ser807 / 811 (Cell Signaling Technology, 8516), anti-E2F1 (HUABIO, ET1701-73), anti-WNT5A (Santa Cruz Biotechnology, sc365370), anti-EHD1 (Proteintech, 24657-1-AP), anti-CCNE1 / 2 (Santa Cruz Biotechnology, sc247), anti-COX-2 (anta Cruz Biotechnology, sc19999), anti-KI67 (Proteintech, 27309-1-AP), anti-O. S151-OPG (customized by Jingjie Biotechnology), anti-nmt1 (Abcam, ab186123), anti-phospho-src-Y416 (Abclonal Technology, AP0452), anti-actb / β-actin (Servicebio, GB15001), anti-gapdh (Servicebio, GB15002 or GB15004), anti-tubub (Sagecreation, 05015), anti-streptavidin (LSBio, LS-C203634), anti-tamra (Abcam, ab171120), secondary antibody (Cell Signaling Technology, 70074 or 7076), etc. The bands were exposed using an enhanced chemiluminescence kit (Life-iLab, AP34L025).

[0315] (5) Immunohistochemical staining (IHC) and hematoxylin and eosin staining (H&E)

[0316] After dewaxing paraffin sections to water, they were immersed in excess sodium citrate antigen retrieval solution, heated in a 100°C water bath for 45 minutes, and then naturally cooled to room temperature to fully expose the antigenic epitopes. After blocking endogenous peroxidase, the sections were blocked. Following incubation with primary and secondary antibodies, DAB staining was performed. DAB reacted with HRP to generate a brownish-yellow positive signal. The staining reaction was terminated when a clear brownish-yellow signal with good contrast was observed at the target protein location under a microscope. After hematoxylin counterstaining and hydrochloric acid-ethanol differentiation to achieve blue reversion, a suitable amount of neutral resin was added for mounting, and the slides were scanned and IHC scored. For H&E staining, after dewaxing to water, the sections were immersed in hematoxylin staining solution for 3 minutes to stain the cell nuclei. After washing with water, the sections were redifferentiated for a few seconds until the cell nuclei appeared a relatively transparent blue-purple. After washing with water, the sections were immersed in eosin staining solution for 5 minutes until the cytoplasm and intercellular matrix appeared pink, thus completing H&E staining.

[0317] (6) Multiple immunohistochemical staining (mIHC)

[0318] After preparing paraffin sections, dewaxing to water, antigen retrieval, endogenous peroxidase blocking, blocking, and antibody incubation, the sections stained with primary antibody were stained with tyrosine fluorescent dye. After incubation at room temperature for 15 min, the paraffin sections were immersed in sodium citrate antigen retrieval solution, heated in a 100°C water bath for 30 min, and then naturally cooled to room temperature. The non-covalently bound primary antibody-secondary antibody-HRP complex could be eluted by the heat retrieval method, while the tyrosine carrying the fluorescent dye was activated by HRP and covalently bound to the corresponding antigen, preventing the fluorescence signal from being eluted, thus achieving the first round of labeling. The second round of labeling was achieved by repeating the endogenous peroxidase blocking to antibody elution step using a second tyrosine fluorescent dye. The third round of labeling was achieved by repeating the endogenous peroxidase blocking to antibody elution step using a third tyrosine fluorescent dye. After counterstaining cell nuclei with DAPI, the sections were mounted and scanned.

[0319] (7) Cell sorting

[0320] Fresh tumor tissue was digested with type I collagenase and dissociated into a single-cell suspension. Tumor epithelial cells, immune cells, fibroblasts, macrophages, and mononuclear cells were then separated by flow cytometry using magnetic beads (CD326 / CD45 / CD163 / anti-fibroblast magnetic beads) or a mononuclear cell separation kit.

[0321] (8) Gene expression intervention

[0322] CRISPR Cas9 gene knockout: The LentiCRISPR v2 plasmid vector was linearized using the restriction endonuclease BsmBI-v2. The digestion products were loaded onto a 1% agarose gel and subjected to horizontal electrophoresis for 20 min. Gel imaging analysis revealed two linear fragments of different sizes, 13kb and 2kb, produced after BsmBI-v2 digestion. The larger fragment was extracted from the gel. The large DNA fragment was purified by gel extraction. Utilizing the characteristic that DNA denatures and unwinds into single strands at high temperatures and binds into double strands according to the base pairing principle at low temperatures, sgRNA double-stranded oligonucleotides were obtained. T4 DNA ligase was used to catalyze the formation of phosphodiester bonds in the DNA to construct a recombinant plasmid. The successfully sequenced recombinant plasmid was packaged with lentivirus, and the viral fluid was used to infect target cells. Gene knockout cells were obtained through puromycin selection and single-clone picking.

[0323] Gene overexpression (or mutant) construction: order overexpression plasmid (or mutant plasmid), obtain viral fluid through lentivirus packaging, infect target cells with viral fluid, and obtain gene overexpression cells (or gene mutant cells) through puromycin screening.

[0324] (9) Co-immunoprecipitation (Co-IP)

[0325] Cellular protein samples were lysed under non-denaturing conditions using a mild lysis buffer to preserve the natural binding interactions within the cells. Cocktail protease inhibitors and PMSF were added to prevent protein degradation. Input protein samples were prepared, and two equal aliquots of protein solution (approximately 1000 μg total protein) were taken: one aliquot was mixed with 1.5 μg of precipitation antibody, and the other aliquot was mixed with an equal volume of isotyped antibody as a control. Both aliquots were placed on a rotary mixer and captured at 4°C for 12 h. 20 μL of Protein A / G magnetic beads were added to 1×PBS, thoroughly resuspended, and then magnetically separated on a magnetic rack. The supernatant was discarded; repeated washing was performed to remove the bead preservation solution. The incubation solution of the precipitation antibody against the target protein was taken and added to the magnetic beads, and the mixture was captured on a rotary mixer at room temperature for 2 h. The binding ability of Protein A / G magnetic beads to IgG was used to further capture the target protein antigen-antibody complex. Wash the magnetic beads several times on a magnetic rack, prepare 1× protein loading buffer, elute the protein complex from the magnetic beads, separate the supernatant into a new EP tube, and heat in a metal bath at 95°C for 10 min to denature the protein. Separate the protein complex on an SDS-PAGE gel, using a detection antibody of a different species than the precipitation antibody to avoid heavy and light chain contamination, in order to determine the IP efficiency and detect protein interactions.

[0326] (10) Immunofluorescence (IF)

[0327] Place a round cell spreader into a 24-well plate and seed with 1×10⁶ cells / well. 4 Cells were incubated at 37°C. After cell fixation, permeabilization, and blocking, the corresponding primary antibody was diluted with blocking buffer, and the cells were covered with the slides and incubated overnight at 4°C. After washing, a suitable fluorescently labeled secondary antibody was selected according to the type of primary antibody and sample characteristics, and incubated at room temperature in the dark for 60 min. After recovering the secondary antibody solution, excess secondary antibody was slowly washed away. Cell nuclei were counterstained with DAPI for 5 min, and the slides were washed. An appropriate amount of anti-fluorescence quencher was added to the slide, the slide was mounted, and a suitable amount of nail polish was applied for fixation. After air drying, the slides were observed and imaged using a fluorescence microscopy system, and finally stored at -20°C. The colocalization signal intensity of the stained dual antigens was quantified using Image J (Scatter J).

[0328] (11) Adjacency Linkage (PLA)

[0329] After cell plating, fixation, and permeabilization, cells were blocked at 37°C for 60 min using the blocking solution provided in the kit. Two primary antibodies from different species were selected to detect two protein targets (a single antibody negative control was also included). The primary antibody solution was used to cover the cell slides, which were then incubated overnight at 4°C in a humidified dark chamber. The oligonucleotide-labeled adjacent probes were diluted to the appropriate working concentration using antibody dilution buffer. After washing the slides, probe solution was added, and the slides were incubated at 37°C for 60 min in a humidified dark chamber to allow binding of the primary antibody. The ligase was diluted to the appropriate working concentration using ligation buffer. The slides were washed with wash buffer A, and ligase solution was added, followed by incubation at 37°C for 30 min in a humidified dark chamber. When a pair of adjacent probes are sufficiently close, the ligase will hybridize complementaryly with the probe pair, forming a closed circular DNA, which serves as a template for rolling circle amplification. The DNA polymerase was diluted to the appropriate working concentration using amplification buffer. The slides were thoroughly washed with wash buffer A for 10 min, and ligation solution was added, followed by incubation at 37°C for 100 min in a humidified dark chamber. DNA polymerase uses adjacent probes as primers and circular DNA formed by ligase as templates to synthesize cascaded sequences through rolling circle amplification to increase signal intensity. Oligonucleotides in the probe hybridize with complementary sequences of the amplicon, forming scattered PLA signals. After thorough washing, DAPI counterstaining, mounting, and imaging are performed.

[0330] (12) O-GlcNAc chemiluminescent labeling method

[0331] In vitro: Cell pellets were lysed using click chemistry cell lysis buffer without EDTA chelating agent. Protein supernatant was collected on ice and quantified using the BCA method to determine the total protein concentration in the supernatant. OPG-Myc fusion protein was immunoprecipitated using Myc-tag antibody and Protein A / G magnetic beads. In vitro chemical enzymatic labeling of the magnetic beads: Azide groups were transferred to O-GlcNAc modified protein under Gal-T1 (Y289L) catalysis for chemical labeling, followed by a click chemistry reaction between the azide and alkyne. The OPG-Myc fusion protein after the click chemistry reaction was eluted with protein loading buffer. The supernatant was separated using a magnetic rack, heated for denaturation, and then analyzed on an SDS-PAGE gel to determine whether O-GlcNAc modification had occurred in vitro.

[0332] In vivo: Cells were co-incubated with tetraacylated N-acetylgalactosamine azide Ac4GalNAz. Ac4GalNAz is metabolized to UDP-GalNAz via the N-acetylgalactosamine salvage pathway, and subsequently converted to uridine diphosphate-N-acetylglucosamine azide UDP-GlcNAz by UDP-galactose 4-epimerase (GALE). This process, similar to UDP-GlcNAc incorporation into O-GlcNAc, modifies the substrate protein, giving it an azide group. Click on the chemical reactions and immunoblotting results in the same results as in "In vitro".

[0333] (13) Identification of O-GlcNAc modification sites in OPG protein

[0334] Immunoprecipitation combined with liquid chromatography-mass spectrometry (IP-LC / MS) was used to detect the O-GlcNAc modification sites of OPG protein. After enriching cellular OPG protein by immunoprecipitation, the complex was separated by SDS-PAGE protein gel chromatography, stained with Coomassie Brilliant Blue at room temperature for 60 min, and then destained. Destaining was stopped after the background became clear, and gel strips were cut according to molecular weight. O-glycopeptide analysis kits were used to release and label O-glycopeptides. The O-glycopeptide sample preparation process involved glycopeptide capture microspheres, a filtration column, a purification column, 2-AB labeling reagent, and a reducing agent. Samples were analyzed using a nanoscale high-performance liquid chromatography system, Easy nLC. TM For separation and cascading, use Orbitrap Exploris 480MS or Q Exactive. TM HF-X was used for mass spectrometry detection.

[0335] The Swiss-Prot Human protein database was used for mass spectrometry data analysis; specific glycosylation sites were retrieved using the pGlyco3.0 glycopeptide search engine. O-GlcNAc modification originated from the GlcNAc group (chemical formula C8H) in the substrate UDP-GlcNAc. 15 NO6) is linked to serine and threonine residues and loses one molecule of H2O, thus resulting in oxygen-linked group modification (chemical formula C8H) on the target peptide. 13 NO5). The mass peak at 204.09 in the secondary mass spectrum represents the [M+H] peak of the O-GlcNAc modified peptide. + Signal.

[0336] (14) Anti-O S151 Development of GlcNAc-OPG polyclonal antibodies

[0337] Animals were immunized with modified peptide sequences, and the peptides that recognize O were then screened and purified.S151 The polyclonal antibody against -GlcNAc-OPG was developed, and all related work was outsourced to Hangzhou Jingjie Biotechnology Co., Ltd. Modified peptide A: PDGFF-(O-GlcNAc)S-NETSSKC; Modified peptide B: CPDGFF-(O-GlcNAc)S-NETSSK(145-157); Unmodified peptide: PDGFFSNETSSK C. After antigen synthesis, the peptides were linked to the carrier hemocyanin to form a covalently coupled complete antigen, which was then used to immunize New Zealand white rabbits. Serum screening and purification were completed by ammonium sulfate precipitation and affinity purification using Protein A / G chromatography. The antibody titers for modified peptides A and B, and the unmodified peptides were tested using affinity chromatography for both modified and unmodified peptides to demonstrate that the antibodies only recognize site-modified peptides.

[0338] (15) Actinomycete ketone tracing experiment

[0339] Cycloheximide (CHX) is a classic inhibitor of protein synthesis. Its mechanism of action is to bind to the 80S ribosome, thereby preventing tRNA translocation during translation. CHX is commonly used to detect the half-life of proteins under different conditions. Cells are treated with CHX, and equal volumes of cell pellets are collected after different treatment times. After cell lysis and centrifugation, the protein supernatant is obtained, and the target proteins in the supernatant are qualitatively or quantitatively detected.

[0340] (16) Enzyme-linked immunosorbent assay

[0341] Serum samples, cellular protein samples, and cell culture supernatant samples were prepared according to standard procedures. Standards were reconstituted using standard diluent or cell culture medium, and added sequentially from high to low concentrations, with appropriate dilution ratios for each sample. After adding the detection antibody, incubation was performed to form a solid-phase antibody-sample-detection antibody double-antibody sandwich complex. Excess sample and detection antibody were thoroughly washed away. HRP-labeled streptavidin was added and incubated to amplify the signal intensity of the double-antibody sandwich complex. After thorough washing, a substrate solution containing tetramethylbenzidine was added. A blue product was generated after HRP treatment. An acidic stop solution was added to stop the colorimetric reaction. Dual-wavelength detection was performed, measuring the absorbance at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm. The target protein concentration of the sample was calculated using the calibrated absorbance value (i.e., the 450 nm measurement minus the 630 nm measurement) and a standard curve.

[0342] (17) Ubiquitination Experiment

[0343] In vivo: A549 WT-OPG-Myc or S151A-OPG-Myc cells, after different treatments, were enriched with endogenous or exogenous OPG proteins by immunoprecipitation, and then the proteins were separated and visualized by SDS-PAGE protein gel.

[0344] In vitro: After briefly centrifuging the recombinant protein at 250×g for 1 min, add the corresponding 1×PBS buffer to dissolve the recombinant protein to the appropriate storage concentration. Prepare the in vitro ubiquitination reaction system and set up a negative control group (lacking E1 ubiquitin activator enzyme or E2 ubiquitin conjugate enzyme, the corresponding volume is made up with double-distilled water). Prepare the system, incubate in a 37℃ water bath for 3 h, and then terminate the reaction. After denaturation by heating in a 95℃ metal bath for 10 min, separate and develop the protein by SDS-PAGE.

[0345] (18) Plate colony formation experiment

[0346] Cells were digested and prepared into single-cell suspensions of uniform concentration. The same number of cells were seeded into each well of a 6-well plate. A single-cell clone containing more than 50 cells, with a visually apparent size between 0.3 mm and 1.0 mm, was ready for fixation and staining. Old culture medium was discarded, and cells were washed with 1×PBS, fixed with 4% paraformaldehyde for 15 min, washed thoroughly with 1×PBS, stained with crystal violet for 20 min, and then washed with double-distilled water. The plates were inverted, air-dried, and photographed. Cell images were processed using software such as ImageJ, and changes in cell proliferation capacity between the treated and control groups were calculated.

[0347] (19) Cell cycle detection

[0348] After normal cells were plated, centrifuged, and the cell pellet collected, they were stained with propidium iodide (PI) at room temperature in the dark for 30 min. Fluorescence intensity was detected by flow cytometry to analyze the distribution of different cell cycle stages in tumor cells under different treatments. Flow cytometry data of the cell cycle were analyzed using ModFit LT 5.0.

[0349] (20) CCK-8 detection

[0350] Cells from different treatment groups were counted and seeded into 96-well plates for normal culture at a seeding density of 2,000 cells / 100 μL of complete medium. Cell killing was treated at a seeding density of 10,000 cells / 100 μL of complete medium, with drug intervention added at the corresponding time points. 100 μL of CCK-8 dilution buffer was added to each well before incubation at 37°C for 30 min. The absorbance at 450 nm was measured using a microplate reader, recorded, and the 96-well plates were returned to the incubator for further culture. The process was repeated at the next detection time point. Data analysis used the absorbance at the starting point of the control group as a reference to calculate the relative fold increase or survival rate of each experimental group.

[0351] (21) 3D Tumor Spheroid Formation Experiment

[0352] Remove the matrix gel in advance and thaw it on ice. Using a pre-cooled tip, pipette 70 μL of matrix gel into each 96-well plate and incubate at 37°C until solidification. Digest the cells to be inoculated, and after cell counting, seed 1,000 cells / 200 μL / well into each 96-well plate. Change the medium every other day. Take 3D images of the tumor spheres under a microscope.

[0353] (22) In vitro O-GlcNAc experiment

[0354] OPG purified proteins, with or without amino acid mutations, were custom-made from MCE. OPG-FLAG purified proteins underwent an in vitro O-GlcNAc reaction system (1 μM OPG purified protein, 0.125 μM OGT purified protein, 10 μM UDP-GlcNAc, 50 mM Tris-HCl, pH 7.5, 1 mM DTT) and were incubated at 37°C for 90 min to achieve O-GlcNAc modification. The OPG proteins from the in vitro O-GlcNAc reaction system were then subjected to FLAG magnetic bead immunoprecipitation, followed by competitive elution with FLAG peptides. The supernatant was collected, ultrafiltered, and freeze-dried to obtain purified protein powder.

[0355] (23) Multi-omics and bioinformatics analysis

[0356] Proteomics identification of proteins interacting with OPG was performed by Oebiotech using immunoprecipitation with an anti-opg antibody (Novus Biologicals, NB100-56505). Transcriptome sequencing of proteins regulated by OGT or OPG genes and purified with exogenously added OPG was performed by Oebiotech. OPG-EHD1 protein docking was performed using PyMOL (3.0.3) and Rosetta (3.2) software.

[0357] (24) Statistical methods

[0358] Statistical analysis and plotting were performed using GraphPad Prism (8.2.1) software. Data are expressed as mean ± standard deviation (mean ± SD). Student's t-test was used for comparisons between two groups; one-way or two-way ANOVA was used for comparisons among multiple groups; and chi-square test was used for comparisons of rates or proportions. A p-value < 0.05 was considered statistically significant. Specific statistical differences are as follows: *p < 0.05,

[0359] **p<0.01, ***p<0.001, ****p<0.0001, NS indicates p>0.05, no significant difference.

[0360] Example 1: O-GlcNAc modification regulates OPG expression in tumor cells.

[0361] To clarify the tumor cell extracellular secretory proteins regulated by O-GlcNAc modification ( Figure 1 A) In human lung adenocarcinoma

[0362] OGT gene knockout strain was constructed in the A549 cell line using CRISPR Cas9 experimental technology. Figure 1 B), and using a human cytokine antibody microarray to detect cytokines secreted by human A549 tumor cells into mouse peripheral blood in a nude mouse CDX model, it was found that serum OPG, IL-4, and CCL5 are very likely directly regulated by OGT ( Figure 1 C). Combined immunoprecipitation of O-GlcNAc antibody and proteomics analysis of tumor cell secreted proteins were performed on tumor tissue samples from three different lung adenocarcinoma patients. Figure 1 D), we identified O-GlcNAc modification-related secretory proteins that are highly expressed in cancer patients, among which OPG, IL-4, and CCL5 were among the most abundant. Figure 1 E). Further investigation using OGT gene knockout cells constructed in primary cells of lung adenocarcinoma A549, lung squamous cell carcinoma NCI-H1588, small cell lung cancer DMS-114, esophageal squamous cell carcinoma KYSE-150, and thymoma patients after surgery revealed that the cytokine OPG was most significantly regulated by OGT. Figure 2 ).

[0363] Multiple immunohistochemical staining of fresh lung adenocarcinoma tissue samples revealed that tumor cells (I) showed the strongest co-localization with OPG, while extratumor cells such as immune cells, macrophages, and fibroblasts (E) showed weaker co-localization with OPG. This indicates that the cytokine OPG is mainly expressed on tumor epithelial cells (I), rather than on immune cells, macrophages, and fibroblasts. Figure 3 AC)(E). Flow cytometry sorting of tumor tissue and isolation of mononuclear cells revealed that tumor cells exhibited stronger OPG expression, further confirming that tumor cells are the cell type most richly expressing OPG. Figure 3 These results indicate that tumor cells regulate the expression level of OPG protein through O-GlcNAc modification.

[0364] Example 2: O-GlcNAc modification occurred inside tumor cells.

[0365] OPG knockout using sgRNA in A549 cells ( Figure 4 A) OPG gene knockout inhibited the proliferation of A549 cells both in vivo and in vitro. Furthermore, OPG gene knockout significantly reduced OPG expression in both intracellular and in vitro tumor cells in nude mouse CDX cells. Figure 4 OGT gene knockout also weakened the proliferation ability of tumor cells both in vivo and in vitro, while exogenous supplementation of purified OPG protein could, to some extent, reverse the inhibitory effect of OGT gene knockout on proliferation. Figure 5 (AB). The results confirmed that OPG is downstream of O-GlcNAc modification regulation.

[0366] To further investigate whether OPG directly underwent O-GlcNAc modification, immunoprecipitation, immunofluorescence, and quantitative and chemiluminescent immunoassays (in vivo and in vitro) were used to confirm the interaction between OPG and O-GlcNAc modification in the A549 cell line. Figure 6 To clarify the specific modification site, immunoprecipitation combined with liquid chromatography-mass spectrometry was used to identify serine at position 151 of the OPG protein. th Serine, S151) is a potential O-GlcNAc modification site ( Figure 7 A). Protein sequence alignment analysis across different species revealed that this site is highly conserved and may have important biological functions. Figure 7 B). Through mutant construction combined with immunoprecipitation, enzyme labeling, and adjacent linkage assays, it was found that the O-GlcNAc modification level of OPG protein was weakened after mutation at the S151 site, thus confirming that the S151 site is the true O-GlcNAc modification site of the OPG protein. Figure 7 CE). Simultaneously, OGT overexpression enhances the interaction between WT-OPG and O-GlcNAc, significantly increasing the OPG protein O-GlcNAc modification level ( Figure 7 F).

[0367] Furthermore, OGA overexpression or OSMI-1 treatment (50 μM, 24 h) in A549 cells significantly reduced the O-GlcNAc modification level in wild-type OPG but not in S151A mutant OPG. Figure 8 (AB) It is evident that the regulation of O-GlcNAc modification-related genes and drug treatment only regulate the O-GlcNAc level of WT-OPG, not S151A-OPG. All of the above results indicate that O-GlcNAc modification occurs in the OPG protein at the S151 site.

[0368] Example 3: O-GlcNAc modification of OPG protein promotes its protein stability.

[0369] To further investigate the specific biological function of O-GlcNAc modification at the S151 site of OPG protein, in vitro clonogenic assays and in vivo nude mouse tumor-bearing experiments revealed that S151A-OPG exhibited weaker tumor proliferation ability both in vitro and in vitro, demonstrating that O-GlcNAc modification at the S151 site promotes the proliferation ability of OPG. Figure 9 AB). OPG is a secreted protein that primarily functions in the matrix. Treatment of tumor cells overexpressing wild-type or S151A mutant OPG with adaptive culture medium revealed that the adaptive medium from S151A-OPG-overexpressing cells lost its exogenous tumor-promoting ability; only wild-type OPG, and not S151A mutant OPG, possessed the ability to promote tumor cell proliferation in the matrix. Figure 9 C). Furthermore, in the CDX model, the levels of OPG inside and outside tumor cells were lower in S151A mutant A549 xenograft tissue. Figure 9 D).

[0370] Given that O-GlcNAc modification often regulates protein stability, subsequent cyclohexane tracking experiments confirmed that OGT overexpression could enhance the O-GlcNAc modification level of wild-type OPG protein, thereby increasing its protein stability, but it could not enhance the O-GlcNAc modification level of S151A mutant OPG protein, therefore it had no effect on the protein stability of mutant OPG. Figure 10 (AC). In addition, the peptide MOTS-c, which does not contain serine or threonine, was selected as a negative control protein that cannot undergo O-GlcNAc modification. These results indicate that O-GlcNAc modification at the S151 site of the OPG protein enhances the stability of the OPG protein.

[0371] IP mass spectrometry indicated a protein-protein interaction between RNF2 and OPG. Figure 11A) may be a potential E3 ubiquitin ligase for OPG protein. This was confirmed by constructing tumor cells overexpressing RNF2 and conducting in vitro and in vivo ubiquitination experiments. RNF2 enhanced OPG ubiquitination. Figure 11 BC). Furthermore, IP experiments further confirmed that O-GlcNAc modification reduces RNF2-mediated OPG ubiquitination in a S151-dependent manner. Figure 11 D) There is competition between O-GlcNAc modification and ubiquitination modification.

[0372] Example 4: Exogenous OPG drives the internal cell cycle through membrane protein EHD1

[0373] To clarify the downstream pathways co-regulated by O-GlcNAc modification and OPG, transcriptome sequencing was performed on OGT gene knockout or OPG gene knockout A549 cells using RNA-seq. A total of 261 genes were found to be co-regulated by O-GlcNAc modification and OPG. Figure 12 A), according to enrichment analysis by the Kyoto Encyclopedia of Genetics and Genomes (KEGG), the cell cycle pathway was among the top ten enriched pathways. Figure 12 B) The gene names annotated for this pathway include

[0374] CCNB1, MYC, CUL1, CCNB2, CDC45, MCM4, and CDC25A. Among them, MYC and MCM4 are important cell cycle regulatory genes and have been shown to directly undergo O-GlcNAc modification; CUL1 has also been shown to be associated with O-GlcNAc modification. To further analyze the regulatory role of exogenous OPG proteins secreted into cell culture supernatant on the cell cycle, transcriptome sequencing was performed again on A549 cells treated with OPG-purified protein medium, confirming that WNT5A is a cell cycle-related gene regulated by OPG. Figure 12 C).

[0375] Flow cytometry analysis of cell cycle and CCK-8 proliferation capacity confirmed that both OGT and OPG gene knockout resulted in G0 / G1 phase arrest in tumor cells. However, gradient refilling of WT-OPG instead of S151A-OPG (with a gradient during viral infection, virus solution:culture medium = 1:1 or 2:1) could, to some extent, reverse the G0 / G1 phase arrest in tumor cells caused by OGT gene knockout. Figure 13 A); similarly, replenishing WT-OPG instead of S151A-OPG can gradually reverse the proliferation inhibition caused by OGT gene knockout. Figure 13B). By detecting the expression of proteins related to the classical cell cycle pathway, it was confirmed that exogenous OPG partially restored the G0 / G1 phase arrest in tumor cells caused by OGT gene knockout by upregulating the CDK2-Rb-E2F1-WNT5A regulatory axis (specifically, the regulated CDK2-Rb-E2F1-WNT5A sequence). Figure 13 CD).

[0376] IP mass spectrometry showed that EHD1 and IQGAP1 are the only two membrane proteins that interact with OPG. Figure 14 A). Considering the well-defined role of IQGAP1 in the cell cycle, the membrane protein EHD1 was the primary focus. Cell cycle validation revealed that the absence of EHD1 resulted in rhOPG losing its cell cycle-promoting effect on tumor cells, fully demonstrating the bridging role of EHD1 in the tumor-promoting capacity of exogenous OPG. Figure 14 B), that is, EHD1 can transmit extracellular OPG signals into the cell, thereby promoting the cell cycle within tumor cells. Figure 15 A). Constructing EHD1 gene knockout cells using CRISPR Cas9 ( Figure 15 B), and after treatment with purified proteins using exogenous OPG, it was found that OPG could activate the CDK2-Rb-E2F1-WNT5A regulatory axis, while knocking out EHD1 completely blocked the activation effect of OPG. Exogenous OPG could not alter the expression of proteins related to G0 / G1 phase cell arrest. Figure 15 C). Simultaneously, exogenous OPG can dose-dependently promote the binding of CCNE1 / 2 to CDK2, and EHD1 knockout can similarly block this phenomenon. Figure 15 D) indicates that exogenous OPG drives the cell cycle by regulating CDK2 and CCNE1 / 2 binding, and this regulation is EHD1-dependent. This phenomenon is related to the tumor cells' EHD1-dependent endocytosis capacity of exogenous OPG. Figure 15 E) This indicates that EHD1 gene knockout inhibits the ability of tumor cells to endocytose exogenous recombinant OPG proteins. These results suggest that extracellular OPG signaling drives internal cell cycle processes through the membrane protein EHD1.

[0377] Example 5: O-GlcNAc-modified OPG promotes cell cycle by enhancing binding to EHD1.

[0378] O-GlcNAc modified WT-OPG, non-O-GlcNAc modified S151A-OPG, WT-rhOPG, and S151A-rhOPG were obtained using in vitro O-GlcNAc modification experiments. The corresponding purified proteins were then obtained through competitive elution. Figure 16 A). After verification of the degree of O-GlcNAc modification and ELISA concentration detection ( Figure 16 AC) was used to co-culture A549OPG gene knockout cells. Cell cycle analysis confirmed that O-GlcNAc modified WT-OPG (i.e., O-WT-rhOPG-FL) had the most significant ability to reverse G0 / G1 phase arrest and inhibit proliferation in A549 OPG gene knockout cells. Figure 16 DE is associated with increased pCDK2 levels during the cell cycle. Figure 16 F).

[0379] By co-incubating OPG purified protein modified with O-GlcNAc in vitro with cell lysate from A549 OPG gene knockout cells, IP results confirmed that the O-GlcNAc-modified WT-OPG (i.e., O-WT-rhOPG-FL) had the strongest binding affinity to the membrane protein EHD1. Figure 17 A). Meanwhile, O-GlcNAc-modified WT-OPG was most significantly endocytosed ( Figure 17 B). Molecular docking correlation analysis results show that O-GlcNAc modified WT-OPG can increase the number of hydrogen bonds between OPG and EHD1, weaken the binding energy between OPG and EHD1, and thus enhance the interaction between the two. Figure 17 CE). Next, the purified OPG protein with mutant hydrogen bond sites was constructed, and after in vitro O-GlcNAc experiments, it was co-incubated again with A549 OPG gene knockout cell lysate. Figure 17 F) confirmed that O-GlcNAc-modified WT-OPG can enhance the interaction between OPG and EHD1. However, after mutation of the relevant hydrogen bond sites, although the O-GlcNAc-mutated OPG can still undergo in vitro O-GlcNAc modification at comparable concentrations, the interaction between O-GlcNAc-modified Y49A-OPG or K88A-OPG and EHD1 is weakened. Figure 17 G) confirms the authenticity of the hydrogen bond sites revealed by molecular docking.

[0380] Exogenous OPG is delivered via the membrane protein EHD1, promoting the endogenous cell cycle by reducing the second messenger cAMP. Figure 18 A). ELISA validation revealed that treatment with O-GlcNAc-modified WT-OPG purified protein significantly inhibited cAMP levels, but this effect disappeared after EHD1 knockout. Figure 18 B) indicates that O-WT-rhOPG-FL has the strongest cAMP-reducing effect, which is EHD1-dependent. Adding dbcAMP to cells enhances cellular cAMP levels, and IP and IB results confirm that cAMP is downstream of OPG regulation; exogenous OPG promotes the internal cell cycle by inhibiting cAMP levels. Figure 18CE). A nude mouse xenograft tumor progression model also found that cAMP levels gradually decreased as the xenograft progressed. Figure 18 These results indicate that O-GlcNAc-modified WT-OPG promotes the cell cycle through cAMP inhibition by enhancing the interaction between OPG and EHD1.

[0381] Example 6: Evaluation of the clinical and translational significance of OPG in tumors

[0382] By matching Ogt floxed Conditional knockout mice, Kras / p53 gene conditional point mutation knock-in mice, and Sftpc CreERT2 / + Ogt KO KPC and Control KPC mice were obtained through genotyping to investigate the role of Ogt in tumorigenesis. At 6 weeks of age, mice were administered tamoxifen (Tam, 10 mg / mL, 75 mg / kg) targeting the deletion of the loxP sequence in type II alveolar epithelial cells, leading to Kras activation and p53 inactivation, with or without Ogt knockout, thereby mediating spontaneous tumorigenesis of LUAD in the mouse lungs. Ninety days after Tam induction, the Ogt KO KPC group had fewer macroscopically visible tumor lesions in the lungs compared to the Control KPC group. Figure 19 A), H&E staining also confirmed that its tumor burden was lighter (A), Figure 19 B). Successful knockout of OGT in mouse lung tissue was validated at the protein level. Figure 19 C).

[0383] A polyclonal antibody that specifically recognizes O-GlcNAc-modified OPG at the S151 site was developed and named anti-O. S151 -GlcNAc-OPG. To verify the antibody's authenticity, we constructed cells with gradient overexpression of OGT or N-myristoyltransferase 1 (NMT1) (the gradient was set during viral infection, with virus solution:culture medium = 1:2, 1:1, or 2:1) to simulate gradually increasing levels of O-GlcNAc or N-myristoylyltransferase modification, and validated the relevant target proteins to confirm the antibody's effectiveness. Figure 19 D). IHC validation revealed that Ogt KO reduced overall and tumor stroma levels of O-GlcNAc, OPG, and O. S151 Expression of -GlcNAc-OPG ( Figure 19 E). In addition to IHC-related staining, primary cells from spontaneously tumorigenic mouse lung tumors also unbiasedly demonstrated the positive regulatory role of OGT-mediated O-GlcNAc modification on OPG. Figure 19F). Furthermore, mouse serum ELISA further illustrates the total OPG and O2 secreted into the bloodstream by Ogt KO KPC mice. S151 -GlcNAc-OPG levels decreased significantly ( Figure 19 G), further confirming that OGT plays a pro-cancer role in tumorigenesis and development by regulating O-GlcNAc modification of OPG protein. Survival analysis revealed that Ogt KO KPC mice had a significantly prolonged overall survival time (G). Figure 20 A) may be related to the upregulation of pCDK2 expression and enhanced proliferation, which suppresses tumor proliferation after Ogt KO. Figure 20 B).

[0384] Hyperglycemic patients provide more O-GlcNAc modification raw materials for tumors, therefore, hyperglycemic tumor patients often exhibit higher O-GlcNAc modification levels. C57BL / 6J mice were divided into two groups, fed a normal diet or a high-sugar, high-fat diet for 5 weeks, respectively. Then, type II diabetic mouse models and their NC mice were constructed by intraperitoneal injection of either STZ-insoluble or STZ-containing citrate-sodium citrate solution (STZ, 10 mg / mL, 40 mg / kg). Fasting blood glucose was measured to verify the success rate of model establishment. LLC cells (5 × 10⁻⁶ cells / mL) were injected via the tail vein. 5 A type II diabetic mouse tail vein lung transfer model was constructed using (150 μL / piece). Figure 21 AB). The results showed that, compared with the NC group mice, the T2DM group mice (type II diabetic mouse model) had a heavier tumor burden (AB). Figure 21 C), and the O-GlcNAc modification level in the tumor parenchyma and stroma of lung metastases is higher ( Figure 21 D). Isolation and culture of primary cells from metastatic tumors also confirmed that T2DM can enhance the O-GlcNAc modification level in tumor cells (D). Figure 21 D) This further clarifies that T2DM promotes tumor progression by regulating O-GlcNAc modification levels. IHC and ELISA confirmed that total OPG and O2 in T2DM mice were significantly lower than normal. S151 The expression levels of -GlcNAc-OPG and pCDK2 were both increased. Figure 21 E). ELISA analysis of mouse serum showed that OPG and O2 levels in the serum of mice in the T2DM group were... S151 The expression of -GlcNAc-OPG was also elevated. Tumors participate in tumorigenesis and development by regulating the O-GlcNAc modification level of the OPG protein.

[0385] KM database analysis revealed that lung adenocarcinoma patients with high OPG expression levels had shorter overall survival times, indicating that high OPG expression was associated with poorer overall survival. Figure 22A). HbA1c ≥ 7.0 indicates poor long-term glycemic control. Experimental results show that OPG and O2 are commonly present in the serum of thoracic tumor patients with HbA1c ≥ 7.0. S151 Upregulation of O-GlcNAc-OPG expression suggests that there may be widespread abnormal expression of O-GlcNAc modification-related proteins in patients with hyperglycemia and tumors. Figure 22 B). The eight-cytokine assay reflects the inflammatory state in a patient's body. Experimental results indicate that the eight-cytokine assay may reflect the levels of OPG and O2. S151 A broader range of cytokine disorders, including -GlcNAc-OPG ( Figure 22 C). Primary tumor cells better reflect the true state of the tumor in the patient's body. By isolating and culturing primary tumor cells, a combined CCK-8 and 3D tumor spheroid formation assay revealed that hyperglycemia and widespread cytokine dysregulation respectively promoted the malignant proliferation and clonogenic capacity of tumor cells. Figure 23 Primary tumor cells with AD, combined with hyperglycemia or widespread cytokine dysregulation, showed higher OGT protein expression and O-GlcNAc modification levels. Figure 23 EF).

[0386] OPG was knocked out in primary tumor cells from patients using CRISPR Cas9 assays, and either WT-OPG or S151A-OPG was exogenously reinjected, respectively, to construct primary tumor cells with OPG protein O-GlcNAc modification specifically knocked out. Figure 24 A), and the knockout efficiency was verified using IB and ELISA. Figure 24 B). Cells were treated with D-glucose and its inactive isoform, L-glucose, respectively. CCK-8 assays and 3D tumorigenesis experiments revealed that only active D-glucose, not inactive L-glucose, could enhance O2. S151 -GlcNAc-OPG wild-type strains have high in vitro proliferation ability, but are susceptible to O S151 -GlcNAc-OPG knockout strains had no effect, meaning that high levels of D-glucose could enhance tumor proliferation, but specific knockout of O-GlcNAc-modified OPG protein could inhibit tumor proliferation. Figure 24 C). Furthermore, subcutaneous tumor-bearing models were constructed in NC nude mice and type II diabetic nude mice. Experimental results showed that hyperglycemia only enhanced O2. S151 -GlcNAc-OPG wild-type plants have high in vivo proliferation capacity, but are susceptible to O S151 -GlcNAc-OPG knockout strains had no effect, meaning that hyperglycemia promotes tumor progression by regulating O-GlcNAc modification at the S151 site of the OPG protein. Figure 24 D).

[0387] Machine learning predicted that celecoxib, an FDA-approved first-line drug for osteoarthritis, may have potential OGT inhibitory effects. Treatment of A549 cells with this drug (50 μM, 24 h) revealed that celecoxib not only inhibited the expression levels of OGT and O-GlcNAc modification in A549 cells, but also inhibited the expression of downstream proteins OPG and O2. S151 -GlcNAc-OPG( Figure 25 A). Celecoxib primarily inhibits OGT at the Q839 site of the OGT protein ( Figure 25 B), and this process is proteasome-dependent. Figure 25 C). Also a COX-2 inhibitor, parecoxib did not exhibit the same O-GlcNAc modification inhibition as celecoxib, confirming that celecoxib's inhibitory function against O-GlcNAc modification is independent of COX-2. Figure 25 D). Carboplatin and pemetrexed (CP) are first-line chemotherapy drugs for advanced lung adenocarcinoma. Metastatic primary tumor cells isolated and cultured from malignant pleural effusions of patients with CP-resistant advanced lung adenocarcinoma, treated alone or in combination with chemotherapy drugs, showed poor responsiveness to CP monotherapy, while CP combined with celecoxib (CPC) restored tumor cell sensitivity to CP in a dose-dependent manner. Figure 26 A).

[0388] To further explore the therapeutic effect of celecoxib on tumors, a PDX model was constructed using freshly resected lung adenocarcinoma tumor tissue from patients. This model better simulates the real microenvironment and drug response in patients while highly preserving tumor heterogeneity. One lung adenocarcinoma tissue sample was selected from patients with hyperglycemia or cytokine disorders, respectively, for the construction and passage of the PDX model. After drug treatment, it was found that compared with the celecoxib-only treatment group, CPC combination therapy could inhibit tumor growth and prolong the survival of mice. Figure 26 BE). Validated by IB, CPC combined treatment successfully inhibited O-GlcNAc modification and OPG / O2 modification in PDX model xenografts. S151 -GlcNAc-OPG level ( Figure 26 FG). For CDX models derived from pleural effusion, the combination of OSMI-1 drugs and CP therapy (CPO) significantly reduced the malignant proliferative capacity of pleural effusion xenografts both in vivo and in vitro. Figure 27 Combined treatment with AB and CPO also inhibited O-GlcNAc modification and OPG and O2 modification in CDX xenografts in pleural effusion. S151 -GlcNAc-OPG level ( Figure 27 C).

[0389] These results demonstrate that O-GlcNAc-modified OPG inhibitors treat tumors by inhibiting secreted proteins such as OPG and enhance the drug resistance of tumor cells.

[0390] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of O-GlcNAc-modified OPG inhibitors in the preparation of drugs that enhance the drug sensitivity of tumor cells, characterized in that, The amino acid sequence of the OPG is shown in SEQ ID NO.1, and the S151 site of the OPG is modified with O-GlcNAc.

2. The application according to claim 1, characterized in that, The drugs mentioned in the tumor cell drug sensitivity test include carboplatin and pemetrexed.

3. The application according to claim 1, characterized in that, The tumor cells include lung adenocarcinoma cells, lung adenocarcinoma cells with hyperglycemia and / or cytokine disorders, lung cancer cells, and human non-small cell lung cancer cells.

4. The application according to claim 1, characterized in that, The drug is a drug that inhibits the CDK2-Rb-E2F1 cell cycle axis.

5. The application according to claim 1, characterized in that, The drug is a drug that inhibits the expression of the membrane protein EHD1.

6. The application according to claim 1, characterized in that, The drug is a drug that inhibits the expression of RNF2 protein.

7. The application according to claim 1, characterized in that, The drug is a drug that inhibits the expression of OGT protein.

8. The application according to claim 8, characterized in that, The drug is one that binds to the Q839 site of the OGT protein.

9. The application according to claim 1, characterized in that, The inhibitor of O-GlcNAc modified OPG is an inhibitor that inhibits the regulation of O-GlcNAc-modified OPG.

10. The application according to claim 1, characterized in that, The inhibitors of O-GlcNAc modified OPG include celecoxib and OSMI-1.

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