Application of UGDH in diagnosis, treatment and prognosis prediction of renal cancer

Abstract

The application discloses application of UGDH in diagnosis, treatment and prognosis prediction of kidney cancer. The application finds that the expression amount of UGDH in kidney cancer tissues is significantly lower than that in normal tissues adjacent to the cancer, and therefore provides application of a reagent for detecting UGDH in preparation of a product for diagnosing kidney cancer and a kit for diagnosing kidney cancer. The application also finds that overexpression of UGDH can inhibit migration and invasion ability of kidney cancer cells, further research finds that UGDH can inhibit kidney cancer metastasis by regulating glycosylation level, and finds that a glycosylation enzyme OGT inhibitor OSMI-1 has an effect of inhibiting migration of kidney cancer cells, and therefore the application also provides application of a promoter of UGDH and OSMI-1 in preparation of a medicine for treating kidney cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically involving the application of UGDH in the diagnosis, treatment and prognosis prediction of renal cell carcinoma. Background Technology

[0002] Renal cell carcinoma (RCC) is a malignant tumor that originates from the renal tubular epithelium. It is a common and fatal disease of the urinary system.

[0003] Currently, the main treatment for localized and locally progressive RCC is surgery, primarily radical nephrectomy and nephron-sparing partial nephrectomy. However, approximately one-quarter of patients develop distant metastases post-surgery. Because early RCC often presents insidiously with no obvious symptoms, about one-third of RCC patients are diagnosed with distant metastases, i.e., advanced / metastatic RCC. Common distant metastases occur in the lungs, lymph nodes, liver, bones, and brain. Patients with metastatic RCC have lost the opportunity for curative surgery. These patients mainly receive systemic therapy, including targeted therapy and immunotherapy, but the treatment outcome for metastatic RCC is not ideal, with a 5-year survival rate of only about 10%. Therefore, elucidating the molecular mechanisms of RCC metastasis and identifying new potential molecular biomarkers and effective therapeutic targets are of great significance for improving the treatment efficacy and prognosis of patients with metastatic RCC. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a biomarker, UGDH, which can enable the diagnosis, treatment, and prognosis prediction of renal cell carcinoma.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of this invention provides any of the following applications:

[0007] 1) The use of UGDH promoters or pharmaceutical compositions containing UGDH promoters in the preparation of medicaments for treating renal cell carcinoma;

[0008] 2) Application of reagents for detecting UGDH in the preparation of products for diagnosing renal cell carcinoma / predicting the prognosis of renal cell carcinoma;

[0009] 3) Applications of UGDH in screening candidate drugs for the treatment of renal cell carcinoma, constructing systems / devices for diagnosing renal cell carcinoma / predicting renal cell carcinoma prognosis, or constructing computer-readable storage media for diagnosing renal cell carcinoma / predicting renal cell carcinoma prognosis;

[0010] 4) Use of UGDH promoters or pharmaceutical compositions containing UGDH promoters in the preparation of agents for the in vitro non-therapeutic purpose of inhibiting the migration and / or invasion of renal cell carcinoma cells;

[0011] 5) The use of OSMI-1 or its pharmaceutically acceptable salts in the preparation of drugs for the treatment of renal cell carcinoma;

[0012] 6) Use of OSMI-1 or its pharmaceutically acceptable salts in the preparation of reagents for the in vitro non-therapeutic purposes to inhibit the migration and / or invasion of renal cell carcinoma cells, or in the screening of drugs for the treatment of renal cell carcinoma.

[0013] Furthermore, the promoter specifically promotes the expression level of UGDH or increases its enzyme activity.

[0014] Furthermore, the expression level of UGDH includes both the mRNA level and the protein level of UGDH.

[0015] Furthermore, the promoter is an UGDH overexpression vector or UGDH protein.

[0016] Furthermore, the overexpression vector includes plasmids, lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors.

[0017] Through extensive and in-depth research, this invention has discovered significant differences in UGDH levels in renal cell carcinoma. Furthermore, it was found that the UGDH content in renal cell carcinoma tissue was significantly lower than that in normal renal cell carcinoma tissue and adjacent cancer tissue. Simultaneously, overexpression of UGDH can inhibit the proliferation, migration, and invasion of renal cell carcinoma cells, suggesting that UGDH could serve as a good biomarker for the diagnosis, treatment, and prognosis of renal cell carcinoma. Additionally, the OGT inhibitor OSMI-1 was found to inhibit the migration of renal cell carcinoma cells, suggesting that it holds promise as a safer and more effective drug for the treatment of renal cell carcinoma.

[0018] UGDH includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed UGDH, as well as any form of UGDH derived from cell-processed sources. The term encompasses naturally occurring variants of UGDH (e.g., splice variants or allelic variants). The term encompasses, for example, the UGDH gene, human UGDH, and UGDH from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in this invention, UGDH is the human gene, with gene ID 7358.

[0019] In this invention, treatment refers to the improvement, prevention, or reversal of a disease or condition or at least one identifiable symptom thereof. In some specific embodiments, treatment refers to the improvement, prevention, or reversal of at least one measurable physiological parameter associated with the disease or condition to be treated, which is not necessarily identifiable in or recognized by mammals. In some embodiments, treatment refers to the suppression or alleviation of a disease or its course, which may be physical, such as certain identifiable severe symptoms. The term "treatment" as used in this invention encompasses diseases in mammals, particularly humans, including: (a) preventing the onset of a disease or condition in individuals susceptible to the disease but not yet diagnosed with it; (b) suppressing a disease, such as halting its progression; or (c) alleviating a disease, such as reducing symptoms associated with the disease.

[0020] In this invention, the term "diagnosis" refers to a predictive process in which the presence, absence, severity, or treatment progress of a disease, ailment, or other medical condition is evaluated. For the purposes of this invention, diagnosis also includes predictive processes for determining treatment outcomes. Similarly, the term "diagnosis" refers to determining whether a treated individual exhibits one or more characteristics of a symptom or disease. The term "diagnosis" includes establishing the presence or absence of a target, such as a target antigen or binding agent, or establishing or otherwise determining one or more characteristics of a symptom or disease, including type, grade, stage, or similar conditions. The term "diagnosis" includes initial diagnosis or testing, prognosis, and monitoring of the symptom or disease. Additionally, the term "monitoring," for example, in "monitoring the progression of a disease or symptom," refers to ongoing diagnostic work on samples obtained from a treated individual who has or is suspected of having a disease or symptom.

[0021] The term "prognosis" and its derivatives refer to the determination or prediction of the course of a disease or condition. The course of a disease or condition can be determined, for example, based on life expectancy or quality of life. "Prognosis" includes determining the time course of a disease or condition with or without treatment. When treatment is considered, prognosis includes determining the effectiveness of treatment for the disease or condition.

[0022] In this invention, the term "promoter" is also called "agonist," referring to any substance or reagent that can enhance the activity of UGDH protein, enhance the stability of the UGDH gene or protein, promote the expression level of UGDH, or increase the effective duration of UGDH protein. As one embodiment of this invention, the "promoter" is a substance or reagent that promotes the expression level of UGDH.

[0023] In this invention, the term "expression level" refers to the amount, accumulation, or rate of a biomarker molecule or genome. Expression level can be expressed, for example, by the amount or rate of synthesis of messenger RNA (mRNA) encoded by a gene, the amount or rate of synthesis of a polypeptide or protein encoded by a gene, or the amount or rate of synthesis of a biomolecule accumulated in a cell or biological fluid. The term "expression level" also refers to the absolute amount or relative amount of a molecule in a sample, determined under steady-state or non-steady-state conditions.

[0024] In this invention, substances that enhance UGDH enzyme activity refer to chemical or biological molecules that can enhance the catalytic activity of UDP-glucose dehydrogenase (UGDH) through direct or indirect mechanisms. Specifically, they can improve the efficiency of the enzyme in converting UDP-glucose (UDP-Glc) into UDP-glucuronic acid (UDP-GlcUA), or indirectly promote its function by regulating its expression, modification, stability, and other aspects.

[0025] In this invention, OSMI-1 can be OSMI-1 itself, or OSMI-1 (hereinafter also referred to as a compound) hydrate, enantiomer, diastereomer, solvate, or crystalline form.

[0026] In this invention, the pharmaceutically acceptable salt refers to an acidic salt formed with inorganic and / or organic acids and a basic salt formed with inorganic and / or organic bases. Additionally, when the compound contains a basic moiety (e.g., but not limited to, pyridine or imidazole) and an acidic moiety (e.g., but not limited to, carboxylic acid), an amphoteric ion can be formed, and the amphoteric ion is contained in the pharmaceutically acceptable salt described in this application. Pharmaceutically acceptable (i.e., non-toxic and physiologically acceptable) salts are preferred, but other salts are also useful. The pharmaceutically acceptable salt of the compound can be formed, for example, by reacting the compound with a certain amount of acid or base in a medium, such as a medium in which salts precipitate or an aqueous medium (fly-dried after the reaction).

[0027] Specific pharmaceutically acceptable salts include those salts that, within the bounds of reliable medical judgment, are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and in proportion to a reasonable benefit or risk. Pharmaceutically acceptable salts (pharmaceutically acceptable salts) are well known in the art. Pharmaceutically acceptable salts of the compounds described in this application include salts derived from suitable inorganic and organic acids and inorganic and organic bases.

[0028] Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or salts formed with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid. Salts formed using methods conventional in the art are also included, such as ion exchange methods. Other pharmaceutically acceptable salts include: adipates, alginates, ascorbic acid salts, aspartate salts, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentylpropionate, diglucuronide, dodecyl sulfate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphates, hemisulfates, heptarates, hydroiodates, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonic acid, and mesylate. Pharmaceutically acceptable salts derived from suitable bases include alkali metals, alkaline earth metals, and ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts.

[0029] In this invention, hydrates refer to compounds that are combined with water.

[0030] In this invention, the solvate refers to a compound or its salt that is bound to a solvent and is typically formed by a solvent decomposition reaction. This physical association may include hydrogen bonding. Common solvents include methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, etc. In some cases, the solvate will be separable, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. Solvates include solvates in solution and separable solvates.

[0031] In this invention, the crystalline form refers to the crystalline form of a compound with a specific crystal packing arrangement. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, photoelectric properties, stability, and solubility. Recrystallization solvents, crystallization rates, storage temperatures, and other factors can lead to the dominance of one crystalline form. Various polymorphs of the compound can be prepared by crystallization under different conditions. In this invention, the crystalline form also includes special crystalline states, such as amorphous states.

[0032] It should be noted that the present invention does not impose any particular limitation on the specific dosage of OSMI-1. Any dosage that can produce corresponding therapeutic and / or preventive effects on subjects or other test subjects (such as in vitro cells, in vitro tissues, in vitro organoids, etc.) for renal cancer is within the protection scope of the present invention.

[0033] Furthermore, the reagent for detecting UGDH is selected from oligonucleotide probes that specifically recognize the UGDH gene, primers that specifically amplify the UGDH gene, or binding agents that specifically bind to the protein encoded by the UGDH gene.

[0034] The term "probe" refers to a molecule that can bind to a specific sequence, subsequence, or other portion of another molecule. Unless otherwise specified, the term "probe" generally refers to a polynucleotide probe that can bind to another polynucleotide (often called a "target polynucleotide") through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. Probes can be labeled directly or indirectly. Hybridization methods include, but are not limited to, solution-phase, solid-phase, mixed-phase, or in situ hybridization assays.

[0035] The term "amplification primer" or "primer" refers to an oligonucleotide that can specifically anneal to a site on an adjacent target RNA or DNA sequence and serve as an initiating primer for DNA synthesis under suitable conditions, under which the synthesis of primer extension products is induced, for example, in the presence of nucleotides and polymerization inducers such as DNA-dependent DNA polymerases, as well as suitable temperature, pH, metal concentration, and salt concentration. Typically, a PCR reaction uses a pair of amplification primers, also known as a "primer pair," consisting of an "upstream" or "forward" primer and a "downstream" or "reverse" primer, which define the region of RNA or DNA to be amplified.

[0036] The term "amplification" refers to a method of replicating a portion of a nucleic acid by applying any of various primer extension reactions, such as PCR. Exemplary primer extension reactions include, but are not limited to, PCR. Unless otherwise stated, "amplification" refers to single replication, or arithmetic, logarithmic, or exponential amplification.

[0037] The term "binding agent" refers to all or part of a protein molecule (protein, protein-like, or protein-containing) that is capable of binding to a membrane protein using specific intermolecular interactions. Protein binding agents are, for example, protein receptors, protein-binding lectins, proteins-specific antibodies, protein-specific peptide bodies, bispecific dual binders, or bispecific antibody forms. More specifically, the term "binding agent" refers to polypeptides, and more specifically, protein domains. A suitable protein domain is an element of the overall protein structure; it is self-stabilizing and folds independently of the rest of the protein chain and is often referred to as a "binding domain." The length of such binding domains varies from about 25 amino acids to 500 amino acids and more. Many binding domains can be classified as folds and are recognizable, identifiable, 3-D structures. Some folds are so common in many different proteins that they have been given specific names.

[0038] Furthermore, OSMI-1 significantly inhibits the migration and invasion abilities of renal cell carcinoma cells.

[0039] Furthermore, the dosage form of the drug includes oral dosage form, parenteral dosage form and / or topical dosage form.

[0040] Furthermore, the dosage forms of the pharmaceutical preparations include solutions, sustained-release formulations, suspensions, granules, tablets, capsules, powders, effervescent tablets, emulsions, syrups, drops, and / or chewable tablets.

[0041] Furthermore, the administration methods of the drug include oral, subcutaneous, intravenous, intramuscular, intra-arterial, intranasal, intrathecal, mucosal, intrapulmonary, and / or rectal administration.

[0042] A second aspect of the present invention provides a product for diagnosing renal cell carcinoma / predicting the prognosis of renal cell carcinoma.

[0043] Furthermore, the product includes reagents capable of detecting UGDH expression levels.

[0044] Furthermore, the reagent is selected from oligonucleotide probes that specifically recognize the UGDH gene, primers that specifically amplify the UGDH gene, or binding agents that specifically bind to the protein encoded by the UGDH gene.

[0045] Furthermore, the products include chips, reagent kits, or nucleic acid membrane strips.

[0046] The term "chip," also known as an "array," refers to a solid support containing linked nucleic acid or peptide probes. Arrays typically contain a variety of different nucleic acid or peptide probes attached to a substrate surface at various known locations. These arrays, also called "microarrays," can generally be produced using mechanosynthesis or photoguided synthesis, which combines photolithography and solid-phase synthesis methods. Arrays can comprise flat surfaces or can be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. Arrays can be packaged in a manner that allows for diagnostic or other manipulation of fully functional devices.

[0047] The term "microarray" refers to a hybridization array element arranged in an ordered manner on a matrix, such as a polynucleotide probe (e.g., an oligonucleotide) or a binder (e.g., an antibody). The matrix can be a solid matrix, such as a glass or silica slide, beads, fiber optic adhesive, or a semi-solid matrix, such as a nitrocellulose membrane. The nucleotide sequence can be DNA, RNA, or any arrangement thereof.

[0048] The term "kit" refers to any delivery system used to deliver materials, including kits used for research and clinical applications.

[0049] The kit described in this invention comprises reagents for detecting the UGDH gene or protein, and one or more substances selected from the group consisting of: containers, instructions for use, positive controls, negative controls, buffers, auxiliaries, or solvents. The components of the kit may be packaged in an aqueous medium or in lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container in which one component can be placed, and preferably, appropriately aliquoted. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container in which the additional components are placed separately. However, different combinations of components may be contained in a single vial. The kit of this invention will also typically include a container for containing the reactants, sealed for commercial sale. Such a container may include injection-molded or blow-molded plastic containers in which the desired vials can be held.

[0050] The kits described in this invention include, but are not limited to, qPCR kits, ELISA kits, Western blot detection kits, immunochromatographic detection kits, immunohistochemical detection kits, flow cytometry analysis kits, and electrochemiluminescence detection kits.

[0051] The term "nucleic acid membrane strip" includes a substrate and an oligonucleotide probe immobilized on the substrate; the substrate can be any substrate suitable for immobilizing oligonucleotide probes, such as nylon membrane, nitrocellulose membrane, polypropylene membrane, glass slide, silicone wafer, micro-magnetic beads, etc.

[0052] A third aspect of the present invention provides a pharmaceutical composition for treating kidney cancer.

[0053] Furthermore, the pharmaceutical composition includes an UGDH promoter and / or OSMI-1 or a pharmaceutically acceptable salt thereof.

[0054] Furthermore, the pharmaceutical composition also includes a second therapeutic agent.

[0055] Furthermore, the second therapeutic agent is another drug used to treat kidney cancer.

[0056] Furthermore, the other drugs used to treat renal cell carcinoma include sunitinib, pazopanib, cabozantinib, lenvatinib, bevacizumab, tesiromoximus, and / or everolimus.

[0057] Furthermore, the promoter specifically promotes the expression level of UGDH or increases its enzyme activity.

[0058] Furthermore, the expression level of UGDH includes both the mRNA level and the protein level of UGDH.

[0059] Furthermore, the promoter is an UGDH overexpression vector or UGDH protein.

[0060] Furthermore, the promoter inhibits the proliferation, migration, and / or invasion of renal cell carcinoma cells.

[0061] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0062] The fourth aspect of the present invention provides any of the following methods:

[0063] 1) A method for screening candidate drugs for treating renal cell carcinoma, the method comprising: treating a culture system expressing or containing the UGDH gene or its encoded protein with a substance to be screened; and detecting the expression or activity of the UGDH gene or its encoded protein in the system; wherein, when the substance to be screened promotes the expression level or activity of the UGDH gene or its encoded protein, the substance to be screened is a candidate drug for treating renal cell carcinoma.

[0064] 2) A method for inhibiting the proliferation, migration, invasion and / or promoting apoptosis of renal cell carcinoma cells in vitro for non-therapeutic purposes, the method comprising the steps of treating renal cell carcinoma cells with an UGDH promoter and / or OSMI-1 or a pharmaceutically acceptable salt thereof;

[0065] Furthermore, the culture system containing the UGDH gene or its encoded protein can be a cell system, wherein the cells can be cells that endogenously express UGDH; or cells that recombinantly express UGDH. The culture system containing the UGDH gene or its encoded protein can also be (but is not limited to) a subcellular system, a solution system, a tissue system, an organ system, or an animal system (such as an animal model).

[0066] As a preferred embodiment of the present invention, the method further includes: conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and identify substances that are truly useful for the treatment of renal cell carcinoma.

[0067] This invention does not impose any particular limitations on the detection methods for UGDH expression and activity. Conventional protein quantification or semi-quantitative detection techniques can be used, such as (but not limited to): immunoprecipitation, SDS-PAGE, Western blotting, ELISA, etc.

[0068] Furthermore, the renal cancer cells include OSRC-2 cells, 786-O cells, 769-P cells, Caki-1 cells, Caki-2 cells, ACHN cells, and A498 cells.

[0069] Furthermore, the renal cell carcinoma cells are selected from Caki-1 cells or OSRC-2 cells.

[0070] A fifth aspect of the present invention provides a system / device for diagnosing renal cell carcinoma / predicting the prognosis of renal cell carcinoma, the system / device comprising:

[0071] Acquisition unit: used to acquire the expression level of UGDH in the sample;

[0072] Processing unit: Based on the expression of UGDH, obtain the diagnostic / prognostic prediction results of renal cell carcinoma.

[0073] If the expression level of UGDH is significantly downregulated compared to normal samples, the diagnosis is renal cell carcinoma.

[0074] A sixth aspect of the present invention provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the system / apparatus described in the fifth aspect of the present invention.

[0075] Advantages and beneficial effects of the present invention:

[0076] The molecular marker UGDH provided by this invention can effectively diagnose renal cell carcinoma. UGDH promoters can achieve the purpose of treating renal cell carcinoma by inhibiting the growth, proliferation and / or migration of renal cell carcinoma cells. In addition, the OGT inhibitor OSMI-1 also has the effect of inhibiting the migration of renal cell carcinoma cells. This invention provides a new direction for the research and development of drugs for the treatment of renal cell carcinoma. Attached Figure Description

[0077] Figure 1 This is a sequencing result image of a mouse model of orthotopic metastasis of renal cell carcinoma; among which, Figure 1 A in the diagram represents a mouse renal cell carcinoma orthotopic metastasis model and a schematic diagram of tissue sampling. Figure 1 B in the image represents an in vivo imaging image of a mouse undergoing in situ transfer. Figure 1 C in the image represents the fluorescence imaging of the primary renal cell carcinoma lesion and the lung metastases after dissection. Figure 1 D in the image represents HE staining, showing the primary lesion (left) and lung metastases (right) in a mouse renal cell carcinoma metastasis model. Figure 1 E in the image represents a heatmap of differentially expressed proteins from proteomics screening of primary renal cell carcinoma lesions and lung metastases. Figure 1 F in the figure represents the volcano plot of differentially expressed proteins from proteomics screening of primary renal cell carcinoma lesions and lung metastases;

[0078] Figure 2 This image shows the expression results of UGDH in cancerous and adjacent tissues of renal cell carcinoma patients; among them, Figure 2 A in Figure 2 In the figure, B represents the immunohistochemical staining results and their statistical graph; Figure 2 C in the figure represents the AUC result (the cancer and adjacent normal tissues were generated by IRS scoring of 15 pairs of immunohistochemical staining results).

[0079] Figure 3The effect of UGDH on the renal cell carcinoma line Caki-1; among which, Figure 3 Figures A and B in the diagram represent the verification results of CaKi-1 cell knockdown of UGDH. Figure 3 Figure C in the figure shows the results of cell migration and invasion experiments with knocked-down UGDH. Figure 3 Figures D and E in the figure represent the verification results of UGDH overexpression in CaKi-1 cells. Figure 3 F in the figure represents the results of cell migration and invasion experiments with overexpression of UGDH;

[0080] Figure 4 The effect of UGDH on the renal cell carcinoma line OSRC-2; among which, Figure 4 Figures A and B in the figure show the verification results of OSRC-2 cell knockdown of UGDH; Figure 4 Figure C in the figure shows the results of cell migration and invasion experiments with knocked-down UGDH. Figure 4 Figures D and E in the figure represent the validation results of OSRC-2 cells overexpressing UGDH. Figure 4 F in the figure represents the results of cell migration and invasion experiments with overexpression of UGDH;

[0081] Figure 5 The figure shows the effect of knocking down UGDH on the tail vein lung metastasis model; among them, Figure 5 In the image, A represents an in vivo imaging image of a mouse model with tail vein transfer. Figure 5 In the figure, B represents the statistical analysis of in vivo imaging images of mice with tail vein transfer model; Figure 5 C in the image represents an in vivo imaging image of lung metastases after dissection of a mouse model with tail vein transfer. Figure 5 In the figure, D represents the statistical analysis of in vivo imaging images of lung metastases after dissection of mice with tail vein transfer model. Figure 5 E in the figure represents the statistical analysis of the number of lung metastases after dissection of mice with tail vein transfer model; Figure 5 F in the figure represents the statistical analysis of the extent of lung metastases after dissection of mice with tail vein transfer model.

[0082] Figure 6 To investigate the effect of UGDH overexpression on a tail vein lung metastasis model; among which, Figure 6 In the image, A represents an in vivo imaging image of a tail vein transfer model mouse overexpressing UGDH; Figure 6 B in the figure is a statistical graph of in vivo imaging of lung metastases in mice with tail vein metastasis model overexpressing UGDH after dissection; Figure 6 C in the figure represents the statistical analysis of the number of lung metastases after dissection in mice with a tail vein metastasis model that overexpresses UGDH; Figure 6 D in the figure represents the statistical analysis of the extent of lung metastases after dissection in mice with a tail vein metastasis model that overexpresses UGDH. Figure 6 E in the image represents an in vivo imaging image of lung metastases in a tail vein metastasis model mouse overexpressing UGDH after dissection. Figure 6 F in the figure represents the statistical analysis of in vivo imaging images of lung metastases in mice with tail vein metastasis model overexpressing UGDH after dissection;

[0083] Figure 7 The impact of OSMI-1 on renal cell carcinoma; among which, Figure 7 In the figure, A represents the successful construction of a mouse tail vein metastatic tumor model; Figure 7 Figure B in the figure represents the control group and mice treated with OSMI-1 via tail vein injection for 2 weeks. Figure 7 In the figure, C represents the anatomical diagram of lung metastases in the control group and 2 weeks after OSMI-1 administration in mice via tail vein injection. Detailed Implementation

[0084] The present invention is further illustrated by the following embodiments, which should not be construed as limiting. The embodiments are merely illustrative and are not intended to limit any aspect described herein. The following embodiments do not limit the invention in any way.

[0085] Unless otherwise specified, the experimental methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0086] The experimental materials and methods used in the following embodiments are described below:

[0087] 1. Selection of cell lines:

[0088] The cell lines required for this study included: human embryonic kidney cells HEK-293T (293T for short), human renal cortical proximal tubule epithelial cells HK2, human renal cell carcinoma OSRC-2, human renal cell carcinoma 786-O, human renal cell carcinoma 769-P, human renal cell carcinoma Caki-1, human renal cell carcinoma Caki-2, human renal cell carcinoma ACHN, and human renal cell carcinoma A498. These RCC cell lines were purchased from the American Type Culture Collection (ATCC). Subsequent experimental studies were conducted after short tandem repeat (STR) identification to confirm the accuracy of the cell lines.

[0089] 2. Selection of laboratory animals:

[0090] The animal experiments conducted in this study were approved by the Laboratory Animal Ethics Committee of Peking University First Hospital. All experimental procedures adhered to the principles of laboratory animal welfare and were supervised by the Laboratory Animal Ethics Committee and the animal laboratory staff. The experimental animals used in this study included two types: (1) 6-week-old male B-NDG mice (purchased from Beijing Biocytogen Biotechnology Co., Ltd.); and (2) 6-week-old BALB / c-nude mice (purchased from Vital Rivers). Both types of mice were purchased from the relevant companies by the animal facility of Peking University First Hospital after the researchers submitted their applications and were housed in a specific pathogen-free (SPF) enclosure.

[0091] 3. Cell culture:

[0092] Cell resuscitation: First, wipe the biosafety cabinet with 75% alcohol and place sterile culture dishes, centrifuge tubes, pipettes, etc. on it. Sterilize the clean bench with UV light for 30 minutes. Prepare a complete culture medium: 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin + basal medium. Mix well and preheat in a 37°C incubator. Preheat the water bath to 37°C. Remove the cells from the liquid nitrogen tank and quickly thaw them in the water bath, shaking constantly to accelerate thawing until only a small piece of ice remains. Stop thawing, spray with alcohol, and transfer to the biosafety cabinet. Wipe the cryovials with alcohol swabs, carefully unscrew the caps, add an appropriate amount of preheated culture medium, mix well, and transfer to centrifuge tubes. Rinse the cryovials with a separate amount of culture medium and transfer the washings to centrifuge tubes. Centrifuge at 700 rpm for 3 minutes at room temperature. Discard the supernatant, add 2 ml of preheated culture medium, gently mix by pipetting, and count the cells. According to the inoculation density and cell state, inoculate an appropriate amount of cells into a culture dish, add an appropriate amount of culture medium, gently shake to mix, and then place in a cell culture incubator at 37°C, 5% carbon dioxide and 90% humidity for culture.

[0093] Cell passage: Observe cell status under a microscope daily, including cell density, morphology, and culture medium color. Replace the culture medium with fresh medium every 1-2 days depending on cell condition. When changing the medium, discard the old medium, add an appropriate amount of sterile phosphate buffered saline (PBS), gently shake the culture dish to wash away residual old medium and cell debris, discard the washing solution, and aspirate any remaining liquid with a wash head. Repeat the above steps twice, then add an appropriate amount of fresh culture medium to the culture dish. Use McCoy's 5A medium to culture Caki-1 and Caki-2 cells, PRMI 1640 medium to culture 786-O cells, and DMEM medium to culture other RCC cell lines and 293T cells. Wipe the outer surface of the culture dish clean with an alcohol swab and return the dish to the cell culture incubator for continued culture. Depending on cell condition and experimental needs, cell passage is generally performed when the cell density reaches 80%-90%. First, discard the old culture medium and aspirate it with a pipette. Add preheated sterile PBS along the wall of the culture dish, gently agitate the dish to wash the cells, discard the washing solution, and aspirate any remaining liquid with a pipette. Repeat this washing process twice. Add an appropriate amount of trypsin (enough to cover the bottom of the culture dish), place the dish in a cell culture incubator to accelerate cell digestion and detach the cells from the culture dish wall. After about 2 minutes, observe under a microscope that the cells appear as scattered sheets that can be suspended and float with the liquid waves. Add sufficient complete culture medium (generally more than twice the volume of trypsin) to stop digestion. Carefully and repeatedly pipette the bottom of the dish to detach the cells from the culture dish wall. Transfer the digestion solution to a centrifuge tube, add an appropriate amount of culture medium to rinse the culture dish, and transfer the washing solution to the centrifuge tube as well. Centrifuge the centrifuge tube at room temperature and 700 rpm for 3 minutes. Discard the supernatant and gently tap the bottom of the centrifuge tube to disperse the cells. Add 1-2 ml of culture medium to resuspend the cells and repeatedly pipette to mix thoroughly. Add 20 μL of cell suspension to the wells of a cell counting chamber and count the cells using a cell counter. According to the experimental requirements, inoculate an appropriate amount of cells into a new culture dish and add an appropriate amount of culture medium. Gently shake the culture dish using the "cross" mixing method to mix the cells. Use a marker to clearly mark the cell type, treatment conditions, and date of the operation on the culture dish, and then place it in a cell culture incubator for culture.

[0094] Cell cryopreservation: Cells exhibiting good morphology and in the logarithmic growth phase under a microscope can be cryopreserved for propagation. After discarding the old culture medium, wash the cells twice with pre-warmed PBS and digest them with trypsin. After thorough digestion, add an appropriate amount of complete culture medium to stop the digestion, and transfer the digestion solution to a centrifuge tube. Centrifuge at 700 rpm for 3 minutes at room temperature, and discard the supernatant. Resuspend the cell pellet in the centrifuge tube with cell cryopreservation solution, carefully pipette to mix, and transfer to cryopreservation tubes. Tighten the caps and clearly label the cell line type, treatment conditions, passage number, cryopreservation time, and other important information. Gradually cool the cells using a programmed cooling box and finally transfer them to a liquid nitrogen tank for long-term storage.

[0095] 4. Plasmid transformation and extraction:

[0096] Plasmid transformation:

[0097] Place 100 μL of DH5α competent cells in an ice bath, add 1 ng of plasmid DNA to the competent cells, gently tap the tube wall to mix, and incubate on ice for 30 min. Preheat a water bath to 42°C, transfer the ice-bathed centrifuge tube to the water bath, and incubate for 2 min. Prepare sterile LB and LA media in advance. The preparation method for LB and LA media is as follows: weigh tryptone, yeast extract, and sodium chloride according to the formula, place them in a clean beaker, add 2 / 3 volume of double-distilled water to the beaker, and dissolve them completely with a glass rod or gel stick. Adjust the pH to 7.2 using 1 mol / L sodium hydroxide (NaOH) solution, then make up the volume, add double-distilled water to the required volume, stir well, and dispense. Affix the autoclave indicator strip, autoclave at 100 Pa for 20 min, and store at 4°C. Take an appropriate amount of LB media, ampicillin, and LB media and mix them at a ratio of 1:1000 to obtain LA media. In a clean bench, add 600 μL of LB medium to each centrifuge tube and shake at 200 rpm for 1 hour at 37°C to revive the bacteria and induce expression of the resistance gene in the plasmid. Separately, prepare 15 ml centrifuge tubes and add 10 ml of LA medium to each tube. Add the LB medium containing the plasmid to the LA medium and shake at 200 rpm for approximately 16 hours at 37°C.

[0098] Plasmid Extraction: We synthesized silencing and overexpression plasmids at Syngene Biotechnology Co., Ltd. The vector information for the silencing plasmid is pLV-hU6-neg / shRNAs-hEF1a-EGFP-2A-Puro / Neo, and the vector information for the overexpression plasmid is pLV-hef1a-mNeongreen-P2A-Puro / Neo-WPRE-CMV-3Xflag. We used a rapid plasmid miniprep kit to extract the plasmids. This kit contains the following components: solution P1, solution P2, solution P5, wash buffer PWT, elution buffer TB, RNase A, TIANRed, adsorption column CP3, and collection tube (2 ml). 2 ml of bacterial culture (previously incubated for approximately 16 h) was added to a 15 ml centrifuge tube and centrifuged at 12000 rpm for 1 min. The supernatant was discarded. 150 μL of solution P1 (containing RNase A and TIANRed) was added to the centrifuge tube containing the bacterial pellet, and the bacterial pellet was resuspended by pipetting. Add 150 μL of solution P2 to the centrifuge tube and gently invert and mix 10 times to completely lyse the bacteria. Add 350 μL of solution P5 to the centrifuge tube and immediately invert and mix 20 times. Then centrifuge at 12000 rpm for 2 min. Transfer the supernatant to the adsorption column CP3 and centrifuge at 12000 rpm for 30 sec. Discard the waste liquid in the collection tube and put the adsorption column CP3 back into the collection tube. Add 300 μL of wash buffer PWT (with anhydrous ethanol added) and centrifuge at 12000 rpm for 1 min. Discard the waste liquid in the collection tube and put the adsorption column CP3 back into the collection tube. Centrifuge again at 12000 rpm for 1 min to completely remove the residual wash buffer from the adsorption column. Transfer the CP3 adsorption column to a clean centrifuge tube. Add 100 μL of elution buffer TB to the center of the adsorption membrane. Centrifuge at 12000 rpm for 1 min. Add the liquid from the centrifuge tube back to the center of the adsorption membrane. Repeat the above steps to obtain the plasmid solution. Measure the concentration of the plasmid solution using a NanoDrop micro-UV spectrophotometer. Label the centrifuge tube wall with the plasmid name, concentration, and operation date. Aliquot and store at -20°C for later use.

[0099] 5. Lentiviral packaging and infection:

[0100] Lentiviral Packaging: We used Lipofectamine 3000 transfection reagent for lentiviral packaging. First, 293T cells were cultured and conditioned to a good condition, with a cell density of approximately 60%-70%. In centrifuge tube A, 250 μL of serum-free Opti-MEM Medium and 7.5 μL of Lipo 3000 reagent were added; in centrifuge tube B, 250 μL of serum-free medium, 10 μL of P3000 reagent, and 5 μg of mixed plasmid DNA (target plasmid: psPAX2:pMD2.G = 2:1:1) were added. Centrifuge tubes A and B were mixed gently by pipetting and incubated at room temperature for 15 min. The mixture was then added to a culture dish containing 293T cells, and an appropriate amount of complete medium was added. The dish was then incubated in a cell culture incubator. After 12 hours of packaging, the culture medium could be changed (without washing the cells) depending on the cell condition. After 48 hours of packaging, the cell culture medium was transferred to a 15 ml centrifuge tube, centrifuged at 1000 rpm for 5 min, and the supernatant was collected. The supernatant virus liquid was filtered through a 0.22 μm filter, aliquoted, and stored at -80℃.

[0101] Lentiviral infection: Culture the cell lines to be infected in six-well plates and adjust them to a good condition with a density of approximately 30%-50%, depending on the cell proliferation rate. Remove the virus from a -80°C freezer and thaw it in a water bath. Add 100 μL of virus, 900 μL of complete culture medium, and 2 μL of Polybrene infection stimulant to each well, gently shake to mix, and then incubate in a cell culture incubator. Set up a blank control group (containing cells and infection stimulant under the same conditions, but without virus) as a control group for drug screening. After 48 days of infection, begin drug screening with puromycin. The initial concentration can be 1-2 μg / ml, and the screening concentration can be adjusted as needed. Once all cells in the blank control group have died, puromycin-resistant surviving cells can be obtained. Subsequently, cellular RNA and proteins can be extracted to verify the infection efficiency as needed.

[0102] 6. Real-time quantitative PCR:

[0103] RNA Extraction: We used the TRIzol method to extract RNA from cell and tissue samples. For adherent cells, the cell culture medium was discarded, and the cells were washed twice with 1X PBS solution, with residual liquid aspirated. An appropriate amount of TRIzol reagent was added to the cell culture dish to cover the cell surface, and the cells were completely detached by pipetting. The mixture was incubated at room temperature for 5 min. For tissue samples, the tissue blocks were first minced, then thoroughly pulverized in a tissue homogenizer. TRIzol reagent was added, and the mixture was vortexed to achieve complete lysis. The mixture was incubated at room temperature for 10 min. 0.2 ml of chloroform was added per ml of TRIzol reagent, and the mixture was rapidly shaken to mix. The mixture was then incubated at room temperature for 3 min. Centrifugation was performed at 4°C and 12000 rpm for 15 min. At this point, the mixture in the tube separated into three layers, with RNA in the upper aqueous phase. Transfer the upper aqueous phase to a new EP tube, yielding approximately 600 μL of aqueous phase per mL of TRIzol reagent. Add isopropanol at a 1:1 ratio, mix thoroughly by inverting, and incubate at room temperature for 10 min. Centrifuge at 4°C and 12,000 rpm for 15 min. Discard the supernatant and aspirate any remaining liquid; a white flocculent RNA precipitate will appear at the bottom of the tube. Add 1 mL of 75% ethanol per mL of TRIzol reagent to wash the RNA precipitate, mix thoroughly by pipetting, and centrifuge at 4°C and 12,000 rpm for 5 min. Repeat the washing once. Air-dry the RNA precipitate for 15 min to completely remove ethanol until the RNA precipitate at the bottom of the tube becomes clear. Add 50 μL of RNase-free water, mix thoroughly by pipetting, and then determine the sample concentration and purity.

[0104] Reverse transcription: RNA was reverse transcribed using the FastKing RT Kit (With gDNase) FastKing cDNA Reverse Transcription Kit. Take an appropriate amount of template RNA as needed and thaw it on ice. Thaw 5X gDNA Buffer, FQ-RT PrimerMix, 10X King RT Buffer, and RNase-Free ddH2O at room temperature. After thawing, place on ice until ready for use. Before use, vortex each solution to mix thoroughly and briefly centrifuge to collect the liquid from the tube wall.

[0105] 7. Western blot analysis of proteins:

[0106] Protein Sample Preparation: Total protein was extracted from cell and tissue samples. For adherent cells, the old culture medium was discarded, the cells were washed twice with PBS, residual liquid was aspirated, an appropriate amount of PBS was added, cells were scraped off using a cell scraper, and transferred to centrifuge tubes. The cells were centrifuged at 4°C and 5000 rpm for 5 min, and the supernatant was discarded to obtain a cell pellet. For tissue samples, the tissue was first minced as finely as possible, and after adding an appropriate amount of PBS, it was thoroughly pulverized in a tissue homogenizer. The pellet was then centrifuged at 4°C and 5000 rpm for 5 min, and the supernatant was discarded. An appropriate amount of protein lysis buffer and protease inhibitor were added, and the mixture was pipetted and lysed in a 4°C rotary mixer for 30 min. Subsequently, the pellet was centrifuged at 4°C and 12000 rpm for 25 min, and the supernatant was transferred to a new EP tube for later use.

[0107] Protein Concentration Determination: We used the BCA protein quantification kit to determine protein concentration. One ampoule of bovine serum albumin (BSA) standard was diluted with double-distilled water. According to experimental requirements, the BCA working solution was prepared at a ratio of Reagent A:Reagent B = 50:1 and thoroughly mixed by pipetting. 200 μL of the working solution was added to each well of a 96-well plate, followed by 10 μL of standard or sample to be tested in each well. The mixture was gently mixed and incubated at 37°C for 30 min. After cooling to room temperature, the absorbance was measured at 562 nm using an automated microplate reader. A standard curve was plotted based on the standard, and the concentration formula was obtained. The concentration of each protein sample was then calculated. An appropriate amount of 5X SDS Loading Buffer was used to balance the protein sample concentrations using double-distilled water, according to the concentration of each protein sample. The protein sample was denatured by heating it in a 98℃ metal bath for 15 min. After it cooled to room temperature naturally, it was briefly centrifuged and then aliquoted and stored at -20℃ for later use.

[0108] Gel preparation: Wash and dry the glass plate used for preparing the gel. Secure the glass plate with a gel holder, ensuring the bottom is tightly fitted to the silicone pad. Prepare the separating gel according to experimental requirements, mix thoroughly, add an appropriate amount of TEMED, mix well, pour the gel, and press the liquid surface with isopropanol. Let it stand at room temperature for about 30 minutes. After the separating gel has fully solidified, discard the isopropanol and rinse three times with double-distilled water. Prepare the stacking gel according to experimental requirements, add an appropriate amount of TEMED, mix thoroughly, pour it on top of the separating gel, insert the sample comb, check for air bubbles, and let it stand at room temperature for 60 minutes.

[0109] SDS-PAGE gel electrophoresis: Clamp the prepared gel in the electrophoresis core, place it in the electrophoresis tank, and add electrophoresis buffer. Carefully and vertically remove the sample comb, and use the sample pipette tip to add the prepared protein samples to each lane in sequence. Add an equal volume of protein marker to each lane, and fill the blank lane with 1X SDS loading buffer. Cover the electrophoresis tank and turn on the power. Perform electrophoresis at a constant voltage of 80 V for the stacking gel and 120 V for the separating gel until the bromophenol blue dye reaches the bottom of the gel. Turn off the power to stop the electrophoresis.

[0110] Wet transfer: Cut a PVDF membrane to an appropriate size and activate it in methanol for 3 min. Place the activated membrane on the gel to construct a "transfer sandwich" structure, i.e., from negative to positive electrode: sponge, filter paper, gel, PVDF membrane, filter paper, sponge. Cover the electrophoresis tank and turn on the power. Electrophoresis at a constant voltage of 120 V for 90 min. The transfer time can be adjusted according to the molecular weight. Ice can be placed around the electrophoresis tank for cooling.

[0111] Antibody incubation: Prepare a 5% blocking buffer using TBST buffer and skim milk powder, ensuring complete dissolution. After successful transfer, block the membrane in the antibody incubation chamber using the blocking buffer, ensuring the membrane is completely immersed. Incubate the chamber on a shaker with low-speed shaking at room temperature for 1 hour. After blocking, wash the membrane with TBST buffer at 80 rpm for 5 minutes each time, changing the TBST buffer and repeating the wash three times. Dilute the primary antibody to the appropriate concentration according to experimental requirements, immerse the membrane in the primary antibody solution, and incubate overnight at 4°C. The next day, recover the primary antibody and wash the membrane with TBST buffer at 80 rpm for 5 minutes each time, changing the TBST buffer and repeating the wash three times. Then, immerse the membrane in the secondary antibody solution and incubate at room temperature for 1 hour. Next, wash the membrane with TBST buffer at 80 rpm for 5 minutes each time, changing the TBST buffer and repeating the wash three times.

[0112] Exposure and color development: Prepare the exposure working solution by mixing reagents A and B of the ECL luminescent solution in equal proportions, and avoid light exposure. Cover the membrane with the working solution and incubate for 1-2 minutes. Perform exposure and color development using an automated gel imaging system, take pictures, save the images, and perform analysis.

[0113] 8. IHC analysis:

[0114] IHC staining: Under the guidance of a urologist, embedded paraffin samples were cut into 3-5 μm sections. After baking in a 70°C oven for 2 hours, dewaxing and hydration were performed, followed by soaking in the following order: xylene I (10 min), xylene II (10 min), anhydrous ethanol I (10 min), anhydrous ethanol II (10 min), 95% ethanol (5 min), 85% ethanol (5 min), and 75% ethanol (5 min). Subsequently, the samples were washed with PBS buffer at 80 rpm / min for 5 min each time on a shaker, with the PBS buffer changed three times. Antigen retrieval was performed using antigen retrieval solution under autoclaving at 110°C for 10 min, followed by natural cooling to room temperature. The samples were then washed three times with PBS buffer on a shaker for 5 min each time. The tissue blocks were then circled with a histochemical pen, and an appropriate amount of endogenous peroxidase inhibitor was added. The samples were incubated at room temperature for 20 min. After inhibition, the samples were washed three times with PBS buffer on a shaker for 5 min each time. Add an appropriate amount of sheep serum to each slide for blocking and incubate at room temperature for 1 hour in a humidified histochemistry chamber. After incubation, discard the serum, add primary antibody as needed, and incubate overnight at 4°C. The next day, remove the slides from the cold storage, allow them to warm to room temperature naturally, and wash them three times with PBS buffer on a shaker for 5 minutes each time. Add an appropriate amount of secondary antibody to each slide and incubate at room temperature for 40 minutes. After incubation, wash three times with PBS buffer on a shaker for 5 minutes each time. Dilute 20X DAB concentrate to 1X to prepare DAB working solution, add an appropriate amount of DAB working solution to the slides, and incubate at room temperature for 5-8 minutes. After sufficient color development, immerse the slides in PBS buffer to stop the color development. Stain the slides in hematoxylin solution for 2-3 minutes, differentiate with 0.1% hydrochloric acid alcohol for 1-3 seconds, and then place them in tap water for "blue re-enhancing" for 30 minutes. The slides were then dehydrated and cleared, and immersed in the following solutions in the following order: 75% ethanol (5 min), 85% ethanol (5 min), 95% ethanol (5 min), anhydrous ethanol II (10 min), anhydrous ethanol I (10 min), xylene II (10 min), and xylene I (10 min). The slides were then mounted with neutral resin. A suitable amount of neutral resin was placed on the slide, covered with a coverslip, and allowed to air dry in a ventilated area. The slides were then examined and analyzed under a microscope.

[0115] IHC scoring: Two experienced pathologists independently score the staining, using criteria including staining intensity and staining extent. Staining intensity score: No staining = 0 points, weak staining = 1 point, moderate staining = 2 points, strong staining = 3 points. Staining extent score: No positive staining = 0 points, 1%-25% = 2 points, 26%-50% = 2 points, 51%-100% = 3 points. The final IHC score is obtained by multiplying the staining intensity score and the staining extent score, ranging from 0 to 9 points.

[0116] 9. Immunofluorescence:

[0117] Remove the culture dish, discard the old culture medium, and aspirate it with an aspirator. Add an appropriate amount of room temperature PBS and wash on a shaker for 5 min, repeating the washing process 3 times. After aspirating the PBS, add an appropriate amount of 4% paraformaldehyde to fix the cells, ensuring the fixative covers the cells. Fix on a shaker at room temperature for 20 min. Discard the paraformaldehyde and wash 3 times with pre-chilled PBS at 4°C, following the same method. After discarding the PBS, block the cells, add an appropriate amount of 1% BSA, and incubate on a shaker at room temperature for 1 h. After discarding the blocking solution, wash 3 times with pre-chilled PBS at 4°C. Except for membrane antigens, permeabilize the cells using Triton X-100. Add an appropriate concentration of primary antibody solution and incubate overnight at 4°C. After discarding the primary antibody solution, wash 3 times with pre-chilled PBS at 4°C. Add secondary antibody solution and incubate at room temperature in the dark for 1 h. After incubation, aspirate the secondary antibody solution and wash 3 times with PBS. Add DAPI and incubate at room temperature for 10 min, followed by 3 washes with PBS. Mount the slide with anti-quenching mounting medium and observe it under a fluorescence microscope.

[0118] 10. Transwell experiment:

[0119] Cells in the logarithmic growth phase were starved for 12 hours, digested with trypsin, and resuspended in serum-free medium. Cells were counted using a cell counter as needed, and the concentration was adjusted to an appropriate level. A 24-well plate was used as the lower chamber; 600-800 μL of complete culture medium was added to each chamber, and 100-200 μL of cell suspension was added to the upper chamber. The cells were then cultured in a cell culture incubator for 24-28 hours. The chambers were removed, washed three times with PBS buffer, and cells that had not penetrated the semipermeable membrane were gently wiped from the inner surface of the chamber with a cotton swab. The cells were washed three more times with PBS buffer, then fixed and stained with 0.5% crystal violet methanol solution and incubated at room temperature for 30 minutes. The chambers were then removed, washed with tap water, and air-dried at room temperature. The results were observed and photographed under a microscope, and the experimental results were calculated and analyzed.

[0120] 11. Experiment on subcapsular orthotopic tumor grafts of the kidney:

[0121] Cells stably expressing luciferase (Luc) and in the logarithmic growth phase were digested and washed twice with PBS buffer. The cells were resuspended in 2% PBS medium, and cell counts were performed. The cell suspension concentration was adjusted according to experimental conditions. The volume of cell suspension inhibited per mouse was approximately 20 μL, and the total number of OSRC-2-Luc cells was approximately 5 × 10⁻⁶. 5Cells were placed on ice for later use. Severely immunodeficient (B-NDG) mice were randomly divided into groups of six. After satisfactory anesthesia, the mice were shaved, and the skin was disinfected with povidone-iodine. A transverse incision of approximately 1 cm was made at the costovertebral angle, the kidney was freed, and cell suspension was aspirated using a microsyringe and injected subcapsularly into the kidney. A circular bulge was formed under the kidney capsule. The needle was slowly withdrawn, and electrocoagulation was performed at the injection site for hemostasis. The kidney was carefully pushed back into the peritoneal cavity, and the peritoneum and skin were sutured in layers. Tumorigenicity was monitored weekly using a small animal in vivo imaging system. A chemiluminescent fluorescein substrate solution (15 mg / mL) was prepared, and 100 μL of the substrate solution was injected intraperitoneally into each mouse. Imaging was performed 3 minutes later, and the signal intensity of the in situ renal and lung metastatic tumors was recorded. Eight weeks after tumor cell inoculation, all mice were euthanized using carbon dioxide asphyxiation. The cadavers were dissected, and the in situ renal tumors and lung metastatic tumors were collected. The weight of the renal tumors was recorded using an electronic scale, and the tumor diameter was measured with calipers. The tumor tissue was fixed and processed for subsequent H&E staining, immunohistochemistry and other experiments.

[0122] 12. Mouse tail vein transfer model:

[0123] The mouse model experiments were conducted according to the National Institutes of Health's "Guideline for the Care and Use of Laboratory Animals" and approved by the review committee of Peking University First Hospital. Mice were kept in pathogen-free conditions with regulated temperature and humidity levels. Eighteen 5-week-old male B-NDG mice lacking mature T cells, B cells, and natural killer (NK) cells were purchased from Biocytogen Genetics Co., Ltd. in Beijing, China. Approximately 1 × 10⁶ mice were used in the experiments. 6 One shUGDH#1-Luc-Caki-1, shUGDH#2-Luc-Caki-1, and / or CON-Luc-Caki-1 cell line was suspended in 200 μl of PBS and injected into the lateral caudal vein of each unanesthetized B-NDG mouse at 5 weeks of age. Thirty days post-injection, mice were anesthetized with isoflurane (Hebei Yiping Pharmaceutical Co., Ltd., China). Ten minutes after D-Luciferin administration, sodium salt (150 mg / kg) was injected intraperitoneally, and cancer cells were detected using the Xenogen IVIS in vivo imaging system (PerkinElmer, MA, USA). The total photon flux per second in the lung and liver regions of each mouse was calculated and reported using in vivo imaging 4.3.1 (PerkinElmer / Caliper).

[0124] Example 1: Application of UGDH in the diagnosis and treatment of renal cell carcinoma

[0125] To verify the significance of the UGDH gene in renal cell carcinoma, we first established a mouse model of orthotopic metastasis of renal cell carcinoma. We then sent lung metastases and the primary tumor to a proteomics database. Based on proteomics, we screened for the molecule UGDH, which is closely related to renal cell carcinoma metastasis. Figure 1 Furthermore, we selected 200 consecutive patients who underwent renal cell carcinoma surgery at the Department of Urology, Peking University First Hospital within the past three years. Postoperative pathological diagnosis confirmed renal cell carcinoma. Paraffin sections of tumor tissue and normal kidney tissue were collected for immunohistochemical staining to detect UGDH protein expression and intracellular localization. Immunohistochemical staining of tumor tissue (T) and adjacent normal tissue (N) from 15 pairs of renal cell carcinoma patients revealed that UGDH expression in renal cell carcinoma was lower than that in adjacent normal tissue. Figure 2 (AB in the text). Further, we performed an AUC analysis and found that the AUC of UGDH in the clinical sample was 0.99, indicating that UGDH has high diagnostic efficacy in renal cell carcinoma. Figure 2 (C in the middle).

[0126] To clarify the effect of UGDH targeting on renal cell carcinoma metastasis, we investigated the impact of UGDH knockdown or overexpression on renal cell carcinoma cell migration. First, we obtained the UGDH mRNA sequence information from the NCBI database. We designed and chemically synthesized three shRNAs and a mock-shRNA negative control sequence that had no homology with any gene. The recombinant adenovirus overexpressing the UGDH gene was custom-made from a biotechnology company (Gikai Gene). Western blotting and qPCR results showed that the Caki-1 cell line stably knocked down and overexpressed with UGDH was successfully constructed. Figure 3 AB in Figure 3 DE in CaKi-1 cells. Cell migration assays showed that knockdown of UGDH in CaKi-1 cells significantly promoted the migration of renal cancer cells, and cell invasion assays showed that knockdown of UGDH in CaKi-1 cells significantly promoted the invasion of renal cancer cells. Figure 3 (C in the text); However, overexpression of UGDH in CaKi-1 cells showed that cell migration assays significantly inhibited renal cell carcinoma cell migration; simultaneously, cell invasion assays showed that overexpression of UGDH significantly inhibited renal cell carcinoma cell invasion (C in the text). Figure 3 The same results were obtained in the OSRC-2 cell line; overexpression of UGDH inhibited renal cell carcinoma migration and invasion, while knockdown of UGDH had the opposite effect. Figure 4 ).

[0127] Furthermore, using a tail vein lung metastasis model, it was demonstrated that knockdown of UGDH promotes tail vein lung metastasis of renal cell carcinoma. The fluorescence, number, and volume of metastatic lesions in the knockdown group were all higher than those in the control group. Figure 5 Overexpression of UGDH significantly inhibited lung metastasis via the tail vein of renal cell carcinoma; the fluorescence, number, and volume of metastatic lesions in the overexpression group were all lower than those in the control group. Figure 6 ).

[0128] Example 2: OGT inhibitor OSMI-1 has therapeutic effects on renal cell carcinoma.

[0129] Furthermore, we investigated the effects of the OGT inhibitor OSMI-1 on renal cell carcinoma. In both in vitro UGDH knockdown renal cell carcinoma and tail vein lung metastasis animal models, the addition of OSMI-1 significantly inhibited renal cell carcinoma, indicating that OSMI-1 can exert a therapeutic effect on renal cell carcinoma. Figure 7 ).

[0130] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Any of the following applications: 1) The use of an UGDH promoter or a pharmaceutical composition containing an UGDH promoter in the preparation of a medicament for treating renal cell carcinoma, characterized in that, The UGDH promoter or pharmaceutical composition containing the UGDH promoter treats renal cell carcinoma by inhibiting the migration and / or invasion of renal cell carcinoma cells, wherein the promoter is an UGDH overexpression vector. 2) The use of an UGDH promoter or a pharmaceutical composition containing an UGDH promoter in the preparation of an agent for the in vitro non-therapeutic purpose of inhibiting the migration and / or invasion of renal cancer cells, characterized in that the cells are selected from Caki-1 cells or OSRC-2 cells, and the promoter is an UGDH overexpression vector.

2. The application according to claim 1, characterized in that, The overexpression vectors include plasmids, lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors.

3. The application according to claim 1, characterized in that, The dosage forms of the drug include oral dosage forms, parenteral dosage forms, and / or topical dosage forms.

4. The application according to claim 1, characterized in that, The dosage forms of the drug include solutions, sustained-release formulations, suspensions, granules, tablets, capsules, powders, effervescent tablets, emulsions, syrups, drops, and / or chewable tablets.

5. The application according to claim 1, characterized in that, The drug can be administered orally, subcutaneously, intravenously, intramuscularly, intra-arterially, intranasally, intrathecally, intrathecally, intramucosally, intrapulmonaryly, and / or rectally.

Images