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

Abstract

The invention discloses application of UGDH in diagnosis, treatment and prognosis prediction of kidney cancer. The invention finds that the expression quantity of UGDH in kidney cancer tissues is obviously lower than that in paracancerous normal tissues, so that the invention 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 invention also finds that overexpression of the UGDH can inhibit the migration and invasion ability of kidney cancer cells, further researches find that the UGDH inhibits the migration of the kidney cancer by regulating the glycosylation level, and finds that a glycosylase OGT inhibitor OSMI-1 has the effect of inhibiting the migration of the kidney cancer cells, so that the invention also provides an accelerant of the UGDH and application of the OSMI-1 in preparation of medicines for treating the kidney cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of UGDH in diagnosis, treatment and prognosis prediction of renal cancer. BACKGROUND

[0002] Renal cell carcinoma (RCC) is a malignant tumor derived from renal tubular epithelium, which is a common and fatal urological disease.

[0003] Currently, the treatment of localized RCC and locally advanced RCC mainly adopts surgical treatment, and the surgical methods mainly are radical nephrectomy and partial nephrectomy for preserving renal units, but about one fourth of the patients will have distant metastasis after the operation. Since the early onset of RCC is occult and has no obvious symptoms, about one third of the patients with RCC have distant metastasis when they are diagnosed, that is, advanced / metastatic RCC, and common distant metastasis occurs in the lung, lymph nodes, liver, bone and brain. The patients with metastatic RCC lose the opportunity for curative surgery, and the patients mainly receive systemic treatment, including targeted therapy, immunotherapy and the like, but the treatment effect of metastatic RCC is not ideal, and the 5-year survival rate of the patients is only about 10%. Therefore, it is of great significance to clarify the molecular mechanism of RCC metastasis and to excavate new potential molecular markers and effective treatment targets for improving the treatment effect of patients with metastatic RCC and improving the prognosis of the patients. SUMMARY

[0004] In order to make up for the deficiency of the prior art, the application provides a biomarker UGDH, which can realize diagnosis, treatment and prognosis prediction of renal cancer.

[0005] In order to achieve the above-mentioned purpose, the application adopts the following technical scheme:

[0006] The first aspect of the application provides the following any application:

[0007] 1) Application of a promoter of UGDH or a pharmaceutical composition containing the promoter of UGDH in preparation of a drug for treating renal cancer;

[0008] 2) Application of a reagent for detecting UGDH in preparation of a product for diagnosing renal cancer / prognosing prognosis of renal cancer;

[0009] 3) Application of UGDH in screening of a candidate drug for treating renal cancer, construction of a system / device for diagnosing renal cancer / prognosing prognosis of renal cancer, or construction of a computer readable storage medium for diagnosing renal cancer / prognosing prognosis of renal cancer;

[0010] 4) Application of a promoter of UGDH or a pharmaceutical composition containing the promoter of UGDH in preparation of a reagent for inhibiting migration and / or invasion of renal cancer cells for in vitro non-treatment purposes;

[0011] 5) Use of OSMI-1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating renal cancer.

[0012] 6) Use of OSMI-1 or a pharmaceutically acceptable salt thereof in the manufacture of a reagent for inhibiting migration and / or invasion of renal cancer cells in vitro for non-therapeutic purposes, or in the manufacture of a medicament for screening a drug for treating renal cancer.

[0013] Further, the promoter specifically promotes the expression level of UGDH or enhances the enzyme activity of UGDH.

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

[0015] Further, the promoter is a UGDH overexpression vector or a UGDH protein.

[0016] Further, the overexpression vector includes a plasmid, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector.

[0017] The present application is based on extensive and in-depth research, and it is found that UGDH is significantly different in renal cancer, and further found that the content of UGDH in renal cancer tissue is significantly lower than that in normal renal cancer tissue and paracancerous tissue; at the same time, overexpression of UGDH can inhibit the proliferation, migration and invasion of renal cancer cells, suggesting that UGDH can be used as a better marker for the diagnosis, treatment and prognosis of renal cancer, and it is found that OGT inhibitor OSMI-1 has the effect of inhibiting the migration of renal cancer cells, suggesting that it is expected to become a safer and more effective drug for the treatment of renal cancer.

[0018] UGDH includes wild type, mutant or fragment thereof. The term encompasses full-length, unprocessed UGDH, as well as any form of UGDH that results from processing in cells. The term encompasses naturally occurring variants of UGDH (e.g., splice variants or allelic variants). The term encompasses, for example, UGDH genes, 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 the present application, UGDH is a human gene with gene ID 7358.

[0019] In the present application, treatment refers to the improvement, prevention, or reversal of a disease or condition or at least one discernible symptom thereof. In certain embodiments, the 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 parameter need not necessarily be discernible by or to the mammal. In some embodiments, the treatment refers to the inhibition or slowing of a disease or disease progression, which inhibition or slowing can be physical, such as the alleviation of some discernible adverse symptom. As used herein, "treatment" of a disease in a mammal, particularly a human, encompasses: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, e.g., arresting its development; or (c) relieving the disease, e.g., causing regression of the symptoms associated with the disease.

[0020] In the present application, the term "diagnosis" refers to a predictive process in which the presence, absence, severity, or course of a disease, disorder, or other medical condition is evaluated. For purposes of the present application, diagnosis also includes predictive processes used to determine the outcome of a treatment. Similarly, the term "diagnosis" refers to the determination of whether a subject exhibits one or more characteristics of a disorder or disease. The term "diagnosis" includes establishing the presence or absence of a target, e.g., a target antigen or binding agent, or establishing or otherwise determining one or more characteristics of a disorder or disease, including type, grade, stage, or the like. The term "diagnosis" includes initial diagnosis or detection, prognosis, and monitoring of a disorder or disease. In addition, the term "monitoring" in, e.g., "monitoring the course of a disease or condition" refers to ongoing diagnosis of a sample obtained from a subject having or suspected of having a disease or condition.

[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, e.g., based on life expectancy or quality of life. "Prognosis" includes the determination of the temporal course of a disease or condition, with or without treatment. In the context of treatment, prognosis includes the determination of the efficacy of a treatment for a disease or condition.

[0022] In the present application, the term "promoter" also referred to as "agonist", refers to any substance or agent that enhances the activity of a UGDH protein, enhances the stability of a UGDH gene or protein, promotes the expression level of UGDH, increases the effective time of action of a UGDH protein, as an embodiment of the present application, the "promoter" is a substance or agent that promotes the expression level of UGDH.

[0023] In the present application, the term "expression level" refers to the amount, accumulation or rate of a biomarker molecule or genome. The expression level can be represented, 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" refers to the absolute amount of a molecule in a sample or the relative amount of the molecule determined under steady-state or non-steady-state conditions.

[0024] In the present application, the substance that enhances the activity of UGDH enzyme refers to a chemical or biological molecule that can enhance the catalytic activity of UDP-glucose dehydrogenase (UGDH) through a direct or indirect mechanism, specifically to improve the efficiency of the enzyme in converting UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcUA), or to indirectly promote the function of the enzyme through regulation of its expression, modification, stability, etc.

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

[0026] In the present application, the pharmaceutically acceptable salt refers to acid salts formed with inorganic and / or organic acids and base salts formed with inorganic and / or organic bases. In addition, when the compound contains a basic moiety (for example, but not limited to, pyridine or imidazole) and an acidic moiety (for example, but not limited to, carboxylic acid), a zwitterion can be formed and the zwitterion is included in the pharmaceutically acceptable salt described in the present application. A pharmaceutically acceptable salt is preferably a pharmaceutically acceptable salt (i.e., non-toxic, physiologically acceptable), but other salts are also useful. The pharmaceutically acceptable salt of the compound can be formed, for example, by reacting the compound with an amount of an acid or a base in a medium, such as a medium in which the salt is precipitated or an aqueous medium (lyophilized after the reaction).

[0027] Specific pharmaceutically acceptable salts include those that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts (pharmaceutically acceptable salts) are well known in the art. The pharmaceutically acceptable salts of the compounds described in the present 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 of inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acid, or organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic or malonic acid, or base salts of amino acids such as arginine. Also included are salts formed when an acidic proton present in the parent compound is replaced by a cation process, for example, ion exchange procedures. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, methylnitrate, methylsulfate, naphthoate, napsylate, nicotinate, nitrate, oxalate, palmitate, pamoate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, polygalacturonate, propionate, stearate, sulfonate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate and valerate salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts. Further pharmaceutically acceptable salts are those in which the counter-ion is pharmaceutically acceptable cations such as Na+, Ca2+, or K+.

[0029] In the present application, hydrate refers to a compound associated with water.

[0030] In the present application, solvate refers to a compound or its salt form associated with a solvent, usually formed by solvolysis reaction. This physical association can include hydrogen bonding. Conventional solvents include methanol, ethanol, acetic acid, DMSO, THF, diethyl ether and the like. In some cases, the solvate will be capable of isolation, for example, where one or more solvent molecules are incorporated in the crystal lattice of the solid state form. Solvates include solution-phase solvates and isolatable solvates.

[0031] In the present application, crystalline form refers to a crystalline form of a compound in a particular crystal packing arrangement. Different crystalline forms often have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. The crystallization solvent, rate of crystallization, storage temperature, and other factors can cause one crystalline form to dominate. Various polymorphs of a compound can be prepared by crystallization under different conditions. In the present application, crystalline form also includes special crystal state, such as amorphous, etc.

[0032] It should be noted that the specific dosage of OSMI-1 used in the present application is not particularly limited, and any dosage that can produce corresponding kidney cancer treatment and / or prevention effect on the subject or other test objects (such as in vitro cells, in vitro tissues, in vitro organoids, etc.) is within the protection scope of the present application.

[0033] Further, the reagent for detecting UGDH is selected from an oligonucleotide probe specifically recognizing UGDH gene, a primer specifically amplifying UGDH gene, or a binding agent specifically binding to a protein encoded by UGDH gene.

[0034] The term "probe" refers to a molecule that binds to a particular sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that binds to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks perfect sequence complementarity to the probe. The probe can be directly or indirectly labeled. Hybridization formats 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 is capable of specifically annealing to a site on a RNA or DNA region adjacent to a target sequence and serving as a point-mutated initiator for DNA synthesis under suitable conditions in which synthesis of a primer extension product is induced, e.g., in the presence of nucleotides and a polymerization-inducing agent such as a DNA-dependent DNA polymerase, and suitable temperature, pH, metal concentration, and salt concentration. Typically, a PCR reaction uses a pair of amplification primers, also referred to as a "primer pair," including an "upstream" or "forward" primer and a "downstream" or "reverse" primer, which define a region of RNA or DNA to be amplified.

[0036] The term "amplification" refers to a process for replicating a portion of a nucleic acid using, for example, any of a variety of primer extension reactions. Exemplary primer extension reactions include, but are not limited to, PCR. Unless otherwise specified, "amplification" refers to single replication, or arithmetic, logarithmic, or exponential amplification.

[0037] The term "binding agent" refers to all or a portion of a proteinaceous (protein, protein-like, or protein-containing) molecule capable of binding to a membrane protein using a particular intermolecular interaction. Binding agents of proteins are, for example, receptors for the protein, lectins that bind the protein, antibodies against the protein, peptide bodies against the protein, bispecific dual binding agents, or bispecific antibody formats. More specifically, the term "binding agent" refers to a polypeptide, more specifically a protein domain. A suitable protein domain is an element of the overall protein structure that is self-stable and folds independently of the rest of the protein chain and is often referred to as a "binding domain." Such binding domains vary in length from about 25 amino acids up to and more than 500 amino acids. Many binding domains can be classified as folds and are recognizable, identifiable, 3-D structures. Some folds are so common among many different proteins that they are given specific names.

[0038] Further, the OSMI-1 significantly inhibited the migration ability and invasion ability of renal cancer cells.

[0039] Further, the dosage form of the medicament includes an oral administration dosage form, a parenteral administration dosage form, and / or a topical administration dosage form.

[0040] Further, the dosage form of the pharmaceutical preparation includes a solution, a sustained release, a suspension, a granule, a tablet, a capsule, a powder, an effervescent, an emulsion, a syrup, a drop, and / or a chewable.

[0041] Further, the administration method of the medicament includes oral, subcutaneous, intravenous, intramuscular, intra-arterial, intranasal, intrathecal, mucosal, intrapulmonary, and / or rectal.

[0042] The second aspect of the present application provides a product for diagnosing / prognosing renal cancer.

[0043] Further, the product includes a reagent capable of detecting the expression level of UGDH.

[0044] Further, the reagent is selected from the group consisting of an oligonucleotide probe specifically recognizing the UGDH gene, a primer specifically amplifying the UGDH gene, or a binding agent specifically binding to a protein encoded by the UGDH gene.

[0045] Further, the product includes a chip, a kit, or a nucleic acid membrane strip.

[0046] The term "chip" also referred to as "array" refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also referred to as "microarrays", can typically be produced using mechanical synthesis methods or light-directed synthesis methods which incorporate a combination of photolithographic methods and solid phase synthesis methods. Arrays can comprise a flat surface, or can be nucleic acids or peptides on beads, gels, polymeric surfaces, fibers such as optical fibers, glass, or any other suitable substrate. Arrays can be packaged in such a way as to allow diagnostic or other manipulation of a fully functional device.

[0047] The term "microarray" is an ordered arrangement of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The substrate can be a solid substrate, e.g., a glass or silica slide, a bead, a fiber optic adhesive, or a semi-solid substrate, e.g., a nitrocellulose membrane. The nucleotide sequences can be DNA, RNA, or any permutation thereof.

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

[0049] The kit of the present application comprises reagents for detecting UGDH gene or protein, and one or more substances selected from the group consisting of a container, an instruction for use, a positive control, a negative control, a buffer, an adjuvant or a solvent. The components of the kit can be packaged in the form of an aqueous medium or in the form of a lyophilized form. The appropriate container in the kit usually comprises at least one vial, test tube, flask, bottle, syringe or other container, in which one component can be placed, and preferably, can be appropriately aliquoted. When there are more than one component in the kit, a second, third or other additional container will also be included in the kit, in which the additional components are placed separately. However, different combinations of components can be contained in one vial. The kit of the present application will also usually comprise a container for containing the reactants, sealed for commercial sale. Such a container can include an injection or blow molded plastic container, in which the desired vials can be retained.

[0050] The kit of the present application includes, but is not limited to, a qPCR kit, an ELISA kit, an immunoblotting detection kit, an immunochromatographic detection kit, an immunohistochemical detection kit, a flow cytometry analysis kit, an electrochemiluminescence detection kit.

[0051] The term "nucleic acid membrane strip" includes a substrate and oligonucleotide probes immobilized on the substrate; the substrate can be any substrate suitable for immobilizing oligonucleotide probes, such as nylon membrane, nitrocellulose membrane, polypropylene membrane, glass sheet, silica gel wafer, microscale magnetic beads, etc.

[0052] The third aspect of the present application provides a pharmaceutical composition for treating renal cancer.

[0053] Further, the pharmaceutical composition comprises a promoter of UGDH, and / or OSMI-1 or a pharmaceutically acceptable salt thereof.

[0054] Further, the pharmaceutical composition further comprises a second therapeutic agent.

[0055] Further, the second therapeutic agent is another drug for treating renal cancer.

[0056] Further, the other drug for treating renal cancer comprises sunitinib, pazopanib, cabozantinib, lenvatinib, bevacizumab, temsirolimus and / or everolimus.

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

[0058] Further, the expression level of UGDH comprises the mRNA level of UGDH and the protein level of UGDH.

[0059] Further, the promoter is a UGDH overexpression vector or a UGDH protein.

[0060] Further, the promoting agent inhibits the proliferation, migration and / or invasion of the renal cancer cells.

[0061] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

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

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

[0064] 2) A method for inhibiting the proliferation, migration, invasion and / or promoting apoptosis of renal cancer cells for non-therapeutic purposes in vitro, the method comprising the step of: treating renal cancer cells with a promoting agent of UGDH and / or OSMI-1 or a pharmaceutically acceptable salt thereof.

[0065] Further, the culture system containing the UGDH gene or the protein encoded by the UGDH gene can be a cell system, and the cell can be a cell endogenously expressing UGDH or a cell recombinantly expressing UGDH. The culture system containing the UGDH gene or the protein encoded by the UGDH gene 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) and the like.

[0066] As a preferred mode of the present application, the method further comprises: performing further cell experiments and / or animal experiments on the obtained potential substances to further select and determine substances that are truly useful for treating renal cancer.

[0067] The method for detecting the expression and activity of UGDH of the present application is not particularly limited. Conventional protein quantification or semi-quantification detection techniques can be used, such as, but not limited to: co-immunoprecipitation, SDS-PAGE method, Western-Blot method, ELISA and the like.

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

[0069] Further, the renal cancer cells are selected from Caki-1 cells or OSRC-2 cells.

[0070] The fifth aspect of the present application provides a system / device for diagnosing / prognosing renal cancer, which comprises:

[0071] The acquisition unit is configured to acquire the expression level of UGDH in the sample.

[0072] The processing unit is configured to obtain the diagnosis / prognosis result of renal cancer according to the expression of UGDH.

[0073] If the expression level of UGDH is significantly down-regulated compared with the normal sample, the diagnosis result is renal cancer.

[0074] The sixth aspect of the present application provides a computer readable storage medium, which stores a computer program, and the computer program is executed by a processor to realize the system / device of the fifth aspect of the present application.

[0075] The present application has the following advantages and beneficial effects:

[0076] The molecular marker UGDH provided by the present application can effectively diagnose renal cancer, and the promoter of UGDH can inhibit the growth, proliferation and / or migration of renal cancer cells, thereby achieving the purpose of treating renal cancer. In addition, the OGT inhibitor OSMI-1 also has the effect of inhibiting the migration of renal cancer cells, and the present application provides a new direction for the research and development of renal cancer treatment drugs. BRIEF DESCRIPTION OF DRAWINGS

[0077] Figure 1 Figure 1 is a sequencing result diagram of a mouse renal cancer orthotopic metastasis model; wherein, Figure 1 Figure 1A is a diagram of a mouse renal cancer orthotopic metastasis model and a schematic diagram of material taking; Figure 1 Figure 1B is a live imaging diagram of a mouse orthotopic metastasis; Figure 1 Figure 1C is a fluorescence imaging diagram of a renal cancer primary lesion and a lung metastasis lesion after dissection; Figure 1 Figure 1D is an HE staining of a mouse renal cancer metastasis model primary lesion (left) and a lung metastasis lesion (right); Figure 1 Figure 1E is a proteinomics screening differential protein co-expression heat map of a renal cancer primary lesion and a lung metastasis lesion; Figure 1 Figure 1F is a proteinomics screening differential protein volcano plot of a renal cancer primary lesion and a lung metastasis lesion;

[0078] Figure 2 Figure 2 is an expression result diagram of UGDH in cancer tissues and para-cancer tissues of renal cancer patients; wherein, Figure 2 Figure 2A, Figure 2 Figure 2B is an immunohistochemical staining result diagram and a statistical diagram thereof; Figure 2 Figure 2C is an AUC result diagram (15 pairs of immunohistochemical staining results of cancer and para-cancer are made by IRS score);

[0079] Figure 3Fig. 1 is a graph showing the effect of UGDH on renal cancer cell line Caki-1; wherein, Figure 3 Fig. 1A and 1B are graphs showing the verification results of knocking down UGDH in CaKi-1 cells; Figure 3 Fig. 1C is a graph showing the migration and invasion experiment results of cells with knocked down UGDH; Figure 3 Fig. 1D and 1E are graphs showing the verification results of overexpressing UGDH in CaKi-1 cells; Figure 3 Fig. 1F is a graph showing the migration and invasion experiment results of cells with overexpressed UGDH;

[0080] Figure 4 Fig. 2 is a graph showing the effect of UGDH on renal cancer cell line OSRC-2; wherein, Figure 4 Fig. 2A and 2B are graphs showing the verification results of knocking down UGDH in OSRC-2 cells; Figure 4 Fig. 2C is a graph showing the migration and invasion experiment results of cells with knocked down UGDH; Figure 4 Fig. 2D and 2E are graphs showing the verification results of overexpressing UGDH in OSRC-2 cells; Figure 4 Fig. 2F is a graph showing the migration and invasion experiment results of cells with overexpressed UGDH;

[0081] Figure 5 Fig. 3 is a graph showing the effect of knocking down UGDH on the tail vein lung metastasis model; wherein, Figure 5 Fig. 3A is a graph showing the live imaging of tail vein metastasis model mice; Figure 5 Fig. 3B is a statistical analysis of the live imaging of tail vein metastasis model mice; Figure 5 Fig. 3C is a graph showing the live imaging of lung metastasis foci of tail vein metastasis model mice after dissection; Figure 5 Fig. 3D is a statistical analysis of the live imaging of lung metastasis foci of tail vein metastasis model mice after dissection; Figure 5 Fig. 3E is a statistical analysis of the number of lung metastasis foci of tail vein metastasis model mice after dissection; Figure 5 Fig. 3F is a statistical analysis of the invasion range of lung metastasis foci of tail vein metastasis model mice after dissection;

[0082] Figure 6 Fig. 4 is a graph showing the effect of overexpressing UGDH on the tail vein lung metastasis model; wherein, Figure 6 Fig. 4A is a graph showing the live imaging of tail vein metastasis model mice with overexpressed UGDH; Figure 6 Fig. 4B is a statistical graph of the live imaging of lung metastasis foci of tail vein metastasis model mice with overexpressed UGDH after dissection; Figure 6 Fig. 4C is a statistical analysis of the number of lung metastasis foci of tail vein metastasis model mice with overexpressed UGDH after dissection; Figure 6 Fig. 4D is a statistical analysis of the invasion range of lung metastasis foci of tail vein metastasis model mice with overexpressed UGDH after dissection; Figure 6 Fig. 4E is a graph showing the live imaging of lung metastasis foci of tail vein metastasis model mice with overexpressed UGDH after dissection;Figure 6 F in Fig. 4 is the statistical analysis of the lung metastasis live imaging graph of the tail vein metastasis model mice overexpressing UGDH after dissection;

[0083] Figure 7 OSMI-1 on renal cell carcinoma; wherein, Figure 7 A in Fig. 2 is a successful construction graph of a mouse tail vein metastasis tumor model; Figure 7 B in Fig. 3 is a graph of the mouse tail vein injection of the control group and OSMI-1 for 2 weeks; Figure 7 C in Fig. 4 is a dissection graph of the lung metastasis of the mouse tail vein injection of the control group and OSMI-1 for 2 weeks; DETAILED DESCRIPTION

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

[0085] The experimental methods in the following examples not specified in the specific conditions, generally in accordance with the conventional conditions or in accordance with the manufacturer's recommended conditions for testing, the following examples of the materials, reagents, etc., if not specified, can be obtained from commercial channels.

[0086] The experimental materials and methods used in the following examples are described as follows:

[0087] 1. Selection of cell lines:

[0088] The cell lines required by the present study include: human embryonic kidney cells HEK-293T cells (referred to as 293T), human renal cortical proximal tubular epithelial cells HK2 cells, human renal carcinoma OSRC-2 cells, human renal carcinoma 786-O cells, human renal carcinoma 769-P cells, human renal carcinoma Caki-1 cells, human renal carcinoma Caki-2 cells, human renal carcinoma ACHN cells, human renal carcinoma A498 cells. The above RCC cell lines are purchased from American Type Culture Collection (ATCC), and after STR identification and determination of the accuracy of the cell lines, subsequent experimental research is carried out.

[0089] 2. Selection of experimental animals:

[0090] The animal experiments carried out in this study were approved by the Experimental Animal Ethics Committee of Peking University First Hospital, and all experimental operations complied with the ethical principles of animal welfare and were supervised by the Experimental Animal Ethics Committee and animal laboratory teachers. The experimental animals used in this study include two types: (1) 6-week-old B-NDG male mice (purchased from Beijing Boaotai Gene Biotechnology Co., Ltd.); (2) 6-week-old BALB / c-nude mice (purchased from Vivotecno Co., Ltd.). Both types of mice were purchased by the animal house of Peking University First Hospital from the relevant company after the researchers submitted an application and were raised in a specific pathogen free (SPF) feeding room.

[0091] 3. Cell culture:

[0092] Cell recovery: First, wipe the biosafety cabinet with 75% alcohol and place sterile culture dishes, centrifuge tubes, pipettes, and other items. Ultraviolet irradiation the clean bench for 30 minutes for disinfection. Prepare complete culture medium, i.e., 10% foetal bovine serum (FBS) + 1% double-antibiotic (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, quickly thaw in the water bath, and constantly shake to accelerate melting. Stop thawing when only a small piece of ice remains, spray with alcohol, and move to the biosafety cabinet. Wipe the cryopreservation tube with an alcohol cotton ball, carefully unscrew the tube cap, add an appropriate amount of preheated culture medium, mix well, and transfer to a centrifuge tube. Add an appropriate amount of culture medium to the cryopreservation tube and transfer the wash to the centrifuge tube. Centrifuge at 700 rpm at room temperature for 3 min, discard the supernatant, add 2 ml of preheated culture medium, mix gently, and count. According to the inoculation density and cell state, inoculate an appropriate amount of cells into the culture dish, add an appropriate amount of culture medium, mix gently, and place in a 37°C, 5% carbon dioxide, and 90% humidity cell incubator for culture.

[0093] Cell passage: Observe the cell status, including cell density, morphology, medium color, etc. under the microscope at a fixed time every day, and replace the fresh medium once every 1-2 days according to the cell status. When the medium needs to be replaced, first discard the old medium, add an appropriate amount of sterile phosphate buffer saline (PBS), gently shake the culture dish to wash the residual old medium and cell debris, discard the washing liquid, and use a washing head to suck the residual liquid. Repeat the above operation twice, then add an appropriate amount of fresh 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 cotton ball, and place the culture dish back into the cell incubator for continued culture. According to the cell status and experimental needs, generally pass the cells when the cell density reaches 80%-90%. First, discard the old culture solution and use a suction head to suck it up, add preheated sterile PBS along the wall of the culture dish, gently shake the culture dish to wash the cells, discard the washing liquid and use a washing head to suck the residual liquid, and wash the cells twice in this way. Add an appropriate amount of trypsin (enough to cover the bottom of the culture dish), place the culture dish in the cell incubator to accelerate cell digestion and make it detach from the wall. After about 2 min, observe under the microscope that the cells are in scattered sheets and can be suspended and floated with liquid waves, add enough complete medium (generally more than 2 times the volume of trypsin) to terminate digestion, carefully blow the bottom of the dish repeatedly to detach the cells from the wall, transfer the digestion solution to a centrifuge tube, add an appropriate amount of medium to wash the culture dish, and transfer the washing liquid to the centrifuge tube. Centrifuge the centrifuge tube at room temperature and 700 rpm for 3 min, discard the supernatant, gently tap the bottom of the centrifuge tube to separate the cells from each other, add 1-2 ml of medium to resuspend the cells, and mix thoroughly by repeatedly blowing. Add 20 μL of cell suspension to the wells of the cell counting plate, and use a cell counter to count the cells. According to the experimental needs, inoculate an appropriate amount of cells into a new culture dish, add an appropriate amount of medium, gently shake the culture dish with the "cross" mixing method, mix the cells, mark the information such as cell type, treatment condition, and operation date on the culture dish with a marker pen, and place it in the cell incubator for culture.

[0094] Cell freezing: when the cell morphology is good under the microscope and in the logarithmic growth phase, the cell can be frozen and stored. After discarding the old culture medium, wash the cells twice with preheated PBS, add trypsin to digest the cells. After sufficient digestion, stop the digestion with an appropriate amount of complete culture medium, and transfer the digestion solution to a centrifuge tube. Centrifuge at room temperature and 700 rpm for 3 min, discard the supernatant. Resuspend the cell pellet in the centrifuge tube with cell freezing solution, mix carefully by blowing, transfer to a freezing tube, tighten the tube cap, and clearly label the cell strain type, treatment condition, passage number, and freezing time. Use the program to gradually cool down, and finally transfer to a liquid nitrogen tank for long-term storage.

[0095] 4. Plasmid transformation and extraction:

[0096] Plasmid transformation:

[0097] Take 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 in an ice bath for 30 min. Preheat the water bath to 42°C, transfer the centrifuge tube from the ice bath to the water bath, and incubate for 2 min. Prepare sterile LB and LA media in advance. The preparation method of LB and LA media is as follows: according to the formula, weigh the tryptone, yeast extract and sodium chloride respectively, and place them in a clean beaker. Add 2 / 3 volume of double distilled water to the beaker, and use a glass rod to completely dissolve it. Adjust the pH to 7.2 using 1 mol / L sodium hydroxide (NaOH) solution, then perform constant volume, add double distilled water to the required volume, mix well, and then perform dispensing. Paste a high-pressure indicator, sterilize in a high-pressure sterilization pot at 100 Pa for 20 min, and store in a 4°C refrigerator. Take an appropriate amount of LB medium, mix ampicillin and LB medium at a ratio of 1:1000 to obtain LA medium. In the clean bench, add 600 μL of LB medium to each centrifuge tube, and incubate at 37°C in a shaker at 200 rpm for 1 h to recover the bacteria and induce the expression of the resistance gene in the plasmid. Prepare 15 ml centrifuge tubes, add 10 ml of LA medium to each centrifuge tube, and add the LB medium containing the plasmid to the LA medium. Shake at 37°C and 200 rpm in a shaker for about 16 h.

[0098] Plasmid extraction: We synthesized the silencing and overexpression plasmids in GenScript Biotech Co., Ltd., the vector information of the silencing plasmid was pLV-hU6-neg / shRNAs-hEF1a-EGFP-2A-Puro / Neo, and the vector information of the overexpression plasmid was pLV-hef1a-mNeongreen-P2A-Puro / Neo-WPRE-CMV-3Xflag. We used the rapid plasmid extraction kit to extract the plasmid, and the kit contained the following components: solution P1, solution P2, solution P5, rinse PWT, elution buffer TB, RNase A, TIANRed, adsorption column CP3, collection tube (2 ml). Take 2 ml of bacteria cultured for about 16 h and add it to a 15 ml centrifuge tube, centrifuge at 12000 rpm for 1 min, discard the supernatant. Add 150 μL of solution P1 (with the addition of RNase A and TIANRed) to the centrifuge tube with bacterial precipitate, resuspend the bacterial precipitate by blowing. Add 150 μL of solution P2 to the centrifuge tube, mix well by turning up and down 10 times to completely lyse the bacteria. Add 350 μL of solution P5 to the centrifuge tube and immediately mix well by turning up and down 20 times, then centrifuge at 12000 rpm for 2 min, transfer the supernatant to the adsorption column CP3, centrifuge at 12000 rpm for 30 sec, discard the waste in the collection tube, and place the adsorption column CP3 back into the collection tube, add 300 μL of rinse PWT (with the addition of anhydrous ethanol), centrifuge at 12000 rpm for 1 min, after discarding the waste in the collection tube, place the adsorption column CP3 back into the collection tube, and centrifuge at 12000 rpm for 1 min again to completely remove the residual rinse from the adsorption column. Transfer the adsorption column CP3 to a clean centrifuge tube, add 100 μL of elution buffer TB to the middle of the adsorption membrane, centrifuge at 12000 rpm for 1 min, and add the liquid in the centrifuge tube to the middle of the adsorption membrane again, repeat the above operation to obtain the plasmid solution. Use the NanoDrop ultraviolet spectrophotometer to detect the concentration of the plasmid solution, mark the plasmid name, concentration, operation date, etc. on the wall of the centrifuge tube, and store it in the -20℃ refrigerator after aliquoting.

[0099] 5. Lentivirus packaging and infection:

[0100] Lentivirus packaging: We use Lipofectamine 3000 transfection reagent for lentivirus packaging. First, culture 293T cells, adjust the cells to be in good condition, and the cell density is about 60%-70%. Add 250 μL of Opti-MEM Medium serum-free medium and 7.5 μL of Lipo 3000 reagent in A centrifuge tube; add 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) in B centrifuge tube. Mix A and B centrifuge tubes, mix gently and incubate at room temperature for 15 min, then add to the culture dish of cultured 293T cells, supplement with an appropriate amount of complete medium, and incubate in the cell culture incubator. After 12 hours of packaging, the culture medium can be changed (without washing the cells) according to the state of the cells. After 48 hours of packaging, the cell culture medium is transferred to a 15 ml centrifuge tube, centrifuged at 1000 rpm for 5 min, and the supernatant is collected. Filter the supernatant virus liquid with a 0.22 μm filter, aliquot and store at -80°C.

[0101] Lentivirus infection: Culture the cell strain that needs to be infected in a six-well plate, and adjust it to be in good condition with a density of about 30%-50%, depending on the cell proliferation speed. Take the virus from the -80°C freezer and thaw it in a water bath. Add 100 μL of virus, 900 μL of complete medium and 2 μL of Polybrene infection-promoting reagent to each well, mix gently and incubate in the cell culture incubator. Set up a blank group (containing cells and infection-promoting reagents under the same conditions, but without virus) for control of the drug screening. After 48 days of infection, start drug screening with puromycin, with an initial concentration of 1-2 μg / ml, adjust the drug screening concentration as needed, and when the blank group cells are all dead, the anti-puromycin surviving cells are obtained. Then extract cell RNA and protein, and verify the infection efficiency as needed for the experiment.

[0102] 6. Real-time fluorescent quantitative PCR:

[0103] Extraction of RNA: We use TRIzol method to extract RNA in cell and tissue samples. For adherent cells, discard the cell culture fluid, wash the cells twice with 1X PBS solution, and aspirate the residual liquid. Add an appropriate amount of TRIzol reagent to the cell culture dish to cover the cell surface, and blow the cells with a pipette to completely detach them, and incubate at room temperature for 5 min. For tissue samples, first cut the tissue block into small pieces, then crush them thoroughly in a tissue crusher, add TRIzol reagent, vortex to fully lyse, and incubate at room temperature for 10 min. Add 0.2 ml chloroform per ml of TRIzol reagent, mix quickly, and incubate at room temperature for 3 min. Centrifuge at 4°C and 12000 rpm for 15 min, at which time the mixture in the tube will separate into three layers, with the RNA in the upper aqueous phase. Transfer the upper aqueous phase to a new EP tube, collect about 600 μL of water phase per ml of TRIzol reagent, add isopropanol at a ratio of 1:1, mix well by inverting the tube, and incubate at room temperature for 10 min, then centrifuge at 4°C and 12000 rpm for 15 min; discard the supernatant and aspirate the remaining liquid, and a white flocculent RNA precipitate appears at the bottom of the tube. Add 75% ethanol 1 ml per ml of TRIzol reagent to wash the RNA precipitate, mix well by blowing, then centrifuge at 4°C and 12000 rpm for 5 min, and repeat the washing once. Dry the RNA precipitate in air for 15 min to completely remove the ethanol, until the RNA precipitate at the bottom of the tube becomes transparent, add 50 μL of RNase-free water, blow to dissolve completely, then detect the sample concentration and purity.

[0104] Reverse transcription: RNA reverse transcription was performed using the FastKing RT Kit (With gDNase) FastKing cDNA Reverse Transcription Kit, and the appropriate amount of template RNA was thawed on ice, 5 X gDNA Buffer, FQ-RT PrimerMix, 10 X King RT Buffer, and RNase-Free ddH2O were thawed at room temperature, and were placed on ice after thawing. Before use, vortex each solution to mix well, and centrifuge briefly to collect the liquid on the tube wall.

[0105] 7. Western blot analysis:

[0106] Protein sample preparation: We extract total protein in cell and tissue samples. For adherent cells, first discard the old culture medium, wash the cells twice with PBS, and aspirate the residual liquid. Add an appropriate amount of PBS, scrape the cells with a cell scraper, and transfer them to a centrifuge tube. Centrifuge at 4°C and 5000 rpm for 5 min, discard the supernatant to obtain the cell pellet. For tissue samples, first cut the tissue as much as possible, add an appropriate amount of PBS, and pulverize the tissue in a tissue pulverizer. Centrifuge at 4°C and 5000 rpm for 5 min, discard the supernatant to obtain the tissue pellet. Add an appropriate amount of protein lysis buffer and protease inhibitor, mix well by blowing, and lyse in a 4°C rotary mixer for 30 min. Then centrifuge at 4°C and 12000 rpm for 25 min, and transfer the supernatant to a new EP tube for standby.

[0107] Protein concentration determination: We use the BCA protein quantitative analysis kit to determine the protein concentration. Take an ampoule of bovine serum albumin (BSA) standard dilution, and use double distilled water as the diluent. According to the experimental requirements, prepare the BCA working solution with reagent A: reagent B = 50:1, and mix well by blowing. Add 200 μL of working solution per well to a 96-well plate, and add 10 μL of standard or sample per well in order. Mix gently and incubate at 37°C for 30 min. After natural cooling to room temperature, use a full-automatic enzyme marker to detect the absorbance value at 562 nm wavelength. According to the standard curve, obtain the concentration formula, and then calculate the concentration of each test protein sample. Take an appropriate amount of 5X SDS Loading Buffer, and dilute the protein sample concentration with double distilled water according to the concentration of each protein sample. Heat the protein sample in a 98°C metal bath for 15 min to denature it, and then store it at -20°C after short centrifugation.

[0108] Glue preparation: Wash and dry the glue preparation glass plate, clamp the glass plate with a glue holder, and tightly attach the bottom to the silica gel pad. Prepare the separation glue according to the experimental requirements, mix well, add an appropriate amount of TEMED, mix well, pour the glue, and press the liquid surface with isopropanol. Let it stand at room temperature for about 30 min. After the separation glue is fully solidified, discard the isopropanol and rinse with double distilled water three times. Prepare the concentrated glue according to the experimental requirements, mix well with an appropriate amount of TEMED, and pour it on the upper layer of the separation glue. Insert the sample comb, check for air bubbles, and let it stand at room temperature for 60 min.

[0109] SDS-PAGE gel electrophoresis: The prepared gel was clamped in the electrophoresis core, placed in the electrophoresis tank, and the electrophoresis buffer was added. Carefully remove the sample comb vertically, use the sample gun head to add the prepared protein sample to each lane in order, and add equal volume of protein Marker to both sides of the lane. Cover the electrophoresis tank cover, turn on the power, concentrate the gel at 80 V, separate the gel at 120 V, and electrophorese until the dye molecule bromophenol blue reaches the bottom of the gel, and then turn off the power to terminate electrophoresis.

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

[0111] Antibody incubation: Prepare a blocking solution with a concentration of 5% in TBST buffer and skimmed milk powder, and dissolve thoroughly for use. After successful transfer, block the membrane in the antibody incubation box with the blocking solution, and make sure the membrane is completely immersed in the blocking solution. Place the incubation box in a low-speed shaking incubator and incubate at room temperature for 1 h. After blocking, wash the membrane with TBST buffer, 5 min each time at 80 rpm / min, replace the TBST buffer, and repeat the washing 3 times. According to the experimental requirements, dilute the primary antibody to the appropriate concentration, immerse the membrane in the primary antibody solution, and incubate at 4°C overnight. The next day, recover the primary antibody and wash the membrane with TBST buffer, 5 min each time at 80 rpm / min, replace the TBST buffer, and repeat the washing 3 times. Then immerse the membrane in the secondary antibody solution and incubate at room temperature for 1 h. Then wash the membrane with TBST buffer, 5 min each time at 80 rpm / min, replace the TBST buffer, and repeat the washing 3 times.

[0112] Exposure and color development: Mix the A and B reagents of ECL luminescent solution in equal proportions to prepare the exposure working solution, and pay attention to avoid light. Cover the working solution on the membrane, incubate for 1-2 min, use the automatic gel imaging system for exposure and color development, take pictures and analyze.

[0113] 8. IHC analysis:

[0114] IHC staining: The embedded paraffin samples were cut into 3-5 pm sections under the guidance of a urological pathologist. After baking the sections in a constant temperature oven at 70 °C for 2 h, the sections were deparaffmized and hydrated, and then immersed in the following solutions in sequence: 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), 75% ethanol (5 min). Subsequently, the sections were washed with PBS buffer for 5 min each time on a shaker, and the PBS buffer was replaced for 3 times. Antigen retrieval was performed using an antigen retrieval solution under the condition of high pressure at 110 °C for 10 min, and the sections were naturally cooled at room temperature after the antigen retrieval. The sections were washed with PBS buffer for 3 times each time on a shaker for 5 min, and then the sections were circled with a histological pen, and an appropriate amount of endogenous peroxidase blocker was added dropwise, and the sections were incubated at room temperature for 20 min. After the blocking was completed, the sections were washed with PBS buffer for 3 times each time on a shaker for 5 min. An appropriate amount of goat serum was added dropwise to each section for blocking, and the sections were incubated in a histological wet box at room temperature for 1 h. After the incubation was completed, the serum was removed, and a primary antibody was added dropwise according to the experimental requirements, and the sections were incubated at 4 °C overnight. On the next day, the sections were taken out from the cold storage, and were naturally warmed to room temperature, and the sections were washed with PBS buffer for 3 times each time on a shaker for 5 min. An appropriate amount of a secondary antibody was added dropwise to each section, and the sections were incubated at room temperature for 40 min. After the incubation was completed, the sections were washed with PBS buffer for 3 times each time on a shaker for 5 min. 20X DAB concentrated solution was diluted to 1X to prepare a DAB working solution, and an appropriate amount of the DAB working solution was added dropwise to the sections, and the sections were incubated at room temperature for 5-8 min, and then the sections were immersed in PBS buffer to terminate the color development. The sections were dyed in hematoxylin dye solution for 2-3 min, and were differentiated in 0.1% hydrochloric acid alcohol for 1-3 s, and then were placed in tap water for “blue returning” for 30 min. Subsequently, the sections were dehydrated and transparentized by immersion in the following solutions in sequence: 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), xylene I (10 min). Then, the sections were mounted with neutral resin, an appropriate amount of the neutral resin was added dropwise to the sections, a cover glass was placed on the sections, and the sections were dried in a ventilated place, and the sections were observed under a microscope and analyzed.

[0115] IHC scoring: Two experienced pathologists independently scored the histology. The scoring criteria included staining intensity and staining extent. Staining intensity score: no staining = 0 point, weak staining = 1 point, moderate staining = 2 points, strong staining = 3 points. Staining extent score: no positive staining = 0 point, 1%-25% = 2 points, 26%-50% = 2 points, 51%-100% = 3 points. The final histology score was obtained by multiplying the staining intensity score and the staining extent score, and the score range was 0-9 points.

[0116] 9. Immunofluorescence:

[0117] Take out the culture dish, discard the old culture medium, and use the suction device to suck it up. Add an appropriate amount of room temperature PBS and rinse for 5 min on the shaker. Wash 3 times. After sucking up the PBS, add an appropriate amount of 4% paraformaldehyde to fix the cells, and make sure the fixing solution covers the cells. Place it on the shaker at room temperature for 20 min. Discard the paraformaldehyde and wash it with 4°C pre-cooled PBS 3 times as above. After discarding the PBS, block the cells and add an appropriate amount of 1% BSA. Incubate at room temperature for 1 h. Discard the blocking solution and wash it with 4°C pre-cooled PBS 3 times. In addition to membrane antigens, Triton X-100 is used to permeabilize the cells. Add an appropriate concentration of primary antibody solution and incubate at 4°C overnight. Discard the primary antibody solution and wash it with 4°C pre-cooled PBS 3 times. Add the secondary antibody solution and incubate at room temperature for 1 h in the dark. After incubation, suck up the secondary antibody solution and wash it with PBS 3 times. Add DAPI and incubate at room temperature for 10 min, then wash it with PBS 3 times. Add an anti-quenching mounting agent and mount it under a fluorescence microscope for observation.

[0118] 10. Transwell experiment:

[0119] Take the cells in the logarithmic growth phase and starve them for 12 h. After trypsin digestion, resuspend the cells in serum-free medium. According to the experimental requirements, use a cell counter to count the cells and adjust the cell concentration to the appropriate level. Use a 24-well plate as the lower chamber, add 600-800 μL of complete medium to each lower chamber, and add 100-200 μL of cell suspension to the upper chamber. Continue to culture in the cell incubator for 24-28 h. Take out the chamber and wash it with PBS buffer 3 times. Gently wipe the cells on the inner surface of the chamber that have not passed through the semi-permeable membrane with a cotton swab. Wash it again with PBS buffer 3 times, then fix and stain it with 0.5% crystal violet methanol solution and incubate at room temperature for 30 min. Take out the chamber, wash it with tap water and air dry at room temperature. Observe and take pictures under a microscope, and calculate and analyze the experimental results.

[0120] 11. Subrenal capsule implantation experiment:

[0121] Stable luciferase (Luc) expression cells in the logarithmic growth phase were digested and washed with PBS buffer 2 times. Resuspend the cells in 2% PBS medium, count the cells, and adjust the cell suspension concentration according to the experimental conditions. The volume of cell suspension per mouse is about 20 μL, and the total number of OSRC-2-Luc cells is about 5×10 5The cells were placed on ice until use. The severely immunodeficient mice B-NDG were randomly divided into groups, with 6 mice in each group. After satisfactory anesthesia, the mice were shaved and the skin was disinfected with iodophor. A transverse incision of about 1 cm was made at the position of the spinal rib angle of the mouse, and the kidney was freed. A microsyringe was used to suck the cell suspension and inject it under the capsule of the mouse kidney. A circular protrusion could be formed under the kidney capsule, and the needle was slowly withdrawn. The electrocautery was used to electrocoagulate and stop bleeding at the injection site. The kidney was carefully pushed back into the abdominal cavity, and the peritoneum and skin were sutured in layers. The tumor formation effect was monitored every week using a small animal live imaging instrument. Chemiluminescent luciferin substrate solution (15 mg / mL) was prepared, and 100 μL of the substrate solution was injected into the abdominal cavity of each mouse. Imaging was performed 3 min later, and the signal intensity of the mouse kidney orthotopic and lung metastatic tumors was recorded. After 8 weeks of tumor cell inoculation, all mice were euthanized using carbon dioxide asphyxiation, and the carcasses were dissected. The kidney orthotopic tumor and lung metastasis tumor were collected, weighed using an electronic balance, and recorded. The length of the tumor was measured using a vernier caliper. The tumor tissue was fixed and processed for subsequent H&E staining, immunohistochemistry, and other tests.

[0122] 12. Mouse tail vein metastasis model:

[0123] Mouse model experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Peking University First Hospital Institutional Animal Care and Use Committee. Mice were maintained under pathogen-free conditions with regulated temperature and humidity levels. Eighteen 5-week-old male B-NDG mice, which lack mature T cells, B cells, and natural killer (NK) cells, were purchased from Baoao Satu Gene Co., Ltd. in Beijing, China. Approximately 1 x 10 6 UGDH#1-Luc-Caki-1, shUGDH#2-Luc-Caki-1, and / or CON-Luc-Caki-1 cells were suspended in 200 μl PBS and injected into the lateral tail vein of each unanesthetized B-NDG mouse at 5 weeks of age. Thirty days after injection, the mice were anesthetized with isoflurane (Yiping Pharmaceutical Co., Ltd., Hebei, China). Ten minutes after D-Luciferin, sodium salt (150 mg / kg) was injected intraperitoneally, and cancer cells were detected using an in vivo imaging system, Xenogen IVIS (PerkinElmer, MA, USA). The total flux of photons per second in the lung and liver regions of each mouse was calculated and reported using Live Image 4.3.1 (PerkinElmer / Caliper).

[0124] Example 1. Application of UGDH in the diagnosis and treatment of renal cancer

[0125] To verify the significance of the UGDH gene in renal cancer, we first established a mouse renal cancer orthotopic metastasis model, and sent the lung metastasis and primary tumor for proteomics. Based on proteomics, the molecule UGDH closely related to renal cancer metastasis was screened out.Figure 1 ). Further, we selected 200 patients who underwent surgical treatment for kidney cancer in the Department of Urology, Peking University First Hospital in the past 3 years, and confirmed by pathology that they were diagnosed with renal cell carcinoma. We took their tumor tissues and normal kidney tissues paraffin sections for immunohistochemical staining to detect the expression of UGDH protein and intracellular localization. Immunohistochemical staining of 15 pairs of cancer tissues (T) and cancer-adjacent tissues (N) of kidney cancer patients showed that the expression of UGDH in kidney cancer was lower than that in cancer-adjacent tissues (P < 0.05, Fig. 1A-B). Further, we performed AUC analysis and found that the AUC of UGDH was 0.99 in the clinical sample, which indicated that UGDH had high diagnostic efficiency in kidney cancer (P < 0.05, Fig. 1C). Figure 2 Figure 2

[0126] To clarify the effect of targeting UGDH on the metastasis of kidney cancer cells, we knocked down or overexpressed UGDH in kidney cancer cells to study the effect on the migration of kidney cancer cells. First, we obtained the sequence information of UGDH mRNA in the NCBI database, and designed and chemically synthesized 3 shRNAs and a mock-shRNA negative control sequence that had no homology with any gene. The recombinant adenovirus for overexpression of UGDH gene was customized from a biological company (Jikai Gene). WB and qPCR experimental results showed that the Caki-1 cell strain with stable knockdown and overexpression of UGDH was successfully constructed (Fig. 2A-B, Fig. 2D-E). Cell migration experiment results showed that knocking down UGDH in CaKi-1 cells significantly promoted the migration of kidney cancer cells, and cell invasion experiment results showed that knocking down UGDH in CaKi-1 cells significantly promoted the invasion of kidney cancer cells (Fig. 3C); however, overexpression of UGDH in CaKi-1 cells significantly inhibited the migration of kidney cancer cells, and cell invasion experiment results showed that overexpression of UGDH significantly inhibited the invasion of kidney cancer cells (Fig. 3F). The same results were obtained in the OSRC-2 cell line, overexpression of UGDH inhibited kidney cancer migration and invasion, and knockdown of UGDH was the opposite (Fig. 4). Figure 3 Figure 3 Figure 3 Figure 3 Figure 4

[0127] Further, it was proved by the tail vein lung metastasis model that in the tail vein lung metastasis model, knocking down UGDH could promote the tail vein lung metastasis of kidney cancer, and the fluorescence, number, and volume of metastatic foci in the knockdown group were higher than those in the control group (Fig. 5); while overexpression of UGDH significantly inhibited the tail vein lung metastasis of kidney cancer, and the fluorescence, number, and volume of metastatic foci in the overexpression group were lower than those in the control group (Fig. 6). Figure 5 Figure 6

[0128] Example 2 OGT inhibitor OSMI-1 has a therapeutic effect on kidney cancer​​​​​​​​​

[0129] Further, we explored the effect of OGT inhibitor OSMI-1 on renal cancer. In both in vitro UGDH-knocked down renal cancer cells and tail vein lung metastasis animal models, the addition of OSMI-1 can significantly inhibit renal cancer, which indicates that OSMI-1 can produce a therapeutic effect on renal cancer. Figure 7 ).

[0130] The above description of the embodiments is only for the purpose of understanding the method of the present application and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. Any of the following applications: 1) The use of UGDH promoters or pharmaceutical compositions containing UGDH promoters in the preparation of medicaments for treating renal cell carcinoma; 2) Application of reagents for detecting UGDH in the preparation of products for diagnosing renal cell carcinoma / predicting the prognosis of renal cell carcinoma; 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; 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; 5) The use of OSMI-1 or its pharmaceutically acceptable salts in the preparation of drugs for the treatment of renal cell carcinoma; 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.

2. The application according to claim 1, characterized in that, The promoter specifically promotes the expression level of UGDH or increases its enzyme activity; Preferably, the expression level of UGDH includes the mRNA level and the protein level of UGDH; Preferably, the promoter is an UGDH overexpression vector or UGDH protein; Preferably, the overexpression vector includes plasmids, lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors.

3. The application according to claim 1, characterized in that, The reagents for detecting UGDH are 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.

4. The application according to claim 1, characterized in that, The OSMI-1 significantly inhibited the migration and invasion abilities of renal cell carcinoma cells; Preferably, the dosage form of the drug includes an oral dosage form, a parenteral dosage form, and / or a topical dosage form; More preferably, the dosage form of the pharmaceutical preparation includes solutions, sustained-release formulations, suspensions, granules, tablets, capsules, powders, effervescent tablets, emulsions, syrups, drops, and / or chewable tablets. Preferably, the administration of the drug includes oral, subcutaneous, intravenous, intramuscular, intra-arterial, intranasal, intrathecal, mucosal, intrapulmonary, and / or rectal administration.

5. A product for diagnosing renal cell carcinoma / predicting the prognosis of renal cell carcinoma, characterized in that, The product includes reagents capable of detecting UGDH expression levels; Preferably, 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. Preferably, the product includes a chip, a reagent kit, or a nucleic acid membrane strip.

6. A pharmaceutical composition for treating renal cell carcinoma, characterized in that, The pharmaceutical composition includes an UGDH promoter and / or OSMI-1 or a pharmaceutically acceptable salt thereof; Preferably, the pharmaceutical composition further comprises a second therapeutic agent; Preferably, the second therapeutic agent is another drug used to treat kidney cancer; More preferably, the other drugs used to treat renal cell carcinoma include sunitinib, pazopanib, cabozantinib, lenvatinib, bevacizumab, tesiromoximus and / or everolimus.

7. The pharmaceutical composition according to claim 6, characterized in that, The promoter specifically promotes the expression level of UGDH or increases its enzyme activity; Preferably, the expression level of UGDH includes the mRNA level and the protein level of UGDH; Preferably, the promoter is an UGDH overexpression vector or UGDH protein; Preferably, the promoter inhibits the proliferation, migration, and / or invasion of renal cell carcinoma cells; Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

8. Any of the following methods: 1) A method for screening candidate drugs for the treatment of renal cell carcinoma, characterized in that, The method includes: 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. 2) A method for inhibiting the proliferation, migration, and invasion of renal cell carcinoma cells and / or promoting apoptosis of renal cell carcinoma cells in vitro for non-therapeutic purposes, characterized in that the method comprises the following steps: treating renal cell carcinoma cells with an UGDH promoter and / or OSMI-1 or a pharmaceutically acceptable salt thereof; Preferably, 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; Preferably, the renal cell carcinoma cells are selected from Caki-1 cells or OSRC-2 cells.

9. A system / device for diagnosing renal cell carcinoma / predicting the prognosis of renal cell carcinoma, characterized in that, The system / device includes: Acquisition unit: used to acquire the expression level of UGDH in the sample; Processing unit: Based on the expression of UGDH, obtain the diagnostic / prognostic prediction results of renal cell carcinoma.

10. A computer-readable storage medium, characterized in that, It stores a computer program that, when executed by a processor, implements the system / device of claim 9.

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