Application of NAT10 in diagnosis and treatment of glioma

Abstract

The invention discloses an application of NAT10 in diagnosis and treatment of glioma. According to the application, high expression of the NAT10 in glioma tissues is found for the first time, the high expression of the NAT10 is positively related to the tumor level and poor prognosis of glioma patients, and proliferation and migration invasion of glioma cells can be inhibited by inhibiting the NAT10. The application provides a new thought for early diagnosis and effective treatment of glioma, and has a wide application prospect clinically.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of NAT10 in diagnosing and treating glioma. Background Art

[0002] Glioma is the most common primary malignant tumor of the human central nervous system. Glioblastoma multiforme (GBM) is the most common type of glioma, accounting for about 46.1%. It is characterized by strong invasiveness, high malignancy, rapid progression, and poor prognosis. With the development of molecular biology technology, molecular diagnosis can achieve more accurate diagnosis and provide safe and effective treatment options for glioma patients. Finding molecular diagnostic markers for glioma and exploring the mechanisms and molecular targets that affect the occurrence and development of glioma are of great significance for the accurate diagnosis and effective treatment of glioma. Summary of the invention

[0003] To make up for the deficiencies of the prior art, the present invention provides the use of NAT10 in the diagnosis and treatment of glioma.

[0004] To achieve the above purpose, the present invention adopts the following technical solution.

[0005] The first aspect of the present invention provides the use of a reagent for detecting the expression level of NAT10 in the preparation of a product for diagnosing glioma / predicting the prognosis of glioma / diagnosing the grade of glioma.

[0006] Furthermore, the reagent is selected from a probe that specifically recognizes the NAT10 gene, a primer that specifically amplifies the NAT10 gene, or a binding agent that specifically binds to a protein encoded by the NAT10 gene.

[0007] Furthermore, the sequences of the primers are shown in SEQ ID NO:7-8.

[0008] Furthermore, the reagent also includes a detectable label.

[0009] Furthermore, the detectable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, and bioluminescent moieties.

[0010] Furthermore, the glioma is glioblastoma.

[0011] A second aspect of the present invention provides a product for diagnosing glioma / predicting glioma prognosis / diagnosing glioma grade, wherein the product comprises a reagent for detecting the expression level of NAT10.

[0012] Furthermore, the product includes a chip, a test kit, a test paper or a nucleic acid membrane strip.

[0013] Furthermore, the chip includes a gene chip and a protein chip.

[0014] Furthermore, the kit includes a gene detection kit and a protein detection kit.

[0015] Furthermore, the kit also includes reagents for detecting the expression level of NAT10 gene or protein by RT-PCR method, biochip detection method, Southern blotting method, in situ hybridization method, and immunoblotting method.

[0016] The third aspect of the present invention provides use of a NAT10 inhibitor in the preparation of a pharmaceutical composition for treating glioma.

[0017] Furthermore, the inhibitors include nucleic acid inhibitors, protein inhibitors, and compounds.

[0018] Furthermore, the inhibitor is selected from nucleic acid inhibitors.

[0019] Furthermore, the nucleic acid inhibitor is selected from shRNA.

[0020] Furthermore, the sequence of the shRNA is shown in any one of SEQ ID NOs: 3-6.

[0021] Furthermore, the glioma is glioblastoma.

[0022] A fourth aspect of the present invention provides a pharmaceutical composition comprising an inhibitor of NAT10.

[0023] Furthermore, the inhibitors include nucleic acid inhibitors, protein inhibitors, and compounds.

[0024] Furthermore, the inhibitor is selected from nucleic acid inhibitors.

[0025] Furthermore, the nucleic acid inhibitor is selected from shRNA.

[0026] Furthermore, the sequence of the shRNA is shown in any one of SEQ ID NOs: 3-6.

[0027] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0028] The fifth aspect of the present invention provides a method for screening candidate drugs for treating glioma, the method comprising: treating a culture system expressing or containing the NAT10 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the NAT10 gene or a protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the NAT10 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating glioma.

[0029] Furthermore, the glioma is glioblastoma.

[0030] The sixth aspect of the present invention provides the use of NAT10 as a target in screening candidate drugs for treating glioma.

[0031] Furthermore, the method for screening candidate drugs for treating glioma comprises: treating a culture system expressing or containing the NAT10 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the NAT10 gene or a protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the NAT10 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating glioma.

[0032] Furthermore, the glioma is glioblastoma.

[0033] The seventh aspect of the present invention provides a method for inhibiting glioma cell proliferation / migration in vitro, comprising administering an inhibitor of NAT10.

[0034] Furthermore, the inhibitor is selected from nucleic acid inhibitors.

[0035] Furthermore, the nucleic acid inhibitor is selected from shRNA.

[0036] Furthermore, the sequence of the shRNA is shown in any one of SEQ ID NOs: 3-6.

[0037] Furthermore, the glioma is glioblastoma.

[0038] Furthermore, the method is a non-therapeutic method.

[0039] An eighth aspect of the present invention provides a method for regulating epithelial-mesenchymal transition markers in vitro, the method comprising administering an inhibitor of NAT10.

[0040] Furthermore, the markers include any one or more of FN1, N-cadherin, Vimentin and Snail.

[0041] Furthermore, the inhibitor is selected from nucleic acid inhibitors.

[0042] Furthermore, the nucleic acid inhibitor is selected from shRNA.

[0043] Furthermore, the sequence of the shRNA is shown in any one of SEQ ID NOs: 3-6.

[0044] Furthermore, the glioma is glioblastoma.

[0045] The ninth aspect of the present invention provides the use of NAT10 in constructing a computational model for diagnosing glioma / predicting glioma prognosis / diagnosing glioma grading.

[0046] Furthermore, the glioma is glioblastoma.

[0047] The tenth aspect of the present invention provides the use of NAT10 in constructing a system / device for diagnosing glioma / predicting glioma prognosis / diagnosing glioma grading.

[0048] Furthermore, the glioma is glioblastoma.

[0049] Advantages and beneficial effects of the present invention:

[0050] This application first discovered that NAT10 is highly expressed in glioma tissues, and that high expression of NAT10 is positively correlated with tumor grade and poor prognosis in glioma patients. Inhibition of NAT10 can inhibit the proliferation, migration and invasion of glioma cells. This application provides new ideas for early diagnosis and effective treatment of gliomas, and has broad application prospects in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 These are the clinical pathological information diagrams of NAT10 in public databases, among which 1A is the NAT10 expression diagram in the CCGA database, 1B is the NAT10 expression diagram in the CPTAC database, and 1C is the patient survival diagram in the CGGA database;

[0052] Figure 2 2A is the correlation diagram between NAT10 and glioma prognosis, among which, 2B is the immunohistochemical score statistical diagram, 2C is the NAT10 protein expression diagram in glioma patient tissues, 2D is the immunohistochemical staining intensity score statistical diagram, and 2E is the overall survival diagram of glioma patients;

[0053] Figure 3 This is a graph showing the relationship between NAT10 staining and clinicopathological characteristics in 428 glioma patients;

[0054] Figure 4 4A is a graph showing the effect of NAT10 on the proliferation of GBM cells in vitro, wherein 4B is a graph showing the knockdown efficiency of NAT10 and the addition of Remodelin (20 μM for 24 h), 4C-F are statistical graphs showing the proliferation ability of cells after knockdown of NAT10 and the addition of Remodelin (20 μM for 24 h), and 4G-J are graphs showing the detection of CCK8 after knockdown of NAT10 and the addition of Remodelin (20 μM for 24 h);

[0055] Figure 55A-I are graphs showing the ability of NAT10 to promote GBM cell migration and invasion in vitro, wherein 5J-K are graphs showing the expression levels of FN1, N-cadherin, Vimentin and Snail after NAT10 knockdown or Remodelin (20 μM for 24 h) treatment in GBM cells, 5L-M are graphs showing the expression levels of FN1, N-cadherin, Vimentin and Snail in GBM cells after Remodelin (20 μM for 24 h) treatment;

[0056] Figure 6 These are diagrams showing that NAT10 promotes GBM malignant growth in vivo, where 6A is a live imaging diagram of transplanted tumor mice, and 6B is a statistical diagram of the fluorescence intensity of transplanted tumor mice. DETAILED DESCRIPTION

[0057] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0058] The present invention provides the use of a reagent for detecting the expression level of NAT10 in preparing a product for diagnosing glioma / predicting the prognosis of glioma / diagnosing the grade of glioma.

[0059] In some embodiments, NAT10 includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed NAT10, any form of NAT10 derived from processing in cells, and naturally occurring variants of NAT10 (e.g., splice variants or allelic variants). The term encompasses, for example, human NAT10 as well as NAT10 from any other vertebrate source, including mammals, such as NAT10 of primates and rodents (e.g., mice and rats), Gene ID: 55226.

[0060] In some embodiments, diagnosis and its variants refer to the discovery, judgment or cognition of the health state or condition of an individual based on one or more signs, symptoms, data or other information related to the individual. The health state of an individual can be diagnosed as healthy / normal (i.e., the absence of a disease or illness), or can be diagnosed as unhealthy / abnormal (i.e., the presence of a disease or illness or an assessment of a characteristic). Diagnosis and its variants include early detection of a disease in relation to a specific disease or illness; the characterization or classification of a disease; the discovery of the progression, cure or recurrence of a disease; the discovery of the response to a disease after treatment or therapy of an individual.

[0061] In some embodiments, prognosis refers to the expectation regarding medical development (e.g., likelihood of long-term survival, disease-free survival rate, etc.), including a positive prognosis or a negative prognosis, wherein the negative prognosis includes disease progression such as recurrence, glioma growth, metastasis, and drug-resistant mortality, and the positive prognosis includes disease remission such as a disease-free state, and disease improvement such as glioma regression or stabilization.

[0062] The reagent is selected from a probe that specifically recognizes the NAT10 gene, a primer that specifically amplifies the NAT10 gene, or a binding agent that specifically binds to a protein encoded by the NAT10 gene.

[0063] In some embodiments, a primer refers to a short nucleic acid sequence, which, as a nucleic acid sequence having a short free 3' terminal hydroxyl group (free 3' hydroxyl group), can form a base pair with a complementary template and serve as a starting point for replicating the template.

[0064] In some embodiments, a probe refers to a molecule that can bind to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, a probe generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as 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. The probe can be directly or indirectly labeled, including primers. Hybridization methods include, but are not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays.

[0065] In some embodiments, a binding agent refers to a naturally occurring or non-naturally occurring molecule that specifically binds to a target. Examples of specific binding agents include, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, and lipids.

[0066] In some embodiments, a binding agent that specifically binds to a protein encoded by the NAT10 gene is, for example, a receptor for protein NAT10, a lectin that binds to protein NAT10, an antibody to protein NAT10, a peptide body to protein NAT10, a bispecific dual binding agent or a bispecific antibody format. Specific examples of specific binding agents are peptides, peptide mimetics, aptamers, spiegelmers, darpins, ankyrin repeat proteins, Kunitz-type domains, antibodies.

[0067] The reagent also includes a detectable label.

[0068] In some embodiments, detectable label refers to a composition that can produce a detectable signal indicating the presence of a target polynucleotide in a determination sample. Suitable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties. Therefore, labeling is any composition that can be detected by a device or method, including but not limited to spectroscopy, photochemistry, biochemistry, immunochemistry, electricity, optics, chemical detection devices or any other suitable device. In some embodiments, labeling can be detected visually without the aid of a device.

[0069] Among them, radioactive isotopes include but are not limited to 3 H. 14 C. 35 S. 125 I. 131 I.

[0070] Enzymes include, but are not limited to, horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase.

[0071] Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors.

[0072] The products include chips, test kits, test strips or nucleic acid membrane strips.

[0073] In some embodiments, a kit refers to a set of components provided in the context of a system for sequencing nucleotides and / or isolating nucleotide sequences and / or diagnosing a subject with a disease or infection based on the presence, absence, and / or amount of expressed nucleotide sequences from a sample or cell.

[0074] In some embodiments, the kit includes a gene detection kit and a protein detection kit, wherein the gene detection kit includes a reagent or a chip for detecting the transcription level of the NAT10 gene, and the protein detection kit includes a reagent or a chip for detecting the expression level of the NAT10 protein.

[0075] In some embodiments, the kit further comprises one or more substances from the following group: a container, a positive control, a negative control, a buffer, a preservative, and a protein stabilizer.

[0076] The kit may also be accompanied by an instruction manual for use of the kit, which describes how to use the kit for testing, and how to use the test results to judge tumor development and select a treatment plan.

[0077] The components of the test kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the test kit usually include at least a vial, test tube, flask, PET bottle, syringe or other container, in which a component can be placed, and preferably, can be appropriately divided. When there is more than one component in the test kit, the test kit will usually also include a second, third or other additional container, in which additional components are placed separately. However, the components of different combinations can be contained in a vial. The test kit of the present application also usually includes a container for accommodating reactants, which is sealed for commercial sale. This container can include a plastic container of injection molding or blow molding, in which required vials can be retained.

[0078] In some embodiments, the chip includes a gene chip and a protein chip. The gene chip includes an oligonucleotide probe targeting the NAT10 gene for detecting the transcription level of the NAT10 gene, and the protein chip includes a specific binding agent for the NAT10 protein.

[0079] In some embodiments, the chip, kit, test paper or nucleic acid membrane strip described in the present application can be used to detect the expression level of multiple genes or proteins including NAT10 gene or protein and their expression products (e.g., multiple genes or proteins related to glioma). Detecting multiple markers of glioma simultaneously can greatly improve the accuracy of glioma diagnosis or prognosis prediction.

[0080] The present invention provides use of an inhibitor of NAT10 in preparing a pharmaceutical composition for treating glioma.

[0081] In some embodiments, the inhibitor refers to any substance that can reduce the activity of NAT10 protein, reduce the stability of NAT10 gene or protein, down-regulate the expression of NAT10 protein, reduce the effective action time of NAT10 protein, or inhibit the transcription and translation of NAT10 gene. These substances can be used in the present invention as substances useful for down-regulating NAT10, and thus can be used to prevent or treat glioma.

[0082] In some embodiments, the inhibitor includes a nucleic acid inhibitor and a protein inhibitor. The nucleic acid inhibitor is selected from: an interfering molecule that uses NAT10 or its transcript as a target sequence and can inhibit NAT10 gene expression or gene transcription, including but not limited to shRNA, siRNA, ribozyme, antisense oligonucleotide, or a construct that can express or form the shRNA, siRNA, ribozyme, antisense oligonucleotide. The protein inhibitor is selected from a substance that specifically binds to the NAT10 protein, such as an antibody or ligand that can inhibit the activity of the NAT10 protein.

[0083] The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0084] In some embodiments, a pharmaceutically acceptable carrier includes a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, which is involved in carrying or transporting the subject chemical from one organ or part of the body to another organ or part of the body. Various carriers are "acceptable" in the sense that they are compatible with the other components of the formulation and will not cause harm to the patient. Some examples of substances that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, etc. Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other nontoxic compatible substances used in pharmaceutical preparations.

[0085] In some embodiments, the pharmaceutical composition can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. Depending on the subject to be treated, the specific mode of administration, the amount of active ingredient that can be combined with a carrier material to produce a single dosage form can be different. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of inhibitor that produces a therapeutic effect. Typically, in addition to 100%, the amount ranges from about 1% to about 99% of the active ingredient, from about 5% to about 70%, or from about 10% to about 30%.

[0086] In the solid dosage forms of the present invention for oral administration (capsules, tablets, pills, lozenges, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or expanders, such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or gum arabic; (3) preservatives. Wetting agents such as glycerol; (4) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retardants such as paraffin; (6) absorption promoters such as quaternary ammonium compounds; (7) wetting agents such as acetyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical composition may also contain a buffer. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugars and high molecular weight polyethylene glycols.

[0087] Tablets can be prepared by compression or molding with one or more auxiliary ingredients. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surfactant or a dispersant. Molded tablets can be prepared by molding a mixture of powdered active ingredients moistened with an inert liquid diluent in a suitable machine.

[0088] In some embodiments, tablets and other solid dosage forms of pharmaceutical compositions, such as lozenges, capsules, pills and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings known in the field of pharmacy, and can also be used, for example, to provide different proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres of the desired release curve (profile), to be formulated so as to provide a sustained or controlled release of the active ingredient therein. They can be sterilized, for example, by filtering through a bacteria-retaining filter, or by introducing a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water, or by introducing some other sterile injectable medium immediately before use. These compositions can also optionally contain an opacifier, and can optionally be a composition that releases the active ingredient only or preferentially in a certain part of the gastrointestinal tract in a delayed manner. Examples of implantable compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microcapsule form, if appropriate, in microcapsule form with one or more of the above-mentioned excipients.

[0089] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0090] Example

[0091] 1. Materials and Methods

[0092] 1) Cell lines

[0093] U87 cells and LN229 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai).

[0094] 2) Animals

[0095] This study used 6-8 week old BALB / c nude mice purchased from Nanjing Jicui Pharmaceutical Co., Ltd. Thirty animals were purchased and housed in a barrier enclosure. All animal experiments followed the relevant regulations of the Experimental Animal Ethics Committee of Xuzhou Medical University.

[0096] 3) Clinical cases

[0097] The tissue microarray chips (TMA) used in the experiment were obtained from 428 glioma patients who underwent surgical treatment in the Affiliated Hospital of Xuzhou Medical University in China from 2008 to 2016. Clinical data information included patient age, gender, tumor grade, pathological type, etc. All patients signed informed consent for the collection of tissue samples for scientific research before surgery. The use of clinical samples was reviewed and approved by the Ethics Review Committee of the Affiliated Hospital of Xuzhou Medical University.

[0098] 4) Cell culture

[0099] U87 and LN229 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin and streptomycin under 5% CO 2 and cultured in a constant temperature incubator at 37°C.

[0100] 5) CCK-8 cell proliferation assay plate: The cells in each treatment group were digested and counted, resuspended in serum-containing medium, and 2000 GBM cells of knockdown NAT10, knockdown NAT10 and control group were inoculated in each well of a 96-well plate. 200 μL PBS liquid was added to the remaining wells on the four sides.

[0101] Detection: After the cells adhered to the wall, culture medium containing CCK-8 detection solution was added to each well (culture medium: CCK-8 solution volume = 9:1), placed in an incubator for 1.5 hours, and then placed in an ELISA reader for detection.

[0102] The absorbance of each sample at a wavelength of 450 nm was measured by an enzyme-labeled instrument. Statistical analysis.

[0103] 6) Cell clone formation experiment Glioma cells in the logarithmic growth phase were obtained, digested with trypsin to prepare single cell suspension, and then the cells were counted.

[0104] Cells of appropriate density were seeded into 6-well plates, and 3 replicate wells were set for each subgroup. An appropriate amount of culture medium containing 10% FBS and 1% PS was added to each well, and the well plate was placed in a 37°C, 5% CO 2 , saturated humidity constant temperature incubator for 2-3 weeks, and replace the culture medium every 2 days.

[0105] When clones visible to the naked eye appear, terminate the culture, discard the old culture medium, add an appropriate amount of 1× PBS solution and gently wash twice, then discard the 1× PBS solution.

[0106] Add an appropriate amount of 4% paraformaldehyde for fixation for 20 min, and discard the 4% paraformaldehyde.

[0107] Add an appropriate amount of crystal violet staining solution and stain for 15 minutes, then discard the crystal violet staining solution.

[0108] Use ddH 2 OAfter washing gently twice, leave it in the air to dry naturally.

[0109] Microscope photos were taken and the number of plate clones formed was recorded. ImageJ software was used to count and calculate the plate clone formation rate. The calculation formula was: plate clone formation rate = (number of clones / number of cells inoculated) × 100%. GraphPadPrism 9.0 was used for statistical analysis.

[0110] 7) Preparation for Transwell migration and invasion experiments: cells were treated accordingly, Matrigel and DMEM were mixed in a ratio of 1:9 in advance and spread into Transwell chambers, 40 μL per chamber, 100 μL of the mixture was added first and then 60 μL was aspirated to evenly distribute the mixture in the chamber, and the mixture was placed in a 4°C display cabinet for 30 min, and then placed in a cell culture incubator for 4-6 h to solidify for use in invasion experiments.

[0111] Take the experimental cells with good growth status for routine digestion and centrifugation, and discard the supernatant.

[0112] Add 1 ml of serum-free medium to resuspend, take a portion of the cell suspension and dilute it as needed before performing the above cell counting.

[0113] After the counting is completed, the corresponding cell system is configured. For example, the system required for U87 cell migration is 10,000 / 200μL, and the system required for invasion is 40,000 / 200μL; the system required for LN229 cell migration is 10,000 / 200μL, and the system required for invasion is 20,000 / 200μL.

[0114] Take a sterile 24-well plate and add 800 μL of culture medium containing 20% ​​fetal bovine serum to the required wells. Three chambers are required for each cell migration and invasion. Then slowly place the chamber into the 24-well plate, paying attention to whether there are bubbles on the contact surface between the chamber and the culture medium to avoid affecting the perforation process of the cells. (The chamber used for the invasion experiment should be aspirated to remove the residual liquid in advance, and the matrix membrane should be hydrated with serum-free culture medium).

[0115] Add 200 μL of the cell suspension of the corresponding system vertically to the upper chamber, then tap the 24-well plate to evenly distribute the cells in the upper chamber and place it in a cell culture incubator (37°C, 5% CO). 2 ) After 48 hours of routine culture, the chamber was harvested.

[0116] First, the chamber was washed twice with 1×PBS at room temperature, and then 800 μL of 4% paraformaldehyde was added to the empty wells of the 24-well plate. The chamber was placed in 4% paraformaldehyde and fixed at room temperature for 20 min.

[0117] After fixation, wash the chamber twice with room temperature 1× PBS, then add 800 μL of crystal violet staining solution to the empty wells of the 24-well plate, and place the chamber in the crystal violet staining solution for staining for 20 minutes.

[0118] After staining, wash the chamber twice with room temperature 1× PBS, and use a cotton swab to gently wipe the remaining crystal violet on the chamber, and place the chamber at room temperature to dry. After the chamber is dried, take pictures using an Olympus microscope and save them, and use Graphpad for statistical analysis.

[0119] 8) Pretreatment of nude mouse intracranial tumors: BALB / c nude mice aged 6-8 weeks were randomly divided into 6 groups, with 5 mice in each group, after being adapted to the environment of the animal center (SPF level) for 1 week; each model was divided into a control group (shNC) and a NAT10 knockdown group (shNAT10).

[0120] Construction of intracranial in situ model: Cell preparation: Prepare the required stable cells in advance, construct the stable cell line as before, and expand the culture; digest the cells with routine trypsin, centrifuge, add an appropriate amount of L15 medium to resuspend the cells to make a single cell suspension, dilute the cells according to a certain ratio, and count the cells under a microscope; aspirate the cells to make the density reach 16×10 7 The nude mice were placed in an air anesthesia cage and anesthetized with flurane. After disinfection of the skin, a 1 cm incision was made along the middle. A drill was used to drill 2 mm to the right of the bregma of the nude mouse skull and 1 mm in front of the bregma and coronal suture. The nude mice were fixed on a stereotaxic device. The cell suspension in the 1.5 ml EP tube was mixed and sucked with a microinjector and injected into the right striatum. Each nude mouse was injected with 5 μL (i.e., 8×10 6 cells), the scalp of the nude mice was sutured after injection, and the inoculation date and cell type were marked on the mouse cage; the growth status of the intracranial tumor was observed 0, 7, 14, and 21 days after the in situ implantation, and potassium luciferin was injected into the groin of the nude mice. After the reaction for 3-5 minutes, the mice were anesthetized and placed in a living imager to observe the fluorescence value and take pictures for analysis; when the nude mice showed cachexia, hemiplegia, mental confusion and other neurological symptoms, they were killed after anesthesia, and the brains of the mice were placed in a neutral fixative for tissue fixation for subsequent use.

[0121] 9) Immunohistochemistry (IHC) staining

[0122] Baking: Place paraffin sections in a 60°C oven and bake overnight.

[0123] Dewaxing and hydration: After the tissue sections are baked, they are dewaxed and placed in xylene I for 30 minutes, and then placed in xylene II for hydration for 30 minutes; then immersed in anhydrous ethanol I and II, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each.

[0124] To remove endogenous peroxidase, add 3% HO to each section. 2 O 2 Cover the tissue and place in a light-tight humidified box for 10 min to remove peroxidase.

[0125] Place in a horizontal shaker and wash with 1× PBS solution for 5 min each time, for a total of 3 times.

[0126] Antigen repair (high temperature and high pressure repair): Prepare EDTA (pH 8.0) or citric acid (pH 6.0) repair solution in advance. The amount should be enough to cover the slices. Heat the pressure cooker with the lid open until boiling, put the slices in, cover the lid, and start the antigen repair process for 3 minutes after the valve starts to spray air.

[0127] After the repair is completed, turn off the furnace to reduce the pressure, and after the pressure returns to normal, open the lid and cool to room temperature. Then wash with 1× PBS solution, 5 minutes each time, for a total of 3 times.

[0128] Blocking: Block the sections with 1× goat serum for 30 min in a humidified chamber.

[0129] Primary antibody incubation: dilute the antibody with 10×PBS according to the antibody usage ratio, add 50 μL of the diluted antibody to each histochemical slide in a wet box, and incubate overnight at 4°C.

[0130] On the next day, the wet box was placed at room temperature for 30 min and washed with 1× PBS solution on a shaker for 5 min each time.

[0131] Secondary antibody incubation: Prepare the corresponding secondary antibody according to the species of the primary antibody, incubate in the strengthening solution at room temperature for 30 minutes, wash twice with PBS, add the secondary antibody, and incubate at room temperature for 30 minutes.

[0132] Prepare DAB colorimetric solution: add 1 drop of solution B to 1 mL of solution A, mix well and store in dark place. Prepare immediately before use.

[0133] DAB color development: shake off the secondary antibody, rinse with PBS, add color development solution to the slice, and observe under the microscope while color development. When brown particles appear on the slice, soak it in distilled water to stop color development.

[0134] Hematoxylin staining: Soak the tissue sections in hematoxylin and pull them up and down 2 to 3 times.

[0135] Dehydration and sealing: Dehydrate in the reverse order of step (1), seal with gum, observe under an optical microscope, and take photos for analysis.

[0136] 10) IHC scoring criteria

[0137] The staining score of NAT10 was scored independently by two professionals in a double-blind manner. The staining was quantified according to the degree of positive cell staining and the percentage of staining. The staining intensity ranged from 0 to 3 (0 = negative, 1 = weak, 2 = moderate, 3 = strong), and the percentage of positive cells was also divided into 4 categories: 1 (0% to 25%), 2 (26% to 50%), 3 (51% to 75%), and 4 (76% to 100%). The IHC score level of NAT10 staining was detected by immunohistochemistry. The score of staining intensity was multiplied by the score of positive cell percentage to calculate the IRS score. According to IRS, it was divided into low (IRS: 0 to 6) and high (IRS: 8 to 12).

[0138] 11) Data analysis

[0139] SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8 were used for statistical analysis. All data are presented as mean ± SEM, and p < 0.05 was considered statistically significant. The chi-square test was used to analyze the correlation factors between NAT10 and glioma patients, and the Kaplan-Meier and LOG-RANK tests were used to evaluate the correlation between NAT10 expression and the survival of glioma patients. The t-test was used to analyze the differences between different groups.

[0140] 2. Experimental results

[0141] NAT10 is highly expressed in gliomas and positively correlated with malignancy and poor prognosis

[0142] In order to explore the role of NAT10 in the malignant progression of glioma, we first searched for the expression of NAT10 in the CPTAC database and the CGGA database. The results showed that high expression of NAT10 in primary glioma was positively correlated with the poor prognosis of patients, and the expression of NAT10 was positively correlated with the grade of glioma ( Figure 1 Then, the expression level of NAT10 in normal human brain tissue and human glioma tissue was detected by immunohistochemistry and western blot. Figure 2 A-2C). It was found that NAT10 was significantly overexpressed in tumor tissues, and these data were consistent with the results of the analysis of the CPTAC database and the CGGA database. The above data show that NAT10 is highly expressed in glioma tissues compared with normal brain tissues.

[0143] Next, the expression of NAT10 in glioma patients was analyzed using glioma tissue microarray (TMA), which contained 428 glioma samples with clinical information and prognostic data. The results of immunohistochemistry showed that the expression of NAT10 was positively correlated with the WHO grade of glioma, but not with gender, age, and pathological classification ( Figure 3 ). It was also found that the expression of NAT10 in patients with high-grade gliomas (grades III and IV) was significantly higher than that in patients with low-grade gliomas (grades I and II) ( Figure 2 D). In addition, the data of this application also show that the overall survival time of glioma patients with high NAT10 expression is shorter than that of patients with low NAT10 expression ( Figure 2 E). In summary, the data of the present application strongly prove that NAT10 is correlated with the malignancy of glioma and the poor overall survival of patients.

[0144] NAT10 promotes GBM cell proliferation in vitro

[0145] The above results indicate that NAT10 is highly expressed in GBM patients. Next, we explored whether NAT10 affects the malignant progression of GBM. In order to study the effect of NAT10 on the proliferation ability of GBM cells, NAT10 shRNA (shRNA sequence is shown in Table 1, primer sequence is shown in Table 2) was used to knock down the expression of NAT10 in LN229 and U87 cells, and then the knockdown efficiency of NAT10 was verified by Western blot experiments. The CCK-8 kit test showed that knocking down NAT10 could inhibit the proliferation ability of LN229 and U87 cells. Similarly, the clone formation experiment also showed that knocking down NAT10 reduced the clone formation ability of LN229 and U87 cells ( Figure 4 ).

[0146] Table 1 shRNA sequences of NAT10

[0147]

[0148] Table 2 NAT10 primer sequences

[0149]

[0150] Remodelin is a small molecule compound that can specifically target and inhibit NAT10. In order to verify the effect of Remodelin on cell proliferation, this inhibitor was added to LN229 cells and U87 cells for culture, and then clone formation experiments and CCK-8 kit tests were also performed. The results showed that Remodelin can inhibit the proliferation of LN229 and U87 cells ( Figure 4 ). The above results suggest that NAT10 can inhibit the proliferation of GBM cells in vitro.

[0151] NAT10 promotes GBM cell migration and invasion in vitro

[0152] This application also evaluates the role of NAT10 in regulating GBM cell migration and invasion. NAT10 was knocked down in LN229 and U87 cells, and then Transwell experiments were performed. The results confirmed that NAT10 knockdown weakened the migration and invasion ability of LN229 and U87 cells. Remodelin was also used to treat GBM cells and then Transwell experiments were performed. The data showed that Remodelin inhibited the migration and invasion ability of LN229 and U87 cells ( Figure 5 ).

[0153] Next, in order to explore the mechanism by which NA10 promotes GBM cell migration and invasion, some classic markers that have been confirmed in epithelial-mesenchymal transition were detected. It was found that in LN229 and U87 cells, after knocking down NAT10, the expression of FN1, N-cadherin, Vimentin and Snail was significantly downregulated. At the same time, it was also found that after Remodelin treatment, the expression of FN1, N-cadherin, Vimentin and Snail in LN229 and U87 cells was also significantly downregulated ( Figure 5 ). In summary, the research of this application suggests that NAT10 increases the invasiveness of GBM cells in vitro through EMT.

[0154] NAT10 promotes GBM progression in vivo

[0155] Next, the effect of NAT10 on the in vivo growth of GBM tumors was studied. First, the same number of U87-shNC, U87-shNAT10#1 and U87-shNAT10#2 cells were injected in situ into the brains of nude mice, and the immunofluorescence intensity was detected and recorded every 7 days. When the mice showed cachexia, hemiplegia, delirium and other neurological symptoms, the mice were killed, and the brain tissue was taken for immunohistochemical analysis. The data showed that the fluorescence intensity of the U87-shNAT10 group was much weaker than that of the U373-shNC group. At the same time, the same number of LN229-shNC, LN229-shNAT10#1 and LN229-shNAT10#2 cells were also injected into the brains of nude mice, and the in vivo growth of the tumor was observed using the same method, and brain tissue was also taken for immunohistochemical analysis. The data showed that LN229

[0156] The fluorescence intensity of the -shNAT10 group was much weaker than that of the LN229-shNC group ( Figure 6 ). These data suggest that NAT10 can promote GBM progression in vivo.

[0157] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of reagents for detecting NAT10 expression levels in the preparation of products for diagnosing gliomas / predicting glioma prognosis / diagnosing glioma grading; Preferably, the reagent is selected from a probe that specifically recognizes the NAT10 gene, a primer that specifically amplifies the NAT10 gene, or a binding agent that specifically binds to a protein encoded by the NAT10 gene; Preferably, the sequence of the primer is as shown in SEQ ID NO:7-8; Preferably, the reagent further comprises a detectable label; Preferably, the detectable label comprises a radioisotope, a nucleotide chromophore, an enzyme, a substrate, a fluorescent molecule, a chemiluminescent moiety, a magnetic particle, a bioluminescent moiety; Preferably, the glioma is glioblastoma.

2. A product for diagnosing glioma / predicting glioma prognosis / diagnosing glioma grading, characterized in that: The product includes a reagent for detecting the expression level of NAT10; Preferably, the product comprises a chip, a test kit, a test paper or a nucleic acid membrane strip; Preferably, the chip includes a gene chip and a protein chip; Preferably, the kit includes a gene detection kit and a protein detection kit; Preferably, the kit further comprises a reagent for detecting the expression level of NAT10 gene or protein by RT-PCR, biochip detection, Southern blotting, in situ hybridization, or immunoblotting.

3. Application of NAT10 inhibitors in the preparation of pharmaceutical compositions for treating gliomas; Preferably, the inhibitor includes a nucleic acid inhibitor, a protein inhibitor, or a compound; Preferably, the inhibitor is selected from nucleic acid inhibitors; Preferably, the nucleic acid inhibitor is selected from shRNA; Preferably, the sequence of the shRNA is shown in any one of SEQ ID NOs: 3-6; Preferably, the glioma is glioblastoma.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an inhibitor of NAT10; preferably, the inhibitor comprises a nucleic acid inhibitor, a protein inhibitor, or a compound; Preferably, the inhibitor is selected from nucleic acid inhibitors; Preferably, the nucleic acid inhibitor is selected from shRNA; Preferably, the sequence of the shRNA is shown in any one of SEQ ID NOs: 3-6; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

5. A method for screening candidate drugs for treating glioma, characterized in that: The method comprises: treating a culture system expressing or containing a NAT10 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the NAT10 gene or a protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the NAT10 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating glioma; Preferably, the glioma is glioblastoma.

6. Application of NAT10 as a target in screening candidate drugs for the treatment of glioma; Preferably, the method for screening candidate drugs for treating glioma comprises: treating a culture system expressing or containing the NAT10 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the NAT10 gene or the protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the NAT10 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating glioma; Preferably, the glioma is glioblastoma.

7. A method for inhibiting glioma cell proliferation / migration in vitro, characterized in that: The method comprises administering an inhibitor of NAT10; Preferably, the inhibitor is selected from nucleic acid inhibitors; Preferably, the nucleic acid inhibitor is selected from shRNA; Preferably, the sequence of the shRNA is shown in any one of SEQ ID NOs: 3-6; Preferably, the glioma is glioblastoma.

8. A method for regulating epithelial-mesenchymal transition markers in vitro, characterized in that: The method comprises administering an inhibitor of NAT10; Preferably, the marker comprises any one or more of FN1, N-cadherin, Vimentin and Snail; preferably, the inhibitor is selected from nucleic acid inhibitors; Preferably, the nucleic acid inhibitor is selected from shRNA; Preferably, the sequence of the shRNA is shown in any one of SEQ ID NOs: 3-6; Preferably, the glioma is glioblastoma.

9. Application of NAT10 in constructing computational models for diagnosing gliomas / predicting glioma prognosis / diagnosing glioma grading; Preferably, the glioma is glioblastoma.

10. Application of NAT10 in constructing a system / device for diagnosing glioma / predicting glioma prognosis / diagnosing glioma grading; Preferably, the glioma is glioblastoma.

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