Application of NAT10 in regulation and control of PIK3R2 and treatment of tumors

Abstract

The invention discloses an application of NAT10 in regulation and control of PIK3R2 and treatment of tumors. It is found for the first time that NAT10 up-regulates ac4C modification of PIK3R2 by directly acting on PIK3R2, the stability of PIK3R2 mRNA is improved, and therefore malignant progression and wettability of glioblastoma are promoted. The invention also proves that the expression of NAT10 and PIK3R2 in glioma patients is positively correlated. According to the application, a new insight is provided for an abnormal EMT mechanism in the glioblastoma, and a theoretical support is also provided for taking the NAT10 as a potential diagnosis and treatment target of the glioblastoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of NAT10 in regulating PIK3R2 and treating tumors. Background Art

[0002] NAT10 is currently the only known acetyltransferase that mediates ac4C modification in eukaryotes. It modifies tRNA with the help of the Tan1 / THUMPD1 adapter to stabilize its structure. It has been reported that it promotes metastasis by regulating the stability of NOTCH3mRNA in cancer, and enhances tumor resistance to chemotherapy and radiotherapy by upregulating MORC2 expression levels through protein acetylation modification in breast cancer. In bladder cancer, the reduction of ac4C modification mediated by NAT10 can inhibit the stability and translation efficiency of BCL9L, SOX4 and AKT1, thereby inhibiting tumor progression. In addition, it also plays a role in promoting tumor progression in breast cancer, gastric cancer, and head and neck squamous cell carcinoma. However, the role of NAT10 in glioblastoma has not been reported, so exploring the role of NAT10 in glioma and its regulatory mechanism is of great significance for the 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 regulating PIK3R2 and in treating glioma.

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

[0005] The first aspect of the present invention provides use of an agent for inhibiting the binding between NAT10 and PIK3R2 in the preparation of a drug for treating tumors.

[0006] Furthermore, the action site of the agent that inhibits the binding between NAT10 and PIK3R2 is on the acetylation site of PIK3R2.

[0007] Furthermore, the tumor is selected from glioma.

[0008] Furthermore, the glioma is selected from glioblastoma.

[0009] A second aspect of the present invention provides a drug for treating a tumor, the drug comprising an agent that inhibits the binding between NAT10 and PIK3R2.

[0010] Furthermore, the action site of the agent that inhibits the binding between NAT10 and PIK3R2 is on the acetylation site of PIK3R2.

[0011] Furthermore, the tumor is selected from glioma.

[0012] Furthermore, the glioma is selected from glioblastoma.

[0013] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0014] The third aspect of the present invention provides a complex comprising NAT10 and PIK3R2.

[0015] Furthermore, the binding site of NAT10 and PIK3R2 is on the acetylation site of PIK3R2.

[0016] The fourth aspect of the present invention provides use of the complex described in the third aspect of the present invention in screening candidate drugs for treating tumors.

[0017] Further, the method for screening candidate drugs for treating tumors includes:

[0018] (1) contacting a substance to be detected with a system containing a complex formed by NAT10 and PIK3R2;

[0019] (2) detecting the binding level of NAT10 and PIK3R2 in the system;

[0020] (3) Select substances that can reduce the binding level of NAT10 and PIK3R2 as candidate drugs for treating or preventing cancer.

[0021] Furthermore, the tumor is selected from glioma.

[0022] Furthermore, the glioma is selected from glioblastoma.

[0023] The fifth aspect of the present invention provides use of the complex described in the third aspect of the present invention in screening agents that inhibit the binding between NAT10 and PIK3R2.

[0024] A sixth aspect of the present invention provides a method for screening candidate drugs for treating tumors, the method comprising:

[0025] (1) contacting a substance to be detected with a system containing a complex formed by NAT10 and PIK3R2;

[0026] (2) detecting the binding level of NAT10 and PIK3R2 in the system;

[0027] (3) Select substances that can reduce the binding level of NAT10 and PIK3R2 as candidate drugs for treating or preventing cancer.

[0028] Furthermore, the tumor is selected from glioma.

[0029] Furthermore, the glioma is selected from glioblastoma.

[0030] A seventh aspect of the present invention provides a method for regulating the expression level of PIK3R2 in a cell, the method comprising changing the expression of NAT10 in the cell.

[0031] Furthermore, the changing the expression of NAT10 in the cell is inhibiting the expression of NAT10 in the cell.

[0032] Furthermore, inhibiting the expression of NAT10 in the cell comprises administering an inhibitor of NAT10 to the cell.

[0033] Furthermore, the inhibitor is selected from nucleic acid inhibitors.

[0034] Furthermore, the nucleic acid inhibitor is shRNA.

[0035] Furthermore, the sequence of the shRNA is shown in any one of SEQ ID NOs: 3-6.

[0036] Furthermore, the cells are glioma cells.

[0037] Furthermore, the glioma cells are glioblastoma cells.

[0038] Furthermore, the glioblastoma cells include U87 cells and / or LN229 cells.

[0039] An eighth aspect of the present invention provides a method for constructing a cell model of PIK3R2 overexpression / underexpression, the method comprising regulating the expression of NAT10 in cells.

[0040] Furthermore, regulating the expression of NAT10 in the cell includes inhibiting the expression of NAT10 in the cell.

[0041] Furthermore, inhibiting the expression of NAT10 in the cell comprises administering an inhibitor of NAT10 to the cell.

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

[0043] Furthermore, the nucleic acid inhibitor is shRNA.

[0044] Furthermore, the sequence of the shRNA is shown in any one of SEQ ID NOs: 3-6.

[0045] A ninth aspect of the present invention provides a method for computer-assisted drug screening based on NAT10 and PIK3R2, the method comprising:

[0046] Obtain NAT10 protein and PIK3R2 protein data;

[0047] Determine the binding sites of the two proteins based on the data of the NAT10 protein and the PIK3R2 protein;

[0048] Candidate drugs targeting the binding sites of the two were obtained through computer-assisted screening.

[0049] Further, the process of computer-assisted screening is:

[0050] Screening of small molecule compounds based on the spatial structure of the binding sites of NAT10 protein and PIK3R2 protein;

[0051] The small molecule compounds obtained by screening are molecularly docked with the binding sites to obtain the scores of the docking molecules;

[0052] The candidate drugs are obtained by sorting according to the scores.

[0053] Further, the process of computer-assisted screening is:

[0054] The molecular structures of NAT10 protein and PIK3R2 protein are obtained and input into the pharmacophore module library for matching, and all the action sites are clustered based on the interaction patterns with NAT10 and PIK3R2 to obtain a pharmacophore model;

[0055] The pharmacophore model is input into a molecular compound database for high throughput screening to obtain candidate drugs.

[0056] Further, the process of computer-assisted screening is:

[0057] Obtaining the molecular structure of a small molecule inhibitor that inhibits NAT10 and / or the molecular structure of a small molecule inhibitor that inhibits PIK3R2;

[0058] Based on the molecular structure of the small molecule inhibitor, a small molecule library with similar structure is obtained by screening;

[0059] Performing molecular docking on the small molecule library with similar structures and the binding site to obtain a score of the docked molecule;

[0060] The candidate drugs are obtained by sorting according to the scores.

[0061] Furthermore, the candidate drugs include protein analogs, antibodies, and RNA drugs.

[0062] The tenth aspect of the present invention provides a system for computer-assisted drug screening based on NAT10 and PIK3R2, the system comprising:

[0063] Acquisition unit: Acquisition of NAT10 protein and PIK3R2 protein data;

[0064] A site determination unit: determining the binding site of the two proteins according to the data of the NAT10 protein and the PIK3R2 protein;

[0065] Screening unit: Obtain candidate drugs targeting the binding sites of the two through computer-assisted screening.

[0066] The eleventh aspect of the present invention provides a device for computer-assisted drug screening based on NAT10 and PIK3R2, the device comprising:

[0067] A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the computer-assisted drug screening method based on NAT10 and PIK3R2 described in the ninth aspect of the present invention is implemented.

[0068] A twelfth aspect of the present invention provides a computer-readable storage medium having a computer program thereon, the computer-readable storage medium comprising:

[0069] When the computer program is executed by a processor, the method for computer-assisted drug screening based on NAT10 and PIK3R2 described in the ninth aspect of the present invention is implemented.

[0070] Advantages and beneficial effects of the present invention:

[0071] This application first discovered that NAT10 directly acts on PIK3R2 to upregulate its ac4C modification, thereby increasing the stability of PIK3R2 mRNA and promoting the malignant progression and invasiveness of glioblastoma. It also confirmed that the expression of NAT10 and PIK3R2 in glioma patients is positively correlated. This application provides new insights into the abnormal EMT mechanism in glioblastoma and provides theoretical support for NAT10 as a potential diagnostic and therapeutic target for glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 is a graph showing that NAT10 positively regulates PIK3R2 expression in GBM, where 1A and 1B are graphs showing changes in RNAac4C levels after knocking down NAT10, 1C is a Venn diagram of RIP-seq and acRIP-seq of Hela and BxCP-3 cells in the database, 1D is a graph showing gene changes after knocking down NAT10, and 1E and 1F are graphs showing changes in PIK3R2 expression levels after knocking down NAT10;

[0073] Figure 2 Figure 2 is the diagram of NAT10 affecting the activation of the PI3K / AKT pathway, where 2A and 2B are the p-AKT (Ser473), PIK3R2 and AKT levels after knocking down NAT10, and 2C and 2D are the p-AKT (Ser473), PIK3R2 and AKT protein level changes after Remodelin (20 μM for 24 h) treatment;

[0074] Figure 3 Figure 3A and 3B are diagrams showing PIK3R2 regulation by ac4C after NAT10 knocked down PIK3R2 by constructing mutant plasmids after predicting ac4C modification sites of PIK3R2 on the PACES website, and then performing RNA-Pull Down to detect specific modification sites, 3C is a diagram showing the relative mRNA abundance of PIK3R2, 3D is a diagram showing the expression of NAT10 and PIK3R2 after NAT10-IP, 3E and 3F are diagrams showing the mRNA levels of PIK3R2 modified by ac4C in GBM cells after knocking down NAT10 by ac4C-RIP, and 3G and 3H are diagrams showing the relative mRNA expression of PIK3R2 after knocking down NAT10 and adding actinomycin D (200 nM);

[0075] Figure 4 NAT10 is involved in the malignant process of GBM through PIK3R2, among which 4A and 4B are

[0076] Protein expression of p-AKT (Ser473), AKT, FN1, N-cadherin, Vimentin and Snail in U87 / LN229-shNC / shNAT10 / PIK3R2 / shNAT10+PIK3R2 cells. 4C-F are

[0077] Figure 4G-H shows the migration and invasion ability of U87 / LN229-shNC / shNAT10 / PIK3R2 / shNAT10+PIK3R2 cells.

[0078] Figure 5 It is the expression diagram of NAT10 and PIK3R2 in in situ tumor tissue detected by immunohistochemistry;

[0079] Figure 6 Figure 6 is a correlation diagram between the NAT10-PIK3R2 cascade reaction and the clinical pathological characteristics of patients with brain glioma. Among them, 6A is a representative diagram of NAT10 and PIK3R2 expression in low-grade and high-grade tumor tissue samples, 6B is an integral and correlation diagram of NAT10 and PIK3R2 staining, 6C is a correlation diagram of NAT10 and PIK3R2 in glioma samples, and 6D is a diagram of the mechanism by which NAT10 directly regulates PIK3R2 through ac4C, leading to EMT and promoting the malignant development of GBM. DETAILED DESCRIPTION

[0080] 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.

[0081] The present invention provides use of an agent for inhibiting the binding between NAT10 and PIK3R2 in preparing a drug for treating tumors.

[0082] In some embodiments, NAT10 / PIK3R2 includes wild type, mutant or fragment thereof. The term encompasses full-length, unprocessed NAT10, any form of NAT10 / PIK3R2 derived from processing in cells, and naturally occurring variants (e.g., splice variants or allelic variants) of NAT10 / PIK3R2. The term encompasses, for example, human NAT10 / PIK3R2 and NAT10 / PIK3R2 from any other vertebrate source, including mammals, such as NAT10 / PIK3R2 of primates and rodents (e.g., mice and rats), NAT10 gene ID: 55226, PIK3R2 gene ID: 5296.

[0083] In some embodiments, agents that inhibit the binding of NAT10 and PIK3R2 include but are not limited to NAT10 inhibitors and PIK3R2 inhibitors, which reduce the binding of NAT10 and PIK3R2 and reduce the content of PIK3R2 in cells, thereby limiting tumor development and achieving the purpose of treatment.

[0084] In a specific embodiment, the action site of the agent that inhibits the binding between NAT10 and PIK3R2 is on the acetylation site of PIK3R2, and the agent that inhibits the binding between NAT10 and PIK3R2 is an inhibitor of NAT10.

[0085] The drug also includes pharmaceutically acceptable excipients.

[0086] In some embodiments, pharmaceutically acceptable excipients include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. These media and solvents are well known in the art for pharmaceutically active substances. Unless any conventional media or medicaments are incompatible with the active ingredient so far, its use in therapeutic compositions is contemplated. In addition, various adjuvants such as those commonly used in the art may be included.

[0087] Examples of pharmaceutically acceptable excipients include sugars, e.g., lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and methylcellulose; powdered yarrow; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa oil; polyols, such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as TWEENS; wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffered solution.

[0088] The choice of pharmaceutically acceptable excipients depends essentially on the way in which the active substance is to be administered.

[0089] The medicine described in the present application is preferably provided in unit dosage form. As used in the present application, a unit dosage form is a composition containing a certain amount of medicine, suitable for administering to animals with a single dose according to good pharmaceutical practice, preferably mammalian subjects. However, the preparation of a single dose or unit dosage form does not mean that the dosage form is administered once a day or once per course of treatment. Some dosage forms are considered to be administered once, twice, three times or more per day, and can be administered with infusion within a period of time (e.g., from about 30 minutes to about 2-6 hours), or administered with continuous infusion, and can be given more than once in a course of treatment, although single administration is not particularly excluded. The technician will recognize that the preparation does not particularly consider the entire course of treatment, and these decisions are left to those therapeutic areas rather than the technicians in the field of formulations.

[0090] The useful medicine described above can be any one of a variety of suitable forms for a variety of routes of administration, for example, for oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intraarterial, intravenous, intramuscular or other parenteral routes of administration. It will be appreciated by the technician that oral and nasal compositions include compositions administered by inhalation and are prepared using available methods. Depending on the desired specific mode of administration, a variety of pharmaceutically acceptable adjuvants well known in the art can be used. Pharmaceutically acceptable adjuvants include, for example, solid or liquid fillers, diluents, cosolvents, surfactants and encapsulating materials. Optional pharmaceutically active materials can be included that do not substantially affect the inhibitory activity of the medicine. The amount of the adjuvant used together with the medicine is sufficient to provide the actual amount of material per unit dose for administering the medicine.

[0091] In some embodiments, treatment / prevention may refer to therapeutic treatment or preventive measures, wherein the goal is to prevent or slow down (mitigate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical outcome. In the present application, treatment / prevention may refer to both treatment and prevention. Beneficial or desired clinical outcomes include, but are not limited to, relieving symptoms; alleviating the extent of a disorder, disorder or disease; stabilizing (i.e., not worsening) the state of a disorder, disorder or disease; delaying the occurrence of a disorder, disorder or disease or slowing its progression; improving a disorder, disorder or disease state; and alleviating (whether partial or complete) (whether detectable or undetectable) or improving or ameliorating a disorder, disorder or disease. Treatment may include causing a clinically significant response without excessive side effects. Treatment also includes a survival period that is prolonged compared to the expected survival period when not receiving treatment.

[0092] The present invention provides a method for computer-assisted drug screening based on NAT10 and PIK3R2, the method comprising:

[0093] Obtain NAT10 protein and PIK3R2 protein data;

[0094] Determine the binding sites of the two proteins based on the data of the NAT10 protein and the PIK3R2 protein;

[0095] Candidate drugs targeting the binding sites of the two were obtained through computer-assisted screening.

[0096] In some embodiments, the molecular libraries used in virtual screening mainly include the following:

[0097] ZINC: contains more than 250 million commercially available compounds for small molecule virtual screening.

[0098] PubChem: contains bioactive substances for small molecule virtual screening.

[0099] DrugBank: Contains drugs and small molecules for drug design and discovery.

[0100] ChEMBL: Contains small molecules and is used for drug discovery and chemical genomics research.

[0101] ChemDB: Contains a large number of known small molecules for chemical genomics research and drug discovery.

[0102] HMDB: Contains a large number of known small molecules for use in chemical genomics research and drug discovery.

[0103] BindingDB: Contains a large number of known small molecules for chemical genomics research and drug discovery.

[0104] SMPDB: Contains a large number of known small molecules for chemical genomics research and drug discovery.

[0105] In addition, there are some commercial databases such as ChemDiv, Enamine, Lifechemicals, Specs, Chembridge, Maybridge, Microsource, Vitas-M and Interbioscreen, which are also often used for virtual screening.

[0106] In some embodiments, in molecular docking, affinity refers to how tightly a molecule binds to a receptor. A high affinity means that the binding is more stable, while a low affinity means that the binding is less stable. Affinity is usually calculated, for example, by calculating the binding free energy (ΔG) or the binding constant (Kd).

[0107] In the process of molecular docking, affinity depends on the interaction between molecules and receptors, including hydrogen bonds, van der Waals forces, electrostatic interactions, etc. These interactions jointly determine the binding mode between molecules and receptors, thus affecting affinity.

[0108] In order to evaluate affinity, some scoring systems or scoring methods are usually used to quantify the interaction between molecules and receptors. These scoring methods are based on different algorithms and physical models, which can reflect the binding energy, interaction type and affinity between molecules and receptors.

[0109] The compounds include protein analogs, antibodies, DNA, and RNA.

[0110] In some embodiments, protein analogs refer to substances that have similar structures and functions to certain proteins in the body. They can mimic the functions of proteins and thus play an important role in the treatment of diseases. For example, insulin is a protein analog used to treat diabetes.

[0111] Antibodies are immunoglobulins produced by plasma cells differentiated from B lymphocytes under antigen stimulation, which can specifically bind to the corresponding antigen. The structure of antibodies is mainly divided into two parts: constant region and variable region. In a given species, the constant regions of different antibody molecules have the same or almost the same amino acid sequence. The variable region is located at the ends of the two arms of the "Y".

[0112] RNA drugs are a class of drugs designed based on the characteristics of RNA and its mechanism of action in cells. RNA can act as messenger RNA (mRNA) to guide protein synthesis in cells, or as microRNA (miRNA) to regulate gene expression.

[0113] Therefore, rationally designed RNA drugs can be used to regulate gene expression in cells to achieve the purpose of treating diseases. For example, many studies are exploring the use of mRNA vaccines to prevent and treat various infectious diseases.

[0114] DNA medicine is a new type of treatment developed based on genetic engineering technology. It aims to achieve therapeutic goals by affecting the structure and function of DNA, interfering with nucleic acid biosynthesis or transcription process.

[0115] 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.

[0116] Example

[0117] 1. Materials and Methods

[0118] 1) Cell lines

[0119] U87 cells, LN229 cells and HEK293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai).

[0120] 2) Animals

[0121] 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.

[0122] 3) RNA pulldown experiment

[0123] A PIK3R2 probe labeled with 3' biotin was designed and synthesized at Nanjing Genewise Co., Ltd. The PIK3R2 probe, i.e., sense probe, and the negative control probe, i.e., antisense probe, were diluted and dissolved to 100 μM using nuclease-free water.

[0124] Thaw the protease inhibitor cocktail and RNAse inhibitor at room temperature in advance, and take out the Western and IP lysis buffer for use. First, mix the protease inhibitor cocktail with the Western and IP lysis buffer at a ratio of 1:99, and then add the RNAse inhibitor so that the concentration of the RNAse inhibitor reaches 1U per microliter. The above mixture is used as the lysis buffer for standby use. This experiment requires at least 20-30 million cells. Prepare the cells in advance, use trypsin to digest the cells, collect the cell pellet by centrifugation, add 1ml lysis buffer, use a 1ml pipette to repeatedly blow the cells, and then use a 1ml syringe to repeatedly aspirate the cell lysis buffer on ice. Finally, place the cell lysis buffer on a 4-degree Ferris wheel for half an hour to achieve the purpose of gentle cell lysis. After the lysis is completed, centrifuge at 13000rpm for 15min. After the centrifugation is completed, aspirate the supernatant, and add 1 microliter of the diluted PIK3R2 probe and antisense probe respectively, and place it on a 4-degree Ferris wheel to rotate for more than 6 hours or overnight to allow the lysis buffer to fully hybridize with the probe. After the hybridization is completed, 50 microliters of streptavidin magnetic beads are added and placed on a 4-degree Ferris wheel for further incubation for more than 3 hours to allow the magnetic beads to fully bind to the biotin probe.

[0125] After incubation, place the EP tube on the magnetic stand, discard the supernatant, add 500 microliters of lysis solution to wash the magnetic beads: place on the Ferris wheel and rotate for 5 minutes, repeat the washing three times. After washing, add 60 microliters of 2×SDS-PAGE buffer loading solution, resuspend the magnetic beads, and heat them in a metal bath at 100℃ for 10 minutes. After boiling the magnetic beads, store them in a negative 20 refrigerator for standby use or directly use them for WB loading.

[0126] 4) RIP experiment

[0127] Thaw the protease inhibitor cocktail and RNAse inhibitor at room temperature in advance, take out the Western and IP lysis buffer, mix the Western and IP lysis buffer with the protease inhibitor cocktail at a ratio of 99:1, and add the RNAse inhibitor to make the final concentration reach 1U per microliter, as the lysis buffer for standby use. Take out the pre-treated cells from the cell culture incubator, digest the cells, collect the cells into a centrifuge tube, centrifuge at 800rpm for 5 minutes, discard the supernatant after the centrifugation, add 1ml of lysis buffer to the cell pellet, use a pipette to repeatedly aspirate the cells and transfer them to a 1.5ml EP tube, place them in liquid nitrogen for repeated freezing and thawing, centrifuge at 13000rpm for 15min after the cell disruption, aspirate the supernatant and transfer it to a new 1.5ml EP tube, add 2 micrograms of the corresponding antibody, and incubate overnight at 4 degrees.

[0128] After the Ferris wheel overnight, on the second day, continue to add 40 microliters of Protein A / G magnetic beads, and continue to rotate the Ferris wheel at 4 degrees for 3 hours to allow for sufficient contact and mixing. Place the EP tube on the magnetic rack, discard the supernatant, add 500 microliters of lysis solution, rotate on the Ferris wheel for 5 minutes to wash the magnetic beads, and repeat the washing 3 times. After washing, add 1 ml of Trizol reagent to the magnetic beads to extract RNA and subsequent related experiments.

[0129] 5) Co-immunoprecipitation (Co-IP)

[0130] The cells were digested with trypsin, and an equal volume of complete medium was added after the cells began to fall off. The culture medium was then pipetted into a 1.5 ml EP tube, centrifuged and the supernatant was discarded.

[0131] Prepare protein IP lysis buffer: Thaw the Protease Inhibitor Cocktail and 100× phosphatase inhibitor in the -20℃ refrigerator at room temperature, and prepare the required protein IP lysis buffer in a ratio of 1:1:100.

[0132] Add an appropriate amount of IP lysis buffer according to the amount of cells (add 400ul IP lysis buffer to each 10cm dish of cells), and repeatedly blow with a 1mL syringe to fully mix the cells. Place the EP tube in a 4℃ rotating mixer for lysis for 30 minutes.

[0133] The cell lysate was centrifuged at 13000 rpm in a 4°C microcentrifuge for 15 minutes. After centrifugation, the supernatant protein lysate was pipetted into a new 1.5 ml EP tube, and the precipitate was avoided as much as possible. The protein concentration was then determined using the Coomassie Brilliant Blue method.

[0134] Prepare Input samples: Use a pipette to draw 100 μl of protein lysate into a new EP tube and mark it. According to the measured protein concentration, add the corresponding volume of IP lysate to dilute it according to the required system, add an appropriate amount of 5× Loading Buffer (the volume ratio of the diluted protein lysate is 1:4), mix it by repeatedly pipetting, put it into a 100℃ constant temperature metal bath, heat it for denaturation for 10 minutes, and after the denaturation is completed, cool the protein sample on ice, and then store it in a -80℃ refrigerator.

[0135] Wash beads: Use scissors to cut the bevel of the pipette tip to facilitate the absorption of magnetic beads. Pipette 20 μL / tube of Flag magnetic beads into the EP tube containing 1 ml IP lysate, rotate the mixer at 4℃ for 5 minutes, then place on the magnetic stand and discard the IP lysate, repeat 3 times (if using Protein A / G magnetic beads, perform this step the next day).

[0136] Add magnetic beads / antibodies: After the magnetic beads are washed, aspirate 100 μL IP lysis buffer to resuspend the Flag magnetic beads, add the magnetic beads to the protein solution according to the proportion, then place the EP tube in a 4°C rotating mixer to incubate the protein solution and magnetic beads overnight (if Protein A / G magnetic beads are used, add the corresponding primary antibody as the IP sample and the required homologous secondary antibody as the IgG sample in this step).

[0137] Wash beads: The next day, take out the EP tube and place it on a magnetic rack, discard the protein lysate, add 1 ml IP lysate, rotate on a rotating mixer at 4°C for 5 minutes, and repeat this step 3 times (if using Protein A / G magnetic beads, perform step 7 here).

[0138] Prepare IP samples: discard the IP lysate in the tube and add 100 μL 1× Loading Buffer (5× Loading Buffer diluted with protein lysate), boil at 100°C for 10 min, then place on a magnetic rack, aspirate the supernatant and transfer it to a new 1.5 ml EP tube, mark it, and then perform electrophoresis or store it at -80°C.

[0139] If using Protein A / G magnetic beads, add an equal amount of Protein A / G magnetic beads washed in the above steps to the IP sample and IgG sample the next day, and rotate on a rotary mixer at 4°C for 3-4 hours. Then wash the beads and prepare the protein sample in the same manner as steps 9 and 10.

[0140] 6) 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).

[0141] Construction of intracranial in situ model

[0142] Cell preparation: Prepare the required stable cells in advance, construct the stable cell line as before, and expand the culture.

[0143] 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 at a certain ratio, and count the cells under a microscope.

[0144] Aspirate the cells to a density of 16 × 10 7 / ml, put it in a 1.5ml EP tube, put it on ice and bring it to the animal room.

[0145] The nude mice were placed in an anesthesia cage and anesthetized with halothane. After the skin was disinfected, an incision of about 1 cm was made along the midline. 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, and the mouse was fixed on a stereotaxic apparatus.

[0146] Mix the cell suspension in the 1.5 ml EP tube, draw up the cell suspension with a microinjector, and inject it into the right striatum. Each nude mouse is injected with 5 μL (i.e. 8×10 6 After injection, the scalp of the nude mouse was sutured and the inoculation date and cell type were marked on the mouse cage.

[0147] The growth status of the intracranial tumor was observed at 0, 7, 14, and 21 days after in situ implantation. Potassium luciferin was injected into the groin of the nude mice. After 3-5 minutes of reaction, the mice were anesthetized and placed in a living imaging device to observe the fluorescence value and take pictures for analysis.

[0148] When the nude mice showed cachexia, hemiplegia, mental confusion and other neurological symptoms, they were killed by systemic circulation after anesthesia, and the mouse brains were taken and placed in a neutral fixative for tissue fixation for subsequent use.

[0149] 7) Immunohistochemistry (IHC) staining

[0150] Baking: Place paraffin sections in a 60°C oven and bake overnight.

[0151] 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.

[0152] Removal of endogenous peroxidase: Add 3% H2O2 to each section to cover the tissue and place in a light-proof humidified box for 10 minutes to remove peroxidase.

[0153] Place in a horizontal shaker and wash with 1× PBS solution for 5 min each time, for a total of 3 times.

[0154] 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.

[0155] After the repair is completed, turn off the furnace and reduce the pressure. After the pressure returns to normal, open the lid and cool to room temperature. Then wash with 1× PBS solution, 5 times each time.

[0156] Blocking: Block the sections with 1× goat serum for 30 min in a humidified chamber.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] Hematoxylin staining: Soak the tissue sections in hematoxylin and pull them up and down 2-3 times.

[0163] Dehydration and sealing: Dehydrate in the reverse order of step (1), seal with gum, observe under an optical microscope, and take photos for analysis.

[0164] 8) IHC scoring criteria

[0165] The staining scores of NAT10 and PIK3R2 were 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 and PIK3R2 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).

[0166] 9) Data analysis

[0167] 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.

[0168] 2. Experimental results

[0169] NAT10 affects AKT pathway activation by regulating PIK3R2. Since NAT10 has been shown to regulate ac4C, it is necessary to clarify whether NAT10 depends on this function to promote the malignant progression of GBM cells. Therefore, NAT10 was knocked down in U87 and LN229 (shRNA sequences are shown in Table 1, and primer sequences are shown in Table 2) and then a dot blot hybridization experiment was performed. The results showed that RNA ac4C modification in U87 and LN229 decreased after knocking down NAT10 ( Figure 1 A, 1B). It can be considered that NAT10 regulates RNA acetylation in GBM cells. In order to find the downstream targets of NAT10 for ac4C modification in GBM cells, we investigated the public database and found the RNA-seq and acRIP-seq data of NAT10 in HELA cells and the RNA-seq and acRIP-seq data of NAT10 in BxPC-3 cells. After the intersection of these four data, we obtained 7 genes ( Figure 1 C). Based on this, we hypothesized whether these seven genes could also be regulated by NAT10 in GBM.

[0170] Table 1 shRNA sequences of NAT10

[0171]

[0172] Table 2 Primer sequences

[0173]

[0174]

[0175] In order to explore the regulatory effect of NAT10 on the above genes in GBM cells, qRT-PCR experiments were performed to detect the changes in the mRNA levels of the selected genes after knocking down NAT10 in U87 cells. The results showed that PIK3R2 was most significantly downregulated after NAT10 knockdown ( Figure 1D), it is speculated that NA10 may affect the malignant progression of GBM by regulating PIK3R2. Then, NAT10 was knocked down in U87 and LN229 cells. The results of qRT-PCR experiments showed that the mRNA level of PIK3R2 was significantly decreased after knocking down NAT10 ( Figure 1 E). Similarly, WB results also confirmed that the expression level of PIK3R2 protein decreased after NAT10 knockdown ( Figure 1 F). The above results indicate that NAT10 has a positive regulatory relationship with the expression of PIK3R2 in GBM.

[0176] Since PIK3R2 is involved in the activation of the PI3K / AKT pathway, it is speculated whether NAT10 can activate the PI3K / AKT pathway by regulating PIK3R2. To verify this hypothesis, WB experiments were performed after knocking down NAT10 in U87 and LN229 cells. The data showed that after NAT10 knockdown, the phosphorylation level of PI3K downstream effector protein AKT at Ser473 site was reduced, which is required for the full activation of AKT, while the level of AKT did not change ( Figure 2 A, 2B). The same results were obtained after treating GBM cells with Remodelin ( Figure 2 C, 2D). The above data indicate that NAT10 may activate the PI3K / AKT pathway in GBM by regulating PIK3R2.

[0177] The cDNA sequence of PIK3R2 was then placed into the PACES (http: / / rnanut.net / paces / ) website for prediction, and two highly conserved acetylation sites were obtained ( Figure 3 A). According to the above conditions, the mutant plasmids Mut1, Mut2 and Mut1+Mut2 of PIK3R2 were constructed, and RNA-Pull Down experiments were performed. The results showed that NAT10 directly bound to both sites, but NAT10 did not bind to them after both sites were mutated, indicating that NAT10 may modify PIK3R2 through these two highly conserved acetylation sites ( Figure 3 B).

[0178] Then, RIP and CO-IP experiments were performed in U87 and LN229 cells to verify that NAT10 and PIK3R2 interact at the mRNA level but not at the protein level ( Figure 3 C, 3D). ac4C-RIP experiments were performed in U87 and LN229 cells with NAT10 knockdown, and qRT-PCR results showed that the mRNA level of PIK3R2 modified by ac4C was decreased ( Figure 3E, 3F). The above results indicate that NAT10 regulates the mRNA level of PIK3R2 through ac4C modification. Previous reports have shown that ac4C modification can regulate the expression of downstream genes by stabilizing mRNA. Subsequently, after knocking down NAT10 in U87 and LN229 cells, actinomycin D was added. qRT-PCR results showed that downregulating NAT10 significantly affected the stability of PIK3R2 ( Figure 3 G, 3H). In summary, NAT10 directly binds to PIK3R2 mRNA and regulates its stability through ac4C.

[0179] NAT10 regulates changes in GBM cell function by upregulating PIK3R2 expression

[0180] Subsequently, a recovery experiment was performed by overexpressing PIK3R2 in U87-shNAT10 and LN229-shNAT10 cells to verify the role of NAT10 in promoting GBM progression by regulating PIK3R2 to affect the PI3K / AKT pathway. The results showed that when PIK3R2 was overexpressed in U87-shNAT10 and LN229-shNAT10 cells, the expression levels of p-AKT (Ser473), FN1, N-cadherin, Vimentin and Snail increased, while there was no significant change in AKT ( Figure 4 A, 4B). CCK-8 detection and clone formation experiments showed that overexpression of PIK3R2 in U87-shNAT10 and LN229-shNAT10 cells enhanced the proliferation ability of tumor cells ( Figure 4 CF).

[0181] In addition, the present application also found through Transwell experiments that overexpression of PIK3R2 in U87-shNAT10 and LN229-shNAT10 cells promoted their migration and invasion abilities ( Figure 4 G-4J). These results indicate that NAT10 needs to regulate PIK3R2 to promote the migration and invasion of GBM cells. The above results indicate that NAT10 needs to affect the PI3K / AKT pathway through PIK3R2 to promote the malignant progression of GBM.

[0182] NAT10 promotes GBM progression in vivo

[0183] Immunohistochemical staining analysis of resected mouse brain tumor sections confirmed that the expression of NAT10 and PIK3R2 in the U87-shNAT10 group was significantly decreased compared with the U87-shNC group ( Figure 5A). The same results were also found in the LN229-shNC group and LN229-shNAT10 group ( Figure 5 B). These in vivo experimental data indicate that NAT10 may promote the malignant growth of GBM through PIK3R2. In summary, the data of this application suggest that the regulation of PIK3R2 by NAT10 plays a key role in the growth of GBM in vivo.

[0184] Relationship between NAT10-PIK3R2 axis and clinicopathological characteristics of glioma patients

[0185] Finally, this application explored the clinical significance of the functions of NAT10 and PIK3R2 in GBM. Immunohistochemistry was used to detect the expression of NAT10 and PIK3R2 in 30 tissue sections of brain gliomas of different grades. The results showed that the expression levels of NAT10 and PIK3R2 in high-grade gliomas were significantly higher than those in low-grade gliomas ( Figure 6 A, 6B). In addition, the present application also found that the expression of NAT10 was positively correlated with that of PIK3R2 ( Figure 6 C). The above results indicate that there is a clear link between the NAT10-PIK3R2 axis and the clinical pathological characteristics of human glioma. Finally, the mechanism diagram summarizes the new findings under the experimental conditions: NAT10 regulates the stability of PIK3R2 through ac4C, leading to EMT and promoting the malignant development of GBM ( Figure 6 D).

[0186] 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. Use of an agent that inhibits the binding between NAT10 and PIK3R2 in the preparation of drugs for treating tumors.

2. The use according to claim 1, characterized in that: The action site of the reagent that inhibits the binding between NAT10 and PIK3R2 is on the acetylation site of PIK3R2; Preferably, the tumor is selected from glioma; Preferably, the glioma is selected from glioblastoma.

3. A drug for treating tumors, characterized in that: The drug includes an agent that inhibits the binding of NAT10 and PIK3R2; Preferably, the action site of the agent that inhibits the binding between NAT10 and PIK3R2 is on the acetylation site of PIK3R2; Preferably, the tumor is selected from glioma; Preferably, the glioma is selected from glioblastoma; Preferably, the drug further comprises a pharmaceutically acceptable excipient.

4. A composite, characterized in that The complex includes NAT10 and PIK3R2; Preferably, the binding site of NAT10 and PIK3R2 is on the acetylation site of PIK3R2.

5. Use of the complex according to claim 4 in screening candidate drugs for treating tumors; Preferably, the method for screening candidate drugs for treating tumors comprises: (1) contacting a substance to be detected with a system containing a complex formed by NAT10 and PIK3R2; (2) detecting the binding level of NAT10 and PIK3R2 in the system; (3) selecting substances that can reduce the binding level of NAT10 and PIK3R2 as candidate drugs for treating or preventing cancer; Preferably, the tumor is selected from glioma; Preferably, the glioma is selected from glioblastoma.

6. Use of the complex according to claim 4 in screening for agents that inhibit the binding between NAT10 and PIK3R2.

7. A method for screening candidate drugs for treating tumors, characterized in that: The method comprises: (1) contacting a substance to be detected with a system containing a complex formed by NAT10 and PIK3R2; (2) detecting the binding level of NAT10 and PIK3R2 in the system; (3) selecting substances that can reduce the binding level of NAT10 and PIK3R2 as candidate drugs for treating or preventing cancer; Preferably, the tumor is selected from glioma; Preferably, the glioma is selected from glioblastoma.

8. A method for regulating the expression level of PIK3R2 in a cell, characterized in that: The method comprises altering the expression of NAT10 in a cell; Preferably, the changing the expression of NAT10 in the cell is inhibiting the expression of NAT10 in the cell; Preferably, the inhibiting the expression of NAT10 in the cell comprises administering an inhibitor of NAT10 to the cell; preferably, the inhibitor is selected from nucleic acid inhibitors; Preferably, the nucleic acid inhibitor is shRNA; Preferably, the sequence of the shRNA is shown in any one of SEQ ID NOs: 3-6; Preferably, the cells are glioma cells; Preferably, the glioma cells are glioblastoma cells; Preferably, the glioblastoma cells include U87 cells and / or LN229 cells.

9. A method for constructing a cell model of PIK3R2 overexpression / underexpression, characterized in that: The method comprises regulating the expression of NAT10 in a cell; Preferably, regulating the expression of NAT10 in the cell comprises inhibiting the expression of NAT10 in the cell; Preferably, the inhibiting the expression of NAT10 in the cell comprises administering an inhibitor of NAT10 to the cell; preferably, the inhibitor is selected from nucleic acid inhibitors; Preferably, the nucleic acid inhibitor is shRNA; Preferably, the sequence of the shRNA is shown in any one of SEQ ID NOs: 3-6.

10. A method for computer-assisted drug screening based on NAT10 and PIK3R2, characterized in that: The method comprises: Obtain NAT10 protein and PIK3R2 protein data; Determine the binding sites of the two proteins based on the data of the NAT10 protein and the PIK3R2 protein; Through computer-assisted screening, candidate drugs targeting the binding sites of the two were obtained; Preferably, the process of computer-assisted screening is: Screening small molecule compounds based on the spatial structure of the binding sites of NAT10 protein and PIK3R2 protein; performing molecular docking of the screened small molecule compounds with the binding sites to obtain scores of the docked molecules; and ranking according to the scores to obtain candidate drugs; Preferably, the process of computer-assisted screening is: The molecular structures of NAT10 protein and PIK3R2 protein are obtained and input into the pharmacophore module library for matching, and all the action sites are clustered based on the interaction patterns with NAT10 and PIK3R2 to obtain a pharmacophore model; The pharmacophore model is input into a molecular compound database for high throughput screening to obtain candidate drugs; preferably, the process of computer-assisted screening is: Obtaining the molecular structure of a small molecule inhibitor that inhibits NAT10 and / or the molecular structure of a small molecule inhibitor that inhibits PIK3R2; Based on the molecular structure of the small molecule inhibitor, a small molecule library with similar structure is obtained by screening; Performing molecular docking on the small molecule library with similar structures and the binding site to obtain scores of docked molecules; and sorting according to the scores to obtain candidate drugs; Preferably, the candidate drugs include protein analogs, antibodies, and RNA drugs.

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