Use of PHF23 acetylation in gastrointestinal stromal tumors

Abstract

The invention discloses an application of PHF23 acetylation in gastrointestinal stromal tumor. The research shows that the K211 site acetylation of the PHF23 protein is obviously increased in gastrointestinal stromal tumors with medium and high recurrence risks, and a gastrointestinal stromal tumor diagnosis product taking the PHF23 as a marker and a drug screening method are provided. A cell function test verifies that PHF23 has an inhibiting effect on proliferation and migration of gastrointestinal stromal tumor cells, and the application of the PHF23 acetylation inhibitor as a gastrointestinal stromal tumor treatment medicine is provided.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the use of PHF23 acetylation in gastrointestinal stromal tumors. Background Technology

[0002] Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract and have a certain potential for malignant transformation. Although targeted therapies such as imatinib have greatly improved the survival of GIST patients, more than half of them eventually relapse or metastasize. Patients with the same relapse risk stratification and the same gene mutation sites also show significant differences in prognosis and sensitivity to targeted therapy, the specific molecular mechanisms of which remain unclear.

[0003] Recent studies have revealed that protein post-translational modifications play a crucial role in the occurrence, development, recurrence, and metastasis of gastrointestinal stromal tumors (GISTs). However, most studies are limited to the post-translational modifications of single proteins and have not yet elucidated the key protein post-translational modifications and key modification sites in GISTs. Parallel analysis of high-throughput data from multiple proteins and multiple sites is needed. Currently, there are no reports that deeply reveal the specific mechanisms of protein post-translational modifications that play a key role in GISTs and explore their molecular mechanisms. This is of great significance for the prevention and treatment of GIST recurrence and metastasis. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides the use of PHF23 acetylation in gastrointestinal stromal tumors.

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

[0006] The first aspect of the present invention provides any of the following substances:

[0007] (1) A PHF23 protein, said protein being acetylated at the PHF23 K211 site;

[0008] (2) A nucleic acid that encodes the protein described in (1);

[0009] (3) A vector comprising the nucleic acid described in (2);

[0010] (4) A host cell comprising the nucleic acid described in (2) or the vector described in (3).

[0011] Furthermore, the K211 site of the protein is mutated from lysine to arginine.

[0012] The second aspect of the invention provides the use of the substance described in the first aspect of the invention or an inhibitor of PHF23 K211 site acetylation in the preparation of a medicament for the prevention / treatment of gastrointestinal stromal tumors.

[0013] Furthermore, the inhibitors of acetylation include promoters of SIRT7.

[0014] Furthermore, the SIRT7 promoter includes a vector for SIRT7 overexpression.

[0015] A third aspect of the present invention provides a medicament for treating gastrointestinal stromal tumors, the medicament comprising the substance described in the first aspect of the present invention or an inhibitor of PHF23 K211 site acetylation.

[0016] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0017] A fourth aspect of the invention provides the use of PHF23 K211 site acetylation as a target in screening candidate drugs for the treatment of gastrointestinal stromal tumors.

[0018] Furthermore, the method for screening candidate drugs for the treatment of gastrointestinal stromal tumors includes: testing the effect of the candidate drug on the acetylation level of the PHF23 K211 site in the sample, wherein, after using the candidate drug, the acetylation level of the PHF23 K211 site decreases, indicating that the candidate drug has the effect of treating gastrointestinal stromal tumors.

[0019] A fifth aspect of the present invention provides a method for screening candidate drugs for the treatment of gastrointestinal stromal tumors, the method comprising: testing the effect of the candidate drug on the acetylation level of the PHF23 K211 site in a sample, wherein, after using the candidate drug, the acetylation level of the PHF23 K211 site decreases, indicating that the candidate drug has the effect of treating gastrointestinal stromal tumors.

[0020] A sixth aspect of the invention provides the use of a reagent for detecting the acetylation level at the PHF23 K211 site in the preparation of products for predicting the risk of recurrence of gastrointestinal stromal tumors.

[0021] Furthermore, the reagent includes an antibody against PHF23 K211.

[0022] Furthermore, the reagents also include reagents used to detect the acetylation level of the PHF23 K211 site by ELISA, Western Blot, immunoprecipitation, mass spectrometry, and immunocytochemistry.

[0023] A seventh aspect of the invention provides a product for predicting the risk of recurrence of gastrointestinal stromal tumors, the product comprising a reagent for detecting the acetylation level at the PHF23 K211 site.

[0024] Furthermore, the product also includes testing reagents.

[0025] Furthermore, the products include reagent kits and test strips.

[0026] The eighth aspect of the present invention provides a method for inhibiting the proliferation / migration of gastrointestinal stromal tumor cells in vitro, the method comprising administering the substance described in the first aspect of the present invention or an inhibitor of PHF23 K211 site acetylation.

[0027] Furthermore, the method described is not for therapeutic purposes.

[0028] A ninth aspect of the present invention provides a method for preparing a specific antibody for detecting acetylation at the PHF23 K211 site, the method comprising:

[0029] (1) Synthesize the acetylated antigenic polypeptide at the PHF23 K211 site;

[0030] (2) Immunize the antigenic polypeptide from (1) to obtain the specific antibody.

[0031] Furthermore, the method also includes post-immunopurification processing.

[0032] A tenth aspect of the present invention provides a system / device for predicting the risk of recurrence of gastrointestinal stromal tumors, the system / device comprising:

[0033] Acquisition Unit: Used to acquire acetylation site data of the PHF23 protein in the sample to be tested;

[0034] Extraction unit: used to extract acetylation data of the target site of PHF23 protein in the sample to be tested, wherein the acetylation of the target site is the acetylation of the PHF23 K211 site;

[0035] Prediction Unit: Based on the acetylation data of the target site PHF23 K211, classification prediction is performed to obtain the classification result of the risk of recurrence of gastrointestinal stromal tumors in the test sample. If the acetylation level of the PHF23 K211 site is high, the classification result of the test sample is high risk of recurrence of gastrointestinal stromal tumors; if the acetylation level of the PHF23 K211 site is low, the classification result of the test sample is low risk of recurrence of gastrointestinal stromal tumors.

[0036] Advantages and beneficial effects of the present invention:

[0037] This application demonstrates that acetylation at the K211 site of the PHF23 protein is significantly elevated in gastrointestinal stromal tumors (GISTs) with intermediate to high risk of recurrence, providing diagnostic products and drug screening methods for GISTs using PHF23 as a biomarker. This application also verifies through cell function assays that PHF23 inhibits the proliferation and migration of GIST cells, providing information on the application of PHF23 acetylation inhibitors as therapeutic agents for GISTs. Attached Figure Description

[0038] Figure 1 This is a comparison of the results of Western blotting analysis of PHF23 acetylation expression in gastrointestinal stromal tumor tissues;

[0039] Figure 2 This is a graph showing the effect of SIRT7 on the expression of PHF23 acetylation;

[0040] Figure 3 This is a diagram showing the results of a cell invasion experiment;

[0041] Figure 4 This is a graph showing the effect of reducing PHF23 acetylation on the proliferation of gastrointestinal stromal tumors in nude mice. 4A shows the growth of subcutaneous xenografts in the posterior axilla of nude mice, and 4B shows the changes in the volume of xenografts in nude mice. Detailed Implementation

[0042] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, 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 pertains.

[0043] This invention provides any of the following substances:

[0044] (1) A PHF23 protein, said protein being acetylated at the PHF23 K211 site;

[0045] (2) A nucleic acid that encodes the protein described in (1);

[0046] (3) A vector comprising the nucleic acid described in (2);

[0047] (4) A host cell comprising the nucleic acid described in (2) or the vector described in (3).

[0048] In some implementations, PHF23 includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed PHF23, as well as any form of PHF23 derived from cells and processed. The term encompasses naturally occurring variants of PHF23 (e.g., splice variants or allelic variants). The term encompasses PHF23 from, for example, human and any other vertebrate sources, including mammalian PHF23, such as primates and rodents (e.g., mice and rats), gene ID: 79142.

[0049] In some embodiments, the vector also includes a transcription promoter and optionally present enhancers, translation signals, and transcription and translation termination signals. Expression vectors used for stable transformation typically have selectable tags that allow selection and retention of transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cell. The vector may also include additional nucleotide sequences operatively linked to the linked nucleic acid molecule, such as epitope tags for localization, like 6-his tags or myc tags, or tags for purification, such as GST fusions; and sequences for guiding protein secretion and / or membrane association.

[0050] This application does not impose any particular limitation on the vector, which can be a vector capable of replicating and / or expressing polynucleotides within eukaryotic or prokaryotic cells, including mammalian cells (e.g., human, monkey, rabbit, rat, hamster, or mouse cells), yeast cells, insect cells, and bacterial cells (e.g., E. coli). Preferably, the vector includes at least one selective marker operably linked to a suitable promoter so that polynucleotides can be expressed within a host cell. For example, the vector may include other vectors conventionally used for, for example, genetic engineering, for introducing polynucleotides such as bacteriophages, plasmids, granules, mini-chromosomes, viruses, or retroviral vectors.

[0051] As an alternative approach in this application, the vector is a virus. Viral vectors are used to introduce non-endogenous nucleic acid sequences encoding target-specific polypeptides. Viral vectors can be retroviral vectors or lentiviral vectors. Viral vectors may also include nucleic acid sequences encoding transduction markers. Suitable viral vectors include RNA virus-based vectors, such as vectors derived from retroviruses, such as those derived from Moloney murine leukemia virus (MLV), and more complex retroviral vectors, such as those derived from lentiviruses. HIV-1-derived vectors belong to this category.

[0052] Viral vectors include retroviruses, adenoviruses, parvoviruses (such as adeno-associated virus), coronaviruses, negative-strand RNA viruses (such as orthomyxoviruses, such as influenza viruses), rhabdoviruses (such as rabies and vesicular stomatitis viruses), paramyxoviruses (such as measles and Sendai viruses), positive-strand RNA viruses (such as piconemaviruses and A viruses), and double-stranded DNA viruses, including adenoviruses, herpesviruses (such as herpes simplex virus types 1 and 2 and Epstein-Barr virus and cytomegalovirus), and poxviruses (such as cowpox, fowlpox, and canarypox). Other viruses include, but are not limited to, norovirus, capsid virus, flavivirus, reovirus, papillomavirus, hepatitis virus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma, mammalian C, B, and D viruses, HTLV-BLV groups, lentiviruses, or foamy viruses.

[0053] As an alternative approach in this application, the vector is an expression vector. The expression vector according to this application can guide the replication and expression of the polynucleotides of this application in the host.

[0054] Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027 pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vectors include pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (EdgeBiosystems), pTriEx-Hygro (Novagen), and pCINeo (Promega). Non-restrictive examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all from Invitrogen). Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos, and various mammalian and avian cells is the multipurpose expression vector pCS2+.

[0055] In some embodiments, the host cell is a cell used to receive, maintain, replicate, and amplify the vector. This includes prokaryotic cells and eukaryotic cells. Eukaryotic cells include, but are not limited to, protist cells and animal cells, including mammalian cells, avian cells, and insect cells; mammalian cells include, but are not limited to, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, 293 cells, 293T cells, and 293F cells.

[0056] Methods for conversion into host cells include any method for introducing nucleic acids into an organism, cell, tissue, or organ, which can be performed using standard techniques selected according to the type of host cell, as is known in the art. These methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated conversion, and conversion mediated by PEG, dextran sulfate, lipofectamine, or drying / inhibition.

[0057] This invention provides the use of the above-mentioned substance or an inhibitor of PHF23 K211 site acetylation in the preparation of a medicament for the prevention / treatment of gastrointestinal stromal tumors.

[0058] The inhibitors of acetylation include promoters of SIRT7.

[0059] In some embodiments, a promoter refers to any substance that can increase the activity of the SIRT7 protein, improve the stability of the SIRT7 gene or protein, upregulate the expression of the SIRT7 protein, increase the effective duration of the SIRT7 protein, or promote the transcription and translation of the SIRT7 gene. Such substances can be used in this application. For example, the promoters include nucleic acid promoters and protein promoters. The promoters include, but are not limited to, vectors or constructs thereof that overexpress SIRT7, the SIRT7 protein or its active peptide.

[0060] The drug also includes a pharmaceutically acceptable carrier.

[0061] In some embodiments, a pharmaceutically acceptable carrier is used to refer to a material that is compatible with the recipient, preferably a mammal, more preferably a human, and is suitable for delivering an active agent to a target site without terminating the activity of the agent. The risk / benefit ratio of the toxicity or side effects (if any) associated with the pharmaceutically acceptable carrier is proportionate.

[0062] Pharmaceutically acceptable carriers include, but are not limited to, diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants. These pharmaceutically acceptable carriers may be used as needed to aid in the stability of the formulation or to contribute to its activity or bioavailability, or to produce an acceptable taste or odor when taken orally. The formulation may be used in this drug in the form of the original compound itself or optionally in the form of a pharmaceutically acceptable salt thereof. The drug thus formulated may be administered as needed by any appropriate method known to those skilled in the art.

[0063] The diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, and water.

[0064] Adhesives include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropylcellulose and hydroxypropylmethylcellulose.

[0065] Surfactants include, but are not limited to, polyethylene oxide sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol.

[0066] Humectants include, but are not limited to, glycerin.

[0067] Adsorption carriers include, but are not limited to, bentonite, silica gel, kaolin and soap clay.

[0068] Lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium dodecyl sulfate.

[0069] Fillers include, but are not limited to, mannitol (granular or powdered), xylitol, sorbitol, maltose, erythritol, microcrystalline cellulose, polysaccharides, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate, and sodium bicarbonate.

[0070] Disintegrants include, but are not limited to, crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropylmethyl, crosylcarboxymethyl cellulose sodium, and soybean polysaccharides.

[0071] This invention provides a product for predicting the risk of recurrence of gastrointestinal stromal tumors, the product comprising a reagent for detecting the acetylation level at the PHF23K211 site.

[0072] The product also includes testing reagents.

[0073] In some embodiments, the detection reagent can be any substance having detectable physical or chemical properties. Such detection reagents are well-developed in the field of immunoassays, and generally, most of any labeling useful in such methods can be applied to the provided methods. Therefore, the labeling can be any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical methods. Detectable reagents include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.), and radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), in particular, radioactive labels (e.g.,157 Gd, 55 Mn, 162 Dy、 52 Cr and 56 Fe), metal ions (e.g., 111 In、 97 Ru、 67 Ga、 68 Ga、 72 As、 89 Zr and 201 Tl), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), electron transfer agents (e.g., including metal-binding proteins and compounds), luminescent and chemiluminescent labels (e.g., luciferin and 2,3-dihydrophtahlazinediones, such as luminol), magnetic beads (e.g., DYNABEADS™), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex).

[0074] The products include reagent kits and test strips.

[0075] In some embodiments, the kit may also include one or more substances selected from the group consisting of: a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, or a solvent.

[0076] The kit may also include instructions for use, which describe how to use the kit for testing and how to use the test results to assess the risk of recurrence of gastrointestinal stromal tumors and select treatment options.

[0077] The kit components can be packaged in an aqueous medium or in lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container for holding one component, and preferably, for appropriate aliquoting. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container for separately holding the additional components. However, different combinations of components may be contained in a single vial. The kit of this application will also typically include a container for containing the reactants, sealed for commercial sale. Such a container may include injection-molded or blow-molded plastic containers for holding the desired vials.

[0078] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0079] Example 1

[0080] I. Experimental Methods

[0081] We selected patients with pathologically confirmed gastrointestinal stromal tumors (GISTs) at the Department of Gastrointestinal Surgery, Peking University People's Hospital, between January 2020 and December 2023, and collected their tumor tissues. None of the patients had received targeted therapy before surgery. Patients with incomplete clinicopathological data or those whose total protein extraction failed quality control were excluded; cases with tumor-induced intestinal perforation, intestinal obstruction, or distant metastasis were also excluded.

[0082] The specimens used were preserved in the specimen bank of the Department of Gastrointestinal Surgery and Surgical Oncology, Peking University People's Hospital. All specimens were flash-frozen in liquid nitrogen within 30 minutes of being excised and then stored at -80°C for an extended period. All specimens were pathologically confirmed as corresponding tumor tissues, and frozen sections were used to further confirm that the tissues used were gastrointestinal stromal tumor tissues before the formal experiments.

[0083] 1. Tissue homogenization and protein extraction

[0084] (1) Wipe the lab bench with a clean cloth before extracting tissue protein, and then wipe the lab bench carefully with 75% alcohol for disinfection. After the alcohol evaporates, carry out the experiment.

[0085] (2) Pre-cool the mortar on ice beforehand, then add liquid nitrogen, repeating 4-5 times to ensure the mortar is fully pre-cooled at a low temperature. Take the frozen tissue sample from the -80℃ freezer, quickly cut the tissue sample with a sterile sharp blade, weigh about 500mg of tissue (the size of a soybean), put it into the pre-cooled mortar, and grind it thoroughly with a grinding stick. During the grinding process, an assistant should intermittently add liquid nitrogen to the mortar to ensure that the liquid nitrogen does not completely evaporate during the grinding process. After the tissue is ground into powder, transfer the tissue to a sterile 50ml centrifuge tube, add the pre-prepared pre-cooled mixture of 10% TCA / acetone, 50mM DTT and 0.1% protease inhibitor to the centrifuge tube, precipitate the tissue powder, centrifuge at 20000g for 10min at 4℃, and discard the supernatant.

[0086] (3) Wash the precipitate three times with a pre-cooled mixture of acetone, DTT, and PMSF. After the precipitate has dried slightly in the air, add protein lysis buffer (8M urea, 2mM EDTA, 10mM DTT) at a ratio of 1mg:10µl and mix thoroughly. Lyse on ice for 1 hour, intermittently vortexing. After lysis, keep the sample on ice and sonicate it on an ultrasonic cell disruptor for 3 minutes each time, sonicating for 1 second followed by a 3-second pause. After sonication, centrifuge at 20,000g for 10 minutes at 4°C, collect the supernatant, freeze one portion at -80°C, and perform SDS-PAGE electrophoresis on the other portion to determine the protein content.

[0087] 2. Detection of protein acetylation modification (immunoprecipitation IP)

[0088] (1) Add 50µl of sodium butyrate (1 M stock solution concentration) and 50µl of nicotinamide (1 M stock solution concentration) to the protein extract and continue to incubate.

[0089] (2) Prepare protein lysis buffer on ice: 1 ml cell protein lysis buffer + 50 µl sodium butyrate + 50 µl nicotinamide + 10 µl protease inhibitor cocktail.

[0090] (3) After 6 hours, remove the cell culture dish and wash the cells twice with PBS.

[0091] (4) Add the prepared protein lysis buffer and lyse on ice for 1 hour, intermittently using a vortex mixer. After lysis, transfer the lysis buffer into a pre-cooled EP tube, keep the EP tube on ice, and place it on an ultrasonic cell disruptor for sonication. Sonicate for 1 second, pause for 3 seconds, and repeat 3 times for 3 minutes each time.

[0092] (5) After the ultrasound is completed, centrifuge at 15,000 rpm for 10 min at 4°C and transfer the supernatant to another new EP tube.

[0093] (6) Add 20µl of ANTI-FLAG®M2 Affinity Gel, seal the tube with EP tube sealing glue, place it on a vertical mixer, and incubate overnight in a 4℃ chromatography cabinet with the tube turned upside down.

[0094] (7) Remove the EP tube, centrifuge at 3500 rpm for 1 min at 4℃, and discard the supernatant.

[0095] (8) Add 1 ml of BC200 lysis buffer to the EP tube, wash the beads twice, centrifuge at 3500 rpm for 1 min at 4°C, and discard the supernatant.

[0096] (9) Add 1 ml of BC100 lysis buffer to the EP tube, wash the beads twice, centrifuge at 3500 rpm for 1 min at 4°C, and discard the supernatant.

[0097] (10) Insert the filter into the small hole of the centrifuge column, add 500µl of BC100 to the EP tube, gently mix the beads and transfer them into the centrifuge column, put the centrifuge column into the collection tube, centrifuge at 3500rpm for 1min at 4℃, and discard the waste liquid in the collection tube.

[0098] (11) Add 500µl of BC100 to the EP tube again, gently mix the beads and transfer them to the centrifuge column, and repeat step (10).

[0099] (12) Plug the outlet of the centrifuge column with a stopper, add 20µl of glycine (storage concentration of 1 M), and let stand at room temperature for 10 min.

[0100] (13) Add 2.5µl of Tris-base (stock solution concentration of 1 M) to the glycine solution, remove the bottom stopper, replace with a new collection tube, and centrifuge at 3500rpm for 1min at 4℃.

[0101] (14) Repeat steps (12) and (13) once.

[0102] (15) The protein collected in the collection tube should be stored at -80°C.

[0103] 3. Western Blot

[0104] (1) Use the BCA method to quantify the protein and adjust the concentration to 4 µg / µl. Mix the protein solution with protein loading buffer (5×) at a ratio of 4:1, boil in boiling water at 100℃ for 10 min, dispense the protein, and store at -80℃ for later use.

[0105] (2) Preparation of separating gel and concentrating gel.

[0106] (3) Add the prepared protein and protein marker to the stacking gel wells. Start with 80V constant voltage electrophoresis. When the bromophenol blue indicator reaches the separating gel, switch to 100V constant voltage electrophoresis until the bromophenol blue indicator reaches the bottom of the separating gel, then stop electrophoresis.

[0107] (4) Transfer: Prepare the transfer solution in advance and pre-cool it in a -20℃ refrigerator. Pry open the two glass plates and carefully cut out the PAGE gel containing the target band and place it in the transfer solution. Open the transfer clamp and assemble the membrane in the following order: (-) black clamp - sponge pad - filter paper - PAGE gel - nitrocellulose (NC) membrane - filter paper - sponge pad - red clamp (+). Note that there should be no air bubbles between each layer. If there are air bubbles, gently remove them with a glass rod.

[0108] (5) Align the positive and negative electrodes of the transfer clamp and place it in the transfer tank. Fill the tank with transfer solution and place it in an ice-water mixture. Connect the electrodes and perform electrophoresis at 70V for 2 hours.

[0109] (6) Take out the transfer tank, mark the front and back of the NC membrane, put the NC membrane into the pre-prepared blocking solution (5% skim milk powder, TBST solution), and block it in a shaker at room temperature for 2 hours.

[0110] (7) Cut the required bands according to the protein marker, mark the front and back sides, put them into an incubation bag, add the corresponding diluted primary antibody, and incubate overnight on a shaker at 4°C.

[0111] (8) The next day, remove the NC membrane, recover the primary antibody from the incubation bag, and wash the NC membrane in TBST 3 times, 10 min each time.

[0112] (9) Add the corresponding secondary antibody labeled with HRP (1:5000) according to the species of the primary antibody and incubate at room temperature on a shaker for 1 hour.

[0113] (10) Wash the NC membrane in TBST three times, 10 min each time.

[0114] (11) Apply ECL method for luminescence and color development. After exposure to light-sensitive X-ray film in a dark room, place the film in the developer and fixer in sequence. Rinse the fixed film with clean water, let it air dry, scan it to the computer, and perform grayscale analysis.

[0115] II. Experimental Results

[0116] Western blotting analysis of PHF23 acetylation expression in human gastrointestinal stromal tumor (GIST) tissues revealed significantly elevated PHF23 acetylation expression in GIST tissues with a high risk of recurrence. Figure 1 ).

[0117] Example 2

[0118] I. Experimental Methods

[0119] The GIST-T1 cells used in this experiment were purchased from the National Biomedical Experimental Cell Resource Bank.

[0120] 1. Neofect-mediated cell transfection

[0121] (1) Before transfection, passage the cells according to the normal cell passage method and place them in a 10cm diameter cell culture dish. 10ml of culture medium needs to be added to this size of culture dish.

[0122] (2) Observe the cell density one day before transfection to confirm that the cell density can reach 70%-80% on the second day of transfection.

[0123] (3) Two hours before transfection, remove the original culture medium from the culture dish and replace it with fresh complete culture medium.

[0124] (4) Mix the plasmid with an appropriate amount of Opti-MEM medium in a certain proportion. Add 5µg Flag-PHF23 WT plasmid or Flag-PHF23 K211R plasmid and 5µg HA-CBP plasmid to 500µl Opti-MEM medium and mix gently. This mixture is labeled as A.

[0125] (5) Add 10µl of Neofect transfection reagent directly to solution A, mix gently, label it as B, and let it stand at room temperature for 20 min.

[0126] (6) Add mixture B to the cell culture dish, gently shake horizontally to mix, and place in a cell culture incubator for culture.

[0127] (7) Protein was extracted according to the method in Example 1 and PHF23 acetylation expression was detected by parallel IP and Western Blot.

[0128] 2. Transwell cell invasion assay

[0129] (1) The day before the experiment, take the ECM matrix gel out of the -20℃ refrigerator and let it melt overnight at 4℃.

[0130] (2) Place the sterile pipette tip box at -20℃ for half an hour to pre-cool.

[0131] (3) Place the Transwell chamber into a sterile 24-well plate.

[0132] (4) Mix the ECM gel with the pre-cooled serum-free culture medium at a ratio of 1:9. Add 40µl of diluted ECM gel to each well. Gently shake the 24-well plate from side to side to ensure that the ECM gel is evenly covered on the semi-permeable membrane. Place the 24-well plate in a 37°C cell culture incubator and incubate for 5 hours.

[0133] (5) Remove the culture plate, carefully aspirate the residual liquid in the upper chamber, add 70µl of the corresponding serum-free culture medium to each well to hydrate the basement membrane, put the 24-well plate into the 37℃ incubator again for 30 min, and then remove it and aspirate the culture medium in the upper chamber.

[0134] (6) Prepare the corresponding cell culture media containing 0.1% fetal bovine serum and 30% fetal bovine serum respectively for later use.

[0135] (7) Digest and centrifuge the experimental cells according to the cell passage procedure, resuspend the cells in a culture medium containing 0.1% fetal bovine serum, count the cells, and adjust the cell density to 2.5 × 10⁻⁶. 5 per ml.

[0136] (8) Use a pipette tip to draw 200µl of cell suspension and add it to the upper chamber of the Transwell chamber. Use sterile forceps to lift the chamber and add 700µl of culture medium containing 30% fetal bovine serum into the well plate. Put it back into the chamber and ensure that there are no air bubbles on the contact surface between the membrane and the culture medium.

[0137] (9) Place the cell culture plate in an incubator and culture for 24-48 hours as usual.

[0138] (10) Remove the culture plate, discard the culture medium in the upper chamber of the small chamber, wash 3 times with PBS, fix with 4% paraformaldehyde for 15 min, and wash 3 times with PBS.

[0139] (11) Stain with 0.1% crystal violet for 20 min, wash 3 times with PBS, and carefully wipe away the cells that have not penetrated the membrane on the upper surface of the chamber with a cotton swab.

[0140] (12) Cell counting: Under the high magnification of the microscope (400×), six fields of view were randomly selected to count the number of cells on the lower surface of the chamber and take pictures.

[0141] II. Experimental Results

[0142] Immunoprecipitation experiments revealed that the K211 site of PHF23 is a key acetylation site of the PHF23 protein, and SIRT7 is a key deacetylation enzyme of PHF23. Figure 2 Transwell assays showed that the invasion ability of GIST-T1 cells overexpressing the Flag-PHF23 K211R plasmid was significantly reduced. Figure 3 ).

[0143] Example 3

[0144] I. Experimental Methods

[0145] This embodiment uses 4-6 week old female nude mice for experiments. All nude mice were purchased from Beijing Vital River Laboratory Animal Co., Ltd.

[0146] PHF23 deacetylation enhances the proliferation of gastrointestinal stromal tumor cells in nude mice.

[0147] The PHF23 K211R stably expressing cell line and the control cell line from Example 2 were inoculated into the posterior axilla of nude mice, a region with rich blood supply. Tumor volume (V = 1 / 2 × a × b) was measured weekly after inoculation. 2 (where a is the major axis and b is the minor axis), the longest and shortest parts of the tumor were measured with calipers, and a tumor volume growth curve was plotted. Nude mice were sacrificed 20 days later and photographed.

[0148] II. Experimental Results

[0149] The tumor volume of the PHF23 low-acetylation group (PHF23 K211R) was significantly smaller than that of the control group PHF23 WT, and the growth rate was also significantly lower. These experiments demonstrate that deacetylation of PHF23 K211 significantly reduces the tumorigenicity of gastrointestinal stromal tumor cells in the subcutaneous tissue of nude mice. Figure 4 ).

[0150] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Any one of the following substances: (1) A PHF23 protein, characterized in that, The protein is acetylated at the PHF23 K211 site; (2) A nucleic acid, characterized in that the nucleic acid encodes the protein described in (1); (3) A vector, characterized in that the vector comprises the nucleic acid described in (2); (4) A host cell, characterized in that the host cell comprises the nucleic acid described in (2) or the vector described in (3); Preferably, the K211 site of the protein is mutated from lysine to arginine.

2. The use of the substance of claim 1 or an inhibitor of PHF23 K211 site acetylation in the preparation of a medicament for the prevention / treatment of gastrointestinal stromal tumors; Preferably, the inhibitor of acetylation includes a promoter of SIRT7; Preferably, the SIRT7 promoter comprises a vector for SIRT7 overexpression.

3. A drug for treating gastrointestinal stromal tumors, characterized in that, The drug comprises the substance of claim 1 or an inhibitor of PHF23 K211 site acetylation; Preferably, the drug further includes a pharmaceutically acceptable carrier.

4. Application of PHF23 K211 acetylation as a target in screening candidate drugs for the treatment of gastrointestinal stromal tumors; Preferably, the method for screening candidate drugs for the treatment of gastrointestinal stromal tumors includes: The effect of the candidate drug on the acetylation level of the PHF23K211 site in the sample was tested. The decrease in the acetylation level of the PHF23K211 site after the candidate drug was used indicated that the candidate drug has the effect of treating gastrointestinal stromal tumors.

5. A method for screening candidate drugs for the treatment of gastrointestinal stromal tumors, characterized in that, The method includes: testing the effect of a candidate drug on the acetylation level of the PHF23K211 site in a sample, wherein, after using the candidate drug, the acetylation level of the PHF23K211 site decreases, indicating that the candidate drug has the effect of treating gastrointestinal stromal tumors.

6. Application of reagents for detecting acetylation levels at the PHF23 K211 site in the preparation of products for predicting the risk of recurrence of gastrointestinal stromal tumors; Preferably, the reagent comprises an antibody against PHF23 K211; Preferably, the reagents also include reagents used to detect the acetylation level of the PHF23 K211 site by ELISA, Western Blot, immunoprecipitation, mass spectrometry, or immunocytochemistry.

7. A product for predicting the risk of recurrence of gastrointestinal stromal tumors, characterized in that, The product includes a reagent for detecting the acetylation level at the PHF23 K211 site; Preferably, the product further includes testing reagents; Preferably, the product includes a reagent kit and test strips.

8. A method for inhibiting the proliferation / migration of gastrointestinal stromal tumor cells in vitro, characterized in that, The method includes administering the substance of claim 1 or an inhibitor of PHF23 K211 site acetylation.

9. A method for preparing a specific antibody for detecting acetylation at the K211 site of PHF23, characterized in that, The method includes: (1) Synthesize the acetylated antigenic polypeptide at the PHF23 K211 site; (2) Immunize the antigenic polypeptide from (1) to obtain the specific antibody; Preferably, the method further includes post-immunopurification treatment.

10. A system / device for predicting the risk of recurrence of gastrointestinal stromal tumors, characterized in that, The system / device includes: Acquisition Unit: Used to acquire acetylation site data of the PHF23 protein in the sample to be tested; Extraction unit: used to extract acetylation data of the target site of PHF23 protein in the sample to be tested, wherein the acetylation of the target site is the acetylation of the PHF23 K211 site; Prediction Unit: Based on the acetylation data of the target site PHF23 K211, classification prediction is performed to obtain the classification result of the risk of recurrence of gastrointestinal stromal tumors in the test sample. If the acetylation level of the PHF23 K211 site is high, the classification result of the test sample is high risk of recurrence of gastrointestinal stromal tumors; if the acetylation level of the PHF23 K211 site is low, the classification result of the test sample is low risk of recurrence of gastrointestinal stromal tumors.

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