Application of MDM2-213 polypeptide in preparation of antitumor drugs

Abstract

The invention discloses application of MDM2-213 polypeptide in preparation of antitumor drugs, and belongs to the technical field of biological drugs. In order to solve the technical problems that in the prior art, drugs targeting MDM2 face hematological toxicity challenge and the curative effect is insufficient, the invention provides application of MDM2-213 polypeptide in preparation of antitumor drugs, and the amino acid sequence of the MDM2-213 polypeptide is shown as SEQ ID NO.1 or SEQ ID NO.8; the MDM2-213 polypeptide can significantly inhibit the proliferation and migration ability of p53 wild type tumor cells, improve the expression level of p53, reduce the ubiquitination level of cells, and protect p53 and play a tumor inhibition role mechanism by destroying the loop balance of MDM2-p53. On the basis of continuous exploration in the field of MDM2 targeted therapy, a new strategy for preparing the antitumor drug by using the MDM2-213 polypeptide is provided, and a potential way is provided for expanding tumor therapy selection.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and in particular relates to the application of MDM2-213 polypeptide in the preparation of antitumor drugs. Background Technology

[0002] Alternative splicing, also known as alternative splicing, is a common post-transcriptional regulatory mechanism. The spliceosome, composed of five small nucleonucleotide proteins (U1, U2, U4, U5, and U6 snRNPs), splices pre-mRNA, retaining exons and removing introns, ultimately producing mature mRNA. This process can generate various mRNA variants, resulting in protein isoforms with different structures and functions. Recent studies have confirmed that aberrant regulation of splicing factors during tumorigenesis leads to aberrant splicing of various molecular precursor mRNAs, such as aberrant splicing of apoptosis proteins like BIN1, BCL2, and Caspase-9, producing functionally abnormal isoforms that regulate tumor progression. Therefore, targeted regulation of alternative splicing is of great significance for exploring and developing new cancer treatments.

[0003] The MDM2-p53 loop is a classic tumor regulatory pathway. p53 protein, as a key tumor suppressor, exerts its tumor-suppressive function by regulating cell cycle progression and cellular stress responses (such as DNA damage, oncogene activation, and hypoxia). In this loop, p53, as a transcription factor, activates the expression of the full-length MDM2 protein (Murine double-minute 2 full-length protein, MDM2-FL), thereby promoting tumor progression. MDM2, as an E3 ubiquitin ligase, inhibits p53 expression by mediating its ubiquitination and degradation, forming a negative feedback mechanism in the MDM2-p53 loop's regulation of tumors. Studies have shown that dysregulation of the MDM2-p53 loop is a key factor driving the progression of various tumors. U2SURP, also known as SR140, is a U2 snRNP-related protein containing the SURP motif and participates in the alternative splicing processes of various molecules as a core splicing factor. Research has found that U2SURP is abnormally expressed in various tumors, determining tumor progression.

[0004] In the existing technology, several drugs targeting MDM2 have entered clinical trials, such as APG-115, Brigimadlin and Navtemadlin, but they have encountered development difficulties due to significant hematological toxicity challenges and insufficient efficacy. Summary of the Invention

[0005] This invention addresses the challenges of hematologic toxicity and insufficient efficacy of existing drugs targeting MDM2 by providing the application of the MDM2-213 peptide in the preparation of antitumor drugs.

[0006] One of the objectives of this invention is to provide the application of MDM2-213 peptide in the preparation of antitumor drugs.

[0007] In a preferred embodiment of the present invention, the amino acid sequence of the MDM2-213 polypeptide is shown in SEQ ID NO.1 or SEQ ID NO.8.

[0008] In a preferred embodiment of the present invention, the tumor is a p53 wild-type colorectal cancer, ovarian cancer, lung cancer, cervical cancer, breast cancer, thyroid cancer, liver cancer, or laryngeal cancer tumor.

[0009] A second objective of this invention is to provide an antitumor drug comprising an MDM2-213 polypeptide and pharmaceutically acceptable excipients; the amino acid sequence of the MDM2-213 polypeptide is shown in SEQ ID NO.1 or SEQ ID NO.8.

[0010] In a preferred embodiment of the present invention, the tumor is a p53 wild-type colorectal cancer, ovarian cancer, lung cancer, cervical cancer, breast cancer, thyroid cancer, liver cancer, or laryngeal cancer tumor.

[0011] In a preferred embodiment of the present invention, the dosage form of the antitumor drug is any one of capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

[0012] Compared with the prior art, the beneficial effects of the present invention are: the present invention provides the application of MDM2-213 polypeptide in the preparation of antitumor drugs, wherein the MDM2-213 polypeptide is the complete MDM2-213 polypeptide with the amino acid sequence shown in SEQ ID NO.1, or the truncated MDM2-213 polypeptide with the amino acid sequence shown in SEQ ID NO.8.

[0013] This invention discovers the tumor-suppressive function of the MDM2-213 peptide in pan-cancer and reveals the influence and regulatory mechanism of the MDM2-213 peptide on the MDM2-p53 loop. As an endogenous small molecule protein, the MDM2-213 peptide has both the advantage of homology and the innovative mechanism of action of "killing two birds with one stone" in vivo, providing a new strategy for the treatment of p53 wild-type tumors and providing a new theoretical basis for the development of small molecule anti-tumor drugs targeting the MDM2-213 peptide.

[0014] This invention also discovered that after knocking down U2SURP, exons 3 and 4 of MDM2 undergo mutually exclusive splicing, generating a large number of MDM2-213 isoform transcripts (which contain exon 4 but not exon 3). The stop codon TAG appears in exon 4 of the isoform MDM2-213 mRNA sequence, causing premature termination of translation of the transcript isoform MDM2-213, ultimately producing a 76-amino acid polypeptide molecule. This polypeptide is a truncated protein with a partial loss of its functional region, giving it a molecular function that is completely different from MDM2-FL.

[0015] This invention, through pan-cancer clinical tissue samples and cell lines, has demonstrated a clear anti-tumor effect against tumors positive for wild-type p53 (WT-p53). Specifically, overexpression of the MDM2-213 peptide significantly inhibited the proliferation and migration of WT-p53 colon cancer HCT-116 cells, WT-p53 human ovarian cancer A2780 cells, WT-p53 human cervical squamous cell carcinoma Siha cells, WT-p53 human non-small cell lung cancer H1299 cells, p53 synonymous mutant human non-small cell lung cancer H1650 cells, and p53 synonymous mutant human non-small cell lung cancer H1568 cells. The study found that the proliferation and migration of p53-deficient (LT-p53) human ovarian cancer SKOV3 cells, p53-mutant human cervical cancer C33A cells, and p53-mutant human pancreatic cancer SW1990 cells were not affected by overexpression of the MDM2-213 peptide. However, after the above tumor cells restored their WT-p53 function by overexpressing p53, the overexpression of the MDM2-213 peptide exerted the ability to inhibit the proliferation and migration of the above tumor cells. It is evident that the ability of the MDM2-213 peptide to inhibit the proliferation and migration of tumor cells depends on WT-p53.

[0016] This invention demonstrates through in vitro and in vivo experiments that the MDM2-213 peptide exerts its anti-tumor effect through a dual mechanism of action: specifically, it stabilizes p53 and promotes its nuclear translocation, thereby increasing the expression level of p53 protein through interaction, significantly promoting p53 nuclear translocation, and reducing the level of cellular ubiquitination, preventing p53 from being degraded by ubiquitination and promoting its nuclear translocation; at the same time, it inhibits the expression of MDM2-FL, targets and induces the degradation of MDM2-FL, breaks the MDM2-p53 negative feedback loop, and relieves p53 inhibition.

[0017] Small molecule peptide drugs, with their high specificity, good activity, strong designability, and relatively low production cost, have shown great potential in tumor targeted therapy, immunotherapy, and as delivery carriers. To improve the druggability of the MDM2-213 peptide, this invention further reduced the molecular weight of the MDM2-213 peptide. Through co-immunoprecipitation (Co-IP) and point-mutation plasmid experiments, the key active sites of MDM2-213 were identified. Subsequently, using ten amino acid residues with high druggability as truncation ranges, a series of overexpression plasmids were constructed targeting the active sites, and their effects on p53 wild-type tumor cells were tested. The study found that the truncated peptide MDM2-213-QV (SEQ ID NO. 8) has similar anti-cancer activity to the MDM2-213 peptide (SEQ ID NO. 1) and is the main functional fragment for the anti-cancer effect of the MDM2-213 peptide. Compared to the MDM2-213 peptide, the truncated peptide MDM2-213-QV has a smaller molecular weight and therefore higher druggability.

[0018] The MDM2-213 peptide and its truncated peptide provided by this invention can exert their tumor-suppressive function in a p53-dependent manner by disrupting the MDM2-p53 loop balance. These findings provide new theoretical support for the application of the MDM2-213 peptide in clinical cancer treatment. Attached Figure Description

[0019] Figure 1 This is a graph showing the immunofluorescence detection results of the MDM2-213 peptide and the truncated fragment of the MDM2-213 peptide in Example 1; Figure 2 The graph shows the results of MDM2-213 mRNA expression level detection in different tumor tissues in Example 1; A represents colon cancer; B represents cervical cancer; C represents ovarian cancer; D represents lung cancer; E represents breast cancer; F represents thyroid cancer; G represents liver cancer; H represents laryngeal cancer; the horizontal axis NC represents the control group and CA represents the experimental group; the vertical axis represents the expression level of MDM2-213. Figure 3 Figure A shows the detection results of MDM2-213 overexpression plasmid in the HCT-116 cell line in Example 2; Figure B shows the Western blot detection results; Figure B shows the statistical results of HIS tag protein expression. Figure 4The graph shows the effect of MDM2-213 overexpression on the proliferation ability of HCT-116 cells in Example 2; A is the colony formation experiment result; B is the colony number statistics, with the vertical axis "Colony Numbers" representing the colony number; C is the CCK-8 experiment result; D is the Spherold cell in vitro spheroidization experiment result; E is the cell body diameter statistics, with the vertical axis "The diameter of the spherold" representing the cell body diameter. Figure 5 The graph shows the effect of MDM2-213 overexpression on the migration ability of HCT-116 cells in Example 2; A is the result of the wound healing experiment; B is the cell migration rate statistics graph, with the vertical axis representing cell migration rate; C is the result of the Transwell cell migration experiment; and D is the cell count statistics graph, with the vertical axis representing cell count. Figure 6 Figure A shows the effect of MDM2-213 overexpression on the apoptosis ability of HCT-116 cells in Example 2; A is a flow cytometry detection graph; B is a statistical graph of the number of apoptotic cells; where NC is the control group, OE-MDM2-213 is the experimental group overexpressing MDM2-213; Q4 is live cells, Q2 is cells with late apoptosis and necrosis, and Q3 is cells with early apoptosis; Figure 7 Figure 1 shows the effect of MDM2-213 peptide and truncated fragment of MDM2-213 peptide on the proliferation ability of HCT-116 cells in Example 2; Figures A and C are statistical graphs of CCK-8 experimental results, with the vertical axis "absorbence of 450 nm" representing the absorbance at a wavelength of 450 nm; Figures B and D are graphs of colony formation experimental results; Figure E is a statistical graph of colony number, with the vertical axis "Colony Numbers" representing the number of colonies. Figure 8 The graph shows the effect of MDM2-213 peptide and truncated fragment of MDM2-213 peptide on the migration ability of HCT-116 cells in Example 2; A and C are graphs of wound healing experiment results, B and D are graphs of wound closure rate statistics, with the vertical axis representing the wound closure rate; E and G are graphs of Transwell cell migration experiment results, F and H are graphs of cell number statistics, with the vertical axis representing the cell number; Figure 9 The images show the detection results of the mouse xenograft model overexpressing MDM2-213 in Example 2; A is the appearance image; B is the tumor volume statistics, with the vertical axis representing tumor volume; C is the tumor weight statistics, with the vertical axis representing tumor weight. Figure 10Figure A shows the detection of the tumor-suppressive mechanism of the MDM2-213 peptide in Example 2; Figure B shows the results of the Co-IP experiment; Figure C shows the results of the Western blot experiment; Figure D shows the statistical graph of protein expression level, where HIS-MDM2-213 is the MDM2-213 peptide with the HIS tag, p53 is the p53 protein, MDM2-FL is the full-length MDM2 protein, UBI is ubiquitin, and the vertical axis represents the expression level; Figure D shows the results of the immunofluorescence experiment. Figure 11 The graph shows the effect of MDM2-213 overexpression on the proliferation ability of A2780 cells in Example 3; A is the Western blot experiment results; B is the protein expression level statistical graph, HIS is the MDM2-213 polypeptide with HIS tag, p53 is the p53 protein, MDM2-FL is the full-length MDM2 protein, UBI is ubiquitin, and the vertical axis is Expression level; C is the CCK-8 experiment results; D is the colony formation experiment results; E is the colony number statistical graph, and the vertical axis is Colony Number. Figure 12 The graph shows the effect of MDM2-213 overexpression on the proliferation and migration of SKOV3 cells in Example 3; A is the CCK-8 experiment result; B is the Transwell experiment result; C is the cell count statistics, with Cell Counts on the vertical axis representing cell number; D is the wound healing experiment result; E is the cell migration rate statistics, with Cell migrationrate on the vertical axis representing cell migration rate. Figure 13 The graph shows the effect of MDM2-213 overexpression on the proliferation and migration of Siha cells in Example 4; A is the RT-qPCR detection result; B is the CCK-8 experiment result; C is the colony formation experiment result; D is the colony number statistics graph, with the ordinate being Colony Number; E is the wound healing experiment result; F is the wound closure rate statistics graph, with the ordinate being Wound Closure Rate; G is the Transwell experiment result, and H is the cell number statistics graph, with the ordinate being Cell Number. Figure 14The graph shows the effect of MDM2-213 overexpression on the proliferation and migration ability of C33A cells in Example 4; A is the RT-qPCR detection result; B is the CCK-8 experiment result; C is the colony formation experiment result; D is the colony number statistics graph, with the vertical axis representing the colony number; E is the wound healing experiment result; F is the wound closure rate statistics graph, with the vertical axis representing the wound closure rate; G is the Transwell experiment result, and H is the cell number statistics graph, with the vertical axis representing the cell count. Figure 15 Figure A shows the effect of MDM2-213 overexpression on the proliferation and migration ability of H1299 cells in Example 5; Figure B shows the RT-qPCR detection results; Figure C shows the CCK-8 assay results; Figure D shows the colony formation assay results; Figure D shows the colony number statistics, with the ordinate being Colony Number; Figure E shows the wound healing assay results; Figure F shows the wound closure rate statistics, with the ordinate being Wound Closure Rate; Figure G shows the Transwell assay results; Figure H shows the cell number statistics, with the ordinate being Cell Number. Figure 16 Figure A shows the effect of MDM2-213 overexpression on the proliferation and migration ability of H1650 cells in Example 5; Figure B shows the RT-qPCR detection results; Figure C shows the CCK-8 assay results; Figure D shows the colony formation assay results; Figure D shows the colony number statistics, with the ordinate being Colony Number; Figure E shows the wound healing assay results; Figure F shows the wound closure rate statistics, with the ordinate being Wound Closure Rate; Figure G shows the Transwell assay results; Figure H shows the cell number statistics, with the ordinate being Cell Number. Figure 17 Figure A shows the effect of MDM2-213 overexpression on the proliferation and migration ability of H1568 cells in Example 5; Figure B shows the results of the Western blot experiment; Figure B shows the results of the CCK-8 assay; Figure C shows the results of the colony formation experiment; Figure D shows the colony number statistics, with the vertical axis representing the colony number; Figure E shows the results of the wound healing experiment; Figure F shows the wound closure rate statistics, with the vertical axis representing the wound closure rate; Figure G shows the results of the Transwell assay; Figure H shows the cell number statistics, with the vertical axis representing the cell count. Figure 18The graph shows the effect of MDM2-213 overexpression on the proliferation and migration of SW1990 cells in Example 6; A is the RT-qPCR detection result; B is the CCK-8 assay result; C is the colony formation assay result; D is the colony number statistics graph, with the ordinate being Colony Number; E is the wound healing assay result; F is the wound closure rate statistics graph, with the ordinate being Wound Closure Rate; G is the Transwell assay result; H is the cell number statistics graph, with the ordinate being Cell Number. Figure 19 A shows the detection of the binding site between the MDM2-213 peptide and WT-p53; A is the Alphafold server prediction result; B is the experimental result of Co-IP and point mutation plasmid. Detailed Implementation

[0020] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0022] The experimental steps involved in the following embodiments: (1) CCK8 cell proliferation experiment: All culture reagents required for the experiment must be brought to room temperature beforehand, and all experimental equipment must be sterilized by UV irradiation for 15-30 minutes beforehand. Digest the cells required for the experiment and count them, then seed 1000 cells / well into a 96-well plate. After the experimental incubation period, add 10... CCK8 enhanced solution was mixed well and cultured in a 37°C cell incubator for 2 hours; absorbance at 450 nm was measured using an ELISA reader.

[0023] (2) Colony formation experiment: The cells used in the experiment were seeded at a rate of 1000 cells per well in a six-well plate; the culture medium was changed every 3 to 4 days; after culturing in an incubator for 10 days, the cells were removed, washed gently with PBS, fixed with methanol at room temperature for 30 minutes, washed gently with PBS and air-dried; 0.5% crystal violet solution was added and stained at room temperature for 15 minutes; excess crystal violet solution was washed off and the cells were air-dried in a cool place; the cells were observed and photographed, and the cell colony count was analyzed and counted using ImageJ software.

[0024] (3) Astroglial cell spheroidization experiment: Prepare target cells in the logarithmic growth phase and in good condition. Perform routine passages the day before the experiment to ensure the cells are in optimal condition. Digest, collect, and count the cells at 1000 cells / well. Thaw the matrix gel in advance and add culture medium to a concentration of 1 mg / L. Quickly mix the matrix gel with the cells and transfer them to small dishes. Place the culture dishes in a 37°C, 5% CO2 cell culture incubator. After the matrix gel solidifies, add an appropriate amount of culture medium. Observe the number and size of the spheroids daily and change the medium every 3 days. Observe and photograph the cells, and use ImageJ software to analyze and count the diameter and number of spheroids.

[0025] (4) Transwell cell migration assay: The cell suspension was prepared at a ratio of 5 × 10⁵ cells / well. 4 Seed cells, using 200 Serum-free culture medium was incubated above the Transwell chamber, with the lower part of the chamber at 600°C. Complete culture medium containing 20% ​​serum was used. After 6 hours of cell adhesion, the cells were treated with additional drugs according to experimental requirements. After culturing in an incubator for 36-48 hours, the cells were removed, the culture medium was discarded, and the cells were rinsed in PBS. After fixation with methanol at room temperature for 30 minutes, the cells were rinsed in PBS. The cells were stained with 0.5% crystal violet solution at room temperature for 3 minutes, and the cells in the upper chamber were gently wiped away with a cotton swab. Excess crystal violet was gently washed away in PBS and the cells were air-dried. After drying, the cells were observed under a microscope, and images were taken in different fields of view. The images were analyzed and counted using ImageJ software.

[0026] (5) Wound healing experiment: Draw 2 to 3 marker lines on the bottom of a 6-well plate using a marker, then seed the cell suspension into the 6-well plate; after the cells have grown to confluence, use 10... Scratch marks were made with the pipette tip; floating cells were washed with PBS buffer and photographed under a microscope; after photographing, the culture medium was replaced with culture medium containing 1% serum to prevent cell proliferation from affecting the experimental results, and the cells were cultured in a 37°C cell incubator; photographs were taken at the same location every 24 hours; the area of ​​the scratch marks was statistically analyzed using ImageJ software to calculate the cell migration rate and perform statistical analysis.

[0027] (6) Flow cytometry detection of apoptosis Seed cells into a plate. When the cell density is suitable, digest the cells and centrifuge at 1300 rpm for 8 minutes, discarding the supernatant. Add 1 mL of pre-chilled PBS, resuspend the cells, centrifuge at 1300 rpm for 8 minutes, discarding the supernatant. Repeat the above steps 3 times. Add 200 mL of pre-chilled PBS. Binding Buffer, resuspend cells, add 10 Annexin-V-FITC, gently mix at room temperature for 15 minutes (protected from light) or at 4°C for 30 minutes (protected from light), and add 5% FITC solution after 25 minutes of reaction. PI; finally add 300 Binding Buffer, tested on the machine.

[0028] (7) Western blot experiment: Assemble the clean glass plate for dispensing glue onto the glue preparation frame correctly, add double distilled water to check for leaks. If the liquid level drops too quickly, it will affect subsequent glue preparation. Prepare SDS-PAGE gel according to Table 1. Prepare and use immediately, or wrap it in plastic wrap and store at 4°C for use within 2 days.

[0029] Table 1

[0030] Electrophoresis: Fix the SDS-PAGE gel on the electrophoresis tank, remove the comb, add electrophoresis buffer (1×) and check for leaks; thaw the test protein and marker at room temperature and place them on ice, shake to mix, and load the samples according to the set order and volume; start electrophoresis at a constant voltage of 80V, and after the markers separate, adjust the voltage to a constant voltage of 120V until the protein is completely separated.

[0031] Transfer: Prepare transfer buffer (1×) in a ratio of double-distilled water:methanol:transfer buffer (10×) = 7:2:1; Place the gel after electrophoresis into the transfer buffer, cut off any parts irrelevant to the experiment, and cut the NC membrane according to the size of the gel (the NC membrane is slightly larger than the gel); Place the sponge, 3 layers of filter paper, gel, NC membrane, 3 layers of filter paper, and sponge into the transfer clamp in sequence, use a roller to remove air bubbles between each layer, clamp the transfer clamp tightly, and transfer to the transfer tank, with the gel at the negative electrode and the NC membrane at the positive electrode. Fill the transfer tank with transfer buffer and place it in an ice basin; Start the transfer apparatus and begin the transfer at a constant current of 250mA.

[0032] Sealing: Prepare a 5% skim milk powder solution (prepared with 1×TBST); after the transfer is completed, rinse the NC membrane surface with TBST to remove the transfer solution, then place it in 5% skim milk powder and seal on a shaker for 1-2 hours.

[0033] Primary antibody incubation: After blocking, wash off the surface milk powder with TBST; dilute the corresponding antibody with TBST and incubate overnight at 4°C; antibody dilution ratios are shown in Table 2.

[0034] Secondary antibody incubation: After the primary antibody incubation is completed, wash the NC membrane with TBST on a shaker for 10 minutes each time, for a total of 3 washes; dilute the corresponding secondary antibody with TBST (antibody dilution ratio is 1:5000 for rabbit antibody; 1:5000 for mouse antibody), and incubate on a shaker at room temperature for 1 hour.

[0035] Photographing and developing: After incubating the secondary antibody for 1 hour, wash the membrane with TBST; prepare the developing solution (according to the instructions); turn on the Tanon chemiluminescence imaging system, place the membrane on the imaging plate, add an appropriate amount of developing solution, take a picture and save it.

[0036] Table 2

[0037] (8) Co-IP experiment: Preparation of cell lysis buffer: Collect cells to be tested, about 3 to 4 culture dishes with a cell density of 90%, and wash 3 times with pre-cooled PBS; prepare cell lysis buffer (RIPA:PS:PMSF=100:2:1), digest on ice for 15 minutes, and then scrape to collect cells; lyse on ice for 30 minutes, mixing by pipetting every 10 minutes; centrifuge at 4°C, 12000 rpm for 20 minutes; collect the supernatant and separate a small portion of the supernatant as the input group.

[0038] Pre-clarification of cell lysate: Resuspend magnetic beads and aspirate 20 μL of the solution. Place the bead mixture in a new centrifuge tube and carefully aspirate the buffer solution using a magnetic separator for 10-15 seconds; add 500 ml of the buffer solution to the magnetic beads. Cell lysis buffer was used to wash the magnetic beads by vortexing. The buffer was carefully aspirated after 10-15 seconds on a magnetic separator. This process was repeated twice. The cell lysis buffer was then mixed with the washed magnetic beads and incubated at room temperature for 20 minutes by vortexing. The supernatant was then separated using a magnetic separator to obtain the pre-clarified cell lysis buffer.

[0039] Immunoprecipitation: Take 200 Cell lysis buffer was added to the primary antibody at a ratio of 1:200, and the mixture was incubated overnight at 4°C by rotation. The magnetic beads were pre-washed (step: add 500 ml of the primary antibody to the magnetic beads). Wash the magnetic beads with cell lysis buffer, vortex for 10-15 seconds using a magnetic separator, carefully aspirate the buffer, repeat twice. Add the immune complex (overnight incubated primary antibody and cell lysis buffer) to the washed magnetic beads and incubate at room temperature for 20 minutes. Separate the magnetic beads using a magnetic separator, discard the supernatant, and rinse the beads with 500 ml of water. Wash the cells five times with cell lysis buffer; add loading buffer, mix well, heat at 95-100℃ for 10 minutes, and analyze the sample using Western blot technology.

[0040] (9) Immunofluorescence experiment: Cells were crawled onto slides, and experiments began when the cell density reached 50%. The culture medium was discarded, and the cells were washed twice with PBS. The cells were fixed with 4% paraformaldehyde for approximately 30 minutes, followed by three washes with PBS on a shaker for 5 minutes each. 0.5% Triton X-100 was prepared and added to each slide for cell permeation for approximately 10 minutes. Excess permeation solution was aspirated after permeation, and goat serum blocking solution was added to each slide to cover the cells. Blocking was performed for 1 hour. After blocking, the cells were washed twice with PBS. Primary antibodies were prepared according to the following ratios (HIS-M, 1:500; HIS-R, 1:500; p53, 1:500; Caspase-3, 1:500), and an appropriate amount was added to cover the cells. The cells were incubated overnight at 4°C for 12 hours. The cells were then recovered. Primary antibody: Wash cells three times with PBS for 5 minutes on a shaker. Prepare secondary antibody according to the specified ratio, add an appropriate amount of secondary antibody to cover cells in a dark room, and incubate at room temperature for 1 hour. Recover secondary antibody: Wash cells three times with PBS for 5 minutes on a shaker. Prepare DAPI working solution according to the specified ratio, add an appropriate amount of DAPI to cover cells in a dark room, and incubate at room temperature for 15 minutes. Remove DAPI: Wash cells three times with PBS for 5 minutes on a shaker. Shake off excess liquid, add anti-fluorescence quencher, mount the slide, and store in a humidified chamber at 4°C. Take photos under a fluorescence microscope.

[0041] (10) Cell protein extraction steps: All experimental steps below should be performed on ice. Discard the cell culture medium. Taking a six-well plate as an example, add 2 mL of pre-chilled PBS to each well and wash twice, then aspirate the PBS. Prepare lysis buffer and add an appropriate amount according to the cell density (per 10 wells). 6 Add 100-200 to the cells Lysis buffer); shake on ice for 15 minutes, sealing with sealing film to prevent water ingress; scrape cells with a cell scraper and collect in centrifuge tubes, incubate on ice for 15 minutes, vigorously shaking and gently centrifuging once every 5 minutes; pre-cool the centrifuge, centrifuge at 4°C, 12000 rpm / min for 30 minutes; remove the EP tube, aspirate the supernatant into a new EP tube, and record the volume of supernatant aspirated; measure the protein concentration according to the BCA protein concentration assay kit instructions; add 5× protein loading buffer; incubate in a 100°C metal bath for 5 minutes, remove and immediately place on ice, then store at -20°C.

[0042] Example 1: Construction of overexpression plasmid The MDM2-213 polypeptide with amino acid sequence SEQ ID NO.1 and the truncated fragments of the MDM2-213 polypeptide with amino acid sequences SEQ ID NO.2-8 were ligated with HIS tag sequences using gene synthesis method to construct overexpression plasmids and obtain fusion proteins, collectively referred to as MDM2-213-HIS, and named as shown in Table 3.

[0043] Table 3

[0044] Effect Experiment: (1) Immunofluorescence experiments were performed on the overexpression plasmids obtained above, and the results are as follows: Figure 1 As shown, the MDM2-213-HIS fusion proteins obtained above are all stably expressed without degradation.

[0045] (2) RNA was extracted from adjacent normal tissue and tumor tissue samples (colon cancer, cervical cancer, ovarian cancer, lung cancer, breast cancer, thyroid cancer, liver cancer and laryngeal cancer) of clinical cancer patients. The expression level of MDM2-213 polypeptide mRNA with nucleotide sequence shown in SEQ ID NO.1 in the above tumor tissues was detected by RT-qPCR using primers MDM2-213-F (SEQ ID NO.9) and MDM2-213-R (SEQ ID NO.10).

[0046] The following experimental procedures were all performed on ice. The Trizol method for extracting RNA from tissues followed these steps: 1) Take a piece of tissue the size of a mung bean and quickly put it into a mortar and grind it (crush it first, then grind it). Add liquid nitrogen while grinding until it becomes powder. Transfer it into a centrifuge tube and immediately add 1 mL of Trizol. 2) Let stand on ice for 5 minutes, add 200 mL of chloroform, shake vigorously and vortex for 30 seconds, then let stand on ice for 5 minutes; 3) Pre-cool the low-temperature centrifuge and centrifuge at 12,000 rpm and 4°C for 15 minutes; aspirate the supernatant, being careful not to aspirate the precipitate, and transfer the supernatant to a new sterile centrifuge tube. Add an equal volume of isopropanol to the supernatant, mix well, and let stand on ice for 15 minutes. Centrifuge at 12,000 rpm and 4°C for 10 minutes; discard the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water) and wash twice. Centrifuge at 12,000 rpm and 4°C for 5 minutes. 4) Discard the supernatant, invert and air dry, add an appropriate amount of DEPC water to dissolve the precipitate; use Nanodrop to measure RNA concentration and purity.

[0047] The RNA obtained above was diluted to 500 ng / Reverse transcription was performed using the SPARKscript II RT Plus Kit (WithgDNA Eraser) (purchased from Cisco Systems, Inc.), entirely on ice. The reverse transcription system was prepared according to the instructions, as shown in Table 4. Before use, all reagents except PrimeScript RT Enzyme Mix were shaken and briefly centrifuged. After preparation, the mixture was shaken and centrifuged to perform the reverse transcription reaction and obtain cDNA. The reverse transcription reaction conditions were: 37°C for 15 minutes; 85°C for 5 seconds; and stored at -20°C.

[0048] Table 4

[0049] Using cDNA diluted 2-fold from the tumor tissue sample as a template, the PCR reaction system was prepared according to Table 5. Two-step PCR was performed using primers MDM2-213-F (SEQ ID NO. 9) and MDM2-213-R (SEQ ID NO. 10) as primers. The reaction program was as follows: pre-denaturation, 95℃, 30 seconds; PCR reaction, 95℃, 5 seconds; 55℃-60℃, 30 seconds; 40 cycles.

[0050] Table 5

[0051] like Figure 2 As shown, compared with the corresponding adjacent normal control (NC) tissues, the expression level of MDM2-213 mRNA was significantly reduced in tumor tissues of colon cancer, cervical cancer, ovarian cancer, lung cancer, breast cancer, thyroid cancer, liver cancer, and laryngeal cancer; indicating that the MDM2-213 peptide is expressed at low levels in tumor tissues.

[0052] Example 2: Application of MDM2-213 peptide in the preparation of anti-HCT-116 human colon cancer tumor drug 1. The MDM2-213 polypeptide with the amino acid sequence shown in SEQ ID NO.1 was linked to the HIS tag sequence by gene synthesis to construct an overexpression plasmid and obtain the fusion protein, abbreviated as: OE-MDM2-213.

[0053] In the HCT-116 cell line, the overexpression of OE-MDM2-213 was detected by HIS antibody after cell transfection. The specific steps are as follows: Ctrl is the blank control group, NC is the plasmid control group, and OE-MDM2-213 is the MDM2-213 overexpression group (SEQ ID NO.1); transfection was started when the cell density reached 40%. The corresponding plasmid was taken out from the -20℃ freezer, and the PEI transfection reagent was taken out from the 4℃ freezer. After brief centrifugation, the cells were placed on ice and transfected according to the PEI (… ): Plasmid ( Transfection was performed using a ratio of 2:1; taking one well of a six-well plate as an example, 100... Add 4% of serum-free and antibiotic-free culture medium PEI, 2 After the plasmid has been left to stand at room temperature for 10-15 minutes, the mixture is dropped into the wells to be transfected. Cell culture medium is added to make up the amount of cell culture medium. The medium is changed after 4 hours of incubation.

[0054] like Figure 3 As shown, a distinct band appeared in the OE-MDM2-213 group in the HCT-116 cell line, and HIS antibody was clearly detected in the OE-MDM2-213 group; indicating that the MDM2-213 overexpression plasmid was successfully constructed in the HCT-116 cell line, and MDM2-213 overexpression was successful.

[0055] 2. To determine the effects of the MDM2-213 polypeptide with the amino acid sequence SEQ ID NO.1 and the truncated fragments of the MDM2-213 polypeptide with the amino acid sequences SEQ ID NO.2-8 on the WT-p53 human colon cancer cell line HCT-116, the effects of MDM2-213 overexpression on cell proliferation, migration and apoptosis were detected by CCK-8 assay, colony formation assay, in vitro spheroidization assay of MMR cells, Transwell cell migration assay, wound healing assay and flow cytometry.

[0056] like Figure 4 As shown, the results of CCK-8, colony formation and in vitro spheroidization experiments of matrix glial cells showed that in the WT-p53 colon cancer HCT-116 cell line, the proliferation ability of tumor cells in the OE-MDM2-213 group was significantly lower than that in the control group, indicating that overexpression of MDM2-213 peptide (SEQ ID NO.1) has the ability to inhibit the proliferation of tumor cells HCT-116.

[0057] like Figure 5 As shown in the Transwell cell migration and wound healing assay, the migration ability of tumor cells in the OE-MDM2-213 group was significantly lower than that in the control group in the WT-p53 colon cancer HCT-116 cell line, indicating that overexpression of MDM2-213 peptide (SEQ ID NO.1) in colon cancer cells has the ability to inhibit the migration of HCT-116 tumor cells.

[0058] like Figure 6As shown, flow cytometry results indicated that in the WT-p53 colon cancer HCT-116 cell line, the number of tumor cells in the OE-MDM2-213 group undergoing early and late apoptosis was significantly higher than that in the control group, suggesting that overexpression of the MDM2-213 peptide (SEQ ID NO.1) in colon cancer cells has the ability to promote apoptosis of HCT-116 tumor cells.

[0059] like Figure 7 As shown in the CCK-8 and colony formation assays, in the WT-p53 colon cancer HCT-116 cell line, there was no significant difference in the proliferation capacity of tumor cells in the OE-MDM2-213-T1, T2, T3, T4, T5, and T6 groups compared with the NC control group; however, the proliferation capacity of tumor cells in the OE-MDM2-213-QV group was significantly lower than that in the NC control group, and similar to that in the OE-MDM2-213 group, indicating that the function of MDM2-213 peptide in inhibiting tumor proliferation mainly depends on the active fragment MDM2-213-QV (SEQ ID NO. 8).

[0060] like Figure 8 As shown in the Transwell cell migration and wound healing assay, in the WT-p53 colon cancer HCT-116 cell line, there was no significant difference in the migration ability of tumor cells in the OE-MDM2-213-T1, T2, T3, T4, T5, and T6 groups compared with the NC control group. However, the migration ability of tumor cells in the OE-MDM2-213-QV group was significantly lower than that in the NC control group, and similar to that in the OE-MDM2-213 group. This indicates that the function of MDM2-213 peptide in inhibiting tumor migration mainly depends on the active fragment MDM2-213-QV (SEQ ID NO.8).

[0061] 3. Animal in vivo experiments Constructing a mouse xenograft model: Establishment of a mouse xenograft model overexpressing MDM2-213 peptide: NC group (n=7), MDM2-213 overexpression group (n=7); 1×10n per mouse 6 100 Prepare the cell suspension (using DPBS), and use a disposable 1 mL syringe; gently pipette the cells to mix them, and then aspirate 100 mL of the solution. Cell suspension; capture immunodeficient mice and inject into the back of the neck; ear tag the mice, measure and record the weight of the mice and the long and short diameters of the tumors every 2 days; when the tumors grow to a suitable size, sacrifice the mice and photograph them according to the experimental groups; carefully separate the mouse tumor tissue, weigh it, and process and preserve it according to the requirements of subsequent experiments.

[0062] like Figure 9As shown, the tumor volume (long and short diameters) and tumor weight of xenograft mice overexpressing MDM2-213 peptide were significantly lower than those of the NC control group, indicating that overexpression of MDM2-213 peptide (SEQ ID NO.1) has the ability to inhibit tumor tissue development.

[0063] 4. To determine the effect of MDM2-213 peptide (SEQ ID NO.1) on the WT-p53 human colon cancer cell line HCT-116, its potential regulatory mechanism was explored by Co-IP, Western blot and immunofluorescence experiments.

[0064] like Figure 10 As shown in Part A, the Co-IP experiment results indicate that the MDM2-213 peptide can interact with p53; as Figure 10 As shown in section B and C, Western blot results indicated that in the WT-p53 colon cancer HCT-116 cell line, the MDM2-213 peptide could increase the expression level of p53 protein, decrease the level of cellular ubiquitination, and inhibit the expression of MDM2-FL; this suggests that the MDM2-213 peptide can exert a tumor-suppressive function by increasing p53 protein expression. Figure 10 As shown in section D, the immunofluorescence assay results indicate that overexpression of MDM2-213 significantly promoted p53 nuclear translocation, revealing that MDM2-213 exerts its anti-tumor effect by promoting p53 nuclear translocation. Therefore, the MDM2-213 peptide exerts its tumor-suppressive function by increasing p53 protein expression.

[0065] Example 3: Application of MDM2-213 peptide in the preparation of anti-A2780 and SKOV3 human ovarian cancer tumor drugs The MDM2-213 polypeptide with the amino acid sequence shown in SEQ ID NO.1 was linked to the HIS tag sequence using a gene synthesis method to construct an overexpression plasmid and obtain a fusion protein, abbreviated as: OE-MDM2-213.

[0066] To determine the effects of the MDM2-213 polypeptide with the amino acid sequence SEQ ID NO.1 and the truncated fragments of the MDM2-213 polypeptide with the amino acid sequences SEQ ID NO.2-8 on the WT-p53 human ovarian cancer cell lines A2780 and SKOV3, the effects of MDM2-213 overexpression on cell proliferation, migration, and apoptosis were detected by Western blot, CCK-8 assay, colony formation assay, Transwell cell migration assay, and wound healing assay.

[0067] like Figure 11As shown in section AB, Western blot results indicated that in the WT-p53 human ovarian cancer A2780 cell line, the MDM2-213 peptide increased p53 protein expression and decreased cellular ubiquitination levels, while simultaneously inhibiting MDM2-FL expression; this suggests that the MDM2-213 peptide can exert tumor suppressive function by increasing p53 protein expression. Figure 11 As shown in section CE, in the WT-p53 human ovarian cancer A2780 cell line, the cell proliferation capacity of the OE-MDM2-213 group was significantly lower than that of the control group; indicating that overexpression of MDM2-213 in human ovarian cancer cells has the ability to inhibit the proliferation of tumor cells A2780.

[0068] like Figure 12 As shown, in the p53-deficient (LT-p53) human ovarian cancer SKOV3 cell line, the OE-MDM2-213 group showed no significant change in cell proliferation and migration compared to the NC control group. However, after overexpressing WT-p53 to rescue p53 function, the OE-MDM2-213+OE-P53 group showed significantly lower cell proliferation and migration than the control group and other groups. This indicates that overexpressing MDM2-213 in the p53-deficient (LT-p53) human ovarian cancer cell line SKOV3 does not have a tumor-suppressive effect. However, overexpressing WT-p53 to rescue p53 function restores its ability to inhibit the proliferation and migration of SKOV3 tumor cells, further demonstrating that the ability of MDM2-213 peptide to inhibit tumor cell proliferation and migration depends on WT-p53.

[0069] Example 4: Application of MDM2-213 peptide in the preparation of anti-Siha and C33A human cervical cancer tumor drugs The MDM2-213 polypeptide with the amino acid sequence shown in SEQ ID NO.1 was linked to the HIS tag sequence using a gene synthesis method to construct an overexpression plasmid and obtain a fusion protein, abbreviated as: OE-MDM2-213.

[0070] To determine the effects of the MDM2-213 polypeptide with the amino acid sequence SEQ ID NO.1 and the truncated fragments of the MDM2-213 polypeptide with the amino acid sequences SEQ ID NO.2-8 on the WT-p53 human cervical cancer cell lines Siha and C33A, the effects of MDM2-213 overexpression on cell proliferation, migration, and apoptosis were detected by RT-qPCR, CCK-8 assay, colony formation assay, Transwell cell migration assay, and wound healing assay.

[0071] like Figure 13 As shown in section A, MDM2-213 was successfully overexpressed in the WT-p53 human cervical squamous cell carcinoma Siha cell line; Figure 13 As shown in section BD, in the WT-p53 human cervical squamous cell carcinoma Siha cell line, the cell proliferation ability of the OE-MDM2-213 group was significantly lower than that of the control group, indicating that overexpression of MDM2-213 in human cervical squamous cell carcinoma cells has the ability to inhibit the proliferation of tumor cells Siha; Figure 13 As shown in the EH section, in the WT-p53 human cervical squamous cell carcinoma Siha cell line, the cell migration ability of the OE-MDM2-213 group was significantly lower than that of the control group, indicating that overexpression of MDM2-213 in human cervical squamous cell carcinoma cells has the ability to inhibit the migration of tumor cells Siha.

[0072] like Figure 14 As shown in section A, MDM2-213 was successfully overexpressed in the p53-mutant human cervical cancer C33A cell line; Figure 14 As shown in section BD, in the p53-mutant human cervical cancer C33A cell line, there was no significant difference in cell proliferation between the OE-MDM2-213 group and the control group, indicating that overexpression of MDM2-213 in p53-mutant human cervical cancer cells does not affect the proliferation ability of p53-mutant human cervical cancer cells C33A; further demonstrating that the function of MDM2-213 peptide in inhibiting tumor cell proliferation depends on WT-p53; Figure 14 As shown in the middle EH section, in the p53-mutant human cervical cancer C33A cell line, there was no significant difference in cell migration ability between the OE-MDM2-213 group and the control group, indicating that overexpression of MDM2-213 in p53-mutant human cervical cancer cells does not affect the migration ability of p53-mutant human cervical cancer cells C33A; further illustrating that the function of MDM2-213 peptide in inhibiting tumor cell migration depends on WT-p53.

[0073] Example 5: Application of MDM2-213 peptide in the preparation of anti-H1299, H1650 and H1568 human lung cancer tumor drugs The MDM2-213 polypeptide with the amino acid sequence shown in SEQ ID NO.1 was linked to the HIS tag sequence using a gene synthesis method to construct an overexpression plasmid and obtain a fusion protein, abbreviated as: OE-MDM2-213.

[0074] To determine the effects of the MDM2-213 polypeptide with the amino acid sequence SEQ ID NO.1 and the truncated fragments of the MDM2-213 polypeptide with the amino acid sequences SEQ ID NO.2-8 on WT-p53 human lung cancer cell lines H1299, H1650 and H1568, the effects of MDM2-213 overexpression on cell proliferation, migration and apoptosis were detected by RT-qPCR, Western blot, CCK-8 assay, colony formation assay, Transwell cell migration assay and wound healing assay.

[0075] like Figure 15 As shown in section A, MDM2-213 was successfully overexpressed in the WT-p53 human non-small cell lung cancer H1299 cell line; Figure 15 As shown in section BD, in the WT-p53 human non-small cell lung cancer H1299 cell line, the cell proliferation capacity of the OE-MDM2-213 group was significantly lower than that of the control group, indicating that overexpression of MDM2-213 in human non-small cell lung cancer cells has the ability to inhibit the proliferation of tumor cells H1299; Figure 15 As shown in the EH section, in the WT-p53 human non-small cell lung cancer H1299 cell line, the cell migration ability of the OE-MDM2-213 group was significantly lower than that of the control group, indicating that overexpression of MDM2-213 in human non-small cell lung cancer cells has the ability to inhibit the migration of tumor cells H1299.

[0076] like Figure 16 As shown in section A, MDM2-213 was successfully overexpressed in the p53 synonymous mutant human non-small cell lung cancer H1650 cell line; Figure 16 As shown in section BD, in the p53 synonymous mutant human non-small cell lung cancer H1650 cell line, the cell proliferation capacity of the OE-MDM2-213 group was significantly lower than that of the control group, indicating that overexpression of MDM2-213 in p53 synonymous mutant human non-small cell lung cancer cells has the ability to inhibit the proliferation of tumor cells H1650; Figure 16 As shown in the EH section, in the p53 synonymous mutant human non-small cell lung cancer H1650 cell line, the migration ability of the OE-MDM2-213 group was significantly lower than that of the control group, indicating that overexpression of MDM2-213 in p53 synonymous mutant human non-small cell lung cancer cells has the ability to inhibit the migration of tumor cells H1650.

[0077] like Figure 17 As shown in section A, MDM2-213 was successfully overexpressed in the p53 synonymous mutant human non-small cell lung cancer H1568 cell line; Figure 17As shown in section BD, in the p53 synonymous mutant human non-small cell lung cancer H1568 cell line, the cell proliferation capacity of the OE-MDM2-213 group was significantly lower than that of the control group, indicating that overexpression of MDM2-213 in p53 synonymous mutant human non-small cell lung cancer cells has the ability to inhibit the proliferation of tumor cells H1568; Figure 17 As shown in the EH section, in the p53 synonymous mutant human non-small cell lung cancer H1568 cell line, the cell migration ability of the OE-MDM2-213 group was significantly lower than that of the control group, indicating that overexpression of MDM2-213 in p53 synonymous mutant human non-small cell lung cancer cells has the ability to inhibit the migration of tumor cells H1568.

[0078] Example 6: Application of MDM2-213 peptide in the preparation of anti-SW1990 human pancreatic cancer tumor drug The MDM2-213 polypeptide with the amino acid sequence shown in SEQ ID NO.1 was linked to the HIS tag sequence using a gene synthesis method to construct an overexpression plasmid and obtain a fusion protein, abbreviated as: OE-MDM2-213.

[0079] To determine the effects of the MDM2-213 polypeptide with the amino acid sequence SEQ ID NO.1 and the truncated fragments of the MDM2-213 polypeptide with the amino acid sequences SEQ ID NO.2-8 on the WT-p53 human pancreatic cancer cell line SW1990, the effects of MDM2-213 overexpression on cell proliferation, migration and apoptosis were detected by RT-qPCR, CCK-8 assay, colony formation assay, Transwell cell migration assay and wound healing assay.

[0080] like Figure 18 As shown in section A, MDM2-213 was successfully overexpressed in the p53-mutant human pancreatic cancer SW1990 cell line; Figure 18 As shown in section BD, in the p53-mutant human pancreatic cancer SW1990 cell line, there was no significant difference in cell proliferation between the OE-MDM2-213 group and the control group, indicating that overexpression of MDM2-213 in p53-mutant human pancreatic cancer cells does not affect the proliferation ability of p53-mutant human pancreatic cancer cells SW1990; further demonstrating that the function of MDM2-213 peptide in inhibiting tumor cell proliferation depends on WT-p53; Figure 18As shown in the middle EH section, in the p53-mutant human pancreatic cancer SW1990 cell line, there was no significant difference in cell migration ability between the OE-MDM2-213 group and the control group, indicating that overexpression of MDM2-213 in p53-mutant human pancreatic cancer cells does not affect the migration ability of p53-mutant human pancreatic cancer cells SW1990; further illustrating that the function of MDM2-213 peptide in inhibiting tumor cell migration depends on WT-p53.

[0081] This invention uses Alphafold server to predict the binding site of peptide MDM2-213 to p53, and verifies the binding site of MDM2-213 peptide (SEQ ID NO.1) to p53 using a p53 point mutation plasmid designed and synthesized by Wuhan Miaoling Biotechnology Co., Ltd. through Co-IP experiments. Figure 19 As shown in Part A, Alphafold server prediction results indicate that the MDM2-213 peptide binds to p53 via a specific site; as shown in Part A. Figure 19 As shown in Part B, the binding of the peptide MDM2-213 to the mutant p53 was detected by Co-IP assay after constructing a p53 point mutant plasmid. In this assay, IP:FLAG represents p53 protein expressed by immunoprecipitation and the FLAG tag fusion, the heavy chain represents the heavy chain, IB:MDM2-213-HIS represents MDM2-213 peptide expressed by Western blot and the HIS tag fusion, p53-1 (W23 G) represents the p53 protein with a tryptophan mutation at position 23 to glycine, p53-2 (H179R) represents the p53 protein with a histidine mutation at position 179 to arginine, and p53-3 (W23 G, H179R) represents a double-point mutant group. The results indicate that the MDM2-213 peptide is linked to the W23 site of WT-p53 through the D75 site.

[0082] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. Application of MDM2-213 peptide in the preparation of antitumor drugs.

2. The application according to claim 1, characterized in that, The amino acid sequence of the MDM2-213 polypeptide is shown in SEQ ID NO.1 or SEQ ID NO.

8.

3. The application according to claim 1, characterized in that, The tumors mentioned are p53 wild-type colorectal cancer, ovarian cancer, lung cancer, cervical cancer, breast cancer, thyroid cancer, liver cancer, and laryngeal cancer.

4. An antitumor drug, characterized in that, The antitumor drug includes an MDM2-213 polypeptide and pharmaceutically acceptable excipients; the amino acid sequence of the MDM2-213 polypeptide is shown in SEQ ID NO.1 or SEQ ID NO.

8.

5. The antitumor drug according to claim 4, characterized in that, The tumors mentioned are p53 wild-type colorectal cancer, ovarian cancer, lung cancer, cervical cancer, breast cancer, thyroid cancer, liver cancer, and laryngeal cancer.

6. The antitumor drug according to claim 4, characterized in that, The dosage form of the antitumor drug is any one of the following: capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

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