Phosphorylation modification interfering peptide targeting foxm1 protein and application thereof

Abstract

The present application relates to a kind of FOXM1 protein phosphorylation modification interfering peptide and its application, belong to the field of biological medicine technology. Specifically, the present application is found in research, the transcriptional activity of FOXM1 protein depends on the condensate formed by its polymerization, destroying the polymerization of FOXM1 can effectively inhibit the occurrence and development of tumor, and the phosphorylation of the 376th amino acid of FOXM1 protein can also inhibit polymerization to some extent, thereby inhibiting the transcriptional activation of FOXM1 protein. Therefore, the present application designs an interfering peptide, and introduces phosphorylation modification in appropriate position, obtains a kind of phosphorylation modification interfering peptide FIP4 targeted to FOXM1 protein polymerization domain, the interfering peptide plays a significant role in inhibiting the transcriptional activation of FOXM1 protein in cell and animal in vivo, and can be used for treating tumor-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a phosphorylation-modified interfering peptide targeting the FOXM1 protein and its applications. Background Technology

[0002] The mammalian transcription factor Forkhead Box M1 (FOXM1) is a member of the Forkhead family of proteins. There are four isoforms of FOXM1: FOXM1A, B, C, and D, generated by alternative splicing. We focus on FOXM1C, referring to it as FOXM1. FOXM1 is mainly composed of an N-terminal autonomous repressor region (NRD), an intermediate DNA-binding region (FHD), a C-terminal transcriptional activation region (TAD), and a disorder region (IDR). The disorder region contains the FOXM1 polymerization domain. FOXM1 is a known proto-oncogene that plays an important role in physiological processes such as cell proliferation, stem cell pluripotency, aging, DNA damage repair, and tumorigenesis. The oncogenic activity of FOXM1 has been extensively studied, with overexpression observed in various malignant tumor cells. As a transcription factor, FOXM1 can influence tumor invasion and metastasis by transcribing and regulating downstream tumor-related genes.

[0003] Since most transcription factors are traditionally considered "undruggable" targets due to their significantly disordered structures and lack of classic small-molecule binding pockets, designing specific interfering peptides to target the polymerization domain of FOXM1 to inhibit its transcriptional activity could offer a novel approach for cancer treatment. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention, based on the discovery that the transcriptional activity of the FOXM1 protein depends on the aggregates formed by its polymerization, and that disrupting FOXM1 polymerization can effectively inhibit tumor development and progression, as well as the new discovery that phosphorylation of amino acid 376 of the FOXM1 protein can also inhibit polymerization to some extent, designed a targeted interfering peptide and introduced phosphorylation modification to obtain a phosphorylated interfering peptide that targets the polymerization domain of the FOXM1 protein. This peptide can significantly inhibit FOXM1 polymerization both in vivo and in vitro, and has great potential in tumor treatment.

[0005] The first objective of this invention is to provide a polypeptide comprising the sequence shown in SEQ ID NO.2 (VPFQIPVLYSSVRP), wherein all amino acids are D-type and the serine at position 10 is phosphorylated.

[0006] Furthermore, the polypeptide also includes an auxiliary sequence fused to the sequence shown in SEQ ID NO.2.

[0007] Furthermore, the auxiliary sequence can be any sequence capable of enhancing the effect of the interfering peptide, including but not limited to sequences for cell membrane penetration, sequences for targeting, sequences for preventing degradation, etc. Preferably, the auxiliary sequence is as shown in SEQ ID NO.3 (RRRQRRKKRG).

[0008] Furthermore, during fusion, there is a linker sequence between the sequence shown in SEQ ID NO.2 and the auxiliary sequence, which may consist of 2 to 5 proline residues.

[0009] Furthermore, the auxiliary sequence is fused to the C-terminus of the sequence shown in SEQ ID NO.2.

[0010] Further, the preferred sequence of the polypeptide is shown in SEQ ID NO.1, wherein all amino acids are D-type and the 10th serine residue is phosphorylated. The preparation method of this phosphorylated interfering peptide targeting FOXM1 protein is as follows:

[0011] S1. Design an interfering peptide to target the amino acid sequence PRVSSYLVPIQFPV located in the polymer domain of the FOXM1 protein.

[0012] S2, Fuse the interfering peptide described in S1 with HIV-TAT (GRKKRRQRRR);

[0013] S3. The interfering peptide fused with HIV-TAT in S2 is converted into an inverse isomer to obtain the amino acid sequence of the interfering peptide.

[0014] S4. The interfering peptide in S3 was synthesized using D-type amino acids as raw materials;

[0015] S5. Phosphorylation modification of the S376 site, which affects FOXM1 polymerization, is introduced into the corresponding site of the interfering peptide obtained in S4, and finally the phosphorylated interfering peptide FIP4 is synthesized.

[0016] A second object of the present invention is to provide a composition containing the polypeptide.

[0017] A third objective of this invention is to provide the use of the said polypeptide or composition in the preparation of targeted formulations for targeting and interfering with the polymerization of FOXM1 in vivo or in vitro, thereby blocking interactions between FOXM1 proteins.

[0018] Furthermore, the sequence of FOXM1 is shown in SEQ ID NO.4.

[0019] A fourth object of the present invention is to provide the use of the said polypeptide or composition in the preparation of antitumor drugs.

[0020] Furthermore, the antitumor drug is used to inhibit the occurrence or development of in situ tumors and / or metastatic tumors, such as lung metastases.

[0021] Furthermore, the tumors include, but are not limited to, cervical cancer, breast cancer, etc.

[0022] By means of the above-described solution, the present invention has at least the following advantages:

[0023] 1. The phosphorylated interfering peptide FIP4, which targets the FOXM1 protein, can better inhibit the polymerization of the FOXM1 protein compared with ordinary interfering peptides, and significantly prevents the transcriptional activation of the FOXM1 protein, which can effectively limit tumor growth and provide a new idea for tumor immunotherapy.

[0024] 2. Protein-protein interactions are usually face-to-face interactions. Compared with traditional small molecule drugs, the interfering peptide FIP4 targeting FOXM1 protein involved in this invention can effectively block the interaction of FOXM1 protein itself.

[0025] 3. The interfering peptide FIP4, which targets the FOXM1 protein, contains the HIV-TAT sequence, which allows the peptide to directly cross the cell membrane and enter the cell to exert its effect without any carrier, thus avoiding the toxic side effects caused by carriers.

[0026] 4. D-amino acids degrade more slowly in animals than natural L-amino acids. Modifying interfering peptides into D-amino acid retrotransforms (DRI) isomers has been shown to have good tolerability and therapeutic effects in previous clinical trials. Therefore, the interfering peptide FIP4 targeting FOXM1 protein is feasible for clinical trials.

[0027] 5. The interfering peptide FIP4, which targets the FOXM1 protein, is a small peptide with a length of only 26 amino acids. According to immunological principles, it does not have immunogenicity and can avoid causing hypersensitivity reactions.

[0028] 6. The interfering peptide FIP4, which targets the FOXM1 protein, can be obtained directly through existing mature peptide synthesis technology, and has the advantages of high purity, controllable quality, and great potential for drug development.

[0029] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following describes the preferred embodiments of the present invention in conjunction with detailed drawings. Attached Figure Description

[0030] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0031] Figure 1 The left image shows the tertiary structure of the FOXM1 protein and the location of phosphorylation modifications, while the right image shows a schematic diagram of the target sequence of the phosphorylation-modified interfering peptide FIP4.

[0032] Figure 2 This is a schematic diagram showing the determination of the molecular weight of the synthesized interfering peptide FIP3 using mass spectrometry (MS).

[0033] Figure 3 This is a schematic diagram illustrating the determination of the purity of the synthesized interfering peptide FIP3 using high performance liquid chromatography (HPLC).

[0034] Figure 4 This is a schematic diagram showing the determination of the molecular weight of the synthesized interfering peptide FIP4 using mass spectrometry (MS).

[0035] Figure 5 This is a schematic diagram illustrating the determination of the purity of the synthesized interfering peptide FIP4 using high-performance liquid chromatography (HPLC).

[0036] Figure 6 The in vitro experiments showed that the interfering peptide FIP4 could significantly inhibit the formation of FOXM1 protein aggregates.

[0037] Figure 7 To discover through immunofluorescence experiments that the interfering peptide FIP4 can significantly inhibit the formation of FOXM1 protein aggregates in cells;

[0038] Figure 8 To demonstrate, using immunoprecipitation, that the interfering peptide FIP4 inhibits the polymerization of FOXM1 protein;

[0039] Figure 9 To detect that the interfering peptide FIP4 can inhibit the weak interaction between FOXM1 proteins using microscale thermophoresis (MST);

[0040] Figure 10 The interference peptide FIP4 was detected by quantitative real-time PCR (qPCR) to inhibit the transcriptional activity of FOXM1 protein.

[0041] Figure 11 The migration assay (left) and invasion assay (right) demonstrate that the interfering peptide FIP4 can inhibit tumor cell migration and invasion.

[0042] Figure 12 This diagram illustrates how subcutaneous injection of the breast cancer cell line MDA-MB-231 into nude mice, along with measurements of tumor growth, demonstrates that the interfering peptide FIP4 inhibits tumor growth.

[0043] Figure 13 This diagram illustrates how the interfering peptide FIP4 inhibits tumor metastasis by injecting BALB / c breast cancer cell line 4T1 into BALB / c mice via tail vein and then detecting lung metastasis of tumor cells using a small animal in vivo imaging system. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0045] The solution involved in this invention is as follows:

[0046] This invention provides a method for preparing a phosphorylation-modified interfering peptide targeting the polymerization domain of the FOXM1 protein, the steps of which are as follows:

[0047] First, an interfering peptide was designed to target amino acids 363-376 within the polymer domain of the FOXM1 protein. The amino acid sequence of this segment is PRVSSYLVPIQFPV, and the natural amino acid is L-type. Short peptides were designed to specifically disrupt the interactions between FOXM1 proteins.

[0048] Secondly, in order to promote the absorption of interfering peptides by cells, an HIV-TAT hydrophilic sequence was fused in, with the amino acid sequence being GRKKRRQRRR. This sequence can enable the peptides to cross the cell membrane and be absorbed by the cells in an energy-independent manner.

[0049] Then, we attempted to improve the stability and efficacy of the peptide in cell and animal experiments by modifying the peptide with DRI. The entire interfering peptide was modified into an inverse isomer, and the amino acid sequence of the interfering peptide FIP3 was designed as VPFQIPVLYSSVRPPPRRRQRRKKRG;

[0050] Next, the interfering peptide FIP3 was synthesized using D-type amino acids as raw materials, with a peptide purity of over 98%.

[0051] Finally, in an attempt to enhance the effect of the peptide, phosphorylation modification at position 376, which inhibits FOXM1 protein polymerization, was designed onto the peptide. The final amino acid sequence of the interfering peptide FIP4 is VPFQIPVLYS(pi)SVRPPPRRRQRRKKRG, with a molecular weight of 3254.81.

[0052] The design concept of this invention is as follows: Given the large interaction area of ​​protein-protein interactions, a single small molecule may not be able to effectively interfere. However, using macromolecular drugs, such as peptides (interfering peptides) with similar interaction surfaces, to interfere with protein-protein interactions may be effective. Furthermore, it is known that the FOXM1 protein regulates multiple oncogenic signaling pathways through polymerization. This invention has discovered that phosphorylation at position 376 of the FOXM1 protein can inhibit the formation of FOXM1 protein aggregates and its transcriptional activity. Therefore, an interfering peptide, FIP4, targeting the FOXM1 protein polymerization domain and phosphorylating at the corresponding site, was artificially designed and synthesized. This interfering peptide can disrupt hydrophobic interactions by binding to the interaction surface of the domain mediating FOXM1 protein polymerization, thereby relieving the promoting effect of FOXM1 protein on tumor occurrence and development, and thus achieving effective treatment of clinical symptoms.

[0053] The FOXM1 sequence involved in this invention is as follows (SEQ ID NO.4):

[0054] MKTSPRRPLILKRRRLPLPVQNAPSETSEEEPKRSPAQQESNQAEASKEVAESNSCKFPAGIKIINHPTMPNTQVVAIPNNANIHSIITALTAKGKESGSSGPNKFILISCGGAPTQPPGLRPQTQTSYDAKRTEVTLETLGPKPAARDVNLPRPPGALCEQKRETCADGEAAGCTINNSLSNIQWLRKM SSDGLGSRSIKQEMEEKENCHLEQRQVKVEEPSRPSASWQNSVSERPPYSYMAMIQFAINSTERKRMTLKDIYTWIEDHFPYFKHIAKPGWKNSIRHNLSLHDMFVRETSANGKVSFWTIHPSANRYLTLDQVFKPLDPGSPQLPEHLESQQKRPNPELRRNMTIKTELPLGARRKMKPLLPRVSSYLVPI QFPVNQSLVLQPSVKVPLPLAASLMSSELARHSKRVRIAPKVLLAEEGIAPLSSAGPGKEEKLLFGEGFSPLLPVQTIKEEEIQPGEEMPHLARPIKVESPPLEEWPSPAPSFKEESSHSWEDSSQSPTPRPKKSYSGLRSPTRCVSEMLVIQHRERRRERSRSRRKQHLLPPCVDEPELLFSEGPSTSRWA AELPFPADSSDPASQLSYSQEVGGPFKTPIKETLPISSTPSKSVLPRTPESWRLTPPAKVGGLDFSPVQTSQGASDPLPDPLGLMDLSTTPLQSAPPLESPQRLLSSEPLDLISVPFGNSSPSDIDVPKPGSPEPQVSGLAANRSLTEGLVLDTMNDSLSKILLDISFPGLDEDPLGPDNINWSQFIPELQ

[0055] Example 1: Synthesis and Detection of the Interfering Peptide Drug FIP3

[0056] The interfering peptide drug FIP3 designed in this invention has the amino acid sequence VPFQIPVLYSSVRPPPRRRQRRKKRG, and the targeting sequence is as follows: Figure 1 As shown, the synthesis was carried out at Jier Biochemical (Shanghai) Co., Ltd. using D-type amino acids as raw materials.

[0057] like Figure 2As shown, the synthesized interfering peptide drug FIP3 was identified as having a molecular weight of 3174.83 using an Agilent-6125B liquid chromatography-mass spectrometry system (Agilent Technologies). HPLC was performed using an Inertsil ODS-SP column (Shimadzu, 4.6 mm × 250 mm) as the stationary phase, with gradient elution using mobile phase A (100% nitrile, 0.1% trifluoroacetic acid) and mobile phase B (100% ultrapure water, 0.1% trifluoroacetic acid). Figure 3 As shown in Table 1, the purity was greater than 98% as determined by HPLC.

[0058] Table 1

[0059] Retention time content(%) Peak area Peak height 10.223 95.35 9736337 928022 9.769 0.8193 83664 6275

[0060] Example 2: Synthesis and Detection of Phosphorylation-Optimized Interfering Peptide Drug FIP4

[0061] The interfering peptide drug FIP4 designed in this invention has the amino acid sequence VPFQIPVLYS(pi)SVRPPPRRRQRRKKRG (SEQ ID NO.1), and the targeting sequence is as follows: Figure 1 As shown, the synthesis was carried out at Jier Biochemical (Shanghai) Co., Ltd. using D-type amino acids as raw materials.

[0062] like Figure 4 As shown, the synthesized interfering peptide drug FIP4 was identified as having a molecular weight of 3254.81 using an Agilent-6125B liquid chromatography-mass spectrometry system (Agilent Technologies). HPLC was performed using an Inertsil ODS-SP column (Shimadzu, 4.6 mm × 250 mm) as the stationary phase, with gradient elution using mobile phase A (100% nitrile, 0.1% trifluoroacetic acid) and mobile phase B (100% ultrapure water, 0.1% trifluoroacetic acid). Figure 5 As shown in Table 2, the purity was greater than 98% as determined by HPLC.

[0063] Table 2

[0064] Retention time content(%) Peak area Peak height 11.498 96.93 4303857 398190 11.323 0.9816 43585 3368

[0065] Example 3: The interfering peptide drug FIP4 significantly inhibited the formation of FOXM1 protein aggregates.

[0066] 1. Experimental materials

[0067] Protein expression plasmid GFP-FOXM1, E. coli BL21 competent cells, isolactose mimic IPTG, E. coli lysate, streptacin magnetic beads (Smart Lifesciences), protein dialysis buffer, FIP3 interfering peptide drug prepared in Example 1, FIP4 interfering peptide drug prepared in Example 2, Zeiss LSM880 ultra-high resolution inverted confocal microscope.

[0068] 2. Experimental Methods

[0069] The plasmid GFP-FOXM1 was transformed into BL21 competent cells, and a large number of E. coli expressing FOXM1 protein were obtained by induction. These E. coli were lysed by sonication, and the FOXM1 protein tagged with GFP was purified in vitro. Then, the GFP-FOXM1 protein was concentrated by dialysis to achieve suitable purity and concentration. The purified protein solution (with or without interfering peptides) was added to a glass slide, covered with a coverslip, and imaged using an LSM880 confocal microscope system (Zeiss).

[0070] 3. Experimental Results

[0071] like Figure 6 As shown, compared with the control group with PBS, FOXM1 protein can form obvious aggregates. Treatment with interfering peptide FIP3 can partially inhibit the formation of aggregates of FOXM1 protein. However, after treatment with interfering peptide FIP4, FOXM1 protein aggregates are almost invisible under the microscope. Therefore, compared with interfering peptide FIP3, phosphorylated interfering peptide FIP4 can significantly inhibit the formation of aggregates of FOXM1 protein in vitro.

[0072] Example 4: The interfering peptide drug FIP4 significantly inhibited the formation of intracellular FOXM1 protein aggregates.

[0073] 1. Experimental materials

[0074] Protein expression plasmid FOXM1-GFP, HeLa cell line (ATCC source), polyethyleneimine (PEI), fetal bovine serum, DMEM medium, penicillin / streptomycin solution (Gibco), FIP3 interfering peptide drug prepared in Example 1, FIP4 interfering peptide drug prepared in Example 2, Zeiss LSM880 ultra-high resolution inverted confocal microscope.

[0075] 2. Experimental Methods

[0076] HeLa cells were seeded in 24-well plates and cell crawling slides were added. When the cell density reached 70%, FOXM1-GFP plasmid was transfected into two wells. After 8 hours, the cells were treated with 50 μM FIP3 / FIP4. After 24 hours, the cells were collected and immunofluorescence experiments were performed. The experimental results were captured using an LSM880 confocal microscope system (Zeiss) under 488 nm laser light.

[0077] 3. Experimental Results

[0078] like Figure 7 As shown, without interfering peptide treatment, FOXM1 protein can form aggregate spots in the nucleus of HeLa cells. However, the addition of interfering peptide FIP3 partially inhibits the formation of FOXM1 protein aggregate spots, and the addition of interfering peptide FIP4 almost completely eliminates the formation of FOXM1 protein aggregate spots in the HeLa cell nucleus. These results indicate that, compared to interfering peptide FIP3, phosphorylated interfering peptide FIP4 can significantly inhibit the formation of FOXM1 protein aggregates within cells.

[0079] Example 5: Treatment of cells with the interfering peptide drug FIP4 can inhibit the polymerization of FOXM1 proteins.

[0080] 1. Experimental materials

[0081] The following were prepared according to Example 2: FIP4 interfering peptide drug, fetal bovine serum, DMEM medium, penicillin / streptomycin solution (Gibco), MDA-MB-231 cell line (ATCC source), FOXM1 antibody, Actin antibody and related secondary antibody (CST).

[0082] 2. Experimental Methods

[0083] MDA-MB-231 cells were seeded in 6-well plates. When the cell density reached 70%, the cells were treated with 50 μM and 100 μM FIP4. After 24 h, the cells were collected and the multimers of FOXM1 protein were detected by semi-denaturing electrophoresis (SDD-PAGE).

[0084] 3. Experimental Results

[0085] like Figure 8 As shown, FOXM1 protein polymerizes without FIP4 treatment, while pretreatment with FIP4 dose-dependently inhibits FOXM1 protein polymerization, demonstrating that FIP4 can inhibit FOXM1 protein polymerization.

[0086] Example 6 uses the interfering peptide drug FIP4 to inhibit the interaction between FOXM1 proteins.

[0087] 1. Experimental materials

[0088] PBST, prokaryotically expressed FOXM1 and GFP-FOXM1 proteins, FIP4 interfering peptide drug prepared according to Example 2, MONOLITH NT.115 system (NanoTemper Technologies).

[0089] 2. Experimental Methods

[0090] Using a MONOLITH NT.115 system, 10 μl of GFP-FOXM1 was mixed with 10 μl of serially diluted FOXM1 at room temperature. PBS was added to the control group, and FIP4 was added to the experimental group. The data were analyzed by plotting the relationship between peptide concentration and liquid-induced fluorescence changes (changes in original fluorescence on the y-axis).

[0091] 3. Experimental Results

[0092] like Figure 9 The analysis showed that polymerization forces could be detected between FOXM1 proteins in the control group, but the interaction forces between FOXM1 proteins disappeared after FIP4 was added to the experimental group, indicating that FIP4 inhibited the aggregation of FOXM1 proteins.

[0093] Example 7: The use of the interfering peptide drug FIP4 significantly attenuated the transcriptional activity of the FOXM1 protein.

[0094] 1. Experimental materials

[0095] FOXM1 plasmid, HeLa cell line (ATCC source), FIP4 interfering peptide drug prepared according to Example 2, polyethyleneimine (PEI), fetal bovine serum, DMEM medium, penicillin / streptomycin solution (Gibco), RNA II kit ( Netherlands), II Q RT SuperMix (R223-01, Novizan), ChamQ Universal SYBR qPCR Master Mix (Q711-02, Novizan). Table 3 lists the primers required for qPCR (synthesized by Genewiz).

[0096] Table 3

[0097]

[0098] 2. Experimental Methods

[0099] HeLa cells were seeded in 6-well plates. When the cell density reached 70%, FOXM1 plasmid was transfected into two wells. After 8 hours, the cells were treated with PBS or 50 μM FIP4, and the cells were collected after 24 hours. RNA was extracted from the cells using an RNA extraction kit, and 1 μg of RNA was reverse transcribed using a reverse transcription kit to obtain cDNA. qPCR experiments were performed using a real-time quantitative PCR kit to detect the content of downstream target genes of FOXM1. Statistical analysis of the results is expressed as mean ± standard deviation (mean ± SEM). Analysis of variance (ANOVA) was used for comparison. p < 0.05 was considered statistically significant, and p < 0.01 was considered highly significant.

[0100] 3. Experimental Results

[0101] like Figure 10 As shown, 1 represents the control group, and 2 and 3 represent the experimental groups. In the experimental groups, without the interfering peptide FIP4, the mRNA levels of FOXM1 downstream target genes CDC25B and CyclinA2 were very high; however, in the groups with the interfering peptide FIP4, the mRNA levels of CDC25B and CyclinA2 were significantly reduced. This demonstrates that the interfering peptide FIP4 has a significant inhibitory effect on the transcription of FOXM1 protein.

[0102] Example 8: The interfering peptide drug FIP4 can significantly inhibit the migration and invasion of tumor cells, exhibiting good anti-tumor effects.

[0103] 1. Experimental materials

[0104] MDA-MB-231 cell line (ATCC source), FIP4 interferon drug prepared according to Example 2, 24-well plate, 8.0 μm pore size polycarbonate membrane, fetal bovine serum, DMEM medium, penicillin / streptomycin solution (Gibco), 4% PFA, 0.5% crystal violet, and matrix gel (BD Biocoat, 354234).

[0105] 2. Experimental Methods

[0106] Two trays of MDA-MB-231 cells were cultured, and when the cell density reached 70%, they were treated with PBS or 50 μM FIP4 for 24 hours.

[0107] In the tumor cell migration assay, polycarbonate membrane-lined chambers were placed in two wells of a 24-well plate. MDA-MB-231 cells treated with PBS or FIP4 were seeded into each chamber. 10% complete culture medium was added to the lower well, and the plate was incubated for 24 hours. The original culture medium was discarded, and 4% PFA was added to the wells. The cells were fixed at room temperature for 20 minutes. The fixative was removed, and the wells were stained with 0.5% crystal violet solution for 2 minutes. Unmigrated cells were wiped from the chambers, and the membrane at the bottom of the chambers was transferred to a glass slide for observation and counting under a microscope.

[0108] In the tumor cell invasion assay, similar to the migration assay, chambers were placed in the wells and their basement membranes were coated with matrix gel. MDA-MB-231 cells, treated with PBS / FIP4 and serum-starved, were seeded into the matrix gel-coated chambers and incubated for 24 hours. Then, 4% PFA was added for fixation, the matrix gel was wiped off, and the cells were stained with 0.5% crystal violet solution for 2 minutes. The membrane at the bottom of the chamber was transferred to a glass slide and observed and counted under a microscope.

[0109] 3. Experimental Results

[0110] like Figure 11 As shown, the left image represents the tumor cell migration experiment, and the right image represents the tumor cell invasion experiment. In both experiments, the number of tumor cells in the group with added interfering peptide FIP4 was significantly less than the number of cells in the control group (without FIP4), indicating that interfering peptide FIP4 significantly inhibits the migration and invasion capabilities of tumor cells.

[0111] Example 9: The interfering peptide drug FIP4 can effectively slow down tumor growth and inhibit tumor development.

[0112] 1. Experimental materials

[0113] Nude mice (purchased from Shanghai Southern Model Biotechnology), MDA-MB-231 cell line (ATCC source), FIP4 interfering peptide drug prepared according to Example 2, surgical instruments, vernier calipers.

[0114] 2. Experimental Methods

[0115] Twelve nude mice were subcutaneously injected with 2×10 6 MDA-MB-231 tumor cells were collected from nude mice and the mice were randomly divided into two groups. The mice were intraperitoneally injected with either PBS (control group) or the interfering peptide drug FIP4 (experimental group) every other day for four weeks. Tumor volume was measured every 2-3 days during this period. The tumor volume was determined using the formula for the volume of an ellipsoid: 1 / 2 × D × d 2 Where D is the longer diameter and d is the shorter diameter. Mice were euthanized 35 days after tumor inoculation, and the subcutaneous solid tumor was surgically removed for measurement and recording.

[0116] 3. Experimental Results

[0117] like Figure 12 As shown, the top left image is a schematic diagram of the nude mouse model construction and a photograph of the nude mouse and its solid tumor on day 35; the top right image is a trend chart of tumor growth; the bottom image shows the volume and weight of the solid tumor in the nude mouse on day 35. It is evident that the nude mice injected with the interfering peptide FIP4 had significantly lower tumor volume, weight, and growth rate than the control group injected with PBS, demonstrating that the interfering peptide drug FIP4 can effectively inhibit tumor growth.

[0118] Example 10: The interfering peptide drug FIP4 can effectively slow down the progression of lung metastasis of mouse breast cancer cells and inhibit tumor development.

[0119] 1. Experimental materials

[0120] Female BALB / c mice (purchased from Shanghai Southern Model Biotechnology), 4T1-Luc cell line, and the interfering peptide drug FIP4 prepared according to Example 2.

[0121] 2. Experimental Methods

[0122] Twelve BALB / c mice were used, and 4T1-Luc cells (2 × 10⁻⁶) were injected via the tail vein. 5 Mice injected with FIP4 (100 μl PBS) were randomly divided into two groups. The control group received an intraperitoneal injection of PBS every other day, while the experimental group received an intraperitoneal injection of FIP4. During this period, tumor cell metastasis was monitored weekly using a bioluminescent reporter imaging system in both groups for three weeks.

[0123] 3. Experimental Results

[0124] like Figure 13 As shown, the upper left image is a schematic diagram of the mouse model construction, the upper right image is a trend diagram of mouse lung metastasis, and the lower image is a bioluminescence imaging image of the mice. In the first week, the metastasis situation in the two groups of mice was similar. From the second week onwards, the lung metastasis signal in the control group mice significantly increased, while the signal intensity in the FIP4-injected experimental group was relatively weaker. This demonstrates that the interfering peptide drug FIP4 has a significant inhibitory effect on lung metastasis of mouse breast cancer cells.

[0125] In summary, the interfering peptide drug FIP4 provided by this invention can inhibit the polymerization and aggregate formation of FOXM1 protein by binding to the polymerization domain of FOXM1 protein, thereby relieving the pro-tumor function of FOXM1 and thus inhibiting the occurrence, development and metastasis of tumors.

[0126] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A polypeptide, characterized in that, The sequence of the polypeptide is shown in SEQ ID NO.1, wherein all amino acids are D-type and the 10th serine is phosphorylated.

2. A composition, characterized in that, It contains the polypeptide of claim 1.

3. The use of the polypeptide of claim 1 or the composition of claim 2 in the preparation of an antitumor drug, characterized in that, The anti-tumor drug is used to treat breast cancer.

4. An antitumor drug, characterized in that, The antitumor drug contains the polypeptide of claim 1 or the composition of claim 2 and is used to treat breast cancer.

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