Polypeptide for targeted blocking of combination of TRIM25 and BRD7 protein and application thereof

Abstract

The invention belongs to the field of biomedicine, and particularly relates to polypeptide for targeted blocking of combination of TRIM25 and BRD7 protein and application of the polypeptide. According to the present invention, based on the minimum binding region PRYSPRY structural domain (439 aa-630 aa) of the TRIM25 and the BRD7 protein, the blocking peptide TB16 capable of specifically blocking the mutual binding of the TRIM25 and the BRD7 protein is screened, such that the ubiquitination degradation of the BRD7 is inhibited, and the stability of the BRD7 tumor inhibition protein is improved. The polypeptide drug TB16 can effectively inhibit breast cancer, nasopharynx cancer, ovarian cancer, lung cancer and liver cancer cell proliferation and breast cancer in-vivo tumor growth, has good anti-solid tumor activity, and is expected to become a new anti-tumor targeting drug.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a polypeptide that targets and blocks the binding of TRIM25 and BRD7 proteins and its application in anti-tumor and BRD7 protein stabilization. Background Art

[0002] Commonly used anti-tumor drugs in clinical practice have disadvantages such as weak targeting, strong adverse reactions, and easy generation of drug resistance. Compared with traditional anti-tumor drugs, polypeptide drugs have the advantages of small molecular weight, strong targeting, high activity, low toxicity, and easy transmembrane absorption. They can directly or indirectly act on tumor cells and participate in the regulation of biological functions such as the growth and apoptosis of tumor cells. In addition, solid-phase chemical synthesis of polypeptides makes the production cost lower than that of other biopharmaceuticals, facilitating large-scale production. Peptide-based therapy is not only highly efficient and low-toxic, but also can increase the sensitivity of tumors to other treatment methods, which will have important value for the clinical treatment of tumors and is a current research hotspot.

[0003] BRD7 is a gene with low expression in nasopharyngeal carcinoma isolated and cloned by methods such as cDNA representational difference analysis and library screening. BRD7 has been proven to be down-regulated in various tumors and plays a tumor suppressor gene function in cancers such as nasopharyngeal carcinoma, breast cancer, ovarian cancer, lung cancer, liver cancer, and osteosarcoma, and is associated with poor prognosis of patients. As a tumor suppressor factor, BRD7 can play a key role in various biological processes such as cell proliferation, cell cycle progression, apoptosis, invasion, and migration by regulating gene expression.

[0004] The present invention discovers that there is a potential interaction between TRIM25 and BRD7, and TRIM25 participates in the malignant progression of various tumors by reducing the protein stability of BRD7. Therefore, developing a polypeptide drug that blocks the interaction between TRIM25 / BRD7 can stabilize the BRD7 protein in tumor cells, thereby exerting an anti-tumor function, which is a promising clinical treatment strategy. The research and development of this polypeptide drug is expected to fill the gap of tumor polypeptide drugs under this mechanism and has very good application prospects. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a polypeptide that targets and blocks the interaction between TRIM25 and BRD7 and its application. The polypeptide that targets and blocks the binding of TRIM25 and the tumor suppressor protein BRD7 can target and stabilize the BRD7 protein to achieve the anti-tumor purpose, which is an effective and feasible solid tumor treatment strategy. It can treat various solid tumors including breast cancer, nasopharyngeal carcinoma, ovarian cancer, lung cancer, and liver cancer.

[0006] In the first aspect of the present invention, there is provided an anti-tumor polypeptide that targets and blocks the interaction between TRIM25 and BRD7 to stabilize the BRD7 protein. The amino acid sequence of the polypeptide is: KVLETFLAKSRPELLE.

[0007] Furthermore, it includes the polypeptide and pharmaceutically acceptable modification types. The polypeptide modification types include at least one of C-terminal modification, N-terminal modification, middle residue modification, and cyclization modification.

[0008] In the present invention, based on the PRYSPRY domain of the interaction between TRIM25 and BRD7, a polypeptide that targets and blocks the binding between TRIM25 and BRD7 and can stabilize the BRD7 protein is screened and prepared as an anti-tumor drug.

[0009] Specifically, the polypeptide inhibits the ubiquitination and degradation of BRD7 mediated by TRIM25 by blocking the binding of the PRYSPRY domain of TRIM25 to BRD7, and stabilizes the BRD7 protein in breast cancer, nasopharyngeal carcinoma, ovarian cancer, lung cancer, and liver cancer, serving as a tumor treatment drug.

[0010] In the second aspect of the present invention, there is provided an application of the above-mentioned polypeptide in the preparation of drugs or preparations for preventing and / or treating solid tumors.

[0011] The solid tumor is a solid tumor with low BRD7 expression, including one or more of breast cancer, nasopharyngeal carcinoma, ovarian cancer, lung cancer, and liver cancer.

[0012] Breast cancer includes at least one of MDA-MB-231 and MCF7; nasopharyngeal carcinoma includes CNE2; ovarian cancer includes A2780; lung cancer includes PC9; liver cancer cells include Hep3B.

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

[0014] In the third aspect of the present invention, there is provided an application of the above-mentioned polypeptide in the preparation of drugs or preparations for stabilizing the BRD7 tumor suppressor protein. The polypeptide can increase the stability of the BRD7 protein by blocking the binding between TRIM25 and the BRD7 protein.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients. The excipients include conventional diluents, fillers, binders, wetting agents, absorption promoters, surfactants, lubricants, and stabilizers in the pharmaceutical field.

[0016] In the application of the present invention, the pharmaceutical dosage form is an oral dosage form and / or an injection dosage form. Among them, it includes but is not limited to tablets, pills, capsules, sprays, granules, powder injections, injections, etc. The dosage of the drug is a pharmaceutically acceptable dosage. The administration methods include but are not limited to intratumoral injection, intravenous injection, or intraperitoneal injection, etc., and oral administration such as pills, capsules, etc.

[0017] Preferably, it has a pharmaceutical dosage form containing a pharmaceutically acceptable drug.

[0018] In the present invention, α-helices in the TRIM25-PRYSPRY domain that binds to BRD7 were used for analysis to synthesize 2 possible blocking peptide sequences, which have the ability to block the binding of TRIM25 to BRD7; the 2 blocking peptides were used in in vitro experiments to screen out polypeptides that target and stabilize the BRD7 protein.

[0019] Advantages of the present invention

[0020] The solution of the present invention provides an anti-tumor polypeptide TB16 that targets and blocks the interaction between TRIM25 and BRD7, which can specifically block the interaction between TRIM25 and BRD7, reverse the increase in the ubiquitination level of BRD7 mediated by TRIM25, and thus stabilize the BRD7 protein. Biological phenotypes confirm the anti-tumor effect and biosafety of this polypeptide drug in breast cancer, nasopharyngeal cancer, ovarian cancer, lung cancer, and liver cancer. Therefore, TB16 can be used to prepare a potential anti-solid tumor drug for treating various tumors such as breast cancer, nasopharyngeal cancer, ovarian cancer, lung cancer, and liver cancer by targeting and stabilizing the BRD7 protein. Brief description of the drawings

[0021] Figure 1 : Results of TRIM25 negatively regulating the stability of BRD7 protein through the ubiquitin-proteasome pathway;

[0022] Figure 1 A: Results showing that the silenced expression of TRIM25 can increase the protein stability of BRD7 in breast cancer MDA-MB-231 and MCF7 cells;

[0023] Figure 1 B: Results showing that treatment with MG132 (proteasome inhibitor) can reverse the downregulation of BRD7 protein mediated by overexpression of TRIM25 in breast cancer MDA-MB-231 and MCF7 cells;

[0024] Figure 1 C: Effects of silenced expression of TRIM25 on the ubiquitination level of BRD7 in breast cancer MDA-MB-231 and MCF7 cells.

[0025] Figure 2: Result of the binding between BRD7 and the PRYSPRY domain of TRIM25;

[0026] Figure 2 A: Schematic diagram of the Flag-TRIM25 domain deletion vector;

[0027] Figure 2 B: Schematic diagram of the immunoprecipitation (Co-IP) experiment to detect the interaction between the TRIM25 PRYSPRY domain and BRD7 in breast cancer cell MCF7;

[0028] Figure 2 C: Results of the Co-IP experiment showing the binding between the TRIM25-PRYSPRY domain and BRD7 in breast cancer cell MCF7;

[0029] Figure 2 D: Schematic diagram of validating the binding between BRD7 and TRIM25-PRYSPRY by molecular docking through the HDOCK website (http: / / hdock.phys.hust.edu.cn).

[0030] Figure 3 : Schematic diagram of designing and synthesizing α-helix polypeptides based on the PRYSRY binding region of BRD7 and TRIM25;

[0031] Figure 3 A: Schematic diagram of the protein structure of TRIM25-PRYSPRY in the PDB website (https: / / www.rcsb.org);

[0032] Figure 3 B: Schematic diagram of analyzing the α-helices present in the TRIM25-PRYSPRY domain and designing and synthesizing two blocking peptides according to their amino acid sequences.

[0033] Figure 4 : Results of the polypeptide drug TB16 derived from the TRIM25 PRYSPRY domain competitively inhibiting the binding of TRIM25 to BRD7 protein and increasing the stability of BRD7 protein;

[0034] Figure 4 A: Western Blot detection of the effects of TB5 and TB16 on the protein expression level of BRD7 in breast cancer cell MDA-MB-231;

[0035] Figure 4 B: Western Blot detection of the effects of TB5 and TB16 on the protein expression level of BRD7 in breast cancer cell MCF7;

[0036] Figure 4C: Effect of TB5 and TB16 on the binding ability of TRIM25 and BRD7 proteins in breast cancer cell line MCF7 detected by Co-IP;

[0037] Figure 4 D: Effect of TB5 and TB16 on the increased ubiquitination level of BRD7 protein mediated by TRIM25 in breast cancer cell line MCF7 detected by Western Blot.

[0038] Figure 5 : Results that the TAT-modified peptide TAT-TB16 can increase the stability of BRD7 protein in nasopharyngeal carcinoma, ovarian cancer, liver cancer and lung cancer;

[0039] Figure 5 A: Effect of TAT-TB16 on the expression level of BRD7 protein in nasopharyngeal carcinoma cell line CNE2 detected by Western Blot;

[0040] Figure 5 B: Effect of TAT-TB16 on the expression level of BRD7 protein in ovarian cancer cell line A2780 detected by Western Blot;

[0041] Figure 5 C: Effect of TAT-TB16 on the expression level of BRD7 protein in lung cancer cell line PC9 detected by Western Blot;

[0042] Figure 5 D: Effect of TAT-TB16 on the expression level of BRD7 protein in liver cancer cell line Hep3B detected by Western Blot.

[0043] Figure 6 : Results that the polypeptide drug TAT-TB16 exerts antitumor effects in breast cancer, nasopharyngeal carcinoma, ovarian cancer, liver cancer and lung cancer;

[0044] Figure 6 A: Effect of TAT-TB16 on the proliferation ability of breast cancer cells (MDA-MB-231, MCF7), nasopharyngeal carcinoma cells (CNE2), ovarian cancer cells (A2780), lung cancer cells (PC9) and liver cancer cells (Hep3B) detected by CCK8;

[0045] Figure 6 B: Effect of TAT-TB16 on the colony formation ability of breast cancer cells MDA-MB-231 and MCF7.

[0046] Figure 7 : Results that the polypeptide drug TAT-TB16 can significantly inhibit the growth of breast cancer xenografts in vivo;

[0047] Figure 7A: Photos of tumor-bearing mice in the normal saline group, TAT group, and TAT-TB16 group;

[0048] Figure 7 B: Results of comparing the transplanted tumor masses dissected from the mice in the normal saline group, TAT group, and TAT-TB16 group after sacrificing the mice;

[0049] Figure 7 C: Growth curves of mouse tumor masses plotted by measuring the size of the transplanted tumors every two days;

[0050] Figure 7 D: Results of comparing the weights of the transplanted tumors in each group;

[0051] Figure 7 E: Changes in the body weights of the mice in the normal saline group, TAT group, and TAT-TB16 group.

[0052] Figure 8 : Results showing that the polypeptide drug TAT-TB16 has no obvious toxic and side effects on the important organs of mice;

[0053] Data are presented as mean ± standard error, , p < 0.05; , p < 0.01; , p < 0.001; ns, no statistical difference. Detailed implementation mode

[0054] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0055] The MDA-MB-231, MCF7, CNE2, A2780, PC9, and Hep3B cell lines used in the present invention are all preserved in the Molecular Genetics Laboratory of the Cancer Research Institute of Central South University. The culture conditions for MDA-MB-231, MCF7, A2780, and Hep3B cells are as follows: DMEM liquid medium containing 10% fetal bovine serum (FBS) and 1% double antibiotics (penicillin, streptomycin), adherent growth in a constant temperature incubator at 37°C and 5% CO2 concentration. The culture conditions for CNE2 and PC9 cells are as follows: 1640 liquid medium containing 10% fetal bovine serum (FBS) and 1% double antibiotics (penicillin, streptomycin), adherent growth in a constant temperature incubator at 37°C and 5% CO2 concentration.

[0056] Example 1, TRIM25 negatively regulates the protein stability of BRD7 through the ubiquitin-proteasome pathway

[0057] 1.1 Experimental protocol:

[0058] (1) Transfect siNC and siTRIM25 into MDA-MB-231 and MCF7 cells respectively. Subsequently, treat the cells with CHX (50 μM), collect the cells at different time points (0, 1, 2, 4 h), and immediately perform protein extraction to detect the protein expression level of BRD7 by Western Blot technology.

[0059] (2) Transfect the empty vector and TRIM25 plasmid into MDA-MB-231 and MCF7 cells. After 24 h of transfection, treat the cells with the proteasome inhibitor MG132 (20 μM) for 4 h, collect the cells, and immediately perform protein extraction to detect the protein expression level of BRD7 by Western Blot technology.

[0060] (3) Transfect siNC, siTRIM25#1, and siTRIM25#2 into MDA-MB-231 and MCF7 cells respectively. After 48 h of transfection, treat the cells with 20 μM of the proteasome inhibitor MG132 for 4 h, then collect the cells and immediately perform protein extraction. Perform a Co-IP experiment using a BRD7 antibody to detect the ubiquitination level of BRD7 by Western Blot technology.

[0061] 1.2 Experimental results:

[0062] The experimental results showed that the silenced expression of TRIM25 could significantly prolong the half-life of BRD7 protein in the two breast cancer cell lines ( Figure 1 A). In addition, the treatment with MG132 reversed the inhibitory effect of TRIM25 overexpression on the BRD7 protein level, indicating that TRIM25 promotes the protein degradation of BRD7 through the proteasome pathway ( Figure 1 B). The results of the ubiquitination experiment showed that the silenced expression of TRIM25 decreased the ubiquitination level of BRD7 in MDA-MB-231 and MCF7 cells ( Figure 1 C). The above results indicate that TRIM25 reduces the protein stability of BRD7 and promotes its ubiquitination degradation in a ubiquitin-proteasome-dependent manner.

[0063] Example 2, BRD7 binds to the PRYSPRY domain of TRIM25

[0064] 2.1 Experimental protocol:

[0065] (1)According to the protein domains of TRIM25, a series of Flag-TRIM25 domain deletion vectors were constructed, including Flag-TRIM25-△RING (deletion of the RING domain), Flag-TRIM25-△Coiled-Coil (deletion of the Coiled-Coil domain), and Flag-TRIM25-△PRYSPRY (deletion of the PRYSPRY domain). In the breast cancer cell line MCF7, the Flag-TRIM25 series of truncations and HA-BRD7 were co-transfected, and Co-IP experiments were performed using the HA antibody to explore the minimum domain of the interaction between TRIM25 and BRD7. Meanwhile, the binding of BRD7 to TRIM25-PRYSPRY was verified by molecular docking using the HDOCK website (http: / / hdock.phys.hust.edu.cn).

[0066] (2)The Flag-TRIM25 PRYSPRY vector was constructed. In the breast cancer cell line MCF7, Flag-TRIM25PRYSPRY and HA-BRD7 were co-transfected, and forward and reverse Co-IP experiments were performed using the HA antibody and Flag antibody respectively to explore the interaction between TRIM25 PRYSPRY and BRD7.

[0067] 2.2 Experimental results:

[0068] The results of Co-IP and Western Blot experiments showed that there was an interaction between BRD7 and the PRYSPRY domain of TRIM25 ( Figure 2 A-C). Using the HDOCK website (http: / / hdock.phys.hust.edu.cn), molecular docking further confirmed the interaction between BRD7 and TRIM25-PRYSPRY ( Figure 2 D). The above results indicated that there was an interaction between TRIM25-PRYSPRY and BRD7, providing a structural basis for the development of polypeptide drugs based on the TRIM25 / BRD7 regulatory axis.

[0069] Example 3, Design and synthesis of α-helical polypeptides based on the PRYSRY binding region of BRD7 and TRIM25

[0070] 3.1 Experimental scheme:

[0071] To further design polypeptides targeting the interaction between TRIM25 and BRD7, we focused on the protein structure. Among them, α-helix, as an important part of the protein secondary structure, endows proteins with stability and morphology through stable hydrogen bonds and helical conformation, and participates in molecular recognition and binding. Therefore, analyzing the α-helix in the protein domain and synthesizing α-helical peptides are important ways and means to design polypeptide drugs that block the interaction between proteins and targets. According to the crystal structure data of TRIM25 PRYSPRY in the PDB database (PDB: 6FLM), the α-helices present in the TRIM25 PRYSPRY domain were analyzed, and α-helical polypeptides were further synthesized.

[0072] 3.2 Experimental results:

[0073] We used the PDB website (https: / / www.rcsb.org) to analyze the α-helices present in the TRIM25-PRYSPRY domain according to the protein structure of TRIM25-PRYSPRY ( Figure 3 A), and found that there were two α-helices. The first α-helix was located at 439aa-454aa, and the amino acid sequence was KVLETFLAKSRPELLE. The second α-helix was located at 537aa-541aa, and the amino acid sequence was GPESR ( Figure 3 B). According to these two amino acid sequences, we synthesized two polypeptides respectively, and named them TB16 (439aa-454aa) and TB5 (537aa-541aa).

[0074] Example 4, the polypeptide drug TB16 derived from the TRIM25 PRYSPRY domain competitively inhibits the binding of TRIM25 to BRD7 and increases the stability of BRD7 protein

[0075] 4.1 Experimental protocol:

[0076] (1) Treat breast cancer cells MDA-MB-231 and MCF7 with different concentrations of TB5 and TB16 (0 µM, 5 µM, 10 µM, 20 µM, 40 µM) respectively. After 24 h of treatment, collect the cells and immediately perform protein extraction. Detect the protein expression level of BRD7 by Western Blot technology.

[0077] (2) Co-transfect HA-BRD7 and Flag-TRIM25 plasmids into MCF7 breast cancer cells. After 24 h of transfection, treat the cells with TB5 (20 µM) and TB16 (20 µM) respectively, and continue to treat for 24 h. Then perform Co-IP experiment using Flag antibody to explore the effects of TB5 and TB16 on the binding ability between TRIM25 and BRD7.

[0078] (3) Co-transfect HA-BRD7, Flag-TRIM25 and Ub plasmids into MCF7 breast cancer cells. After 24 h of transfection, treat the cells with TB5 (20 µM) and TB16 (20 µM) for 24 h respectively, and then treat the cells with 20 µM MG132 for 4 h. Then perform Co-IP experiment using BRD7 antibody and detect the ubiquitination level of BRD7 by Western Blot technology.

[0079] 4.2 Experimental results:

[0080] The results showed that in two breast cancer cell lines, MDA-MB-231 and MCF7, different concentrations of TB5 had no obvious effect on the protein expression level of BRD7, while with the increase of TB16 concentration, the protein expression level of BRD7 gradually increased ( Figure 4 A - B). The results of CO-IP experiment showed that in MCF7 cells, compared with the TB5 treatment group, the protein binding ability between TRIM25 and BRD7 in the TB16 treatment group was significantly weakened ( Figure 4 C). In addition, in MCF7 cells, overexpression of TRIM25 could promote the ubiquitination level of BRD7, while TB16 could reverse the promoting effect of TRIM25 overexpression on the ubiquitination level of BRD7 ( Figure 4 D). The above results indicated that in breast cancer cells, TB16 could inhibit the protein interaction between TRIM25 and BRD7, thereby inhibiting the increase of BRD7 ubiquitination level mediated by TRIM25, and thus targeting and stabilizing the BRD7 protein.

[0081] Example 5, The TAT-modified peptide TAT-TB16 of the polypeptide drug TB16 can increase the protein stability of BRD7 in nasopharyngeal carcinoma, ovarian cancer, liver cancer and lung cancer

[0082] To increase the cell membrane permeability of TB16, we introduced a transmembrane peptide TAT (amino acid sequence: GRKKRRQRRRPPQ) before the amino acid sequence of TB16 and further synthesized TAT-TB16.

[0083] 5.1 Experimental protocol:

[0084] Nasopharyngeal carcinoma cells CNE2, ovarian cancer cells A2780, liver cancer cells PC9, and lung cancer cells Hep3B were treated with different concentrations of TAT-TB16 (0 µM, 10 µM, 20 µM, 40 µM), respectively. After 24 h of treatment, the cells were collected and protein extraction was carried out immediately. The protein expression level of BRD7 was detected by Western Blot technology.

[0085] 5.2 Experimental results:

[0086] The results of Western Blot experiments showed that in nasopharyngeal carcinoma cells CNE2, ovarian cancer cells A2780, liver cancer cells PC9, and lung cancer cells Hep3B, with the increase in the treatment concentration of TAT-TB16, the protein expression level of BRD7 gradually increased ( Figure 5 A-D). The above results indicate that TAT-TB16 can target and stabilize the BRD7 protein in a variety of tumor cells.

[0087] Example 6, the polypeptide drug TAT-TB16 can exert antitumor effects in vitro cultured cells such as breast cancer, nasopharyngeal carcinoma, ovarian cancer, liver cancer, and lung cancer

[0088] 6.1 Experimental protocol:

[0089] CCK8 experiment:

[0090] (1) Using various in vitro cultured cells such as breast cancer cells (MDA-MB-231, MCF7), nasopharyngeal carcinoma cells (CNE2), ovarian cancer cells (A2780), liver cancer cells (PC9), and lung cancer cells (Hep3B), different tumor cells were seeded into 96-well plates at 1000 cells / well.

[0091] (2) After the cells adhered completely, MDA-MB-231, MCF7, CNE2, A2780, PC9, and Hep3B cells were treated with 100 µM of TAT-TB16, and the control group was the TAT treatment group.

[0092] (3) Six time points of 0 d, 1 d, 2 d, 3 d, 4 d, and 5 d were set. Subsequently, 10% CCK8 solution was added to each well and incubated in an incubator at 37 °C for 2 h. The absorbance value was measured using an enzyme-linked immunosorbent assay (ELISA) reader (wavelength 450 nm).

[0093] (4) Analyze the data and plot the graph: Use GraghPad Prism 9.5.0 software to draw the cell proliferation curve.

[0094] Colony formation assay:

[0095] (1)Count the breast cancer cells (MDA-MB-231, MCF7) in good growth condition, seed them at 500 cells / well in a 12-well plate. After small colonies grow out, co-culture the tumor cells with TAT (100 µM) or TAT-TB16 (100 µM) for 3 days, and then change to complete medium and continue culturing for 10 - 14 days.

[0096] (2)Discard the medium, wash 3 times with 1×PBS for 5 minutes each time, and then add 4% paraformaldehyde to fix at room temperature for 1 h.

[0097] (3)Aspirate the fixative, wash 3 times with 1×PBS for 5 minutes each time. Then add crystal violet staining solution to each well and stain at room temperature for 30 min.

[0098] (4)Aspirate the crystal violet staining solution, rinse with distilled water until there is no residual crystal violet, air dry, and then use a scanner to scan and record the images.

[0099] (5)Count the number of valid cell clones in each group and analyze the data.

[0100] 6.2 Experimental results:

[0101] The results of the CCK8 experiment showed that in two breast cancer cell lines, compared with the TAT group, TAT-TB16 could inhibit the cell proliferation ability of breast cancer, nasopharyngeal carcinoma, ovarian cancer cells, liver cancer cells, and lung cancer cells ( Figure 6 A); the results of the colony formation experiment showed that compared with the TAT group, TAT-TB16 could significantly inhibit the colony formation ability of breast cancer cells ( Figure 6 B). The above results indicated that TAT-TB16 exerted anti-tumor effects in multiple tumors including breast cancer, nasopharyngeal carcinoma, ovarian cancer, liver cancer, and lung cancer.

[0102] Example 7, the polypeptide drug TAT-TB16 could significantly inhibit the growth of breast cancer xenografts in vivo and had no obvious tissue and organ toxicity

[0103] 7.1 Experimental protocol:

[0104] Purchase 21 4 - 6-week-old female BALB / C nude mice from Hunan Slack Jingda Experimental Animal Co., Ltd., and all nude mice passed the quality inspection. Animal feeding and related operations were completed in the Animal Experiment Center of Hunan Cancer Hospital under specific pathogen-free (SPF) conditions.

[0105] (1)Prepare MCF7 breast cancer cells in good growth condition, and wash the collected cells 3 times with pre-cooled physiological saline.

[0106] (2)Prepare 3×10 6150 μL of cell suspension containing cells and 50 μL of Matrigel was inoculated subcutaneously at the upper part of the right anterior axilla of nude mice.

[0107] (3)Starting from the 4th day after injection, the length (L) and width (W) of the subcutaneous tumor in nude mice were measured with a vernier caliper every 2 days. The results were recorded and the tumor volume was calculated using the following formula: Volume = L × W 2 / 2; At the same time, the body weight of the mice was recorded.

[0108] (4)When the tumor volume of the mice reached 30 - 50 mm 3 ³, the mice were randomly divided into 3 groups with 7 mice in each group. In the experimental group, each mouse was injected with 15 mg / kg of TAT or TAT - TB16 into the tumor, and the control group was injected with normal saline. The injection was performed once every 2 days for a total of 6 times.

[0109] (5)On the 18th day of feeding, the mice were euthanized by cervical dislocation. The tumor was removed, photographed, and the tumor volume and weight were recorded.

[0110] (6)Three mice were randomly selected from each group. The heart, liver, spleen, lung, and kidney were dissected, dehydrated, fixed, paraffin - embedded, sectioned, and then HE - stained to observe the tissue status of each organ.

[0111] 7.2 Experimental results:

[0112] The results of the in - vivo nude mouse xenograft tumor model showed that compared with the normal saline group and the TAT group, the tumor growth of mice in the TAT - TB16 treatment group was significantly inhibited, and the tumor weight was significantly reduced ( Figure 7 A - D), indicating that TAT - TB16 can significantly inhibit the growth of tumors in vivo, thus showing an anti - tumor effect in vivo. In addition, during the treatment process, the body weight of each group of mice was not affected ( Figure 7 E). According to the HE - staining results, there was no obvious damage to the tissues of each organ in the mice of the TAT - TB16 treatment group, indicating that TAT - TB16 has certain in - vivo biological safety ( Figure 8 ). The above results indicate that TAT - TB16 can significantly inhibit the growth of breast cancer cells and has no obvious organ toxicity.

Claims

1. A polypeptide that targets and blocks the binding of TRIM25 to BRD7 protein, characterized in that, The amino acid sequence of the polypeptide is: KVLETFLAKSRPELLE, or also includes pharmaceutically acceptable polypeptide modification types.

2. The polypeptide according to claim 1, characterized in that, The polypeptide modification types include one or more of C-terminal modification, N-terminal modification, middle residue modification, and cyclization modification.

3. Use of the polypeptide according to claim 1 or 2 in the preparation of a drug or preparation for preventing and / or treating solid tumors.

4. The application according to claim 3, characterized in that, The solid tumor is a solid tumor with low BRD7 expression, including one or more of breast cancer, nasopharyngeal carcinoma, ovarian cancer, lung cancer, and liver cancer.

5. The application according to claim 4, wherein Breast cancer cells include at least one of MDA-MB-231 and MCF7; nasopharyngeal cancer cells include CNE2; ovarian cancer cells include A2780; lung cancer cells include PC9; liver cancer cells include Hep3B.

6. The application according to claim 3, characterized in that The drug also includes pharmaceutically acceptable excipients.

7. Use of the polypeptide according to claim 1 or 2 in the preparation of a drug or preparation for enhancing the stability of BRD7 tumor suppressor protein, characterized in that, The polypeptide can increase the stability of BRD7 protein by blocking the binding of TRIM25 to BRD7 protein.

8. The application according to claim 7, wherein The drug also includes pharmaceutically acceptable excipients.

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