Application of macrophage MTDH inhibitor in preparation of medicine for preventing and treating lung metastasis of malignant tumors

Abstract

The invention belongs to the technical field of biomedicine, and discloses application of a macrophage MTDH inhibitor in preparation of a medicine for inhibiting angiogenesis and treating malignant tumor metastasis, and the macrophage MTDH inhibitor is a substance for knocking out or knocking down the MTDH level. The invention discloses an application of a macrophage MTDH inhibitor in prevention and treatment of malignant tumor metastasis, and aims to knock out or down-regulate the macrophage MTDH through the inhibitor, and influence the generation of blood vessels in lung tissues by regulating TSP-1, so as to inhibit the lung metastasis of malignant tumors. The modified macrophage provided by the invention can be used as a novel immune cell therapy, and has important clinical significance.

Description

Technical Field

[0001] The invention belongs to the field of medicine and biology, and relates to the application of a macrophage MTDH inhibitor in the preparation of a drug for preventing and treating lung metastasis of a malignant tumor. Background Art

[0002] Tumor metastasis is the main cause of death in cancer patients. The colonization of cancer cells in distant organs is a key step in distant metastasis. The local microenvironment of distant organs determines whether cancer cells can colonize. In particular, macrophages in distant organs acquire immunosuppressive functions and play a key role in the formation of metastatic lesions. Preclinical and clinical studies have shown that targeting tumor-associated macrophages can effectively inhibit distant metastasis of tumors. Therefore, macrophages are potential anti-tumor targets, and finding key targets based on macrophages has important clinical significance and application prospects.

[0003] The cancer metastasis gene MTDH, or Metadherin, is overexpressed in a variety of tumors and is associated with low survival rates, high metastasis risks, and chemotherapy resistance in patients with a variety of tumors. It is considered to be the culprit for the spread or metastasis of cancer cells. Many studies have shown that the danger of MTDH protein depends not only on its presence in tumor cells, but also on its activity in tumor stromal cells. Studies have confirmed that macrophage-derived MTDH also significantly promotes the growth, invasion, metastasis, and vascular mimicry of head and neck squamous cell carcinoma, liver cancer, etc. through a variety of mechanisms. However, there are currently no reports on the regulation of pre-metastatic microenvironment formation and tumor metastasis treatment by macrophage-specific MTDH gene knockout based on macrophage conditional MTDH gene knockout mouse models. Summary of the invention

[0004] Aiming at the problems existing in the traditional cancer metastasis prevention and treatment process, the present invention proposes the use of a substance that reduces the expression of macrophage MTDH gene in the preparation of a drug for preventing and treating malignant tumor lung metastasis.

[0005] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions:

[0006] The first aspect of the present invention provides a use of a macrophage MTDH inhibitor in the preparation of a drug for preventing and treating lung metastasis of malignant tumors. The MTDH gene sequence is shown in SEQ ID NO:1.

[0007] Preferably, the macrophage MTDH inhibitor is a substance that inhibits or reduces the MTDH level.

[0008] More preferably, the substance that inhibits MTDH level is selected from gene knockout; the substance that reduces MTDH level is selected from interfering RNA, and the sequence of the interfering RNA is shown in SEQ ID NO:2.

[0009] The tumor is colorectal cancer and / or melanoma. The target of colorectal cancer treatment is MC-38 cells, and the target of melanoma treatment is B16-F10 cells.

[0010] A second aspect of the present invention provides use of a macrophage MTDH inhibitor in the preparation of a drug for inhibiting angiogenesis.

[0011] Preferably, the macrophage MTDH inhibitor is a substance that inhibits or reduces the MTDH level.

[0012] More preferably, the substance that inhibits MTDH level is selected from gene knockout; and the substance that reduces MTDH level is selected from interfering RNA.

[0013] The third aspect of the present invention provides a use of a macrophage MTDH inhibitor in the preparation of a drug for promoting the expression and secretion of angiogenesis inhibitory molecule TSP-1.

[0014] Preferably, the macrophage MTDH inhibitor is a substance that inhibits or reduces the MTDH level.

[0015] More preferably, the substance that inhibits MTDH level is selected from gene knockout; and the substance that reduces MTDH level is selected from interfering RNA.

[0016] MTDH, also known as AEG-1, is located on mouse chromosome 15. A total of 12 exons have been identified, starting from the start codon ATG of exon 1 and ending with the stop codon TGA of exon 12. Exons 3 and 4 of the macrophage MTDH gene are used as conditional knockout regions.

[0017] Compared with the prior art, the advantages and positive effects of the present invention are:

[0018] The myeloid-specific MTDH knockout mice designed in the present invention significantly inhibit the lung metastasis of colorectal cancer and melanoma, providing a new intervention target for clinical treatment.

[0019] The use of the myeloid-specific MTDH knockout mice designed by the present invention can exclude the influence of systemic or other tissue cells, etc., and provide a good guarantee for studying the role of macrophage MTDH in distant tumor metastasis and its influence on angiogenesis.

[0020] This invention is the first to explore in vivo and co-culture systems in mice how macrophage MTDH knockout regulates TSP-1 to promote angiogenesis and metastasis of malignant tumors in lung tissue, and further confirms in human cells that macrophage MTDH knockdown significantly inhibits angiogenesis and the transendothelial invasion ability of tumor cells. Related content has not been reported at home or abroad. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the result of mouse genotype identification and knockout efficiency verification. Figure 1 A is the electrophoresis diagram of genotype identification of tail DNA samples of macrophage conditional MTDH gene knockout mice and wild-type mice; Figure 1 B is a diagram showing the validation of the knockout effect of MTDH in peritoneal macrophages (PMs) using immunofluorescence. Figure 1 C: Western blot was used to detect the expression level of MTDH protein in peritoneal macrophages of the two groups of mice.

[0022] Figure 2 The effect of myeloid-specific MTDH knockout on lung metastasis of MC-38 colorectal cancer and B16-F10 melanoma in mice. Figure 2 A is a picture of mouse lung tissue after tail vein injection of MC-38 cells to establish a lung metastasis model; Figure 2 B is the HE staining image of lung tissue in the mouse model of MC-38 colorectal cancer cell lung metastasis; Figure 2 C is Figure 2 Statistical chart of B; Figure 2 D is a picture of mouse lung tissue after tail vein injection of B16F10 cells to establish a lung metastasis model; Figure 2 E is the HE staining image of lung tissue in the B16-F10 melanoma cell lung metastasis mouse model.

[0023] Figure 3 The effect of myeloid-specific MTDH knockout on angiogenesis in mouse lung tissue. Figure 3 A is the use of immunofluorescence to detect capillaries (endothelial cell marker CD31) in a colorectal cancer mouse model; Figure 3 B is a statistical analysis of the number of capillaries in the lung metastases of the two groups of mice in Figure A; Figure 3 C is a statistical analysis of the number of capillaries in the lung interstitium of the two groups of mice in Figure A; Figure 3 D is a mouse model of melanoma cell lung metastasis, in which immunofluorescence was used to detect capillaries in the lung interstitium (endothelial cell marker CD31); Figure 3 E is a statistical analysis of the number of capillaries in lung metastases of the two groups of mice in Figure D; Figure 3 F is the statistical analysis of the number of capillaries in the lung interstitium of the two groups of mice in Figure D.

[0024] Figure 4 The effect of in vitro macrophage MTDH knockout on endothelial cell angiogenesis. Figure 4 A is the effect of MTDH knockout in mouse peritoneal macrophages on angiogenesis in mouse brain microvascular endothelial cell line (bEND.3); Figure 4B is a statistical analysis of the relative number of tubes formed by bEND.3 cells in Figure A; Figure 4 C is a scratch assay to detect the effect of MTDH knockout in mouse peritoneal macrophages on the migration ability of bEND.3.

[0025] Figure 5 The effect of MTDH knockout in macrophages on TSP-1 expression. Figure 5 A is the expression level of TSP-1 in the lung interstitium of two groups of mice in the colorectal cancer mouse model, detected by immunofluorescence; Figure 5 B is the expression of TSP-1 mRNA level in peritoneal macrophages of two groups of mice detected by quantitative PCR; Figure 5 C is the expression of TSP-1 protein level in the supernatant of peritoneal macrophages of the two groups of mice detected by Western blot.

[0026] Figure 6 The effect of MTDH knockout in macrophages on TSP-1 transcriptional regulation. Figure 6 A shows peritoneal macrophages from two groups of mice treated with the p53 inhibitor PFT-a, and quantitative PCR was used to detect the expression of TSP-1, p21, and Bax (two known target genes regulated by p53 transcription); Figure 6 B is the cellular localization of MDM2, the main inhibitor of p53, in peritoneal macrophages of the two groups of mice detected by immunofluorescence; Figure 6 C is the application of co-immunoprecipitation to confirm that macrophage MTDH knockout inhibited the MDM2-p53 interaction.

[0027] Figure 7 The effect of MTDH knockdown in macrophages derived from the human monocytic cell line THP-1 on endothelial cell angiogenesis. Figure 7 A is the effect of MTDH knockdown in THP-1-derived macrophages on angiogenesis in human umbilical vein endothelial cells (HUVEC); Figure 7 B is the statistical analysis of the relative number of tubes formed by HUVEC cells in Figure A; Figure 7 C is a transendothelial cell invasion experiment of colorectal cancer cells labeled with green fluorescence; Figure 7 D is a statistical analysis of the number of invading colorectal cancer cells in Figure C. DETAILED DESCRIPTION

[0028] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0029] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments of the following disclosure.

[0030] The main raw materials or reagents used in the following examples are as follows:

[0031] Culture medium: RPMI 1640 culture medium: Beijing Solebow Technology Co., Ltd., 90023; phosphate buffered saline (PBS): White Shark Biotechnology Co., Ltd., BL302A; DMEM high glucose medium: Thermo Fisher Scientific, C11995500BT; red blood cell lysis buffer: White Shark Biotechnology Co., Ltd., BL503A; fetal bovine serum: Shanghai Xiaopeng Biotechnology Co., Ltd., C04002-500.

[0032] Antibodies: MTDH antibody: Shanghai Abi Biotech Co., Ltd., CY7187; CD8 antibody: Beijing Bioson Biotech Co., Ltd., bs-0648R; CD31 antibody: Wuhan Sanying Biotech Co., Ltd., 28083-1-AP; CD206 antibody: Wuhan Sanying Biotech Co., Ltd., 18704-1-AP; TSP-1 antibody: MedChemexpress Biotech, USA, HY-P83530; P53 antibody: CST, USA, 2524; MDM2 antibody: Shanghai Abi Biotech Co., Ltd., AY2958; internal reference GAPDH antibody: Shanghai Biotech Co., Ltd., AF2819; HRP-conjugated secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG (H+L)): Shanghai Biotech Co., Ltd., A0352; HRP-conjugated secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG (H+L)): Shanghai Biotech Co., Ltd., A0350.

[0033] Reagents: TBE electrophoresis buffer: Wuhan Saiweier Biotechnology Co., Ltd., G3002; Nucleic acid dye Goldview: White Shark Biotechnology Co., Ltd., BS357A; Thioglycollate broth: Beijing Solebold Technology Co., Ltd., LA8740; D2000 DNA Marker (100-2000bp): White Shark Biotechnology Co., Ltd., BL102A; 4% paraformaldehyde / universal tissue fixative: White Shark Biotechnology Co., Ltd., BL539A; Phorbol12-myristate 13-acetate (PMA): MedChemexpress Biotechnology Co., Ltd., USA, HY-18739; Triton X-100: White Shark Biotechnology Co., Ltd., BS084; Pifithrin-αhydrobromide (PFT-α): MedChemexpress Biotechnology Co., Ltd., USA, HY-15484; RIPA lysis buffer (strong): Beyotime, P0013B; protease inhibitor cocktail (100×PIC): Beijing Solebow Technology Co., Ltd., P6730; protein phosphatase inhibitor cocktail: Beijing Solebow Technology Co., Ltd., P1260; skim milk: White Shark Biotechnology Co., Ltd., BS102; high concentration gold medal matrix gel (Matrigel): Mogengel & ABW, 0827245; 4% paraformaldehyde: White Shark Biotechnology Co., Ltd., BL539A; crystal violet staining solution (0.1%): White Shark Biotechnology Co., Ltd. BL802A; OCT frozen section embedding agent: SAKURA, USA, 4583; bovine serum albumin BSA: Beijing Solebow Technology Co., Ltd., A8010.

[0034] Kits: ① Blood / tissue / cell / bacteria genomic DNA extraction kit, manufacturer: Vazyme, DC112-01, specifically including Buffer ACL, Proteinase K, Buffer BCL, Buffer WA, Buffer WB, Elution Buffer. ② Accurate Taq Master Mix (dye plus): Hunan Aikerui Bioengineering Co., Ltd., AG11010, specifically including 2X Accurate Taq Master Mix (dye plus) RNase-free water. ③ SDS-PAGE gel preparation kit: White Shark Biotechnology Co., Ltd., BL508A, specifically including 30% Acr-Bis (29:1), 1MTris-HCLpH8.8, 1MTris-HCLpH6.8, 10% SDS, APS (dry powder) and TEMED. ④ Ultra-sensitive ECL chemiluminescence ready-to-use substrate (femtogram level): Wuhan Boster Bioengineering Co., Ltd., AR1191. ⑤ Reverse transcription kit: Beijing Kangwei Century Biotechnology Co., Ltd., CW2582M, including 10×gDNA Eraser Buffer, gDNA Eraser, HiFiScript, 200U / μ, Primer Mix, 5×ScriptRT Buffer, RNase-Free Water. ⑥ Quantitative PCR kit UltraSYBR Mixture (CW0957, Beijing Kangwei Century Biotechnology Co., Ltd.), including 2×UitraSYBR Mixture, RNase-Free Water. ⑦ Co-IP kit: Boxin Biotechnology, Bes3011, including cell lysis buffer, protease inhibitor, protein A / G-MagBeads, elution buffer, IgG.

[0035] Transwell cell culture chamber, for 24-well plates, 8.0um, 6.5mm, polycarbonate membrane chamber: Corning, 3422. PVDF (polyvinylidene fluoride) membrane: Thermo Fisher Scientific, LC2002.

[0036] The rest of the materials, instruments or processing procedures not specifically described are all commonly used reagents or operations in bioengineering or genetic engineering experiments. The percentages of substances not specifically described in the following examples are by mass.

[0037] Example 1

[0038] 1. Construction of macrophage conditional MTDH gene knockout mouse model

[0039] The gene MTDH, also known as AEG-1, is located on mouse chromosome 15, and a total of 12 exons have been identified, starting from the start codon ATG of exon 1 and ending with the stop codon TGA of exon 12 (Transcript Mtdh-201: ENSMUST00000022865). In this example, exons 3 and 4 are selected as conditional knockout regions (cKO regions). The deletion of this region can lead to the loss of function of the mouse MTDH gene.

[0040] (1) Mouse breeding

[0041] Mtdhflox / flox mice (the loxP insertion position is from exon 3 to 4, i.e., amino acids 161-248 (skknkkksksdakavqnssrhdgkevdegawetkishrekrqqrkrdkvltdsgsldstipgieniitvtteqlttasfpvgskknkg) were purchased from Saiye (Suzhou) Biotechnology Co., Ltd., and the DNA sequence is: tcaaagaaaaataagaagaaatcaaagtcagatgctaaagcagtgcaaaacagttcacgccatgat ggaaaggaagttgatgaaggagcctgggaaactaaaattagtcacagagagaaacgacaacagcgtaaacgtgataaagtgctgactgattctggttcattggattcaactatccctgggatagaaaatatcatcacagttaccaccgagcaacttacaactgcatcatttcctgttggttccaagaagaataaag and Lyz2-cre (Cre sequence inserted downstream of Lyz2 promoter, as shown in SEQ ID NO: 3) transgenic mice were cross-bred; MTDH fl / fl The F1 generation mice were obtained by hybridization with Lyz2-cre transgenic mice, and the F1 generation mice were self-fertilized and identified to obtain myeloid-specific MTDH knockout (MTDH - / - ) mice, whose genotype is MTDH fl / fl :Lyz2-Cre+.

[0042] (2) Gene identification

[0043] Extraction of MTDH - / -Mouse genomic DNA (Novozyme FastPure Blood / Cell / Tissue / Bacteria DNAIsolation Mini Kit-DC112): On the first day, cut off 0.5 cm of the mouse tail tip (also applicable to toes), put it into a 1.5 ml Eppendorf tube, add 200 μl Buffer ACL, 20 μl Proteinase K, vortex to mix, and place in a 56°C water bath until complete enzymatic hydrolysis, digest overnight. On the second day, add 200 μl Buffer BCL, shake and mix; add 150 μl of anhydrous ethanol, shake and mix, centrifuge at 8000 rpm for 20 seconds to collect the liquid on the inner wall of the tube cap. Place the FastPure gDNA MiniColumnsII adsorption column in the collection tube and transfer the liquid on the inner wall of the tube cap to the adsorption column. Centrifuge at 12,000 rpm (13,400 × g) for 1 minute, discard the filtrate, and place the adsorption column in the collection tube. Add 500μl Buffer WA to the adsorption column along the tube wall, centrifuge at 12,000rpm (13,400×g) for 1min. Discard the filtrate and place the adsorption column in a collection tube. Add 600μl Buffer WB along the tube wall, centrifuge at 12,000rpm (13,400×g) for 1min, and discard the filtrate. Place the adsorption column in a collection tube. Centrifuge the empty column at 12,000rpm (13,400×g) for 2min. Transfer the adsorption column to a new 1.5ml centrifuge tube. Add 200μl Elution Buffer to the center of the adsorption column membrane, centrifuge at 12,000rpm (13,400×g) to collect DNA. Store at 4℃ for short-term and -20℃ for long-term.

[0044] The PCR system was prepared according to the instructions of the Accurate Taq Master Mix (dye plus) kit, with a final volume of 25 μl. The components and contents of the system are as follows:

[0045]

[0046] Primer information and PCR procedures are as follows:

[0047] ①MTDH-floxp-forward primer:

[0048] 5'-TCCTTTGGAAAATGATGGTGGATTG-3';

[0049] MTDH-floxp-reverse primer:

[0050] 5'-GGTAGAATAGGTTCTACTGTGTCATGT-3'.

[0051] PCR conditions: 94°C for 30 s, 60°C for 35 s, 72°C for 35 s, for a total of 35 cycles.

[0052] ②Lyz2Cre-forward primer: 5'-CCCAGAAATGCCAGATTACG-3';

[0053] Lyz2Cre-reverse primer: 5′-CTTGGGCTGCCAGAATTTCTC-3′.

[0054] PCR conditions: 94°C for 30 s, 60°C for 35 s, 72°C for 35 s, for a total of 35 cycles.

[0055] (3) Electrophoresis

[0056] Weigh 2g of agarose and dissolve it in a conical flask, add 100ml of 1XTBE (2% gel), heat it in a microwave oven on medium-low heat for 3min until the agarose is completely melted, take it out and shake it well. When the agarose solution cools to 55°C, add 10μl of nucleic acid dye Goldview (X10000), mix it well, pour the agarose solution gently into the gel plate, and insert the comb. After the agarose gel is completely cooled and solidified, gently pull out the comb and add 500ml of TEB electrophoresis buffer to the electrophoresis tank. Add the mouse tail PCR product and D2000 DNA Marker to the sample slot, add 10-20μ1 to each slot, and record the order and amount of sample spotting. Install the electrode wires, cover the electrophoresis tank, turn on the power, select the appropriate voltage (4-10V / cm, the distance is the position of the two electrophoresis tank electrodes, the electrophoresis time is about 30min, and the total voltage of the electrophoresis tank is 120V), start electrophoresis, and the DNA sample moves from the negative electrode to the positive electrode. When the bromophenol blue moves to 1-2cm from the front of the gel, cut off the power. Take out the sample, observe it under a 254nm ultraviolet lamp, and record the electrophoresis pattern under the ultraviolet lamp. The genotype of the tail DNA sample of wild-type mice is extracted in the same way and tested.

[0057] (4) Verification of successful knockout of the macrophage MTDH gene

[0058] From wild-type mice and macrophage conditional MTDH knockout mice (MTDH - / - ), peritoneal macrophages were extracted respectively, and the expression of MTDH in peritoneal macrophages was detected by immunofluorescence and Western blot to verify the knockout effect of MTDH in macrophages and prove that the transformation of macrophages was successful.

[0059] Extraction and culture of peritoneal macrophages from mouse peritoneal cavity: 1 mL of 3% thioglycolate broth was injected intraperitoneally each time, and the peritoneal cavity was lavaged twice with pre-cooled PBS after 3 days. The mice were killed by dislocation of the neck, and the whole mice were soaked in 75% alcohol for 5 minutes. The mice were taken out and placed in a stainless steel square plate. After the peritoneal wall was scrubbed with 70% alcohol, 5 mL of normal saline was injected into the peritoneal cavity, and the mouse abdomen was gently rubbed several times for 5 minutes. The lower abdominal skin was lifted with forceps and the peritoneum was exposed by dissection. The needle of the syringe was inserted into the gap to extract the peritoneal fluid, and then the peritoneal fluid was injected into the centrifuge tube. The collected cells were centrifuged at 1000g, 8000rpm, and 4℃ for 5 minutes, the supernatant was discarded, 10 mL of red blood cell lysis buffer was added, incubated at room temperature for 5 minutes, and the supernatant was removed by centrifugation. RPMI 1640 culture medium containing 10% fetal bovine serum was added to resuspend the cells for inoculation. The culture dish was incubated at 37°C for 2 h, and then the non-adherent cells were washed away. The remaining adherent cells were macrophages.

[0060] Immunofluorescence was used to detect the expression and localization of MTDH in the extracted and cultured peritoneal macrophages. Sterile slides were spread on culture dishes, and cell suspensions were seeded into the culture dishes. After 2 hours of inoculation, the cell sample slides were placed on glass slides, fixed with 4% paraformaldehyde at room temperature for 15 minutes, and permeabilized with 0.2% Triton X-100 at room temperature for 5 minutes after immersion. After immersion, 3% BSA was blocked for 60 minutes. The blocking solution was aspirated, anti-mouse / human MTDH antibody was added, and incubated at 4°C overnight. The next day, the primary antibody was aspirated, and after immersion, immunofluorescence secondary antibody (volume ratio 1:10000) was added and incubated at room temperature for 1 hour. DAPI was incubated in the dark for 5 minutes. Excess DAPI was washed off, and anti-fluorescence quenching sealing solution was dropped on the glass slide, and the coverslip with cells was covered, and green fluorescence was observed under a fluorescence microscope.

[0061] Protein immunoblotting was used to detect the protein expression of MTDH in peritoneal macrophages, and MTDH gene knockout in peritoneal macrophages was identified at the protein level. Peritoneal macrophages were lysed with RIPA buffer containing a mixture of protease inhibitors (100×) and a mixture of protein phosphatase inhibitors (100×) to extract protein lysates. After sufficient lysis, centrifuge at 10000-14000g for 3-5min, and the supernatant was taken for protein immunoblotting detection. SDS-PAGE gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. The lysed supernatant of peritoneal macrophages was subjected to SDS-PAGE gel electrophoresis at 100V for 90min; after the electrophoresis, the gel was taken out and the transfer sandwich was assembled: sponge / 3 layers of filter paper / gel / PVDF membrane / 3 layers of filter paper / sponge, and the electrotransfer conditions were 150V for 3h. After the transfer, the PVDF membrane was removed, washed with 1XTBST, and blocked in 5% skim milk at room temperature for 1h. After washing with 1XTBST, the membrane was incubated with MTDH antibody or internal reference GAPDH antibody at 4°C overnight. The next day, after washing with 1XTBST, the membrane was blocked with HRP-coupled secondary antibody at room temperature for 1h. After washing with 1XTBST, the ECL ultrasensitive kit instructions were followed, and freshly prepared ECL chemiluminescent solution was used for development and detection. The multifunctional automatic chemiluminescence / fluorescence image analysis system was used for detection, and Image J software was used for analysis.

[0062] The results are as follows Figure 1 As shown, Figure 1 A is the results of mouse tail phenotype identification of macrophage conditional MTDH knockout mice and wild-type mice; Figure 1 B is the use of immunofluorescence to detect the knockout effect of MTDH in peritoneal macrophages; Figure 1 C is the use of Western blot to detect the expression level of MTDH protein in the peritoneal macrophages of the two groups of mice, indicating that the transformation of macrophages was successful.

[0063] Example 2

[0064] Using mouse transplant tumor experiments, the metastatic tumors in the lung tissues of MTDH gene knockout mice and wild-type mice were compared, demonstrating that the macrophage conditional MTDH gene knockout group significantly inhibited the number and size of metastatic foci in the lung tissues of mice.

[0065] Construction of mouse transplant tumor model: 4-6 week old conditional knockout mice and wild-type mice were selected, and 200 μl of MC-38 and B6F10 cells were injected through the tail vein, with the cell amount of 3*105 / 200 μl, to construct a lung metastasis model. After 17 days, the mice were killed, and the lung tissue was removed and fixed with 4% paraformaldehyde for 24 hours. Dehydration, transparency, wax immersion, embedding and sectioning were routinely performed, and the sections were HE stained according to the following operations.

[0066] ① Baking: Bake the slices in a 65℃ oven for 2 hours.

[0067] ② Dewaxing and hydration: conventional xylene for 10 min; xylene II for 10 min; xylene III for 10 min; anhydrous ethanol for 5 min twice; 95% ethanol for 5 min; 80% ethanol for 5 min; 70% ethanol for 5 min; rinse with running water.

[0068] ③ Staining: Place the slices in hematoxylin for staining at room temperature for 3 minutes, rinse with running water, and observe the staining under a microscope. Differentiate with 1% hydrochloric acid ethanol for 3 seconds, and rinse with tap water to turn blue.

[0069] ④ Dehydration: Stain the sections in eosin for 3 minutes; 80% ethanol for 1 minute, 95% ethanol for 1 minute; anhydrous ethanol for 1 minute; xylene for 1 minute; and place in a fume hood to dry.

[0070] ⑤ Seal the slides with neutral gum, observe under a microscope, take photos, and count the number of metastatic lesions in the lung tissue.

[0071] The results are as follows Figure 2 As shown, Figure 2 The number and size of metastatic foci in lung tissue after tail vein injection of MC-38 and B16F10 tumor cells were compared between macrophage conditional MTDH gene knockout mice and wild-type mice. Figure 2 A is a picture of the mouse lung tissue 17 days after the tail vein injection of MC-38 cells to construct a colorectal cancer lung metastasis mouse model. Compared with wild-type mice, the lung metastasis foci of macrophage knockout MTDH mice were significantly reduced (observed with the naked eye); Figure 2 B is in the mouse model of colorectal cancer MC-38 cell lung metastasis. HE staining further confirmed that the number of metastatic foci in the lung tissue of knockout mice was significantly less than that of wild-type mice. Figure 2 C is Figure 2 Statistical chart of B. Figure 2 D is a picture of the lung tissue of mice constructed by tail vein injection of B16F10 cells for melanoma lung metastasis. Compared with wild-type mice, the lung metastasis of macrophage knockout MTDH mice was significantly reduced (observed with the naked eye); Figure 2 E is the result of HE staining in a melanoma lung metastasis mouse model, which further confirmed that the number of metastatic foci in the lung tissue of knockout mice was significantly less than that of wild-type mice. Figure 2 It can be seen that conditional MTDH gene knockout in macrophages can significantly inhibit the lung metastasis of colorectal cancer and melanoma.

[0072] Example 3

[0073] In the mouse models of colorectal cancer and melanoma lung metastasis, comparison of vascular markers in the lung tissues of knockout and wild-type mice demonstrated that the macrophage conditional MTDH gene knockout group significantly inhibited angiogenesis in lung tissues.

[0074] Five-week-old macrophage conditional MTDH knockout mice and wild-type mice were prepared. 300,000 mouse colorectal cancer cells MC-38 and melanoma cells B16F10 were injected into the tail vein of each mouse. The mice were killed by cervical dislocation on the 17th day. Fresh lung tissues were fixed in 4% paraformaldehyde, dehydrated with 30% sucrose, and prepared into frozen sections after OCT embedding. Immunofluorescence staining was performed with vascular endothelial cell marker CD31 antibody to compare the infiltration degree of CD31 cells in the lung tissues of the two groups.

[0075] The experimental results are as follows Figure 3 As shown, Figures AC are the number of capillaries (vascular marker CD31) in the lung tissue of knockout mice and wild-type mice and their statistical graphs in a mouse model of colorectal cancer lung metastasis. Figure 3 In the figure, A is a representative image of CD31 immunofluorescence staining; B is a statistical analysis of the number of blood vessels in lung metastases; C is a statistical analysis of the number of blood vessels in the pulmonary interstitium. DF is the number of capillaries and their statistical graphs in the melanoma B16-F10 cell lung metastasis mouse model. D is a representative image of CD31 immunofluorescence staining; E is a statistical analysis of the number of blood vessels in lung metastases; F is a statistical analysis of the number of blood vessels in the pulmonary interstitium. Statistical analysis used unpaired t-test to compare the mean values ​​of the two groups, and the data are expressed as mean ± SEM. From the results, compared with wild-type mice, conditional MTDH gene knockout in macrophages significantly inhibited angiogenesis in lung metastases and pulmonary interstitium.

[0076] Example 4

[0077] The experimental subjects of this example are mouse peritoneal macrophage culture and co-culture, verifying that MTDH gene knockout peritoneal macrophages can significantly inhibit angiogenesis.

[0078] The same steps as in Example 1 were used to extract peritoneal macrophages from knockout mice and wild-type mice, inoculated into cell culture dishes, and replaced with RPMI1640 medium containing 10% serum after 2 hours of adhesion. The supernatant of the conditioned medium was collected after 24 hours to analyze the effect of the conditioned medium of the two groups of peritoneal macrophages on the angiogenesis ability of mouse brain microvascular endothelial cells bEND.3. The matrix gel was placed on ice overnight to melt. After the matrix gel melted, 50 μL of matrix gel was added to each well of the 96-well plate and incubated at 37°C for 30 minutes. The mouse brain microvascular endothelial cell line bEND.3 (Wuhan Pronocell Life Science Technology Co., Ltd., CL-0598) was taken out of the incubator for trypsin digestion and cell counting. 2×10 4Each cell was inoculated in 100 μL of conditioned medium, then inoculated on Matrigel and cultured in an incubator for 6 h. The tube formation of cells was observed by an inverted microscope and photographed. Statistical analysis was performed using GraphPadPrism 8, and the mean values ​​of the two groups were compared using an unpaired t-test. Data are expressed as mean ± SEM.

[0079] 80X104 bEND.3 cells were inoculated into each well of a 12-well plate and cultured in an incubator for 24 hours. After the cells were fully spread, a 20ul pipette tip was used to make cell scratches perpendicular to the well plate and line, so that the scratches intersected with the marked line. After the scratches were completed, a microscope was used to take a photo as a 0h control. The old culture medium was aspirated, and the cells were washed three times with sterile PBS, and then replaced with the supernatant of peritoneal macrophages from knockout mice and wild-type mice, and photographed with a microscope after 36 hours.

[0080] The results are as follows Figure 4 As shown, Figure A shows the effect of the supernatant of peritoneal macrophages from knockout mice and wild-type mice on the angiogenesis of bEND.3; Figure B shows the statistical analysis of the relative number of tubes formed by bEND.3 cells in the two groups in Figure A. Figure C shows that the supernatant of peritoneal macrophages from knockout mice significantly inhibited the migration ability of bEND.3 cells as confirmed by the scratch experiment.

[0081] Example 5

[0082] The experimental subjects of this example are lung tissues of knockout mice and wild-type mice and mouse peritoneal macrophages to verify the effect of macrophage MTDH knockout on TSP-1 expression.

[0083] Five-week-old macrophage conditional MTDH knockout mice and wild-type mice were prepared. 300,000 mouse colorectal cancer cells MC-38 were injected into each mouse through the tail vein. The mice were killed by cervical dislocation on the 17th day. Fresh lung tissues were fixed in 4% paraformaldehyde, dehydrated with 30% sucrose, and prepared into frozen sections after OCT embedding. Immunofluorescence staining was performed with TSP-1 antibody to compare the expression of TSP-1 in the lung tissues of the two groups.

[0084] The same steps as in Example 1 were used to extract peritoneal macrophages from knockout mice and wild-type mice, inoculated in 6 cm cell culture dishes, and quantitative PCR was used to detect the difference in the expression of TSP-1 mRNA levels in the two groups of peritoneal macrophages. ① Extraction of total RNA: After adding 1 ml of Trizol, repeatedly blow with a gun or shake violently to lyse the cells. The Trizol lysate of the above tissue or cells was transferred to an EP tube and placed at room temperature for 5 minutes. In the above EP tube, 0.2 ml of chloroform was added, the EP tube cover was covered, and it was turned upside down 6 to 8 times. After being placed at room temperature for 3 minutes, 12000 rpm, 4 ° C, centrifuged for 15 minutes. The upper aqueous phase was placed in a new EP tube, and isopropanol was added according to the amount of 0.5 ml of isopropanol per 1 ml of TRIZOL, placed at room temperature for 10 minutes, and centrifuged at 12000 rpm, 4 ° C for 10 minutes. Discard the supernatant, add 1 ml of 75% ethanol for washing, and centrifuge at 12000 rpm, 4 ° C for 5 minutes. Dissolve the RNA precipitate with RNase-free water. ② Reverse transcription: Take samples and perform reverse transcription according to the kit operation manual (reverse transcription kit, Takara). Specific steps: First, mix the following substances to prepare reaction solution 1:

[0085]

[0086] Reaction conditions: incubate at 42°C for 2 minutes.

[0087] Then the following substances were mixed to prepare reaction solution 2:

[0088]

[0089] Reaction conditions: incubate at 42°C for 15 min and at 85°C for 5 min.

[0090] ③ Quantitative PCR: According to the instructions of the UltraSYBR Mixture kit, a quantitative PCR system was prepared with a final volume of 50 μl. The components and contents of the system are as follows:

[0091]

[0092] The reaction conditions are as follows:

[0093]

[0094] Primer information is as follows:

[0095] ①TSP-1 forward primer:

[0096] 5'-CTAGGTGTCCTGTTCCTGTTG-3';

[0097] TSP-1 reverse primer:

[0098] 5'-AAGGAAGCCAGGAAGATGAAG-3'.

[0099] Statistical analysis was performed using GraphPad Prism 8. Unpaired t-test was used to compare the means of the two groups, and the data are expressed as mean ± SEM.

[0100] The same steps as in Example 1 were used to extract peritoneal macrophages from knockout mice and wild-type mice, inoculated in 6 cm cell culture dishes, and protein immunoblotting was used to detect the protein expression of TSP-1 in peritoneal macrophages. The peritoneal macrophages were lysed with RIPA buffer containing a mixture of protease inhibitors (100×) and a mixture of protein phosphatase inhibitors (100×) to extract protein lysates, which were lysed on ice for 15 minutes. After sufficient lysis, the supernatant was centrifuged at 10000-14000g for 3-5 minutes, and the supernatant was taken for protein immunoblotting detection. SDS-PAGE gel was prepared according to the instructions of the SDS-PAGE gel preparation kit, and the lysed supernatant of peritoneal macrophages was subjected to SDS-PAGE gel electrophoresis at 100V for 90min; after the electrophoresis was completed, the gel was taken out and the transfer sandwich was assembled: sponge / 3 layers of filter paper / gel / PVDF membrane / 3 layers of filter paper / sponge, and the electrotransfer conditions were 150V for 3h. After the transfer, the PVDF membrane was removed, washed with 1XTBST, and blocked in 5% skim milk at room temperature for 1h. After washing with 1XTBST, the membrane was incubated with TSP-1 antibody or internal reference GAPDH antibody at 4°C overnight. The next day, after washing with 1XTBST, the membrane was blocked with HRP-coupled secondary antibody at room temperature for 1h. After washing with 1XTBST, according to the instructions of the ECL ultrasensitive kit, freshly prepared ECL chemiluminescent solution was used for development and detection, and the detection was performed using a multifunctional automatic chemiluminescence / fluorescence image analysis system, and analyzed using Image J software.

[0101] The experimental results are as follows Figure 5 Shown, Panel A is a representative image of immunofluorescence staining of TSP-1 in lung tissues of knockout mice and wild-type mice in a mouse model of colorectal cancer lung metastasis.

[0102] Figure 5 B is the expression level of TSP-1 mRNA in peritoneal macrophages of knockout mice and wild-type mice detected by quantitative PCR; Figure 5 CWestern blot confirmed that TSP-1 expression in peritoneal macrophages of knockout mice was significantly increased. The results confirmed that conditional MTDH gene knockout in macrophages significantly promoted the expression of TSP-1 in the lung interstitium of mice with lung metastasis.

[0103] Example 6

[0104] The experimental subjects of this example are peritoneal macrophages of knockout mice and wild-type mice, and quantitative PCR, immunofluorescence and immunoprecipitation are used to verify the transcriptional regulation of TSP-1 gene by MTDH in macrophages.

[0105] The same steps as in Example 1 were used to extract peritoneal macrophages from knockout mice and wild-type mice, inoculated in cell culture dishes, and p53 inhibitor PFT-α (10 umol) or the same volume of solvent DMSO was added to the culture dish for stimulation for 24 hours. Peritoneal macrophages were collected, and the expression of TSP-1 gene mRNA level was detected using the same steps of quantitative PCR in Example 5. Pifithrin-α (PFT-α) is a commonly used p53 inhibitor that inhibits p53-dependent p53 response gene transcription. Primer information is as follows:

[0106] ①p21 forward primer:

[0107] 5′-GTCCAATCCTGGTGATGTCC-3′;

[0108] p21 reverse primer:

[0109] 5′-GTTTTCGGCCCTGAGATGT-3′.

[0110] ②BAX forward primer:

[0111] 5'-ATGAAGACAGGGGCCTTTTGCTA-3';

[0112] BAX reverse primer:

[0113] 5'-TCAGCCCATCTTCTCCAGATGGT-3'.

[0114] The same steps as in Example 1 were used to extract the peritoneal macrophages of knockout mice and wild-type mice, and the localization of the protein level of MDM2, the main inhibitor of p53 protein in the extracted and cultured peritoneal macrophages, was detected by immunofluorescence: the sterile slide was spread on the culture dish, and the peritoneal macrophage suspension of the extracted knockout mice and wild-type mice was seeded into the culture dish. After 2 hours of inoculation, the cell sample slide was placed on the slide, fixed with 4% paraformaldehyde at room temperature for 15 minutes, and permeabilized with 0.2% Triton X-100 at room temperature for 5 minutes after immersion, and blocked with 3% BSA for 60 minutes after immersion. The blocking solution was aspirated, anti-mouse / human MDM2 antibody was added, and incubated overnight at 4°C. The next day, the primary antibody was aspirated, and after immersion, the immunofluorescence secondary antibody (volume ratio 1:10000) was added dropwise and incubated at room temperature for 1 hour. DAPI was incubated in the dark for 5 minutes. Excess DAPI was washed off, and anti-fluorescence quenching sealing solution was dropped on the slide, and the cover glass with cells was covered, and the green fluorescence was observed under a fluorescence microscope.

[0115] The same steps as in Example 1 were used to extract peritoneal macrophages from knockout mice and wild-type mice, and the interaction between p53 and MDM2 in peritoneal macrophages was detected using an immunoprecipitation kit. According to the instructions of the immunoprecipitation kit, peritoneal macrophages were lysed with RIPA buffer containing protease inhibitors and phosphatase inhibitors to extract protein lysates, 4°C, 13000g, 15min, and the supernatant was collected; 100 μL of the supernatant was taken as the Input group, and 900 μL of the supernatant was taken as the IP group; the Input group was stored at -20°C for use, and p53 antibody or IgG was added to the IP group tube, and incubated overnight at 4°C with a vertical mixer. Protein-A / G magnetic beads were prepared by washing, and 100 μL of Protein A / G-MagBeads suspension was added to the EP tube of the IP group. The beads were shaken on a vertical mixer at 4°C for 1 h. The beads were washed and collected by static adsorption on a magnetic rack for 3 times, and the supernatant was discarded. Elution buffer was added, and the beads were denatured and eluted in a boiling water bath for 10 min. The beads were statically adsorbed on a magnetic rack, and the supernatant was collected. Western blot was used to detect whether there was a difference in the expression level of MDM2 in the precipitates of peritoneal macrophages between knockout mice and wild-type mice.

[0116] The results are as follows Figure 6 As shown, Figure 6 A Quantitative PCR results showed that p53 inhibitors prevented the high expression of TSP-1, p21, and Bax induced by MTDH knockout in macrophages (p21 and Bax are known target genes of p53); Figure 6 B Immunofluorescence results showed that MTDH knockout in macrophages inhibited the nuclear localization of MDM2, the main inhibitor of p53 protein, suggesting that MTDH knockout in macrophages may affect the interaction between p53 and MDM2 to regulate the expression of TSP-1; Figure 6 The results of C co-immunoprecipitation technology (CoIP) showed that knocking out MTDH in macrophages significantly inhibited the interaction between p53 protein and MDM2 protein.

[0117] Example 7

[0118] The experimental subjects of this example are human monocytic cell line THP-1 and human umbilical vein endothelial cells HUVEC (Wuhan Pronocell Life Science Co., Ltd., CL-0675). Interfering RNA was used to knock down the expression of MTDH in THP-1-derived macrophages to verify whether the macrophages with MTDH gene knockdown affect the tubular formation ability of HUVEC.

[0119] The human MTDH knockout lentivirus and control lentivirus were purchased from GeneCare. The sequence of shRNA targeting human MTDH was (CCAAGTCAAATACCAAGCAAA). The lentiviral system was used to infect human mononuclear cells THP-1 to construct a cell line with stable MTDH knockdown. The cells were induced to differentiate into macrophages with phorbol ester PMA for 24 hours and named Mac-shMTDH. The control group cells were Mac-NC. The culture medium of Mac-shMTDH and Mac-NC was collected to observe the effects of the conditioned medium of the two groups on the angiogenesis ability of human umbilical vein endothelial cells HUVEC. 2×10 4 HUVEC cells were inoculated into Matrigel and cultured in an incubator for 6 h. The tube-forming ability of HUVEC cells on Matrigel was observed by an inverted microscope and photographed. GraphPadPrism 8 was used for statistical analysis, and the unpaired t-test was used to compare the mean values ​​of the two groups. The data are expressed as mean ± SEM.

[0120] Transendothelial cell invasion assay: ① HUVEC cells were seeded in the upper chamber of the Transwell chamber in advance and cultured in an incubator at 37 degrees. After 24 hours, they were washed three times with 1XPBS and starved overnight with DMEM medium. The collected culture medium of Mac-shMTDH and Mac-NC was added to the Transwell chamber and stimulated for 48 hours. ② Lentiviral vector-labeled green fluorescent protein (GFP, sequence as shown in SEQ ID NO: 4) was used to label colorectal cells MC-38. 200 μl of green fluorescent labeled MC-38 cell suspension (5×10 5 ③Take out the chamber, remove the liquid in the upper chamber, fix with 4% paraformaldehyde for 20 minutes, stain with 0.1% crystal violet for 15 minutes, immediately transfer to 1×PBS to rinse until there is no excess dye, invert and air dry, observe the cell invasion ability under an inverted microscope, decolorize with 75% ethanol, dry, wash with PBS, and stain again. ④Take pictures under the microscope, randomly select 3 high-power fields to count and take the average value, and perform statistical analysis.

[0121] The results are as follows Figure 7As shown, Figure A shows the effect of MTDH knockdown macrophages and control group cells on angiogenesis of human umbilical vein endothelial cells HUVEC; Figure B shows the statistical analysis of the relative number of HUVEC cells in the two groups in Figure A. The results confirmed that macrophage knockout or knockdown of MTDH significantly inhibited angiogenesis; Figure C shows that MTDH knockdown macrophages significantly inhibited the transendothelial cell invasion ability of colorectal cancer cells in the transendothelial cell invasion experiment; Figure D shows the statistical analysis of the number of colorectal cells invading in Figure C.

[0122] From the results, the present invention inhibits angiogenesis in lung tissue by specifically knocking out MTDH in macrophages, thereby significantly inhibiting the number and size of tumor metastases in lung tissue. The present invention aims to transform macrophages and propose a new type of immune cell therapy. By establishing a mouse model in which the MTDH gene is specifically knocked out in macrophages, the mouse transplant tumor experiment shows that after the macrophages are transformed, the formation of blood vessels in the lung tissue is significantly reduced, thereby inhibiting the formation of lung tissue metastases. Human cell experiments also further confirmed that inhibiting macrophage MTDH significantly inhibited the angiogenesis of HUVEC and the transendothelial invasion ability of tumor cells. The results of this study provide strong support for the innovation of clinical tumor treatment strategies. Compared with the traditional method of directly intervening in the MTDH gene in tumor cells, this targeted macrophage treatment is more feasible. The transformed macrophages can synergize with anti-tumor small molecule drugs or immune checkpoint inhibitors (such as PD-1 or PD-L1 antibodies) to achieve anti-cancer effects. The mouse transplant tumor animal model screening platform is expected to become an important cornerstone for future tumor resistance research and develop new anti-cancer strategies for tumor patients with existing drug resistance.

[0123] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes and apply it to other fields. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Use of a macrophage MTDH inhibitor in the preparation of a drug for inhibiting angiogenesis and preventing and treating lung metastasis of malignant tumors, wherein the MTDH gene sequence is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The macrophage MTDH inhibitor is a substance that reduces the MTDH level. The substance that reduces the MTDH level is a gene knockout material and / or interfering RNA. The sequence of the interfering RNA is shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that: The malignant tumor is colorectal cancer and / or melanoma.

4. The use according to claim 3, characterized in that: The method for preventing and treating lung metastasis of malignant tumors includes inhibiting angiogenesis in lung tissue.

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