WO2024050861A1 - Use of runx2 inhibitor in preparing medicament for combating malignant phyllodes tumors of breast - Google Patents
Use of runx2 inhibitor in preparing medicament for combating malignant phyllodes tumors of breast Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
Definitions
- the invention relates to the field of medical biotechnology, and specifically relates to the application of RUNX2 inhibitors in the preparation of drugs against malignant phyllodes tumors of the breast.
- Phyllodes tumor is a rare fibroepithelial tumor, accounting for 0.3% to 1% of all breast tumors. According to existing literature research, borderline and malignant phyllodes tumors not only grow rapidly, but are also prone to local recurrence and distant metastasis. Blood metastasis is the most common way of metastasis, and lung and bone are the most common metastasis sites. The recurrence rate of malignant phyllodes tumors of the breast is as high as 53.1%, and the metastasis rate is as high as 43.1%. Once recurrence and metastasis occur, the patient will die within a short period of time. The mortality rate of malignant phyllodes tumors is approximately 16.3%.
- breast phyllodes tumors The treatment of breast phyllodes tumors is mainly surgical. Malignant phyllodes tumors have poor clinical prognosis due to their high recurrence rate and high metastasis rate. Unlike breast cancer, adjuvant treatments such as chemotherapy and radiotherapy are not effective after surgery for malignant phyllodes tumors of the breast. There are currently no studies reporting clinical benefit from targeted therapy and immunotherapy. Moreover, because phyllodes tumors of the breast are relatively rare, there is a lack of large-scale clinical data and basic research, especially research on specific molecular markers and treatment targets, and no effective progress has been made, further resulting in the lack of research or screening. Progress in effective treatments has stalled.
- RUNX2 is an important member of the RUNX family of transcription factors and is named because it contains a runt domain.
- the biological role of RUNX2 is mainly as a specific transcription factor for osteogenic differentiation, regulating the transcription of genes such as type I collagen, osteomodulin, osteocalcin, col1a1, col1a2, bone sialoprotein (BSP) and fibronectin. It plays an important role in the formation and differentiation of osteoblasts, differentiation and maturation of chondrocytes, the formation and resorption of osteoclasts, and the synthesis of bone matrix proteins.
- BSP bone sialoprotein
- RUNX2 is related to the biological behavior of malignant phyllodes tumors of the breast.
- the present invention discloses the technical application of RUNX2 as a biological behavioral marker and drug target of malignant phyllodes tumors of the breast.
- RUNX2 is highly expressed in malignant breast phyllodes tumor cells and promotes the progression of malignant breast phyllodes tumors. Knocking down the expression of RUNX2 can inhibit the proliferation, migration, and invasion of malignant phyllodes tumors of the breast.
- RUNX2 can be used as a marker and drug target for malignant phyllodes tumors of the breast, and has high application value in the auxiliary diagnosis, screening and preparation of targeted therapeutic drugs for breast phyllodes tumors. The following applications are thus exposed:
- RUNX2 as a marker in screening or preparing drugs against malignant breast phyllodes tumors.
- detection of RUNX2 expression in test tumor cells can be used as a means to evaluate drug efficacy.
- RUNX2 as a drug target in screening or preparing drugs against malignant phyllodes tumors of the breast.
- Drugs targeting RUNX2 as a therapeutic target can be prioritized for screening as potential drugs against breast malignant phyllodes tumors.
- the drug CADD522 inhibits the binding of RUNX2 to DNA.
- the invention further discloses the application of the RUNX2 inhibitor CADD522 in the preparation of drugs against malignant phyllodes tumors of the breast.
- the present invention has the following beneficial effects:
- the present invention discloses for the first time that the overexpression of RUNX2 in breast malignant phyllodes tumor specimens is significantly related to the biological behavior of breast malignant phyllodes tumors. Knocking down the expression of RUNX2 can inhibit the proliferation, migration, and invasion of breast malignant phyllodes tumors. It is believed that RUNX2 can be used as a marker and therapeutic target for screening drugs against malignant phyllodes tumors of the breast, and has high application value in the screening and preparation of drugs targeting malignant phyllodes tumors of the breast.
- CADD522 can significantly inhibit the proliferation, migration, and invasion of breast malignant phyllodes tumor cells in vitro, and can significantly inhibit the growth of tumors in vivo.
- CADD522 has great potential clinical application value in the development of targeted therapeutic drugs for breast phyllodes tumors.
- Figure 1 A is a histogram of RUNX2 mRNA expression levels in benign and malignant breast phyllodes tumor tissues; B is a protein immunoblot gel imaging image of RUNX2 protein expression test in benign and malignant breast phyllodes tumor tissues; C is benign , RUNX2 expression levels in paraffin sections of malignant phyllodes tumors and RUNX2 expression levels in paraffin sections of multiple recurrences of tumor tissue from the same patient.
- Figure 2 A is a graphical representation of the test results of knocking down the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01 (i.e., the cell line HJP-0320, disclosed in the patent application with publication number CN111019898A) and changing the cell proliferation ability.
- SYSH-MPT-01 i.e., the cell line HJP-0320, disclosed in the patent application with publication number CN111019898A
- B knocks down the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01, and the results of the test on changes in cell colony formation ability
- C shows the knockdown of the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01, Graphical representation of cell cycle change test results
- D is a graphic representation of the knockdown of the RUNX2 gene in the breast malignant phyllodes tumor cell line SYSH-MPT-01, and the results of a cell migration and invasion ability change test
- E is a graphic representation of the knockdown of the breast malignant phyllodes tumor cell line Illustration of the test results of the RUNX2 gene of SYSH-MPT-01 and the changes in cell collagen contraction ability.
- FIG. 3 A is a graphical representation of the test results of changes in cell proliferation ability overexpressing RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01 (i.e., the cell line GLK-1010, disclosed in the patent application with publication number CN111019897A) ; B is a graphical representation of the test results of the overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01, and the changes in the clonogenic ability of the cells; C is the overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01 RUNX2, a graphical representation of the test results of cell cycle changes; D is a graphical representation of the test results of overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01, and the changes in cell migration and invasion capabilities; E is a graphic representation of the test results of the benign breast phyllodes tumor RUNX2 is overex
- Figure 4 is a graphical representation of the results of a trial on the impact of RUNX2 on the prognosis of patients with breast phyllodes tumors.
- Figure 5 is a graphical representation of the results of a test to knock down RUNX2 in malignant phyllodes tumor cells on subcutaneous tumor formation ability and tumor growth; A is the change in tumor volume at the corresponding time point, and B is a picture of the tumors in each group.
- FIG. 6 is a diagram showing the effect of CADD522 on the malignant phyllodes tumor cell lines SYSH-BPT-01 and SYSH-MPT-02 (i.e., the cell line LJ-0429, disclosed in the patent application with publication number CN111019899A);
- A is CADD522 The survival rate curve acting on the malignant phyllodes tumor cell line, illustrating the determination of the half inhibitory concentration (IC50) of CADD522 on the malignant phyllodes tumor cell line;
- B is the growth of the malignant phyllodes tumor cell line SYSH-MPT-01 inhibited by CADD522 , showing a dose- and time-dependent diagram;
- C is a diagram showing the effect of CADD522 on the migration and invasion ability of breast malignant phyllodes tumor cells.
- Figure 7 is a diagram showing the effect of CADD522 on malignant phyllodes tumors in vivo.
- A is the inhibition of tumor growth by intraperitoneal injection of CADD522 in the PDX model of malignant phyllodes tumors, and the changes in tumor volume at corresponding time points.
- B is the pictures of tumors in each group.
- Example 1 Analysis of RUNX 2 expression profile of breast phyllodes tumors
- RUNX2-specific primers to perform fluorescence quantitative PCR to detect the expression levels of RUNX2 mRNA in different types of breast phyllodes tumor tissues.
- Reverse transcription to synthesize cDNA Reverse transcription to synthesize cDNA: Add 1 ⁇ g of template RNA and 4 ⁇ l of reverse transcriptase SuperScript II mix (containing Buffer, dNTP, HiScript II reverse transcriptase, RNase, Random primers/Oligo dT) in the PCR tube.
- Real-time quantitative PCR amplification Dilute the template cDNA 3 times and mix well for later use. Each experimental group was set up with 3 parallel tubes. Reaction system: 1 ⁇ l cDNA, 5 ⁇ l SYBR green dye, 0.3 ⁇ l forward primer, 0.3 ⁇ l reverse primer, 3.4 ⁇ l ddH 2 O, centrifuge and mix. Reaction conditions: 95°C for 5min, 95°C for 30 seconds, 55°C-60°C (depending on the annealing temperature) for 30s, a total of 40 cycles. GAPDH was used as an internal reference to analyze the relative transcription levels of genes. The primer sequences are as follows:
- Example 2 Effects of knocking down RUNX2 on the proliferation, migration, invasion, cell cycle and collagen contraction ability of malignant phyllodes tumor cells
- siRNA The sequence of siRNA is as follows:
- Example 6 Effect of RUNX2 small molecule inhibitor CADD522 on IC50, proliferation, migration and invasion of malignant phyllodes tumor cells
- the RUNX2 small molecule inhibitor CADD522 inhibits the migration and invasion of malignant phyllodes tumor cells
- the RUNX2 small molecule inhibitor CADD522 inhibits PDX tumor growth
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Abstract
Use of RUNX2 as a marker and a treatment target in screening for and preparing a medicament for combating malignant phyllodes tumors of the breast, and use of the RUNX2 inhibitor CAD522 in preparing a medicament for combating malignant phyllodes tumors of the breast. It has been disclosed that the overexpression of RUNX2 is related to the biological behaviors of malignant phyllodes tumors of the breast, and knocking down RUNX2 expression can inhibit the proliferation, migration, and invasion of malignant phyllodes tumors of the breast. Experimental evidence has shown that the RUNX2-specific small-molecule inhibitor CAD522 can inhibit the proliferation, migration, and invasion of malignant phyllodes tumor cells of the breast in vitro and can inhibit tumor growth in vivo.
Description
本发明涉及医药生物技术领域，具体涉及RUNX2抑制剂在制备抗乳腺恶性叶状肿瘤药物中的应用The invention relates to the field of medical biotechnology, and specifically relates to the application of RUNX2 inhibitors in the preparation of drugs against malignant phyllodes tumors of the breast.
乳腺叶状肿瘤(Phyllodes tumor,PT)是一种罕见的纤维上皮性肿瘤，占所有乳腺肿瘤的0.3％至1％。根据现有的文献研究，交界性、恶性叶状肿瘤不仅生长快，而且极易局部复发和远处转移，血液转移是最常见的转移方式，肺、骨是最常见的转移部位。乳腺恶性叶状肿瘤的复发率高达53.1％，转移率高达43.1％。一旦出现复发、转移，患者会在短期内死亡，恶性叶状肿瘤的死亡率约为16.3％。乳腺叶状肿瘤的治疗主要是手术治疗，恶性叶状肿瘤由于其具有高复发率、高转移率等特点，其临床预后差。不同于乳腺癌，乳腺恶性叶状肿瘤术后的辅助治疗手段如化疗、放疗等治疗效果均不佳，靶向治疗及免疫治疗目前也无研究报道具有临床获益。且由于乳腺叶状肿瘤较为罕见，缺乏大规模的临床数据与基础研究，特别在特异性分子标志物和治疗靶点方面的研究更为匮乏，一直没有取得有效的进展，进一步造成在寻求或筛选有效治疗药物方面停滞不前。Phyllodes tumor (PT) is a rare fibroepithelial tumor, accounting for 0.3% to 1% of all breast tumors. According to existing literature research, borderline and malignant phyllodes tumors not only grow rapidly, but are also prone to local recurrence and distant metastasis. Blood metastasis is the most common way of metastasis, and lung and bone are the most common metastasis sites. The recurrence rate of malignant phyllodes tumors of the breast is as high as 53.1%, and the metastasis rate is as high as 43.1%. Once recurrence and metastasis occur, the patient will die within a short period of time. The mortality rate of malignant phyllodes tumors is approximately 16.3%. The treatment of breast phyllodes tumors is mainly surgical. Malignant phyllodes tumors have poor clinical prognosis due to their high recurrence rate and high metastasis rate. Unlike breast cancer, adjuvant treatments such as chemotherapy and radiotherapy are not effective after surgery for malignant phyllodes tumors of the breast. There are currently no studies reporting clinical benefit from targeted therapy and immunotherapy. Moreover, because phyllodes tumors of the breast are relatively rare, there is a lack of large-scale clinical data and basic research, especially research on specific molecular markers and treatment targets, and no effective progress has been made, further resulting in the lack of research or screening. Progress in effective treatments has stalled.
RUNX2是转录因子RUNX家族的重要成员，其因含有runt结构域而被命名。RUNX2的生物学作用主要是作为成骨分化的特异性转录因子，调控一型胶原、骨调素、骨钙蛋白、col1a1、col1a2、骨涎蛋白(BSP)和纤维连接蛋白等基因的转录，在成骨细胞形成和分化、软骨细胞分化和成熟、破骨细胞的形成和吸收及骨基质蛋白的合成中起到重要作用。但未见有RUNX2与乳腺恶性叶状肿瘤生物学行为相关的报道。RUNX2 is an important member of the RUNX family of transcription factors and is named because it contains a runt domain. The biological role of RUNX2 is mainly as a specific transcription factor for osteogenic differentiation, regulating the transcription of genes such as type I collagen, osteomodulin, osteocalcin, col1a1, col1a2, bone sialoprotein (BSP) and fibronectin. It plays an important role in the formation and differentiation of osteoblasts, differentiation and maturation of chondrocytes, the formation and resorption of osteoclasts, and the synthesis of bone matrix proteins. However, there are no reports that RUNX2 is related to the biological behavior of malignant phyllodes tumors of the breast.
发明内容Contents of the invention
为克服以上技术问题，本发明公开了RUNX2作为乳腺恶性叶状肿瘤生物学行为标志物及药物靶点相关的技术应用。In order to overcome the above technical problems, the present invention discloses the technical application of RUNX2 as a biological behavioral marker and drug target of malignant phyllodes tumors of the breast.
发明人在临床样本和体外细胞实验中中发现，RUNX2在乳腺恶性叶状肿瘤细胞中高表达，促进乳腺恶性叶状肿瘤的进展。敲低RUNX2的表达可以抑制乳腺恶性叶状肿瘤的增殖、迁移、侵袭。RUNX2可作为乳腺恶性叶状肿瘤标志物及药物 靶点，在乳腺叶状肿瘤辅助诊断，筛选和制备靶向治疗药物开发中具有较高的应用价值。由此公开了以下应用：The inventors found in clinical samples and in vitro cell experiments that RUNX2 is highly expressed in malignant breast phyllodes tumor cells and promotes the progression of malignant breast phyllodes tumors. Knocking down the expression of RUNX2 can inhibit the proliferation, migration, and invasion of malignant phyllodes tumors of the breast. RUNX2 can be used as a marker and drug target for malignant phyllodes tumors of the breast, and has high application value in the auxiliary diagnosis, screening and preparation of targeted therapeutic drugs for breast phyllodes tumors. The following applications are thus exposed:
1.RUNX2作为标志物在筛选或制备抗乳腺恶性叶状肿瘤药物中的应用。在筛选乳腺恶性叶状肿瘤药物中，可通过检测试验肿瘤细胞的RUNX2表达作为其中的评价药效手段。1. The application of RUNX2 as a marker in screening or preparing drugs against malignant breast phyllodes tumors. In screening drugs for malignant phyllodes tumors of the breast, detection of RUNX2 expression in test tumor cells can be used as a means to evaluate drug efficacy.
2.RUNX2作为药物靶点在筛选或制备抗乳腺恶性叶状肿瘤药物中的应用。可优先筛选针对RUNX2作为治疗靶点的药物作为潜在的抗乳腺恶性叶状肿瘤药物。2. The application of RUNX2 as a drug target in screening or preparing drugs against malignant phyllodes tumors of the breast. Drugs targeting RUNX2 as a therapeutic target can be prioritized for screening as potential drugs against breast malignant phyllodes tumors.
例如RUNX2抑制剂在制备抗乳腺恶性叶状肿瘤药物中的应用。For example, the application of RUNX2 inhibitors in the preparation of drugs against malignant phyllodes tumors of the breast.
药物CADD522，具有抑制RUNX2与DNA结合的作用。本发明进一步公开RUNX2抑制剂CADD522在制备抗乳腺恶性叶状肿瘤药物中的应用。The drug CADD522 inhibits the binding of RUNX2 to DNA. The invention further discloses the application of the RUNX2 inhibitor CADD522 in the preparation of drugs against malignant phyllodes tumors of the breast.
与现有技术相比，本发明具有如下的有益效果：Compared with the prior art, the present invention has the following beneficial effects:
1.本发明首次公开乳腺恶性叶状肿瘤标本RUNX2的过表达与乳腺恶性叶状肿瘤的生物学行为显著相关，敲低RUNX2的表达可以抑制乳腺恶性叶状肿瘤的增殖、迁移、侵袭。认为RUNX2可作为筛选抗乳腺恶性叶状肿瘤药物的标志物和治疗靶点，在筛选和制备靶向抗乳腺恶性叶状肿瘤药物方面均具有较高应用价值。1. The present invention discloses for the first time that the overexpression of RUNX2 in breast malignant phyllodes tumor specimens is significantly related to the biological behavior of breast malignant phyllodes tumors. Knocking down the expression of RUNX2 can inhibit the proliferation, migration, and invasion of breast malignant phyllodes tumors. It is believed that RUNX2 can be used as a marker and therapeutic target for screening drugs against malignant phyllodes tumors of the breast, and has high application value in the screening and preparation of drugs targeting malignant phyllodes tumors of the breast.
2.经试验证明，RUNX2特异性小分子抑制剂CADD522在体外可以显著抑制乳腺恶性叶状肿瘤细胞的增殖、迁移、侵袭，在体内可以显著抑制肿瘤的生长。CADD522在乳腺叶状肿瘤靶向治疗药物开发中具有很好潜在临床应用价值。2. Experiments have proven that the RUNX2-specific small molecule inhibitor CADD522 can significantly inhibit the proliferation, migration, and invasion of breast malignant phyllodes tumor cells in vitro, and can significantly inhibit the growth of tumors in vivo. CADD522 has great potential clinical application value in the development of targeted therapeutic drugs for breast phyllodes tumors.
图1:A是良性和恶性乳腺叶状肿瘤组织中RUNX2的mRNA表达水平的柱状图；B是良性和恶性乳腺叶状肿瘤组织中RUNX2蛋白表达试验的蛋白免疫印迹凝胶成像图；C是良、恶性叶状肿瘤石蜡切片中的RUNX2表达水平及同一患者多次复发的肿瘤组织石蜡切片中RUNX2表达水平情况图示。Figure 1: A is a histogram of RUNX2 mRNA expression levels in benign and malignant breast phyllodes tumor tissues; B is a protein immunoblot gel imaging image of RUNX2 protein expression test in benign and malignant breast phyllodes tumor tissues; C is benign , RUNX2 expression levels in paraffin sections of malignant phyllodes tumors and RUNX2 expression levels in paraffin sections of multiple recurrences of tumor tissue from the same patient.
图2：A是敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01(即细胞系HJP-0320,公开于公布号为CN111019898A的专利申请中)的RUNX2基因，细胞增殖能力改变试验结果图示；B敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2基因，细胞克隆形成能力改变试验结果图示；C是敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2基因，细胞周期改变试验结果图示；D是敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2基因，细胞迁移和侵袭能力改变试验结果图 示；E是敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2基因，细胞胶原收缩能力改变试验结果图示。Figure 2: A is a graphical representation of the test results of knocking down the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01 (i.e., the cell line HJP-0320, disclosed in the patent application with publication number CN111019898A) and changing the cell proliferation ability. ; B knocks down the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01, and the results of the test on changes in cell colony formation ability; C shows the knockdown of the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01, Graphical representation of cell cycle change test results; D is a graphic representation of the knockdown of the RUNX2 gene in the breast malignant phyllodes tumor cell line SYSH-MPT-01, and the results of a cell migration and invasion ability change test; E is a graphic representation of the knockdown of the breast malignant phyllodes tumor cell line Illustration of the test results of the RUNX2 gene of SYSH-MPT-01 and the changes in cell collagen contraction ability.
图3：A是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01(即细胞系GLK-1010,公开于公布号为CN111019897A的专利申请中)中过表达RUNX2，细胞增殖能力改变试验结果图示；B是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01中过表达RUNX2，细胞的克隆形成能力改变试验结果图示；C是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01中过表达RUNX2，细胞周期的改变试验结果图示；D是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01中过表达RUNX2，细胞迁移及侵袭能力改变试验结果图示；E是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01中过表达RUNX2，细胞胶原收缩能力改变试验结果图示。Figure 3: A is a graphical representation of the test results of changes in cell proliferation ability overexpressing RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01 (i.e., the cell line GLK-1010, disclosed in the patent application with publication number CN111019897A) ; B is a graphical representation of the test results of the overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01, and the changes in the clonogenic ability of the cells; C is the overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01 RUNX2, a graphical representation of the test results of cell cycle changes; D is a graphical representation of the test results of overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01, and the changes in cell migration and invasion capabilities; E is a graphic representation of the test results of the benign breast phyllodes tumor RUNX2 is overexpressed in the cell line SYSH-BPT-01, and the results of the test on changes in the collagen contraction ability of the cells are shown graphically.
图4是RUNX2对乳腺叶状肿瘤患者预后影响试验结果图示。Figure 4 is a graphical representation of the results of a trial on the impact of RUNX2 on the prognosis of patients with breast phyllodes tumors.
图5是是敲降恶性叶状肿瘤细胞的RUNX2，对皮下成瘤能力及肿瘤生长影响试验结果图示；A是相应时间点肿瘤体积的变化情况,B是各组肿瘤图片。Figure 5 is a graphical representation of the results of a test to knock down RUNX2 in malignant phyllodes tumor cells on subcutaneous tumor formation ability and tumor growth; A is the change in tumor volume at the corresponding time point, and B is a picture of the tumors in each group.
图6是CADD522对恶性叶状肿瘤细胞系SYSH-BPT-01，SYSH-MPT-02(即细胞系LJ-0429,公开于公布号为CN111019899A的专利申请中)作用试验效果图示；A是CADD522作用于恶性叶状肿瘤细胞系的存活率曲线，确定CADD522对恶性叶状肿瘤细胞系的半数抑制浓度(IC50)的图示；B是CADD522抑制恶性叶状肿瘤细胞系SYSH-MPT-01的生长，呈剂量和时间依赖性图示；C是CADD522对乳腺恶性叶状肿瘤细胞的迁移、侵袭能力影响图示。Figure 6 is a diagram showing the effect of CADD522 on the malignant phyllodes tumor cell lines SYSH-BPT-01 and SYSH-MPT-02 (i.e., the cell line LJ-0429, disclosed in the patent application with publication number CN111019899A); A is CADD522 The survival rate curve acting on the malignant phyllodes tumor cell line, illustrating the determination of the half inhibitory concentration (IC50) of CADD522 on the malignant phyllodes tumor cell line; B is the growth of the malignant phyllodes tumor cell line SYSH-MPT-01 inhibited by CADD522 , showing a dose- and time-dependent diagram; C is a diagram showing the effect of CADD522 on the migration and invasion ability of breast malignant phyllodes tumor cells.
图7是CADD522对体内恶性叶状肿瘤试验效果图示。A是在恶性叶状肿瘤PDX模型中，腹腔注射CADD522对肿瘤生长的抑制，相应时间点肿瘤体积的变化情况图示,B是各组肿瘤图片。Figure 7 is a diagram showing the effect of CADD522 on malignant phyllodes tumors in vivo. A is the inhibition of tumor growth by intraperitoneal injection of CADD522 in the PDX model of malignant phyllodes tumors, and the changes in tumor volume at corresponding time points. B is the pictures of tumors in each group.
下面结合实施例对本发明作进一步的说明。The present invention will be further described below in conjunction with the examples.
实施例1：乳腺叶状肿瘤RUNX 2表达谱分析Example 1: Analysis of RUNX 2 expression profile of breast phyllodes tumors
1.采用RUNX2特异性引物，进行荧光定量PCR，检测不同类型乳腺叶状肿瘤组织中RUNX2mRNA表达水平。1. Use RUNX2-specific primers to perform fluorescence quantitative PCR to detect the expression levels of RUNX2 mRNA in different types of breast phyllodes tumor tissues.
(1)分别选取3例良性叶状肿瘤组织标本、10例恶性叶状肿瘤组织标本。按以下步骤进行实验：将组织用冷冻研磨机研碎，加入Trizol裂解液，用移液器 将裂解物转移至1.5mlEP管中，反复吹打或振荡以裂解细胞。室温静置5min，按每1ml Trizol裂解液加入0.2ml氯仿，用力振荡15s，室温静置2-3min后，12000x g 4℃离心15min，取上层水相至新EP管中，加入等体积异丙醇沉淀RNA；12000x g 4℃离心10min；加入75％乙醇清洗，7500x g 4℃离心5min，弃上清；室温下超静台内晾干RNA沉淀，溶解于适量Rnase-free水中，测定RNA浓度及纯度。反转录合成cDNA：反转录合成cDNA：在PCR管中加入模板RNA 1μg，4μl反转录酶SuperScript II mix(含Buffer、dNTP、HiScript II逆转录酶、RNA酶、Random primers/Oligo dT)，补充RNase-free水至20μl，65℃ 5min，冰上放置5min后加入5×buffer 8μl及0.1M DTT 2μl，混匀后补加ddH2O至40μl，反应条件：50℃ 15min，85℃ 15s，4℃保存。(1) Select 3 benign phyllodes tumor tissue samples and 10 malignant phyllodes tumor tissue samples respectively. Carry out the experiment according to the following steps: Grind the tissue with a freezing grinder, add Trizol lysis solution, transfer the lysate to a 1.5ml EP tube with a pipette, and pipe or shake repeatedly to lyse the cells. Let stand at room temperature for 5 minutes. Add 0.2ml of chloroform per 1ml of Trizol lysis solution. Shake vigorously for 15 seconds. After standing at room temperature for 2-3 minutes, centrifuge at 12000xg and 4℃ for 15 minutes. Take the upper aqueous phase and put it into a new EP tube. Add an equal volume of isopropyl. Alcohol precipitate RNA; centrifuge at 12000xg 4℃ for 10min; add 75% ethanol to wash, centrifuge at 7500xg 4℃ for 5min, discard the supernatant; dry the RNA precipitate in an ultra-quiet stage at room temperature, dissolve it in an appropriate amount of RNase-free water, and measure the RNA concentration. and purity. Reverse transcription to synthesize cDNA: Reverse transcription to synthesize cDNA: Add 1μg of template RNA and 4μl of reverse transcriptase SuperScript II mix (containing Buffer, dNTP, HiScript II reverse transcriptase, RNase, Random primers/Oligo dT) in the PCR tube. , add RNase-free water to 20μl, 65℃ for 5min, place on ice for 5min, then add 5×buffer 8μl and 0.1M DTT 2μl, mix and add ddH2O to 40μl, reaction conditions: 50℃ 15min, 85℃ 15s, 4 Store at ℃.
实时定量PCR扩增：将模板cDNA稀释3倍并混匀备用。每个实验组设置3个平行管。反应体系：cDNA 1μl，SYBR green染料5μl，正向引物0.3μl，反向引物0.3μl，ddH
2O 3.4μl，离心混匀。反应条件：95℃ 5min，95℃ 30秒，55℃-60℃(视退火温度而定)30s，共40个循环。以GAPDH作为内参分析检测基因的相对转录水平。引物序列如下：
Real-time quantitative PCR amplification: Dilute the template cDNA 3 times and mix well for later use. Each experimental group was set up with 3 parallel tubes. Reaction system: 1μl cDNA, 5μl SYBR green dye, 0.3μl forward primer, 0.3μl reverse primer, 3.4μl ddH 2 O, centrifuge and mix. Reaction conditions: 95℃ for 5min, 95℃ for 30 seconds, 55℃-60℃ (depending on the annealing temperature) for 30s, a total of 40 cycles. GAPDH was used as an internal reference to analyze the relative transcription levels of genes. The primer sequences are as follows:
RUNX2 Forward：5’-CGCCTCACAAACAACCACAG-3’(SEQ ID NO.1)RUNX2 Forward: 5’-CGCCTCACAAAACAACCACAG-3’(SEQ ID NO.1)
RUNX2 Reverse：5’-TCACTGTGCTGAAGAGGCTG-3’(SEQ ID NO.2)RUNX2 Reverse：5’-TCACTGTGCTGAAGAGGCTG-3’(SEQ ID NO.2)
定量分析mRNA相对表达水平。统计数据根据三次重复实验结果计算平均值，显著性差异由T-检验来判断，以P<0.05定义为有统计学差异。Quantitative analysis of relative mRNA expression levels. The statistical data were averaged based on the results of three repeated experiments, and significant differences were judged by T-test, with P<0.05 defined as statistical differences.
(2)实验结果：如图1(A)所示，RUNX2在良性叶状肿瘤组织中低表达，在恶性叶状肿瘤组织高表达，恶性中RUNX2mRNA的表达水平约为良性叶状肿瘤组织的25倍。(2) Experimental results: As shown in Figure 1(A), RUNX2 is lowly expressed in benign phyllodes tumor tissues and highly expressed in malignant phyllodes tumor tissues. The expression level of RUNX2 mRNA in malignant phyllodes tumor tissues is approximately 25% of that in benign phyllodes tumor tissues. times.
2.采用蛋白免疫印迹(Western blot)实验检测不同类型叶状肿瘤组织中RUNX2蛋白表达水平。2. Use Western blot experiment to detect RUNX2 protein expression levels in different types of phyllodes tumor tissues.
(1)实验方法：SDS-PAGE电泳、转膜、封闭之后，采用RUNX2特异性抗体作为一抗孵育，进一步采用辣根过氧化物酶标二抗孵育之后进行凝胶成像分析。(1) Experimental method: After SDS-PAGE electrophoresis, transfer, and blocking, RUNX2-specific antibody was used as the primary antibody to incubate, and horseradish peroxidase-labeled secondary antibody was further incubated for gel imaging analysis.
(2)实验结果:如图(1)B显示，10例恶性叶状肿瘤组织中RUNX2蛋白表达量较高，而在3例良性叶状肿瘤组织中RUNX2蛋白不表达或低表达。(2) Experimental results: As shown in Figure (1) B, RUNX2 protein expression was high in 10 cases of malignant phyllodes tumor tissues, while RUNX2 protein was not expressed or had low expression in 3 cases of benign phyllodes tumor tissues.
3.使用RUNX2的特异性抗体，进行免疫组织化学染色，评估良、乳腺恶性叶 状肿瘤组织中RUNX2蛋白水平。3. Use RUNX2-specific antibodies to perform immunohistochemical staining to evaluate RUNX2 protein levels in benign and malignant breast phyllodes tumor tissues.
(1)实验方法：实验所采用病理切片均来自于临床确诊病例。按以下步骤进行石蜡包埋组织免疫组化染色：60℃烘烤处理，入预热二甲苯脱蜡两次，每次5min；脱蜡后的切片经100％-95％-80％-70％-50％乙醇梯度及蒸馏水水化，每个梯度放置5min；0.01M(pH6.0)柠檬酸缓冲液高压热修复10min，自然冷却后PBS洗三次，每次5min；入0.3％过氧化氢溶液处理30min，消除内源性过氧化物酶活性；PBS洗三次，每次5min；10％羊血清37℃封闭1h；滴加羊血清稀释一抗工作液(AA4)，4℃孵育过夜，PBS清洗；加入生物素标记二抗工作液，37℃孵育20min，PBS清洗三次；加入辣根过氧化物酶标记链霉亲和素，37℃孵育20min，PBS清洗三次；滴加DAB显色液，室温避光显色2min，PBS洗去多余显色液；苏木素复染，蒸馏水清洗；50％-70％-80％-90％-100％-100％乙醇梯度逐级脱水，每个梯度5min；中性树脂封片；显微成像系统拍片。(1) Experimental methods: The pathological sections used in the experiment are all from clinically confirmed cases. Follow the following steps to perform immunohistochemical staining of paraffin-embedded tissue: bake at 60°C, dewax in preheated xylene twice, 5 minutes each time; dewax sections after 100%-95%-80%-70% -Hydrate with 50% ethanol gradient and distilled water, leave each gradient for 5 minutes; high-pressure heat repair with 0.01M (pH6.0) citric acid buffer for 10 minutes, wash with PBS three times, 5 minutes each time after natural cooling; add 0.3% hydrogen peroxide solution Treat for 30 minutes to eliminate endogenous peroxidase activity; wash three times with PBS, 5 minutes each time; block with 10% sheep serum at 37°C for 1 hour; add sheep serum to dilute the primary antibody working solution (AA4), incubate at 4°C overnight, and wash with PBS ; Add biotin-labeled secondary antibody working solution, incubate at 37°C for 20 minutes, and wash three times with PBS; add horseradish peroxidase-labeled streptavidin, incubate at 37°C for 20 minutes, and wash three times with PBS; add DAB chromogenic solution dropwise, and wash at room temperature. Protect from light and develop color for 2 minutes, wash away excess chromogenic solution with PBS; counterstain with hematoxylin and wash with distilled water; dehydrate step by step with 50%-70%-80%-90%-100%-100% ethanol gradient, each gradient is 5 minutes; medium The slides were sealed with plastic resin and filmed using a microscopic imaging system.
(2)实验结果:如图1(C)所示：RUNX2在乳腺恶性叶状肿瘤细胞中高表达，在良性中低表达(C)。随着肿瘤复发次数的增加，RUNX2在组织中的表达量也同步增加(C)。(2) Experimental results: As shown in Figure 1(C): RUNX2 is highly expressed in malignant breast phyllodes tumor cells and low in benign cells (C). As the number of tumor recurrences increases, the expression of RUNX2 in tissues also increases simultaneously (C).
实施例2：敲低RUNX2对恶性叶状肿瘤细胞增殖、迁移、侵袭、细胞周期及胶原收缩能力的影响Example 2: Effects of knocking down RUNX2 on the proliferation, migration, invasion, cell cycle and collagen contraction ability of malignant phyllodes tumor cells
1.敲低RUNX2对恶性叶状肿瘤细胞增殖的影响1. Effect of knocking down RUNX2 on the proliferation of malignant phyllodes tumor cells
(1)实验方法：接种恶性叶状肿瘤细胞于96孔板和6孔板中，采用1ipo3000瞬时转染siRNA，至乳腺恶性叶状肿瘤细胞中，敲低RUNX2的表达接种敲低RUNX2表达及未进行基因敲低的对照组细胞，采用CCK8法，测试不同时间点(1-4天)细胞存活率，绘制细胞增殖曲线。接种敲低RUNX2表达及未进行基因敲低的对照组细胞至60mm中皿的完全培养基中，每皿200个细胞，培养14天后，计数克隆形成数量。(1) Experimental method: Inoculate malignant phyllodes tumor cells in 96-well plates and 6-well plates, use 1ipo3000 to transiently transfect siRNA into breast malignant phyllodes tumor cells, knock down the expression of RUNX2 and inoculate to knock down the expression of RUNX2 and not For the cells in the control group that underwent gene knockdown, the CCK8 method was used to test the cell survival rate at different time points (1-4 days) and draw the cell proliferation curve. Cells from the control group with knockdown of RUNX2 expression and no gene knockdown were inoculated into complete culture medium in a 60 mm medium dish, with 200 cells per dish. After 14 days of culture, the number of clones formed was counted.
siRNA的序列如下：The sequence of siRNA is as follows:
sense:GGACGAGGCAAGAGTTTCA,(SEQ ID NO.3)sense:GGACGAGGCAAGAGTTTCA,(SEQ ID NO.3)
antisense:CCAAATTTGCCTAACCAGA(SEQ ID NO.4)antisense:CCAAATTTGCCTAACCAGA(SEQ ID NO.4)
(2)实验结果:如图2(A-B)所示。(2) Experimental results: As shown in Figure 2(A-B).
2.敲低RUNX2对恶性叶状肿瘤细胞周期的影响2. Effects of knocking down RUNX2 on the cell cycle of malignant phyllodes tumors
(1)实验方法：接种敲低表达及对照细胞，培养24h后，收集细胞，用预冷的PBS洗细胞2-3次，离心(1500rpm，4min)弃掉上清，在沉淀中加入少量PBS，重悬细胞，再将重悬细胞加入到4℃预冷的70％冰乙醇中固定，封口膜封口，4℃过夜。PBS洗两次离心去上清液(2000rpm 4min)。加入100ul 100ug/ml RNase A和0.2％Triton X-100重悬细胞。加入400ul 50ug/ml PI，涡旋振荡混匀，室温避光孵育30min。流式细胞仪检测细胞周期，一般计数10万个细胞，在激发波长488nm波长处检测红色荧光，而后用FlowJo软件分析细胞周期时相分布，分析时，使用FL2-w和FL2-A显示，去除粘连在一起的细胞。(1) Experimental method: Inoculate knockdown expression and control cells, culture for 24 hours, collect the cells, wash the cells 2-3 times with pre-cooled PBS, centrifuge (1500rpm, 4min), discard the supernatant, and add a small amount of PBS to the sediment. , resuspend the cells, then add the resuspended cells to 70% ice-cold ethanol pre-cooled at 4°C to fix, seal with parafilm, and leave at 4°C overnight. Wash twice with PBS and centrifuge to remove the supernatant (2000rpm 4min). Add 100ul 100ug/ml RNase A and 0.2% Triton X-100 to resuspend the cells. Add 400ul 50ug/ml PI, vortex to mix, and incubate at room temperature in the dark for 30 minutes. Flow cytometry detects the cell cycle. Generally, 100,000 cells are counted. Red fluorescence is detected at the excitation wavelength of 488nm. Then FlowJo software is used to analyze the cell cycle phase distribution. During analysis, FL2-w and FL2-A are used to display and remove Cells that stick together.
(2)实验结果：如图2(C)敲低RUNX2后的细胞阻滞在G1期。(2) Experimental results: As shown in Figure 2(C), cells after knocking down RUNX2 were arrested in the G1 phase.
3.敲低RUNX2对恶性叶状肿瘤细胞迁移、侵袭能力的影响3. Effects of knocking down RUNX2 on the migration and invasion abilities of malignant phyllodes tumor cells
(1)实验方法：将敲低RUNX2表达及对照组细胞以2x10
4/孔的密度接种于Transwell板(Costar)上层小室无血清培养基中或预铺Matrigel薄层上层培养室中，下层为含16％胎牛血清的完全培养基，每组设置三个重复孔；37℃培养箱分别培养8小时或24小时后，用4％多聚甲醛固定15min，小心擦去膜上层细胞，利用0.5％结晶紫对下室细胞进行染色，显微镜下计数发生迁移或侵袭的细胞数目。
(1) Experimental method: cells with knockdown RUNX2 expression and control group were seeded at a density of 2x10 4 /well in serum-free culture medium in the upper chamber of Transwell plate (Costar) or in a pre-paved Matrigel thin layer upper culture chamber, and the lower layer was containing Complete culture medium with 16% fetal bovine serum, each group has three replicate wells; after culturing in a 37°C incubator for 8 hours or 24 hours, fix it with 4% paraformaldehyde for 15 minutes, carefully wipe off the cells on the upper layer of the membrane, and use 0.5% Crystal violet stains the cells in the lower chamber, and the number of cells that migrate or invade is counted under a microscope.
(2)实验结果；如图2(D)显示，RUNX2敲低表达组细胞迁移及侵袭能力显著下降。(2) Experimental results; Figure 2(D) shows that the cell migration and invasion abilities in the RUNX2 knockdown expression group were significantly reduced.
4.敲低RUNX2对恶性叶状肿瘤胶原收缩能力的影响4. Effect of knocking down RUNX2 on collagen shrinkage ability of malignant phyllodes tumors
(1)实验方法：用含乙酸和NaOH的DMEM培养基稀释Ⅰ型鼠尾胶原，叶状肿瘤细胞悬液与稀释后的鼠尾胶原1:1混合后，接种到24孔板中，加入含血清的培养基4小时后，换上无血清DMEM培养基孵育过夜，使胶与孔壁分离，8小时后观察、测量收缩后的直径。(1) Experimental method: Dilute type I rat tail collagen with DMEM medium containing acetic acid and NaOH. Mix the phyllodes tumor cell suspension with the diluted rat tail collagen at a ratio of 1:1 and then inoculate it into a 24-well plate. After 4 hours of serum culture medium, replace with serum-free DMEM medium and incubate overnight to separate the gel from the well wall. Observe and measure the diameter after shrinkage after 8 hours.
(2)实验结果如图2(E)所示。(2) The experimental results are shown in Figure 2(E).
实施例3.过表达RUNX2对叶状肿瘤细胞增殖、迁移、侵袭、细胞周期及胶原收缩能力的影响Example 3. Effects of overexpression of RUNX2 on proliferation, migration, invasion, cell cycle and collagen contraction ability of phyllodes tumor cells
1.过表达RUNX2对良性乳腺叶状肿瘤细胞增殖的影响1. Effects of overexpression of RUNX2 on the proliferation of benign breast phyllodes tumor cells
(1)实验方法:接种原代良性叶状肿瘤细胞至96孔板和6孔板中，使用转染试剂lipo3000和p3000转染RUNX2过表达质粒(4μg/孔)，转染4-6小时后更 换正常完全培养基，收集细胞提取RNA样品，检测RUNX2过表达水平。接种过表达RUNX2及未进行处理的对照组细胞，采用CCK8法，测试不同时间点(1-4天)细胞存活率，绘制细胞增殖曲线。接种过表达RUNX2及未进行处理的对照组细胞至60mm中皿的完全培养基中，每皿200个细胞，培养14天后，计数克隆形成数量。(1) Experimental method: Inoculate primary benign phyllodes tumor cells into 96-well plates and 6-well plates, use transfection reagents lipo3000 and p3000 to transfect RUNX2 overexpression plasmid (4μg/well), 4-6 hours after transfection The normal complete medium was replaced, cells were collected to extract RNA samples, and the overexpression level of RUNX2 was detected. Cells overexpressing RUNX2 and untreated control cells were inoculated, and the CCK8 method was used to test cell survival rates at different time points (1-4 days) and draw cell proliferation curves. Cells overexpressing RUNX2 and untreated control cells were inoculated into complete culture medium in a 60 mm medium dish, with 200 cells per dish. After 14 days of culture, the number of colonies formed was counted.
(2)实验结果如图3(A-B)所示。(2) The experimental results are shown in Figure 3(A-B).
2.过表达RUNX2对良性叶状肿瘤细胞周期的影响2. Effects of overexpression of RUNX2 on the cell cycle of benign phyllodes tumors
(1)实验方法：接种过表达及对照细胞，培养24h后，收集细胞，用预冷的PBS洗细胞2-3次，离心(1500rpm，4min)弃掉上清，在沉淀中加入少量PBS，重悬细胞，再将重悬细胞加入到4℃预冷的70％冰乙醇中固定，封口膜封口，4℃过夜。PBS洗两次离心去上清液(2000rpm 4min)。加入100ul 100ug/ml RNase A和0.2％Triton X-100重悬细胞。加入400ul 50ug/ml PI，涡旋振荡混匀，室温避光孵育30min。流式细胞仪检测细胞周期，一般计数10万个细胞，在激发波长488nm波长处检测红色荧光，而后用FlowJo软件分析细胞周期时相分布，分析时，使用FL2-w和FL2-A显示，去除粘连在一起的细胞。(1) Experimental method: Inoculate overexpression and control cells, culture for 24 hours, collect the cells, wash the cells 2-3 times with pre-cooled PBS, centrifuge (1500rpm, 4min), discard the supernatant, and add a small amount of PBS to the sediment. Resuspend the cells, then add the resuspended cells to 70% ice-cold ethanol pre-cooled at 4°C to fix, seal with parafilm, and leave at 4°C overnight. Wash twice with PBS and centrifuge to remove the supernatant (2000rpm 4min). Add 100ul 100ug/ml RNase A and 0.2% Triton X-100 to resuspend the cells. Add 400ul 50ug/ml PI, vortex to mix, and incubate at room temperature in the dark for 30 minutes. Flow cytometry detects the cell cycle. Generally, 100,000 cells are counted. Red fluorescence is detected at the excitation wavelength of 488nm. Then FlowJo software is used to analyze the cell cycle phase distribution. During analysis, FL2-w and FL2-A are used to display and remove Cells that stick together.
(2)实验结果：如图3(C)过RUNX2后的细胞活跃在S期。(2) Experimental results: As shown in Figure 3(C), the cells after passing RUNX2 are active in the S phase.
3.过表达RUNX2对乳腺良性叶状肿瘤细胞迁移和侵袭能力的影响3. Effects of overexpression of RUNX2 on the migration and invasion ability of benign breast phyllodes tumor cells
(1)实验方法：将过表达RUNX2及未进行处理的对照组细胞以1*10
4/孔的密度接种于Transwell板(Costar)上层小室无血清培养基中或预铺Matrigel薄层上层培养室中，下层为含16％胎牛血清的完全培养基，每组设置三个重复孔；37℃培养箱分别培养8小时或24小时后，小心擦去膜上层细胞，利用结晶紫对下室细胞进行染色，显微镜下计数发生迁移或侵袭的细胞数目。
(1) Experimental method: Inoculate overexpressed RUNX2 and untreated control cells at a density of 1*10 4 /well in serum-free medium in the upper chamber of Transwell plate (Costar) or pre-pave Matrigel thin layer in the upper culture chamber The middle and lower layers are complete culture medium containing 16% fetal bovine serum. Each group has three replicate wells. After culturing in a 37°C incubator for 8 hours or 24 hours, carefully wipe off the cells on the upper layer of the membrane and use crystal violet to clean the cells in the lower chamber. Stain and count the number of cells that migrate or invade under a microscope.
(2)实验结果如图3(D)所示。(2) The experimental results are shown in Figure 3(D).
4.过表达RUNX2对恶性叶状肿瘤胶原收缩能力的影响4. Effect of overexpression of RUNX2 on collagen shrinkage ability of malignant phyllodes tumors
(1)实验方法：用含乙酸和NaOH的DMEM培养基稀释Ⅰ型鼠尾胶原，叶状肿瘤细胞悬液与稀释后的鼠尾胶原1:1混合后，接种到24孔板中，加入含血清的培养基4小时后，换上无血清DMEM培养基孵育过夜，使胶与孔壁分离，8小时后观察、测量收缩后的直径。(1) Experimental method: Dilute type I rat tail collagen with DMEM medium containing acetic acid and NaOH. Mix the phyllodes tumor cell suspension with the diluted rat tail collagen at a ratio of 1:1 and then inoculate it into a 24-well plate. After 4 hours of serum culture medium, replace with serum-free DMEM medium and incubate overnight to separate the gel from the well wall. Observe and measure the diameter after shrinkage after 8 hours.
(2)实验结果如图3(E)所示。(2) The experimental results are shown in Figure 3(E).
实施例4.RUNX2对乳腺叶状肿瘤患者预后的影响Example 4. Effect of RUNX2 on the prognosis of patients with breast phyllodes tumors
1.RUNX2对乳腺叶状肿瘤患者预后的影响1. The impact of RUNX2 on the prognosis of patients with breast phyllodes tumors
(1)实验方法：取237例乳腺叶状肿瘤患者的新鲜冰冻切片，进行免疫组化染色，由两位病理科医生分别阅读切片，根据染色强度和染色阳性比例，分为RUNX2高表达组和RUNX2低表达组，评估RUNX2表达与总生存期OS的影响，无进展生存期DFS的影响。(1) Experimental method: Take fresh frozen sections of 237 patients with breast phyllodes tumors and perform immunohistochemical staining. Two pathologists will read the sections respectively and divide them into RUNX2 high expression group and RUNX2 high expression group according to the staining intensity and staining positive ratio. In the RUNX2 low expression group, evaluate the impact of RUNX2 expression on overall survival (OS) and progression-free survival (DFS).
(2)实验结果：如图4(A-B)所示，RUNX2高表达组的OS和DFS显著低于RUNX2低表达组。(2) Experimental results: As shown in Figure 4(A-B), the OS and DFS of the RUNX2 high-expression group were significantly lower than those of the RUNX2 low-expression group.
实施例5.敲低RUNX2基因影响小鼠体内肿瘤的生长Example 5. Knockdown of RUNX2 gene affects tumor growth in mice
1.敲低RUNX2基因抑制小鼠皮下移植瘤成瘤及生长的影响1. Effects of knocking down RUNX2 gene on inhibiting tumorigenesis and growth of subcutaneous transplanted tumors in mice
(1)实验方法：构建乳腺恶性叶状肿瘤皮下移植瘤模型：取恶性叶状肿瘤细胞、稳定敲降RUNX2的叶状肿瘤细胞，稀释至5x10
7/ml，取100μl混匀的细胞悬液，于对照组小鼠、实验组小鼠分别于左前肢腋窝部皮下乳房脂肪垫处接种，每4天测量一次肿瘤长径和短径，根据公式V＝1/2(L×W
2)计算移植瘤平均体积。待对照组肿瘤体积达到直径1.5cm时处死小鼠，并绘制肿瘤生长变化曲线。
(1) Experimental method: Construct a breast malignant phyllodes tumor subcutaneous transplant tumor model: Take malignant phyllodes tumor cells and phyllodes tumor cells with stable RUNX2 knockdown, dilute them to 5x10 7 /ml, take 100 μl of mixed cell suspension, Mice in the control group and mice in the experimental group were inoculated into the subcutaneous mammary fat pad of the left forelimb axilla respectively. The long and short diameters of the tumors were measured every 4 days, and the transplantation was calculated according to the formula V=1/2 (L×W 2 ). Average tumor volume. When the tumor volume in the control group reached 1.5 cm in diameter, the mice were sacrificed, and the tumor growth curve was drawn.
(2)实验结果：如图5(A-B)所示，与对照组相比，接种稳定敲降RUNX2叶状肿瘤细胞小鼠的肿瘤成瘤及生长明显被抑制。(2) Experimental results: As shown in Figure 5(A-B), compared with the control group, the tumor formation and growth of mice inoculated with stable knockdown of RUNX2 phyllodes tumor cells were significantly inhibited.
实施例6.RUNX2小分子抑制剂CADD522对恶性叶状肿瘤细胞的IC50、增殖、迁移、侵袭的影响Example 6. Effect of RUNX2 small molecule inhibitor CADD522 on IC50, proliferation, migration and invasion of malignant phyllodes tumor cells
1.RUNX2小分子抑制剂CADD522在恶性叶状肿瘤细胞中的IC501. IC50 of the RUNX2 small molecule inhibitor CADD522 in malignant phyllodes tumor cells
(1)实验方法：接种乳腺恶性叶状肿瘤细胞至96孔板，接种密度为5000个/孔，24h后加入RUNX2小分子抑制剂CADD522，设置浓度梯度为200μM、100μM、50μM、25μM、12.5μM、6.125μM、3.0625μM，培养72小时，采用CCK8试剂检测细胞活力，绘制细胞IC50曲线。(1) Experimental method: Inoculate breast malignant phyllodes tumor cells into a 96-well plate at a seeding density of 5000 cells/well. Add the RUNX2 small molecule inhibitor CADD522 24 hours later and set the concentration gradient to 200 μM, 100 μM, 50 μM, 25 μM, and 12.5 μM. , 6.125μM, 3.0625μM, culture for 72 hours, use CCK8 reagent to detect cell viability, and draw the cell IC50 curve.
(2)实验结果如图6(A)所示。(2) The experimental results are shown in Figure 6(A).
2.RUNX2小分子抑制剂CADD522对恶性叶状肿瘤细胞增殖的影响2. Effect of RUNX2 small molecule inhibitor CADD522 on the proliferation of malignant phyllodes tumor cells
(1)实验方法：接种乳腺恶性叶状肿瘤细胞至96孔板，接种密度为5000个/孔，24h后加入RUNX2小分子抑制剂CADD522，接种密度为3000个/孔，分别培养24、48、72、96小时，采用CCK8试剂检测细胞活力，绘制细胞增殖曲线。(1) Experimental method: Inoculate breast malignant phyllodes tumor cells into a 96-well plate at a seeding density of 5000 cells/well. After 24 hours, add the RUNX2 small molecule inhibitor CADD522 at a seeding density of 3000 cells/well and culture for 24, 48, and At 72 and 96 hours, CCK8 reagent was used to detect cell viability and draw a cell proliferation curve.
(2)实验结果如图6(B)所示。(2) The experimental results are shown in Figure 6(B).
2.RUNX2小分子抑制剂CADD522抑制恶性叶状肿瘤细胞迁移和侵袭2. The RUNX2 small molecule inhibitor CADD522 inhibits the migration and invasion of malignant phyllodes tumor cells
(1)实验方法：将恶性肿瘤细胞以1*104/孔的密度接种于Transwell板(Costar)上层小室无血清培养基中或预铺Matrigel薄层上层培养室中，下层为含16％胎牛血清的完全培养基，接种细胞的同时加入RUNX2小分子抑制剂CADD522共培养，每组设置三个重复孔；37℃培养箱分别培养8小时或24小时后，小心擦去膜上层细胞，利用结晶紫对下室细胞进行染色，显微镜下计数发生迁移或侵袭的细胞数目。(1) Experimental method: Inoculate malignant tumor cells at a density of 1*104/well in serum-free culture medium in the upper chamber of Transwell plate (Costar) or in a pre-paved Matrigel thin layer upper culture chamber, and the lower layer contains 16% fetal bovine Serum's complete culture medium was used to inoculate the cells and the RUNX2 small molecule inhibitor CADD522 was added for co-culture. Three replicate wells were set for each group. After culturing in a 37°C incubator for 8 hours or 24 hours, the cells on the upper layer of the membrane were carefully wiped off and the crystals were used. Stain the cells in the lower chamber with purple, and count the number of cells that have migrated or invaded under a microscope.
实验结果如图6(C)所示。The experimental results are shown in Figure 6(C).
实施例7.使用RUNX2抑制剂影响小鼠体内肿瘤的生长Example 7. Using RUNX2 inhibitors to affect tumor growth in mice
2.RUNX2小分子抑制剂CADD522抑制PDX肿瘤生长2. The RUNX2 small molecule inhibitor CADD522 inhibits PDX tumor growth
(1)实验方法：构建乳腺恶性叶状肿瘤PDX模型：取乳腺恶性叶状肿瘤组织，将其剪成直径约1mm组织块，在距离左前肢腋窝部皮下乳房脂肪垫约2cm处做3mm切口，套管针通过皮下隧道将3-4个小组织块送至皮下脂肪垫。待肿瘤生长至100m3左右时，对荷瘤小鼠进行随机分组，每组5只，分别腹腔注射RUNX2小分子抑制剂CADD522及对照试剂(DMSO)，注射剂量为10mg/kg和20mg/kg，每周注射两次，连续注射8次。持续监测肿瘤大小，根据公式V＝1/2(L×W2)计算移植瘤平均体积。待对照组肿瘤体积达到直径1.5cm时停止注射，并绘制肿瘤生长变化曲线。(1) Experimental method: Construct a PDX model of breast malignant phyllodes tumor: Take the breast malignant phyllodes tumor tissue, cut it into tissue pieces with a diameter of about 1mm, and make a 3mm incision about 2cm away from the subcutaneous breast fat pad in the axilla of the left forelimb. The trocar delivers 3-4 small tissue pieces through the subcutaneous tunnel to the subcutaneous fat pad. When the tumor grew to about 100 m3, the tumor-bearing mice were randomly divided into groups, with 5 mice in each group. The RUNX2 small molecule inhibitor CADD522 and the control reagent (DMSO) were intraperitoneally injected with 10 mg/kg and 20 mg/kg, respectively. Two injections per week, 8 consecutive injections. The tumor size was continuously monitored, and the average volume of the transplanted tumor was calculated according to the formula V=1/2 (L×W2). The injection was stopped when the tumor volume in the control group reached 1.5cm in diameter, and the tumor growth curve was drawn.
(2)实验结果：如图7(A-B)所示，与对照组相比，RUNX2小分子抑制剂CADD522注射组肿瘤生长明显被抑制。(2) Experimental results: As shown in Figure 7(A-B), compared with the control group, tumor growth in the RUNX2 small molecule inhibitor CADD522 injection group was significantly inhibited.
Claims (4)
- RUNX2作为标志物在筛选或制备抗乳腺恶性叶状肿瘤药物中的应用。Application of RUNX2 as a marker in screening or preparing drugs against malignant phyllodes tumors of the breast.
- RUNX2作为药物靶点在筛选或制备抗乳腺恶性叶状肿瘤药物中的应用。Application of RUNX2 as a drug target in screening or preparing drugs against malignant phyllodes tumors of the breast.
- RUNX2抑制剂在制备抗乳腺恶性叶状肿瘤药物中的应用。Application of RUNX2 inhibitors in the preparation of drugs against malignant phyllodes tumors of the breast.
- CADD522在制备抗乳腺恶性叶状肿瘤药物中的应用。Application of CADD522 in the preparation of drugs against malignant phyllodes tumors of the breast.
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| WO2016149667A1 (en) * | 2015-03-19 | 2016-09-22 | University Of Maryland , Baltimore | Runx2 transcription factor inhibitors and uses thereof |
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| US20220065865A1 (en) * | 2018-12-21 | 2022-03-03 | Uea Enterprises Limited | Methods of treatment and diagnosis of tumours |
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| US20220065865A1 (en) * | 2018-12-21 | 2022-03-03 | Uea Enterprises Limited | Methods of treatment and diagnosis of tumours |
| WO2021048292A1 (en) * | 2019-09-11 | 2021-03-18 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for treating melanoma |
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| KIM MYOUNG SOOK, GERNAPUDI RAMKISHORE, CEDEÑO YESSENIA CEDEÑO, POLSTER BRIAN M., MARTINEZ RAMON, SHAPIRO PAUL, KESARI SANTOSH, NUR: "Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis", ONCOTARGET, IMPACT JOURNALS LLC, UNITED STATES, vol. 11, no. 43, 27 October 2020 (2020-10-27), United States , pages 3863 - 3885, XP093146967, ISSN: 1949-2553, DOI: 10.18632/oncotarget.27743 * |
| KIM MYOUNG SOOK, GERNAPUDI RAMKISHORE, CHOI EUN YONG, LAPIDUS RENA G., PASSANITI ANTONINO: "Characterization of CADD522, a small molecule that inhibits RUNX2-DNA binding and exhibits antitumor activity", ONCOTARGET, IMPACT JOURNALS LLC, UNITED STATES, vol. 8, no. 41, 19 September 2017 (2017-09-19), United States , pages 70916 - 70940, XP093134573, ISSN: 1949-2553, DOI: 10.18632/oncotarget.20200 * |
| ZUO ZHONGKUN, YE FEI, LIU ZIRU, HUANG JIANGSHENG, GONG YI: "MicroRNA‑153 inhibits cell proliferation, migration, invasion and epithelial‑mesenchymal transition in breast cancer via direct targeting of RUNX2", EXPERIMENTAL AND THERAPEUTIC MEDICINE, SPANDIDOS PUBLICATIONS, GR, vol. 18, 5 April 2019 (2019-04-05), GR , XP093146968, ISSN: 1792-0981, DOI: 10.3892/etm.2019.7470 * |
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