CN115851932A - Application of RUNX2 inhibitor in preparation of medicine for resisting breast malignant phyllode tumor - Google Patents
Application of RUNX2 inhibitor in preparation of medicine for resisting breast malignant phyllode tumor Download PDFInfo
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Abstract
The invention discloses application of RUNX2 as a marker and a therapeutic target in screening and preparing a breast malignant phylloporphic tumor resisting medicine. In particular to the application of a RUNX2 inhibitor CADD522 in preparing a medicament for resisting breast malignant phyllode tumor. The invention discloses that the over-expression of RUNX2 is obviously related to the biological behavior of the breast malignant phylliform tumor for the first time, and the reduction of the expression of RUNX2 can inhibit the proliferation, migration and invasion of the breast malignant phylliform tumor. Experiments prove that the RUNX2 specific small molecule inhibitor CADD522 can obviously inhibit the proliferation, migration and invasion of breast malignant phylliform tumor cells in vitro and can obviously inhibit the growth of tumors in vivo. The CADD522 has good potential clinical application value in the development of drugs for targeted therapy of breast phyllodes tumors.
Description
Technical Field
The invention relates to the technical field of medical biology, in particular to application of a RUNX2 inhibitor in preparation of a medicine for resisting breast malignant phyllode tumor
Background
Breast Phyllodes Tumor (PT) is a rare fibroepithelial tumor, accounting for 0.3% to 1% of all breast tumors. According to the existing literature research, the junctional and malignant phyllodes tumors not only grow fast, but also are easy to recur locally and metastasize at a distance, blood metastasis is the most common metastasis mode, and the lung and the bone are the most common metastasis parts. The recurrence rate of the breast malignant phylliform tumor is as high as 53.1 percent, and the metastasis rate is as high as 43.1 percent. Once recurrence and metastasis occur, the patient dies in a short period of time, and the mortality rate of malignant leaf tumors is about 16.3%. The treatment of the breast phylliform tumor is mainly surgical treatment, and the clinical prognosis of the malignant phylliform tumor is poor due to the characteristics of high recurrence rate, high metastasis rate and the like. Different from breast cancer, the postoperative adjuvant treatment means of malignant lobular tumor of breast such as chemotherapy, radiotherapy and the like have poor treatment effects, and no research report currently shows that targeted therapy and immunotherapy have clinical benefit. Moreover, the breast phyllodes tumors are rare, lack of large-scale clinical data and basic research, and particularly lack of research on specific molecular markers and therapeutic targets, and have not been effectively developed all the time, thus further causing a delay in finding or screening effective therapeutic drugs.
RUNX2 is an important member of the RUNX family of transcription factors, named for its inclusion of the runt domain. The biological effect of RUNX2 is mainly as a specific transcription factor for osteogenic differentiation, regulating and controlling the transcription of genes such as collagen type, osteoprotegerin, osteocalcin, col1a1, col1a2, bone Sialoprotein (BSP) and fibronectin, and playing an important role in osteoblast formation and differentiation, chondrocyte differentiation and maturation, osteoclast formation and resorption and bone matrix protein synthesis. However, the report that RUNX2 is related to the biological behavior of breast malignant phyllodes tumor is not found.
Disclosure of Invention
In order to overcome the technical problems, the invention discloses the technical application of RUNX2 serving as a biological behavior marker of breast malignant phyllode tumors and a medicine target point.
The inventor finds that RUNX2 is highly expressed in breast malignant phyllodes tumor cells and promotes the development of the breast malignant phyllodes tumor in clinical samples and in vitro cell experiments. The expression of knocking down RUNX2 can inhibit the proliferation, migration and invasion of the breast malignant phylliform tumor. The RUNX2 can be used as a mammary malignant phyllode tumor marker and a drug target, and has higher application value in auxiliary diagnosis, screening and preparation of targeted therapeutic drugs for mammary phyllode tumors. The following applications are hereby disclosed:
application of RUNX2 as a marker in screening or preparing medicines for resisting breast malignant leaf tumors. In screening drugs for malignant lobular tumors of the breast, the expression of RUNX2 in test tumor cells can be detected as a means for evaluating the drug effect.
Application of RUNX2 as a drug target in screening or preparing drugs for resisting breast malignant phyllodes tumor. The drug aiming at the RUNX2 as a therapeutic target can be preferentially screened as a potential drug for resisting the breast malignant phyllodes tumor.
For example, the application of the RUNX2 inhibitor in preparing the medicine for resisting the breast malignant phylliform tumor.
The drug CADD522 has the function of inhibiting the combination of RUNX2 and DNA. The invention further discloses application of the RUNX2 inhibitor CADD522 in preparation of a medicine for resisting malignant phyllopoid tumor of mammary gland.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention discloses that the overexpression of the RUNX2 in a breast malignant phylliform tumor specimen is obviously related to the biological behavior of the breast malignant phylliform tumor for the first time, and the reduction of the expression of the RUNX2 can inhibit the proliferation, migration and invasion of the breast malignant phylliform tumor. The RUNX2 can be used as a marker and a treatment target for screening the medicines for resisting the breast malignant phyllode tumor, and has higher application value in the aspects of screening and preparing the targeted medicines for resisting the breast malignant phyllode tumor.
2. Experiments prove that the RUNX2 specific small molecule inhibitor CADD522 can obviously inhibit the proliferation, migration and invasion of breast malignant phylliform tumor cells in vitro and can obviously inhibit the growth of tumors in vivo. The CADD522 has good potential clinical application value in the development of targeted therapeutic drugs for breast phylliform tumors.
Drawings
FIG. 1A is a bar graph of the mRNA expression levels of RUNX2 in benign and malignant breast phyllode tumor tissue; b is the imaging picture of protein immunoblotting gel of RUNX2 protein expression test in benign and malignant breast phyllode tumor tissues; c is a graphical representation of RUNX2 expression levels in paraffin sections of good and malignant leaf tumors and in paraffin sections of multiple relapsed tumor tissues from the same patient.
FIG. 2: a is a gene of RUNX2 for knocking down a malignant mammary lobular tumor cell line SYSH-MPT-01 (namely, a cell line HJP-0320, disclosed in a patent application with the publication number of CN 111019898A), and the test result of the change of the cell proliferation capacity is shown in a figure; b, knocking down the RUNX2 gene of a mammary malignant phylliform tumor cell line SYSH-MPT-01, and graphically displaying the result of a cell clone forming capability change test; c is the RUNX2 gene of a knocking-down mammary malignant phyllode tumor cell line SYSH-MPT-01, and the cell cycle change test result is shown in a figure; d is RUNX2 gene of knocking down breast malignant phylliform tumor cell line SYSH-MPT-01, and the test result of cell migration and invasion capacity change is shown in the figure; e is the RUNX2 gene of a knocking-down breast malignant phyllode tumor cell line SYSH-MPT-01, and the experimental result of the change of the collagen contractility of cells is shown in a figure.
FIG. 3: a is a graphical representation of the results of experiments on the overexpression of RUNX2 in the benign lobular tumor cell line SYSH-BPT-01 of the breast (cell line GLK-1010, published in the patent application No. CN 111019897A), the alteration of cell proliferation potency; b is the over-expression of RUNX2 in the breast benign leaf tumor cell line SYSH-BPT-01, and the results of the clone forming ability change test of the cells are shown in the figure; c is the overexpression of RUNX2 in the breast benign phyllode tumor cell line SYSH-BPT-01, and the test results of the change of the cell cycle are shown in a graph; d is the over-expression of RUNX2 in the breast benign leaf tumor cell line SYSH-BPT-01, and the test results of the change of cell migration and invasion capacity are shown in a graph; e is a graphical representation of the results of experiments on the overexpression of RUNX2 in the mammary benign leaf tumor cell line SYSH-BPT-01, and the change in the collagen contractile capacity of the cells.
FIG. 4 is a graphical representation of the results of experiments on the effect of RUNX2 on the prognosis of breast leaf tumor patients.
FIG. 5 is a graphic representation of the results of experiments on the effect of knocking down RUNX2 in malignant phyllodes tumor cells on subcutaneous tumorigenicity and tumor growth; a is the change in tumor volume at the corresponding time point, and B is the tumor image for each group.
FIG. 6 is a graphical representation of the effect of CADD522 on malignant phyllodes tumor cell line SYSH-BPT-01, SYSH-MPT-02 (i.e., cell line LJ-0429, disclosed in the patent application with publication number CN 111019899A); a is a graph of the survival rate curve of CADD522 on malignant phyllodes tumor cell line, determining half maximal inhibitory concentration (IC 50) of CADD522 on malignant phyllodes tumor cell line; b is CADD522 inhibits the growth of the malignant phyllode tumor cell line SYSH-MPT-01, shown in a dose-and time-dependent graph; c is a graphical representation of the effect of CADD522 on the migration and invasion capacity of malignant lobular tumor cells in the breast.
FIG. 7 is a graphical representation of the effect of CADD522 on malignant phyllodes in vivo. A is the inhibition of tumor growth by intraperitoneal injection of CADD522 in a malignant phyllode PDX model, and the change situation of the tumor volume at the corresponding time point is shown in a graph, and B is a picture of each group of tumors.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: RUNX2 expression profiling analysis of mammary leaf tumor
1. And carrying out fluorescent quantitative PCR by using a RUNX2 specific primer to detect the expression level of the RUNX2mRNA in different types of breast phylliform tumor tissues.
(1) 3 benign leaf tumor tissue specimens and 10 malignant leaf tumor tissue specimens were selected. The experiment was carried out as follows: the tissue was ground with a cryomill, trizol lysate was added, the lysate was transferred to a 1.5ml EP tube with a pipette, and the cells were lysed by repeated pipetting or shaking. Standing at room temperature for 5min, adding 0.2ml chloroform into each 1ml Trizol lysate, shaking for 15s, standing at room temperature for 2-3min, centrifuging at 12000x g at 4 deg.C for 15min, collecting the upper water phase to a new EP tube, and adding equal volume of isopropanol to precipitate RNA; centrifuging at 12000x g and 4 ℃ for 10min; adding 75% ethanol, cleaning, centrifuging at 7500x g and 4 deg.C for 5min, and removing supernatant; and (3) drying the RNA precipitate in an ultra-static platform at room temperature, dissolving the RNA precipitate in a proper amount of RNase-free water, and measuring the concentration and the purity of the RNA. Reverse transcription to synthesize cDNA: reverse transcription to synthesize cDNA: adding template RNA 1. Mu.g, 4. Mu.l reverse transcriptase SuperScript II mix (containing Buffer, dNTP, hiScript II reverse transcriptase, RNase, random primers/Oligo dT), supplementing RNase-free water to 20. Mu.l, standing at 65 ℃ for 5min on ice, adding 5 XBuffer 8. Mu.l and 0.1M DTT 2. Mu.l, mixing uniformly, supplementing ddH2O to 40. Mu.l, and reacting under the following conditions: 50 ℃ 15min,85 ℃ 15s, and 4 ℃ storage.
Real-time quantitative PCR amplification: the template cDNA was diluted 3-fold and mixed well for use. 3 parallel tubes were set for each experimental group. Reaction system: cDNA 1. Mu.l, SYBR green dye 5. Mu.l, forward primer 0.3. Mu.l, reverse primer 0.3. Mu.l, ddH 2 O3.4. Mu.l, and mixing by centrifugation. The reaction conditions are as follows: 95 ℃ for 5min,95 ℃ for 30 seconds, 55 ℃ to 60 ℃ (depending on the annealing temperature) for 30s, for 40 cycles. Relative transcript levels of the genes were determined using GAPDH as an internal control assay. The primer sequences are as follows:
RUNX2 Forward：5’-CGCCTCACAAACAACCACAG-3’(SEQ ID NO.1)
RUNX2 Reverse：5’-TCACTGTGCTGAAGAGGCTG-3’(SEQ ID NO.2)
mRNA relative expression levels were quantified. Statistical data mean values were calculated from triplicate experiments, significance differences were judged by T-test, with P <0.05 defined as statistical differences.
(2) The experimental results are as follows: as shown in FIG. 1 (A), RUNX2 was expressed at a low level in benign leaf tumor tissue and at a high level in malignant leaf tumor tissue, and the expression level of RUNX2mRNA in malignant leaf tumor tissue was about 25 times that in benign leaf tumor tissue.
2. And detecting the expression level of the RUNX2 protein in different types of leaf tumor tissues by adopting a protein immunoblotting (Western blot) experiment.
(1) The experimental method comprises the following steps: after SDS-PAGE electrophoresis, membrane conversion and sealing, a RUNX2 specific antibody is adopted as a primary antibody for incubation, and a horseradish peroxidase-labeled secondary antibody is further adopted for incubation and then gel imaging analysis is carried out.
(2) As shown in FIG. 1 (B), the expression level of RUNX2 protein was higher in 10 cases of malignant leaf tumor tissues, while the expression level of RUNX2 protein was not or was low in 3 cases of benign leaf tumor tissues.
3. Immunohistochemical staining was performed using antibodies specific for RUNX2 to assess RUNX2 protein levels in benign and mammary malignant leaf tumor tissues.
(1) The experimental method comprises the following steps: the pathological sections used in the experiment are all from clinically confirmed cases. The paraffin-embedded tissue immunohistochemical staining is carried out according to the following steps: baking at 60 deg.C, and dewaxing twice with preheated xylene for 5min each time; subjecting the dewaxed slices to 100% -95% -80% -70% -50% ethanol gradient and distilled water hydration, and placing each gradient for 5min; performing high-pressure heat repair with 0.01M (pH6.0) citric acid buffer solution for 10min, naturally cooling, and washing with PBS for 5min for three times; treating with 0.3% hydrogen peroxide solution for 30min to remove endogenous peroxidase activity; washing with PBS for three times, 5min each time; blocking 10% sheep serum at 37 ℃ for 1h; adding dropwise goat serum to dilute primary antibody working solution (AA 4), incubating overnight at 4 ℃, and washing with PBS; adding biotin-labeled secondary antibody working solution, incubating for 20min at 37 ℃, and washing with PBS for three times; adding horse radish peroxidase labeled streptavidin, incubating for 20min at 37 ℃, and washing for three times by PBS; dripping DAB color development solution, developing at room temperature in a dark place for 2min, and washing off redundant color development solution by PBS; performing hematoxylin counterstaining and washing with distilled water; gradually dehydrating with 50% -70% -80% -90% -100% -100% ethanol gradient, each gradient for 5min; sealing a neutral resin sheet; the microscopic imaging system takes pictures.
(2) As shown in FIG. 1 (C): RUNX2 is highly expressed in breast malignant phyllodes cells and low expressed in benign (C). With the increase in the number of tumor recurrences, the expression level of RUNX2 in tissues was also increased simultaneously (C).
Example 2: effect of knocking down RUNX2 on proliferation, migration, invasion, cell cycle and collagen contraction capacity of malignant phyllode tumor cells
1. Effect of knockdown of RUNX2 on malignant phylloplastic tumor cell proliferation
(1) The experimental method comprises the following steps: inoculating malignant phylliform tumor cells into a 96-well plate and a 6-well plate, adopting 1ipo3000 to transiently transfect siRNA into the malignant phylliform tumor cells of mammary gland, reducing the expression of RUNX2, inoculating cells of a control group with reduced RUNX2 expression and without gene reduction, adopting a CCK8 method, testing the cell survival rate at different time points (1-4 days), and drawing a cell proliferation curve. Cells of the control group with knocked-down RUNX2 expression and without gene knock-down were inoculated into a complete culture medium in a 60mm medium dish at 200 cells per dish, and after 14 days of culture, the number of colony formations was counted.
The sequences of the sirnas are as follows:
sense:GGACGAGGCAAGAGTTTCA,(SEQ ID NO.3)
antisense:CCAAATTTGCCTAACCAGA(SEQ ID NO.4)
(2) The results of the experiment are shown in FIG. 2 (A-B).
2. Effect of knockdown of RUNX2 on cell cycle of malignant phyllodes
(1) The experimental method comprises the following steps: inoculating and knocking down the expression and control cells, culturing for 24h, collecting the cells, washing the cells for 2-3 times by using precooled PBS, centrifuging (1500rpm, 4min) to remove supernatant, adding a small amount of PBS into the precipitate, re-suspending the cells, adding the re-suspended cells into precooled 70% glacial ethanol at 4 ℃ for fixation, sealing by using a sealing film, and standing overnight at 4 ℃. The supernatant was centrifuged twice with PBS (2000rpm for 4 min). Add 100ul 100ug/ml RNase A and 0.2% Triton X-100 to resuspend the cells. 400ul 50ug/ml PI was added, vortexed, mixed well, and incubated at room temperature in the dark for 30min. Flow cytometry measured cell cycle, typically 10 ten thousand cells, detected red fluorescence at 488nm excitation wavelength, and then analyzed by FlowJo software for cell cycle phase distribution, as shown by FL2-w and FL2-a, to remove the cells that were stuck together.
(2) The experimental results are as follows: cell arrest after knockdown of RUNX2 is at G1 as shown in FIG. 2 (C).
3. Effect of knocking down RUNX2 on migration and invasion capacity of malignant phylliform tumor cells
(1) The experimental method comprises the following steps: knockdown of RUNX2 expression andcontrol cells at 2X10 4 The density of each hole is inoculated in a serum-free culture medium of an upper chamber of a Transwell plate (Costar) or a pre-paved Matrigel thin layer upper culture chamber, the lower layer is a complete culture medium containing 16% fetal bovine serum, and each group is provided with three repeat holes; after culturing in an incubator at 37 ℃ for 8 hours or 24 hours respectively, fixing with 4% paraformaldehyde for 15min, carefully wiping off the cells on the upper layer of the membrane, staining the cells in the lower chamber with 0.5% crystal violet, and counting the number of the migrated or invaded cells under a microscope.
(2) Experimental results; as shown in fig. 2 (D), RUNX2 knockdown expression set cells were significantly reduced in migration and invasion capacity.
4. Effect of knocking down RUNX2 on collagen contractility of malignant phyllodes tumors
(1) The experimental method comprises the following steps: diluting type I rat tail collagen by using a DMEM medium containing acetic acid and NaOH, mixing a leaf-shaped tumor cell suspension with the diluted rat tail collagen 1, inoculating the mixture into a 24-well plate, adding a serum-containing medium for 4 hours, then, incubating overnight by using a serum-free DMEM medium, separating the gel from the hole wall, and observing and measuring the diameter after shrinkage after 8 hours.
(2) The results of the experiment are shown in FIG. 2 (E).
Example 3 Effect of overexpression of RUNX2 on proliferation, migration, invasion, cell cycle and collagen contractile Capacity of leaf tumor cells
1. Effect of overexpression of RUNX2 on proliferation of benign mammary leaf tumor cells
(1) The experimental method comprises the steps of inoculating primary benign leaf tumor cells into a 96-well plate and a 6-well plate, transfecting RUNX2 overexpression plasmids (4 mu g/well) by using transfection reagents lipo3000 and p3000, replacing a normal complete culture medium after 4-6 hours of transfection, collecting cells, extracting an RNA sample, and detecting the overexpression level of RUNX 2. Cells of over-expressed RUNX2 and untreated control groups were inoculated, and cell viability was measured at different time points (1-4 days) by the CCK8 method to prepare cell proliferation curves. The over-expressed RUNX2 and untreated control cells were inoculated into a complete medium in a 60mm petri dish at 200 cells per dish and the number of colonies formed was counted after 14 days of culture.
(2) The results of the experiment are shown in FIG. 3 (A-B).
2. Effect of overexpression of RUNX2 on the cell cycle of benign leaf tumors
(1) The experimental method comprises the following steps: inoculating over-expressed and control cells, culturing for 24h, collecting cells, washing the cells with precooled PBS for 2-3 times, centrifuging (1500rpm, 4min) to remove supernatant, adding a small amount of PBS into the precipitate, resuspending the cells, adding the resuspended cells into precooled 70% glacial ethanol at 4 ℃ for fixation, sealing with a sealing film, and standing overnight at 4 ℃. The supernatant was centrifuged twice with PBS (2000rpm for 4 min). Add 100ul 100ug/ml RNase A and 0.2% Triton X-100 to resuspend the cells. 400ul 50ug/ml PI was added, vortexed, mixed, and incubated at room temperature in the dark for 30min. Flow cytometry was used to measure cell cycle, typically 10 tens of thousands of cells were counted, red fluorescence was detected at 488nm excitation wavelength, and then cell cycle phase distribution was analyzed using FlowJo software, using FL2-w and FL2-a to show that adherent cells were removed during analysis.
(2) The experimental results are as follows: the cells after RUNX2 passage shown in FIG. 3 (C) were active in S phase.
3. Effect of overexpression of RUNX2 on migration and invasion capabilities of breast benign leaf tumor cells
(1) The experimental method comprises the following steps: overexpression of RUNX2 and untreated control cells were treated with 1 x10 4 The density of each hole is inoculated in a serum-free culture medium of an upper chamber of a Transwell plate (Costar) or a pre-paved Matrigel thin layer upper culture chamber, the lower layer is a complete culture medium containing 16% fetal bovine serum, and each group is provided with three repeat holes; after culturing in an incubator at 37 ℃ for 8 hours or 24 hours respectively, the cells on the upper layer of the membrane are carefully wiped off, the cells on the lower chamber are stained with crystal violet, and the number of the migrated or invaded cells is counted under a microscope.
(2) The results of the experiment are shown in FIG. 3 (D).
4. Effect of overexpression of RUNX2 on collagen contractility of malignant phylloplastic tumors
(1) The experimental method comprises the following steps: diluting type I rat tail collagen by using a DMEM medium containing acetic acid and NaOH, mixing a leaf-shaped tumor cell suspension with the diluted rat tail collagen 1, inoculating the mixture into a 24-well plate, adding a serum-containing medium for 4 hours, then, incubating overnight by using a serum-free DMEM medium, separating the gel from the hole wall, and observing and measuring the diameter after shrinkage after 8 hours.
(2) The results of the experiment are shown in FIG. 3 (E).
Example 4 Effect of RUNX2 on the prognosis of patients with mammary lobular tumours
Effect of RUNX2 on the prognosis of patients with mammary lobular tumors
(1) The experimental method comprises the following steps: two pathologists read the sections separately and divide them into RUNX2 high expression group and RUNX2 low expression group according to the staining intensity and staining positive proportion, and evaluate the influence of RUNX2 expression and total survival period OS, and the influence of DFS without progression.
(2) The experimental results are as follows: as shown in FIG. 4 (A-B), the OS and DFS of RUNX2 high expression group were significantly lower than those of RUNX2 low expression group.
Example 5 knocking down the RUNX2 Gene affects tumor growth in mice
1. Knocking down RUNX2 gene to inhibit the influence of the growth and the tumorigenesis of mice subcutaneous transplantation tumor
(1) The experimental method comprises the following steps: constructing a subcutaneous transplantation tumor model of the malignant phyllodes tumor of the mammary gland: taking malignant phyllodes tumor cells and phyllodes tumor cells for stably knocking down RUNX2, and diluting to 5x10 7 And/ml, inoculating 100 μ L of the mixed cell suspension to left forelimb axillary subcutaneous breast fat pad of control group mouse and experimental group mouse, measuring tumor major diameter and minor diameter every 4 days, and calculating according to formula V =1/2 (L × W) 2 ) Mean volume of the transplanted tumors was calculated. When the tumor volume of the control group reached 1.5cm in diameter, the mice were sacrificed and a tumor growth curve was plotted.
(2) The experimental results are as follows: as shown in FIG. 5 (A-B), tumor formation and growth of mice inoculated with stably knockdown RUNX2 phyllode tumor cells were significantly inhibited compared to the control group.
Example 6 Effect of the RUNX2 Small molecule inhibitor CADD522 on the IC50, proliferation, migration, invasion of malignant leaf-like tumor cells
IC50 of RUNX2 small molecule inhibitor CADD522 in malignant phyllodes tumor cells
(1) The experimental method comprises the following steps: and (2) inoculating the breast malignant phylliform tumor cells to a 96-well plate, wherein the inoculation density is 5000 cells/well, adding a RUNX2 small molecule inhibitor CADD522 after 24h, setting concentration gradients of 200 mu M, 100 mu M, 50 mu M, 25 mu M, 12.5 mu M, 6.125 mu M and 3.0625 mu M, culturing for 72 h, detecting the cell viability by using a CCK8 reagent, and drawing a cell IC50 curve.
(2) The results of the experiment are shown in FIG. 6 (A).
Effect of RUNX2 Small molecule inhibitor CADD522 on malignant phyllode tumor cell proliferation
(1) The experimental method comprises the following steps: and (2) inoculating the breast malignant phylliform tumor cells to a 96-well plate, wherein the inoculation density is 5000 cells/well, adding a RUNX2 small molecule inhibitor CADD522 after 24 hours, the inoculation density is 3000 cells/well, culturing for 24 hours, 48 hours, 72 hours and 96 hours respectively, detecting the cell activity by adopting a CCK8 reagent, and drawing a cell proliferation curve.
(2) The results of the experiment are shown in FIG. 6 (B).
Inhibition of malignant phyllodes tumor cell migration and invasion by RUNX2 small molecule inhibitor CADD522
(1) The experimental method comprises the following steps: inoculating malignant tumor cells into a serum-free culture medium of an upper chamber of a Transwell plate (Costar) or a pre-paved Matrigel thin-layer upper culture chamber at the density of 1 × 104/hole, wherein the lower layer is a complete culture medium containing 16% fetal bovine serum, and a RUNX2 small-molecule inhibitor CADD522 is added for co-culture while inoculating the cells, and three repeated holes are formed in each group; after culturing in an incubator at 37 ℃ for 8 hours or 24 hours respectively, the cells on the upper layer of the membrane are carefully wiped off, the cells on the lower chamber are stained with crystal violet, and the number of the migrated or invaded cells is counted under a microscope.
The results of the experiment are shown in FIG. 6 (C).
Example 7 use of RUNX2 inhibitors to affect tumor growth in mice
Inhibition of PDX tumor growth by CADD522, a small molecule inhibitor of RUNX2
(1) The experimental method comprises the following steps: constructing a mammary gland malignant phyllode tumor PDX model: taking malignant lobular tumor tissue of mammary gland, shearing the malignant lobular tumor tissue into tissue blocks with the diameter of about 1mm, making 3mm incision at a position about 2cm away from subcutaneous breast fat pad at axillary fossa of left forelimb, and delivering 3-4 small tissue blocks to the subcutaneous fat pad through a subcutaneous tunnel by a trocar. When the tumor grows to about 100m < 3 >, tumor-bearing mice are randomly grouped, 5 mice in each group are respectively injected with a RUNX2 small molecular inhibitor CADD522 and a contrast reagent (DMSO) in the abdominal cavity, the injection doses are 10mg/kg and 20mg/kg, the injection is twice per week and 8 times of continuous injection are carried out. Tumor size was monitored continuously and mean volume of transplanted tumors was calculated according to formula V =1/2 (L × W2). When the tumor volume of the control group reaches 1.5cm in diameter, the injection is stopped, and a tumor growth change curve is drawn.
(2) The experimental results are as follows: as shown in FIG. 7 (A-B), tumor growth was significantly inhibited in the RUNX2 small molecule inhibitor CADD 522-injected group compared to the control group.
Claims (4)
- Application of RUNX2 as a marker in screening or preparing medicines for resisting breast malignant leaf tumors.
- Application of RUNX2 as a drug target in screening or preparing drugs for resisting breast malignant phyllodes tumor.
- Application of RUNX2 inhibitor in preparing medicine for treating malignant lobular tumor of breast is provided.
- Application of CADD522 in preparing medicine for treating malignant lobular tumor of breast.
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| CN202211092785.0A CN115851932A (en) | 2022-09-08 | 2022-09-08 | Application of RUNX2 inhibitor in preparation of medicine for resisting breast malignant phyllode tumor |
| PCT/CN2022/118895 WO2024050861A1 (en) | 2022-09-08 | 2022-09-15 | Use of runx2 inhibitor in preparing medicament for combating malignant phyllodes tumors of breast |
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