Diagnostic reagent for breast phyllodes tumor and application thereof

Abstract

The present application relates to the field of medicine biotechnology, especially the application of RUNX2 as a marker in the preparation of a breast malignant phyllodes tumor diagnosis or detection reagent. The reagent can assist conventional biopsy according to the expression level of RUNX2 in different grades of breast phyllodes tumor, and can diagnose, differentially diagnose, grade and prognostically analyze the breast phyllodes tumor. The RUNX2 detection primer sequence and detection antibody of the present application are important components of the diagnosis or detection reagent or kit, so that the detection of breast malignant phyllodes tumor is more simple, fast and accurate, has good clinical application value, helps to further improve the diagnosis level of breast malignant phyllodes tumor, and is expected to become the first diagnosis and detection kit for breast malignant phyllodes tumor.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, specifically to a diagnostic reagent for phyllodes tumors of the breast and its application. Background Technology

[0002] Phyllodes tumor (PT) is a rare fibroepithelial tumor of the breast, accounting for 0.3% to 1% of all breast tumors. Currently, commonly used preoperative diagnostic methods for PT include breast ultrasound, mammography, breast MRI, preoperative fine-needle aspiration of the mass, and core needle biopsy. However, even using all these methods, the preoperative diagnostic accuracy for PT is only about 76%, and postoperative pathological examination remains the gold standard for diagnosis. Furthermore, due to individual differences and the lack of clear standards and cutoff values, morphological diagnosis of PT remains challenging. Therefore, the pathological diagnosis of PT has a degree of subjectivity and is insufficient to predict the clinical prognosis of individual patients.

[0003] According to existing literature, borderline malignant phyllodes tumors not only grow rapidly but are also prone to local recurrence and distant metastasis. Hematogenous metastasis is the most common mode of metastasis, with the lungs and bones being the most frequent 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 or metastasis occurs, patients will die within a short period of time, with a mortality rate of approximately 16.3%. The main treatment for phyllodes tumors of the breast is surgery. Due to their high recurrence and metastasis rates, malignant phyllodes tumors have a poor clinical prognosis. Unlike breast cancer, adjuvant therapies after surgery for malignant phyllodes tumors, such as chemotherapy and radiotherapy, are not very effective. Targeted therapy and immunotherapy have not yet been reported to have clinical benefits. Furthermore, because phyllodes tumors of the breast are relatively rare, there is a lack of large-scale clinical data and basic research, and the development of specific molecular markers for their diagnosis, as well as related diagnostic reagents and corresponding drugs, is even more lacking. Therefore, it is urgent to find specific molecular markers for phyllodes tumors of the breast, which will play a vital role in improving preoperative diagnostic rates and patient prognosis.

[0004] RUNX2 is an important member of the RUNX family of transcription factors, named for its runt domain. Its biological function is primarily as a specific transcription factor for osteogenic differentiation, regulating the transcription of genes such as type I collagen, osteomodulation, osteocalcin, col1a1, col1a2, osteosialin (BSP), and fibronectin. It plays a crucial role in osteoblast formation and differentiation, chondrocyte differentiation and maturation, osteoclast formation and resorption, and the synthesis of bone matrix proteins. However, there are no reports of RUNX2 being associated with the biological behavior of malignant phyllodes tumors of the breast. Summary of the Invention

[0005] To overcome the above technical problems, this invention discloses the technical application of RUNX2 as a biological behavior marker for malignant phyllodes tumors of the breast.

[0006] The inventors discovered in clinical samples and in vitro cell experiments that RUNX2 is expressed at low levels in benign phyllodes tumors and at high levels in malignant phyllodes tumors, with the mRNA expression level of RUNX2 in malignant tumors being approximately 25 times that in benign phyllodes tumors. The expression level of RUNX2 in tissues also increases with the number of tumor recurrences. The expression level of RUNX2 mRNA in malignant tumors is positively correlated with the proliferation, migration, and invasion capabilities of malignant phyllodes tumor cells. This demonstrates that RUNX2 can serve as a biomarker for the diagnosis or detection of malignant phyllodes tumors of the breast. Based on the expression level of RUNX2 in phyllodes tumors of different grades, and in conjunction with routine tissue biopsies, the diagnosis, differential diagnosis, grading, and prognostic analysis of phyllodes tumors of the breast can be performed.

[0007] This leads to the following applications:

[0008] Application of RUNX2 as a biomarker in the preparation of diagnostic or detection reagents (including kits) for malignant phyllodes tumors of the breast.

[0009] Preferably, the application includes: the use of RUNX2 as a biomarker in the preparation of reagents for early screening or prognostic prediction of malignant phyllodes tumors of the breast.

[0010] Preferably, the diagnostic or detection reagent includes specific RUNX2 primers or RUNX2 antibodies.

[0011] Diagnostic or detection methods may include: detecting the RUNX2 expression level of malignant phyllodes tumor specimens of the breast at various biological stages using qRT-PCR or immunohistochemistry, establishing a detection model, obtaining standard data for diagnosis or detection, and making a judgment based on the RUNX2 expression level of the malignant phyllodes tumor specimens of the breast.

[0012] The diagnostic or testing reagents or kits are used as follows: the expression level of RUNX2 in malignant phyllodes tumor specimens of the breast can be detected by qRT-PCR or immunohistochemistry, and the biological status of the malignant phyllodes tumor specimens of the breast can be judged based on the RUNX2 expression level.

[0013] The preferred specific primer sequences for RUNX2 are:

[0014] Forward Primer: 5'-CGCCTCACAAAACAACCACAG-3'; (Sequence Listing SEQ ID NO.1)

[0015] Reverse Primer: 5'-TCACTGTGCTGAAGAGGCTG-3'; (SEQ ID NO.2)

[0016] The preferred antibodies for RUNX2 are: MBL Anti-Runx2#D130-3, Abcam recombinant Anti-RUNX1 / AML1+RUNX3+RUNX2 antibody [EPR3099]#ab92336.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. This invention discloses for the first time that the overexpression of RUNX2 in malignant phyllodes tumor specimens of the breast is significantly correlated with the biological behavior of malignant phyllodes tumors of the breast. It can be used as a biomarker for the diagnosis and detection of malignant phyllodes tumors of the breast and for screening anti-malignant phyllodes tumor drugs. It has high clinical application value in early screening and diagnosis, prognosis prediction and screening of targeted anti-tumor drugs.

[0019] 2. The experimental results of this invention demonstrate that RUNX2 is expressed at low levels in benign phyllodes tumors and at high levels in malignant phyllodes tumors. The expression level of RUNX2 mRNA in malignant tumors is approximately 25 times that in benign phyllodes tumors. The difference is very significant, demonstrating the high specificity of RUNX2 as a biomarker, and providing strong theoretical support for the feasibility and accuracy of the RUNX2 biomarker-related diagnostic reagents or kits of this invention.

[0020] 3. The RUNX2 detection primer sequence and detection antibody of the present invention can be used as important components of the RUNX2 detection kit, thereby making the detection of malignant phyllodes tumors of the breast simpler, faster and more accurate, with good clinical application value, helping to further improve the diagnostic level of malignant phyllodes tumors of the breast, and is expected to become the first diagnostic detection kit for malignant phyllodes tumors of the breast. Attached Figure Description

[0021] Figure 1 A: Bar chart showing the mRNA expression levels of RUNX2 in benign and malignant phyllodes tumor tissues of the breast; B: Western blot gel imaging of RUNX2 protein expression in benign and malignant phyllodes tumor tissues of the breast; C: Illustration of RUNX2 expression levels in paraffin sections of benign and malignant phyllodes tumors and paraffin sections of tumor tissues from multiple recurrences in the same patient.

[0022] Figure 2A shows the results of an experiment demonstrating altered cell proliferation by knocking down the RUNX2 gene in the SYSH-MPT-01 malignant phyllodes tumor cell line (i.e., cell line HJP-0320, disclosed in patent application CN111019898A); B shows the results of an experiment demonstrating altered cell colony formation ability by knocking down the RUNX2 gene in the SYSH-MPT-01 malignant phyllodes tumor cell line; C shows the results of an experiment demonstrating altered cell cycle by knocking down the RUNX2 gene in the SYSH-MPT-01 malignant phyllodes tumor cell line; D shows the results of an experiment demonstrating altered cell migration and invasion ability by knocking down the RUNX2 gene in the SYSH-MPT-01 malignant phyllodes tumor cell line; E shows the results of an experiment demonstrating altered cell collagen contraction ability by knocking down the RUNX2 gene in the SYSH-MPT-01 malignant phyllodes tumor cell line.

[0023] Figure 3 A: Illustration of the results of an experiment showing changes in cell proliferation capacity after overexpression of RUNX2 in the benign phyllodes tumor cell line SYSH-BPT-01 (i.e., cell line GLK-1010, disclosed in patent application publication number CN111019897A); B: Illustration of the results of an experiment showing changes in cell colony formation capacity after overexpression of RUNX2 in the benign phyllodes tumor cell line SYSH-BPT-01; C: Illustration of the results of an experiment showing changes in cell cycle after overexpression of RUNX2 in the benign phyllodes tumor cell line SYSH-BPT-01; D: Illustration of the results of an experiment showing changes in cell migration and invasion capacity after overexpression of RUNX2 in the benign phyllodes tumor cell line SYSH-BPT-01; E: Illustration of the results of an experiment showing changes in cell collagen contraction capacity after overexpression of RUNX2 in the benign phyllodes tumor cell line SYSH-BPT-01.

[0024] Figure 4 Illustration of the prognostic effects of RUNX2 on patients with phyllodes tumors of the breast.

[0025] Figure 5 The following is a graphic illustration of the effect of knocking down RUNX2 in the malignant phyllodes tumor cell line SYSH-MPT-01 on subcutaneous tumorigenesis and tumor growth: A shows the changes in tumor volume at the corresponding time points, and B shows the tumor images of each group. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments.

[0027] Example 1: RUNX2 expression profile analysis in phyllodes tumors of the breast

[0028] 1. RUNX2 specific primers were used to perform real-time quantitative PCR to detect the expression level of RUNX2 mRNA in different types of phyllodes tumor tissues of the breast.

[0029] (1) Three benign phyllodes tumor tissue specimens and ten malignant phyllodes tumor tissue specimens were selected. The experiment was carried out according to the following steps: The tissues were ground with a cryogenic grinder, Trizol lysis buffer was added, and the lysate was transferred to a 1.5 ml EP tube with a pipette. The cells were repeatedly lysed by pipetting or shaking. After standing at room temperature for 5 min, 0.2 ml of chloroform was added to each 1 ml of Trizol lysis buffer, and the mixture was shaken vigorously for 15 s. After standing at room temperature for 2-3 min, the mixture was centrifuged at 12000 x g at 4℃ for 15 min. The upper aqueous phase was transferred to a new EP tube, and an equal volume of isopropanol was added to precipitate RNA. The mixture was centrifuged at 12000 x g at 4℃ for 10 min. After washing with 75% ethanol, the mixture was centrifuged at 7500 x g at 4℃ for 5 min, and the supernatant was discarded. The RNA precipitate was dried in an ultrafiltration unit at room temperature, dissolved in an appropriate amount of RNase-free water, and the RNA concentration and purity were determined. Reverse transcription to synthesize cDNA: Add 1 μg template RNA, 4 μl SuperScript II reverse transcriptase mix (containing buffer, dNTPs, HiScript II reverse transcriptase, RNase, Random primers / Oligo dT) to a PCR tube, add RNase-free water to 20 μl, incubate at 65℃ for 5 min, place on ice for 5 min, then add 8 μl of 5× buffer and 2 μl of 0.1M DTT, mix well, and add ddH2O to 40 μl. Reaction conditions: 50℃ for 15 min, 85℃ for 15 s, store at 4℃.

[0030] Real-time quantitative PCR amplification: Dilute the template cDNA 3-fold and mix well. Set up 3 parallel tubes for each experimental group. Reaction system: 1 μl cDNA, 5 μl SYBR green dye, 0.3 μl forward primer, 0.3 μl reverse primer, 3.4 μl ddH2O, centrifuge and mix. Reaction conditions: 95℃ for 5 min, 95℃ for 30 s, 55℃-60℃ (depending on annealing temperature) for 30 s, for a total of 40 cycles. Use GAPDH as an internal control to analyze and detect the relative transcription level of the gene. Primer sequences are as follows:

[0031] RUNX2 Forward: 5'-CGCCTCACAAACAACCACAG-3'; (SEQ ID NO.1)

[0032] RUNX2 Reverse: 5'-TCACTGTGCTGAAGAGGCTG-3'. (Sequence Listing SEQ ID NO.2)

[0033] Quantitative analysis of relative mRNA expression levels was performed. Statistical data were calculated based on the average of three replicate experiments. Significant differences were determined using a t-test, with P < 0.05 defined as statistically significant.

[0034] (2) Experimental results: such as Figure 1 As shown in (A), RUNX2 is expressed at low levels in benign phyllodes tumor tissues and at high levels in malignant phyllodes tumor tissues. The expression level of RUNX2 mRNA in malignant phyllodes tumor tissues is approximately 25 times that in benign phyllodes tumor tissues.

[0035] 2. The expression level of RUNX2 protein in different types of phyllodes tumor tissues was detected by Western blot.

[0036] (1) Experimental method: After SDS-PAGE electrophoresis, membrane transfer and blocking, RUNX2 specific antibody was used as the primary antibody for incubation, and then horseradish peroxidase-labeled secondary antibody was used for incubation before gel imaging analysis.

[0037] (2) Experimental results: such as Figure 1 (B) shows that RUNX2 protein expression was high in 10 malignant phyllodes tumor tissues, while RUNX2 protein was not expressed or was low in 3 benign phyllodes tumor tissues.

[0038] 3. Immunohistochemical staining was performed using a specific antibody against RUNX2 to assess the RUNX2 protein level in benign and malignant phyllodes tumors of the breast.

[0039] (1) Experimental methods: The pathological sections used in the experiment were all from clinically confirmed cases. Immunohistochemical staining of paraffin-embedded tissues was performed according to the following steps: baking at 60℃, dewaxing twice in preheated xylene for 5 min each time; after dewaxing, the sections were hydrated in a gradient of 100%-95%-80%-70%-50% ethanol and distilled water, with each gradient incubated for 5 min; autoclaving and heat repair in 0.01M (pH 6.0) citrate buffer for 10 min, followed by natural cooling and washing three times with PBS for 5 min each time; treatment with 0.3% hydrogen peroxide solution for 30 min to eliminate endogenous peroxidase activity; washing three times with PBS for 5 min each time; blocking with 10% sheep serum at 37℃ for 1 h. Add sheep serum to dilute the primary antibody working solution (AA4), incubate overnight at 4°C, and wash with PBS; add biotin-labeled secondary antibody working solution, incubate at 37°C for 20 min, and wash three times with PBS; add horseradish peroxidase-labeled streptavidin, incubate at 37°C for 20 min, and wash three times with PBS; add DAB chromogenic solution, develop at room temperature in the dark for 2 min, and wash away excess chromogenic solution with PBS; counterstain with hematoxylin, and wash with distilled water; dehydrate stepwise with 50%-70%-80%-90%-100%-100% ethanol gradient, 5 min for each gradient; mount with neutral resin; and take images using a microscopic imaging system.

[0040] (2) Experimental results: such as Figure 1 As shown in (C): RUNX2 is highly expressed in malignant phyllodes tumor cells of the breast, and lowly expressed in benign tumors (C). With the increase of tumor recurrence, the expression level of RUNX2 in tissues also increases synchronously (C).

[0041] Example 2: Effects of RUNX2 knockdown on proliferation, migration, invasion, cell cycle, and collagen contraction in malignant phyllodes tumor cells

[0042] 1. Effect of RUNX2 knockdown on the proliferation of malignant phyllodes tumor cells

[0043] (1) Experimental methods: Malignant phyllodes tumor cells were seeded in 96-well and 6-well plates. shRNA was transiently transfected into breast malignant phyllodes tumor cells using 1 ipo 3000. RUNX2 expression was knocked down. Control cells with knocked-down RUNX2 expression and those without gene knockdown were seeded. Cell viability at different time points (1-4 days) was tested using the CCK8 assay, and cell proliferation curves were plotted. Control cells with knocked-down RUNX2 expression and those without gene knockdown were seeded into 60 mm medium-sized dishes of complete culture medium, 200 cells per dish. After 14 days of culture, the number of colonies formed was counted.

[0044] The shRNA sequence is as follows:

[0045] shRUNX2 #1:GGACGAGGCAAGAGTTTCA; (SEQ ID NO.3)

[0046] shRUNX2 #2 :CCAAATTTGCCTAACCAGA. (Sequence Listing SEQ ID NO.4)

[0047] (2) Experimental results: such as Figure 2 As shown in (AB).

[0048] 2. Effects of RUNX2 knockdown on the cell cycle of malignant phyllodes tumors

[0049] (1) Experimental Methods: Knockdown expression and control cells were seeded and cultured for 24 h. Cells were then collected, washed 2-3 times with pre-chilled PBS, centrifuged (1500 rpm, 4 min), and the supernatant was discarded. A small amount of PBS was added to the pellet to resuspend the cells. The resuspended cells were then fixed in 70% ice-cold ethanol pre-chilled at 4°C, sealed with sealing film, and incubated overnight at 4°C. The cells were washed twice with PBS and centrifuged to remove the supernatant (2000 rpm, 4 min). 100 μL of 100 μg / ml RNase A and 0.2% Triton X-100 were added to resuspend the cells. 400 μL of 50 μg / ml PI was added, vortexed to mix, and incubated at room temperature in the dark for 30 min. Cell cycle was detected by flow cytometry. Generally, 100,000 cells were counted, and red fluorescence was detected at an excitation wavelength of 488 nm. The cell cycle phase distribution was then analyzed using FlowJo software. FL2-w and FL2-A were used for visualization, and cells that were stuck together were removed.

[0050] (2) Experimental results: such as Figure 2 (C) Cells arrested in G1 phase after knockdown of RUNX2.

[0051] 3. Effects of RUNX2 knockdown on the migration and invasion abilities of malignant phyllodes tumor cells

[0052] (1) Experimental method: RUNX2 knockdown cells and control group cells were used in a 2x10 4 The cells were seeded at a density of / wells in the upper chamber of a Transwell (Costar) plate in serum-free medium or in the upper chamber of a pre-coated Matrigel layer, with the lower chamber containing complete medium with 16% fetal bovine serum. Each group had three replicate wells. After incubation at 37°C for 8 hours or 24 hours, the cells were fixed with 4% paraformaldehyde for 15 minutes. The cells on the membrane were carefully wiped off, and the cells in the lower chamber were stained with 0.5% crystal violet. The number of cells that had migrated or invaded was counted under a microscope.

[0053] (2) Experimental results; such as Figure 2(D) shows that the RUNX2 knockdown group had significantly reduced cell migration and invasion abilities.

[0054] 4. Effect of RUNX2 knockdown on collagen contraction ability in malignant phyllodes tumors

[0055] (1) Experimental method: Type I rat tail collagen was diluted with DMEM medium containing acetic acid and NaOH. The phyllodes tumor cell suspension was mixed with the diluted rat tail collagen at a ratio of 1:1 and then seeded into 24-well plates. After adding serum-containing medium for 4 hours, the medium was replaced with serum-free DMEM medium and incubated overnight to allow the collagen to separate from the well wall. The diameter after shrinkage was observed and measured after 8 hours.

[0056] (2) Experimental results are as follows Figure 2 As shown in (E).

[0057] Example 3. Effects of RUNX2 overexpression on proliferation, migration, invasion, cell cycle, and collagen contraction in phyllodes tumor cells.

[0058] 1. Effect of RUNX2 overexpression on the proliferation of benign breast phyllodes tumor cells

[0059] (1) Experimental methods: Primary benign phyllodes tumor cells were seeded into 96-well and 6-well plates. RUNX2 overexpression plasmid (4 μg / well) was transfected using lipo3000 and p3000 transfection reagents. After 4-6 hours of transfection, the medium was replaced with normal complete medium. Cells were collected, RNA samples were extracted, and the RUNX2 overexpression level was detected. Cell viability at different time points (1-4 days) was tested using the CCK8 assay, and cell proliferation curves were plotted. RUNX2 overexpression cells and untreated control cells were seeded into 60 mm medium dishes of complete medium, 200 cells per dish. After 14 days of culture, the number of colonies formed was counted.

[0060] (2) Experimental results are as follows Figure 3 As shown in (AB).

[0061] 2. Effects of RUNX2 overexpression on the cell cycle of benign phyllodes tumors

[0062] (1) Experimental Methods: Overexpressing and control cells were inoculated and cultured for 24 hours. Cells were then collected, washed 2-3 times with pre-chilled PBS, centrifuged (1500 rpm, 4 min), and the supernatant was discarded. A small amount of PBS was added to the pellet to resuspend the cells. The resuspended cells were then fixed in pre-chilled 70% ice-cold ethanol at 4°C, sealed with sealing film, and incubated overnight at 4°C. The cells were washed twice with PBS and centrifuged to remove the supernatant (2000 rpm, 4 min). 100 μL of 100 μg / ml RNase A and 0.2% Triton X-100 were added to resuspend the cells. 400 μL of 50 μg / ml PI was added, vortexed to mix, and incubated at room temperature in the dark for 30 min. Cell cycle was detected by flow cytometry. Generally, 100,000 cells were counted, and red fluorescence was detected at an excitation wavelength of 488 nm. The cell cycle phase distribution was then analyzed using FlowJo software. FL2-w and FL2-A were used for visualization, and cells that were stuck together were removed.

[0063] (2) Experimental results: such as Figure 3 (C) Cells overexpressing RUNX2 are active in S phase.

[0064] 3. Effects of RUNX2 overexpression on the migration and invasion abilities of benign phyllodes tumor cells of the breast.

[0065] (1) Experimental method: Cells overexpressing RUNX2 and untreated control cells were subjected to a 1*10 4 The cells were seeded at a density of / wells in the upper chamber of a Transwell (Costar) plate in serum-free medium or in the upper chamber of a pre-coated Matrigel layer, with the lower chamber containing complete medium containing 16% fetal bovine serum. Each group had three replicate wells. After incubation at 37°C for 8 hours or 24 hours, the cells on the membrane were carefully wiped off, and the cells in the lower chamber were stained with crystal violet. The number of cells that had migrated or invaded was counted under a microscope.

[0066] (2) Experimental results are as follows Figure 3 As shown in (D).

[0067] 4. Effects of RUNX2 overexpression on collagen contraction ability in malignant phyllodes tumors

[0068] (1) Experimental method: Type I rat tail collagen was diluted with DMEM medium containing acetic acid and NaOH. The phyllodes tumor cell suspension was mixed with the diluted rat tail collagen at a ratio of 1:1 and then seeded into 24-well plates. After adding serum-containing medium for 4 hours, the medium was replaced with serum-free DMEM medium and incubated overnight to allow the collagen to separate from the well wall. The diameter after shrinkage was observed and measured after 8 hours.

[0069] (2) Experimental results are as follows Figure 3 As shown in (E).

[0070] Example 4. The impact of RUNX2 on the prognosis of patients with phyllodes tumors of the breast.

[0071] 1. The impact of RUNX2 on the prognosis of patients with phyllodes tumors of the breast

[0072] (1) Experimental method: Fresh frozen sections of 237 patients with phyllodes tumors of the breast were taken and immunohistochemically stained. Two pathologists read the sections separately. Based on the staining intensity and the proportion of positive staining, the patients were divided into a high RUNX2 expression group and a low RUNX2 expression group. The effects of RUNX2 expression on overall survival (OS) and progression-free survival (DFS) were evaluated.

[0073] (2) Experimental results: such as Figure 4 As shown in (AB), the OS and DFS of the RUNX2 high expression group were significantly lower than those of the RUNX2 low expression group.

[0074] Example 5: Knocking down the RUNX2 gene affects tumor growth in mice.

[0075] 1. Effects of RUNX2 gene knockdown on tumorigenesis and growth in mice

[0076] (1) Experimental methods: Construction of a subcutaneous xenograft model of malignant phyllodes tumor of the breast: malignant phyllodes tumor cells and phyllodes tumor cells with stable RUNX2 knockdown were obtained and diluted to 5x10. 7 / ml, take 100μl of well-mixed cell suspension and inoculate it into the subcutaneous mammary fat pad of the left forelimb axilla in mice of the control group and experimental group, respectively. Measure the long and short diameters of the tumor every 4 days, and calculate the value according to the formula V=1 / 2(L×W). 2 The average volume of the transplanted tumor was calculated. Mice in the control group were sacrificed when the tumor volume reached 1.5 cm in diameter, and the tumor growth curve was plotted.

[0077] (2) Experimental results: such as Figure 5 As shown in (AB), compared with the control group, tumor formation and growth were significantly inhibited in mice inoculated with stably knocked-down RUNX2 phyllodes tumor cells.

[0078] In summary, this invention identifies RUNX2 as a specific molecular marker for malignant phyllodes tumors of the breast. This marker is highly expressed in malignant phyllodes tumor cells and promotes the progression of malignant phyllodes tumors. Knockdown of RUNX2 expression can inhibit the proliferation, migration, and invasion of malignant phyllodes tumors. RUNX2, as a marker for malignant phyllodes tumors of the breast, has significant clinical application value in the development of auxiliary diagnostic methods for phyllodes tumors.

Claims

1. Substances for detecting RUNX2 biomarker expression levels in the preparation of prognostic tests for malignant phyllodes tumors of the breast. Application in reagents.

2. The application as described in claim 1, characterized in that, The application described is based on the qRT-PCR detection method, and its detection reagent includes specific RUNX2 primers, the sequence of which is: Forward Primer: 5'-CGCCTCACAAAACAACCACAG-3'; Reverse Primer: 5'-TCACTGTGCTGAAGAGGCTG-3'.

3. The application as described in claim 1, characterized in that, The application is based on an immunohistochemical detection method, and its detection reagent includes RUNX2 antibody, which is either MBL Anti-Runx2 antibody or Abcam recombinant Anti-RUNX1 / AML1+RUNX3+RUNX2 antibody.

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