CN116083567A

CN116083567A - Diagnostic reagent for lobular tumor of mammary gland and application thereof

Info

Publication number: CN116083567A
Application number: CN202211092794.XA
Authority: CN
Inventors: 聂燕; 宋尔卫; 雷蓉; 黄红颜; 贺诗施; 艾力菲热·伊力哈木
Original assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Current assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Priority date: 2022-09-08
Filing date: 2022-09-08
Publication date: 2023-05-09
Also published as: WO2024050860A1

Abstract

The invention relates to the technical field of medical biology, in particular to an application of RUNX2 serving as a marker in preparation of a reagent for diagnosing or detecting malignant lobular tumor of mammary gland. The reagent can assist conventional tissue biopsy according to the expression level of RUNX2 in different grading breast phylliform tumors, and can be used for diagnosis, differential diagnosis, grading and prognosis analysis of the breast phylliform tumors. The RUNX2 detection primer sequence and the detection antibody are important components of a diagnosis or detection reagent or kit, so that the detection of the malignant lobular tumor of the mammary gland is simpler, more convenient, quicker and more accurate, has good clinical application value, is favorable for further improving the diagnosis level of the malignant lobular tumor of the mammary gland, and is hopeful to become the first diagnosis and detection kit for the malignant lobular tumor of the mammary gland.

Description

Diagnostic reagent for lobular tumor of mammary gland and application thereof

Technical Field

The invention relates to the technical field of medical biology, in particular to a diagnostic reagent for breast phylliform tumor and application thereof.

Background

Lobular tumor (PT) of the breast is a rare fibrous epithelial tumor accounting for 0.3% to 1% of all breast tumors. The preoperative diagnosis of the lobular tumor of the breast, which is commonly used in clinic at present, comprises mammary gland color ultrasound, molybdenum target, mammary gland nuclear magnetic resonance, small needle puncture of a lump before operation, hollow needle puncture and the like, but the preoperative diagnosis rate of the lobular tumor is only about 76 percent by integrating all the diagnosis methods, and the postoperative pathological histological examination is the gold standard of diagnosis. Also, because of individual differences, lack of clear criteria and thresholds, diagnosis of foliar tumors based on morphology remains challenging. Thus, the pathohistological diagnosis of phylliform tumors is somewhat subjective and insufficient to predict the clinical prognosis of an individual patient.

According to the existing literature researches, the borderline and malignant leaf tumors grow fast, are easy to relapse locally and metastasize remotely, blood metastasis is the most common metastasis mode, and lung and bone are the most common metastasis sites. The recurrence rate of malignant lobular tumor of mammary gland is up to 53.1%, and the metastasis rate is up to 43.1%. Once recurrent, metastatic, patients die in a short period of time, with a mortality rate of approximately 16.3% for malignant leaf tumors. The treatment of the lobular tumor of the mammary gland is mainly surgical treatment, and the malignant lobular tumor has poor clinical prognosis due to the characteristics of high recurrence rate, high metastasis rate and the like. Unlike breast cancer, the auxiliary treatment means such as chemotherapy, radiotherapy and the like after breast malignant phylliform tumor operation have poor treatment effects, and no research report has clinical benefit in targeted treatment and immunotherapy at present. And because the breast phylliform tumor is rare, and lacks large-scale clinical data and basic research, the research and development of specific molecular markers for diagnosis, related diagnostic reagents and corresponding medicaments are clear and are deficient. Therefore, it is needed to find specific molecular markers of breast phylliform tumor, which has important functions for improving the preoperative diagnosis rate and improving the prognosis of patients.

RUNX2 is an important member of the RUNX family of transcription factors, which is named for its runt domain. The biological effect of RUNX2 is mainly used as a specific transcription factor for osteogenic differentiation, regulates and controls transcription of genes such as collagen type I, bone modulin, osteocalcin, col1a1, col1a2, bone Sialoprotein (BSP), fibronectin and the like, and plays an important role in osteoblast formation and differentiation, chondrocyte differentiation and maturation, osteoclast formation and absorption and bone matrix protein synthesis. However, there is no report that RUNX2 is associated with the biological behavior of malignant lobular tumors of the breast.

Disclosure of Invention

In order to overcome the technical problems, the invention discloses a technical application of RUNX2 as a biological behavior marker of malignant lobular tumor of mammary gland.

The inventors found in clinical samples and in vitro cell experiments that RUNX2 was low expressed in benign leaf tumor tissue, high expressed in malignant leaf tumor tissue, and mRNA expression level of RUNX2 in malignancy was about 25 times that of benign leaf tumor tissue. As the number of tumor recurrence increases, the expression level of RUNX2 in tissues increases simultaneously. The expression level of RUNX2mRNA in malignancy has a positive correlation with proliferation, migration and invasion capacity of breast malignant leaf tumor cells. RUNX2 has been shown to be useful as a marker for diagnosing or detecting malignant phylliform tumors of the breast. According to the expression level of RUNX2 in different classified breast phylliform tumors, the diagnosis, differential diagnosis, classification and prognosis analysis of the breast phylliform tumors can be carried out by assisting conventional tissue biopsy.

The following applications are thus disclosed:

the RUNX2 is used as a marker in the preparation of a reagent (including a kit) for diagnosing or detecting malignant lobular tumor of mammary gland.

Preferably, the application comprises: the RUNX2 is used as a marker in the preparation of early screening or prognosis prediction detection reagents for breast malignant phylliform tumors.

Preferably, the diagnostic or detection reagent comprises a specific RUNX2 primer or RUNX2 antibody.

The method of diagnosis or detection may be: detecting RUNX2 expression levels of breast malignant leaf tumor specimens in various biological states by qRT-PCR or immunohistochemical methods, establishing a detection model, obtaining standard data for diagnosis or detection, and judging according to the RUNX2 expression levels of the breast malignant leaf tumor specimens to be detected.

The use method of the diagnosis or detection reagent or the kit comprises the following steps: the RUNX2 expression level of the breast malignant leaf tumor specimen can be detected by qRT-PCR or immunohistochemical method, and the biological condition of the breast malignant leaf tumor specimen can be judged according to the RUNX2 expression level.

The preferred specific primer sequences for RUNX2 are:

forward Primer:5'-CGCCTCACAAACAACCACAG-3'; (sequence Listing SEQ ID NO. 1)

Reverse Primer:5'-TCACTGTGCTGAAGAGGCTG-3'; (sequence Listing SEQ ID NO. 2)

Preferred antibodies to RUNX2 are: MBL Anti-Runx2#D130-3, abcam recombinant Anti-RUNX1/AML1+RUNX3+RUNX2 antibody [ EPR3099] #ab92336.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention discloses that the overexpression of the breast malignant phyllopoietic tumor specimen RUNX2 is obviously related to the biological behavior of the breast malignant phyllopoietic tumor for the first time, can be used as a marker for diagnosing and detecting the breast malignant phyllopoietic tumor and screening medicaments for resisting the breast malignant phyllopoietic tumor, and has higher clinical application value in aspects of early screening diagnosis, prognosis prediction, screening targeted medicaments for resisting the tumor and the like.

2. The test result of the invention proves that RUNX2 is low-expressed in benign leaf tumor tissues, high-expressed in malignant leaf tumor tissues, and the expression level of RUNX2mRNA in malignant is about 25 times that of benign leaf tumor tissues. The difference is very obvious, and the RUNX2 has strong specificity as a marker, so that a powerful theoretical support is provided for the feasibility and the accuracy of the RUNX2 marker related diagnostic detection reagent or the kit.

3. The RUNX2 detection primer sequence and the detection antibody can be used for important components of an RUNX2 detection kit, so that the detection of the malignant lobular tumor of the mammary gland is simpler, more convenient, quicker and more accurate, has good clinical application value, is favorable for further improving the diagnosis level of the malignant lobular tumor of the mammary gland, and is expected to become the first diagnosis detection kit for the malignant lobular tumor of the mammary gland.

Drawings

FIG. 1:A is a bar graph of mRNA expression levels of RUNX2 in benign and malignant breast phylliform tumor tissues; b: is a western blotting gel imaging chart of RUNX2 protein expression test in benign and malignant breast phylliform tumor tissues; c: is a graphical representation of the RUNX2 expression levels in paraffin sections of benign and malignant leaf tumors and in paraffin sections of tumor tissue from multiple recurrences of the same patient.

Fig. 2: a is RUNX2 gene of a breast malignant phylliform tumor cell line SYSH-MPT-01 (namely a cell line HJP-0320, disclosed in a patent application with publication number CN 111019898A), and a cell proliferation capacity change test result diagram; b knocking down RUNX2 gene of a breast malignant phylliform tumor cell line SYSH-MPT-01, and carrying out cell clone formation capacity change test result illustration; c is a diagram of the result of a cell cycle change test of knocking down RUNX2 gene of a breast malignant phylliform tumor cell line SYSH-MPT-01; d is a diagram of the result of a test for knocking down RUNX2 gene of a breast malignant phylliform tumor cell line SYSH-MPT-01 and changing cell migration and invasion capacity; e is a diagram of the result of an experiment for knocking down RUNX2 gene of a breast malignant phylliform tumor cell line SYSH-MPT-01 and changing cell collagen contraction capacity.

Fig. 3: a is a graphical representation of the results of a cell proliferation potency change assay for overexpressing RUNX2 in a breast benign phylliform tumor cell line SYSH-BPT-01 (i.e., cell line GLK-1010, disclosed in patent application publication No. CN 111019897A); b is a graph showing the result of a test for the change of the clonogenic capacity of cells by over-expressing RUNX2 in a breast benign phylliform tumor cell line SYSH-BPT-01; c is a graphical representation of the results of an assay for over-expression of RUNX2 in a breast benign phylliform tumor cell line SYSH-BPT-01, and for a change in cell cycle; d is a graph showing the result of an experiment for over-expressing RUNX2 in a breast benign phylliform tumor cell line SYSH-BPT-01 and changing the cell migration and invasion capacities; e is a graphical representation of the results of an assay for the altered collagenous contractile capacity of cells over-expressing RUNX2 in the breast benign phyllostatic tumor cell line SYSH-BPT-01.

Fig. 4: the result of the RUNX2 prognostic influence test on patients with breast phylliform tumors is shown in the figure.

Fig. 5: RUNX2 of a malignant leaf tumor cell line SYSH-MPT-01 is knocked down, and the test result is shown on subcutaneous tumorigenicity and tumor growth; a is the change of tumor volume at the corresponding time point, and B is the tumor picture of each group.

Detailed Description

The invention is further illustrated below with reference to examples.

Example 1: analysis of RUNX2 expression Profile of mammary tumor

1. And (3) carrying out fluorescent quantitative PCR by using RUNX2 specific primers, and detecting RUNX2mRNA expression levels in different types of breast phylliform tumor tissues.

(1) 3 benign foliate tumor tissue specimens and 10 malignant foliate tumor tissue specimens were respectively selected. The experiment was performed as follows: the tissues were crushed with a freeze grinder, added to Trizol lysate, transferred to a 1.5ml ep tube with a pipette, and repeatedly blown or shaken to lyse the cells. Standing at room temperature for 5min, adding 0.2ml chloroform per 1ml Trizol lysate, shaking vigorously for 15s, standing at room temperature for 2-3min, centrifuging at 12000x g at 4deg.C for 15min, collecting upper water phase into new EP tube, adding equal volume isopropanol, and precipitating RNA; centrifuging 12000x g at 4 ℃ for 10min; adding 75% ethanol for cleaning, centrifuging at 7500 Xg at 4deg.C for 5min, and discarding supernatant; and (3) airing RNA precipitate in an ultra-static table at room temperature, dissolving the RNA precipitate in a proper amount of RNase-free water, and measuring the concentration and purity of RNA. Reverse transcription synthesis of cDNA: reverse transcription synthesis of cDNA: 1. Mu.g of template RNA, 4. Mu.l of reverse transcriptase SuperScript II mix (containing Buffer, dNTP, hiScript II reverse transcriptase, RNase, random primers/Oligo dT) was added to the PCR tube, RNase-free water was added to 20. Mu.l, the mixture was left on ice for 5min at 65℃and then 5 Xbuffer 8. Mu.l and 0.1M DTT 2. Mu.l were added thereto, and ddH2O was added to 40. Mu.l after mixing, the reaction conditions: storing at 50deg.C for 15min, 85deg.C for 15s, and 4deg.C.

Real-time quantitative PCR amplification: the template cDNA was diluted 3-fold and mixed well for use. Each experimental group was provided with 3 parallel tubes. The reaction system: 1 μl of cDNA, 5 μl of SYBR green dye, 0.3 μl of forward primer, 0.3 μl of reverse primer, and ddH ₂ O3.4. Mu.l, and the mixture was centrifuged and homogenized. Reaction conditions: 95℃for 5min,95℃for 30 seconds, 55℃to 60℃for 30s (depending on the annealing temperature), 40 cycles total. The relative transcript levels of the genes were detected using GAPDH as an internal control assay. The primer sequences were as follows:

RUNX2 Forward:5'-CGCCTCACAAACAACCACAG-3'; (sequence Listing SEQ ID NO. 1)

RUNX2 Reverse:5'-TCACTGTGCTGAAGAGGCTG-3'. (sequence Listing SEQ ID NO. 2)

Relative mRNA expression levels were quantitatively analyzed. Statistical data mean values were calculated from the results of three replicates, with significant differences determined by T-test, defined as statistical differences at P < 0.05.

(2) Experimental results: as shown in FIG. 1 (A), RUNX2 was low-expressed in benign leaf tumor tissue, high-expressed in malignant leaf tumor tissue, and the expression level of RUNX2mRNA in malignancy was about 25-fold that in benign leaf tumor tissue.

2. And detecting RUNX2 protein expression levels in different types of leaf tumor tissues by adopting a Western blot (Western blot) experiment.

(1) The experimental method comprises the following steps: after SDS-PAGE electrophoresis, membrane transfer and blocking, RUNX2 specific antibodies are used as primary antibodies for incubation, and gel imaging analysis is carried out after further horseradish peroxidase-labeled secondary antibodies are used for incubation.

(2) As shown in FIG. 1 (B), the RUNX2 protein was expressed in 10 cases of malignant foliar tumor tissues at a high level, whereas RUNX2 protein was not expressed or was expressed at a low level in 3 cases of benign foliar tumor tissues.

3. Immunohistochemical staining was performed using antibodies specific for RUNX2 to assess RUNX2 protein levels in benign breast malignant foliar tumor tissues.

(1) The experimental method comprises the following steps: the pathological sections adopted in the experiment are all from clinically definite diagnosis cases. The paraffin-embedded tissue immunohistochemical staining was performed as follows: roasting at 60 ℃, and dewaxing by preheating dimethylbenzene twice for 5min each time; the dewaxed slice is hydrated by 100% -95% -80% -70% -50% ethanol gradient and distilled water, and each gradient is placed for 5min; autoclaving with 0.01M (pH 6.0) citric acid buffer solution for 10min, naturally cooling, and washing with PBS for three times each for 5min; treating with 0.3% hydrogen peroxide solution for 30min to eliminate endogenous peroxidase activity; PBS is washed for three times, each time for 5min; blocking 10% sheep serum at 37deg.C for 1 hr; dropping sheep 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 horseradish peroxidase labeled streptavidin, incubating for 20min at 37 ℃, and washing with PBS for three times; dripping DAB color development liquid, developing for 2min at room temperature in dark, and washing off excessive color development liquid by PBS; counterstaining with hematoxylin and washing with distilled water; gradient step-by-step dehydration is carried out on 50% -70% -80% -90% -100% ethanol, and each gradient is 5min; a neutral resin sealing piece; a microscopic imaging system takes a picture.

(2) The experimental results are shown in FIG. 1 (C): RUNX2 is highly expressed in breast malignant leaf tumor cells and is poorly expressed in benign medium (C). As the number of tumor recurrence increases, the expression level of RUNX2 in the tissue increases simultaneously (C).

Example 2: effects of knock-down RUNX2 on proliferation, migration, invasion, cell cycle and collagen contractility of malignant leaf tumor cells

1. Effect of knockdown RUNX2 on proliferation of malignant leaf tumor cells

(1) The experimental method comprises the following steps: inoculating malignant leaf tumor cells in a 96-well plate and a 6-well plate, transiently transfecting shRNA (short hairpin ribonucleic acid) by adopting 1ipo3000, inoculating control group cells with RUNX2 expression knocked down and gene knockdown not to be knocked down into breast malignant leaf tumor cells, testing cell survival rates at different time points (1-4 days) by adopting a CCK8 method, and drawing a cell proliferation curve. Cells of the control group, which had knocked down RUNX2 expression and did not undergo gene knockdown, were inoculated into complete medium in 60mm dishes of 200 cells per dish, and after culturing for 14 days, the number of clone formations was counted.

The shRNA sequence is as follows:

shRUNX2 ^#1 GGACGAGGCAAGAGTTTCA; (sequence Listing SEQ ID NO. 3)

shRUNX2 ^#2 CCAAATTTGCCTAACCAGA. (sequence Listing SEQ ID NO. 4)

(2) The experimental results are shown in FIG. 2 (A-B).

2. Effect of knockdown RUNX2 on malignant leaf tumor cell cycle

(1) The experimental method comprises the following steps: knocking down the expression and control cells, culturing for 24h, collecting cells, washing the cells with precooled PBS for 2-3 times, centrifuging (1500 rpm,4 min), discarding supernatant, adding a small amount of PBS into the precipitate, resuspending the cells, adding the resuspension cells into 70% ice ethanol precooled at 4 ℃ for fixation, sealing with a sealing film, and standing at 4 ℃ for overnight. The supernatant was removed by centrifugation twice with PBS (2000 rpm for 4 min). 100ul 100ug/ml RNase A and 0.2% Triton X-100 were added to resuspend cells. 400ul 50ug/ml PI was added, vortexed and mixed well, incubated at room temperature in the dark for 30min. Flow cytometry detects cell cycle, typically counting 10 ten thousand cells, detecting red fluorescence at excitation wavelength 488nm, then analyzing the cell cycle phase distribution with FlowJo software, using FL2-w and FL2-a to show that adherent cells are removed.

(2) Experimental results: cells following the RUNX2 knockdown were arrested in G1 phase as shown in fig. 2 (C).

3. Effect of knockdown RUNX2 on malignant foliar tumor cell migration and invasiveness

(1) The experimental method comprises the following steps: knock down of RUNX2 expression and control cells to 2X10 ⁴ Density of wells/wells inoculated in serum-free medium in the upper chamber of a Transwell plate (Costar) or in the upper culture chamber of a pre-laid Matrigel sheet, the lower layer being complete medium containing 16% fetal bovine serum, three replicate wells being provided per group; after incubation in an incubator at 37℃for 8 hours or 24 hours, respectively, the cells in the upper layer of the membrane were carefully wiped off by fixing with 4% paraformaldehyde for 15 minutes, and the cells in the lower chamber were stained with 0.5% crystal violet, and the number of cells that had migrated or invaded was counted under a microscope.

(2) Experimental results; as shown in fig. 2 (D), RUNX2 knock-down expression sets showed a significant decrease in cell migration and invasion capacity.

4. Effect of knockdown RUNX2 on collagen contractility of malignant phylliform tumor

(1) The experimental method comprises the following steps: diluting type I rat tail collagen with DMEM medium containing acetic acid and NaOH, mixing the foliate tumor cell suspension with diluted rat tail collagen in a ratio of 1:1, inoculating into a 24-well plate, adding a serum-containing medium for 4 hours, replacing the serum-free DMEM medium for overnight incubation, separating the gel from the wall of the well, and observing and measuring the diameter after shrinkage after 8 hours.

(2) The experimental results are shown in FIG. 2 (E).

Example 3 Effect of over-expressed RUNX2 on the proliferation, migration, invasion, cell cycle and collagen contractility of leaf tumor cells

1. Effect of over-expression of RUNX2 on proliferation of benign breast phylliform tumor cells

(1) The experimental method comprises inoculating primary benign leaf tumor cells into 96-well plates and 6-well plates, transfecting RUNX2 overexpression plasmid (4 μg/well) with transfection reagents lipo3000 and p3000, changing normal complete medium after 4-6 hours of transfection, collecting cell extract RNA sample, and detecting RUNX2 overexpression level. Cells of the control group which are over-expressed RUNX2 and not treated are inoculated, the survival rate of the cells at different time points (1-4 days) is tested by adopting a CCK8 method, and a cell proliferation curve is drawn. The cells of the control group, which had overexpressed RUNX2 and had not been treated, were inoculated into 60mm medium, 200 cells per dish, and after 14 days of culture, the number of clone formations was counted.

(2) The experimental results are shown in FIG. 3 (A-B).

2. Effect of over-expression of RUNX2 on benign phylliform tumor cell cycle

(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 (1500 rpm,4 min), discarding supernatant, adding a small amount of PBS into the precipitate, resuspending the cells, adding the resuspending cells into precooled 70% ice ethanol at 4deg.C for fixation, sealing with a sealing film, and standing overnight at 4deg.C. The supernatant was removed by centrifugation twice with PBS (2000 rpm for 4 min). 100ul 100ug/ml RNase A and 0.2% Triton X-100 were added to resuspend cells. 400ul 50ug/ml PI was added, vortexed and mixed well, incubated at room temperature in the dark for 30min. Flow cytometry detects cell cycle, typically counting 10 ten thousand cells, detecting red fluorescence at excitation wavelength 488nm, then analyzing the cell cycle phase distribution with FlowJo software, using FL2-w and FL2-a to show that adherent cells are removed.

(2) Experimental results: cells after overexpressing RUNX2 were active in S phase as shown in fig. 3 (C).

3. Effect of over-expression of RUNX2 on migration and invasion capacity of benign lobular tumor cells of mammary glands

(1) The experimental method comprises the following steps: the cells of the untreated control group which overexpressed RUNX2 were expressed at 1 x10 ⁴ Density seeding of wellsIn a Transwell plate (Costar) upper cell serum-free medium or a pre-laid Matrigel thin layer upper cell, the lower layer is a complete medium containing 16% fetal bovine serum, and three duplicate wells are arranged 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 were carefully wiped off, the cells in the lower chamber were stained with crystal violet, and the number of cells that had migrated or invaded was counted under a microscope.

(2) The experimental results are shown in FIG. 3 (D).

4. Effect of over-expressed RUNX2 on collagen contractility of malignant phylliform tumor

(2) The experimental results are shown in FIG. 3 (E).

EXAMPLE 4 influence of RUNX2 on prognosis of patients with lobular breast tumors

Effect of runx2 on prognosis of patients with lobular breast tumors

(1) The experimental method comprises the following steps: fresh frozen sections of 237 patients with mammary gland phylliform tumor are taken for immunohistochemical staining, the sections are respectively read by two pathologists, and are divided into RUNX2 high expression groups and RUNX2 low expression groups according to the staining intensity and the staining positive proportion, and the influence of RUNX2 expression and total survival OS and the influence of non-progressive survival DFS are evaluated.

(2) Experimental results: as shown in fig. 4 (a-B), the OS and DFS of the RUNX2 high expression group were significantly lower than those of the RUNX2 low expression group.

Example 5 knock down of the RUNX2 Gene affects tumor growth in mice

1. Effects of knock-down of the RUNX2 Gene on inhibition of mouse subcutaneous transplantation tumor formation and growth

(1) The experimental method comprises the following steps: constructing a mammary gland malignant leaf tumor subcutaneous transplantation tumor model: taking malignant leaf tumor cells, stably knocking down RUNX2 leaf tumor cells, and diluting to 5x10 ⁷ 100 μl/ml was taken and mixedThe homogenized cell suspension was inoculated in control mice and experimental mice at the left anterior axillary subcutaneous breast fat pad, respectively, and tumor long and short diameters were measured every 4 days, according to the formula v=1/2 (l×w ² ) The average volume of the transplanted tumors was calculated. Mice were sacrificed when the control tumor volume reached 1.5cm in diameter and tumor growth curves were drawn.

(2) Experimental results: as shown in FIG. 5 (A-B), tumor formation and growth of mice inoculated with the stable knockdown RUNX2 leaf tumor cells were significantly inhibited compared to the control group.

In conclusion, RUNX2 is identified as a specific molecular marker of the malignant lobular tumor of the breast, and the marker is highly expressed in cells of the malignant lobular tumor of the breast, so that the progress of the malignant lobular tumor of the breast is promoted. The expression of the RUNX2 can inhibit proliferation, migration and invasion of malignant phylliform tumor of breast. RUNX2 is used as a breast malignant phylliform tumor marker and has remarkable clinical application value in the development of breast phylliform tumor auxiliary diagnosis.

Claims

Application of RUNX2 as a marker in preparation of a reagent for diagnosing or detecting malignant lobular tumor of mammary gland.
2. The application of claim 1, wherein the application comprises:

(1) Application of RUNX2 serving as a marker in preparation of early screening detection reagent for breast malignant phylliform tumor;

(2) Application of RUNX2 as a marker in preparation of a breast malignant phylliform tumor prognosis prediction reagent.
3. The use according to claim 1 or 2, wherein the diagnostic or detection reagent comprises a specific RUNX2 primer or RUNX2 antibody.
4. The use according to claim 3, wherein the diagnostic or detection reagent comprises the following RUNX2 primer, the sequence of which is:

Forward Primer：5’-CGCCTCACAAACAACCACAG-3’；

Reverse Primer：5’-TCACTGTGCTGAAGAGGCTG-3’。
5. the use according to claim 3, wherein the diagnostic or detection reagent comprises the following RUNX2 antibodies: MBL Anti-Runx2#D130-3, abcam recombinant Anti-RUNX1/AML1+RUNX3+RUNX2 antibody [ EPR3099] #ab92336.
6. The use according to claim 1, wherein the diagnostic or detection reagent is used in the following way: RUNX2 expression levels of breast malignant foliar tumor specimens were detected using qRT-PCR or immunohistochemical methods.