CN113929764B - Breast phylliform tumor molecular marker CD146 and application thereof - Google Patents

Breast phylliform tumor molecular marker CD146 and application thereof Download PDF

Info

Publication number
CN113929764B
CN113929764B CN202111200916.8A CN202111200916A CN113929764B CN 113929764 B CN113929764 B CN 113929764B CN 202111200916 A CN202111200916 A CN 202111200916A CN 113929764 B CN113929764 B CN 113929764B
Authority
CN
China
Prior art keywords
tumor
breast
leu
glu
val
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111200916.8A
Other languages
Chinese (zh)
Other versions
CN113929764A (en
Inventor
宋尔卫
阎锡蕴
刘丹
聂燕
陈洁雯
徐庆喆
陈雪晖
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Biophysics of CAS
Sun Yat Sen Memorial Hospital Sun Yat Sen University
Original Assignee
Institute of Biophysics of CAS
Sun Yat Sen Memorial Hospital Sun Yat Sen University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Biophysics of CAS, Sun Yat Sen Memorial Hospital Sun Yat Sen University filed Critical Institute of Biophysics of CAS
Priority to CN202111200916.8A priority Critical patent/CN113929764B/en
Publication of CN113929764A publication Critical patent/CN113929764A/en
Application granted granted Critical
Publication of CN113929764B publication Critical patent/CN113929764B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/6854—Immunoglobulins

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Food Science & Technology (AREA)
  • Hospice & Palliative Care (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Toxicology (AREA)
  • General Engineering & Computer Science (AREA)

Abstract

The invention discloses a breast phylliform tumor molecular marker CD146 and application thereof, and relates to the technical field of tumor molecular biology. The breast phylliform tumor molecular marker CD146 provided by the invention comprises a CD146 gene with the sequence homology of more than 80% with SEQ ID NO.1 or a CD146 protein with the sequence homology of more than 80% with SEQ ID NO.2 or SEQ ID NO. 3. The differential diagnosis, grading and prognosis prediction analysis of the lobular tumor of the mammary gland can be assisted by detecting the expression level of the lobular tumor CD 146. Aiming at a CD146 target, the expression level of the CD146 or a signal path mediated by the CD146 is regulated and controlled by an inhibitory antibody or a small molecular medicine, so that the growth, migration and malignant progress of the breast phylliform tumor cells can be effectively inhibited, and a new way is provided for diagnosis and treatment of the breast phylliform tumor.

Description

Breast phylliform tumor molecular marker CD146 and application thereof
Technical Field
The invention relates to the technical field of tumor molecular biology, in particular to a breast phylliform tumor molecular marker CD146 and application thereof.
Background
Lobular tumor (PT) of the breast is a rare fibroepithelial tumor with a incidence of less than 1% of breast tumors. World health organization (World Health Organization, WHO) classifies lobal tumors as benign, borderline and malignant based on their histological properties including mesenchymal cell number, mesenchymal cell allotype, interstitial hyperproliferation and tumor boundary characteristics [2]. Currently, imaging examinations: breast ultrasound and molybdenum targets have limited accuracy. Pre-operative needle biopsies are difficult to accurately histological grade due to the presence of heterogeneity in different parts of the tumor. Therefore, the gold standard of current diagnosis is still a post-operative histopathological examination. However, phylliform tumors exhibit a morphological continuity from benign to malignant. At present, the cutoff value of the pathological diagnosis variable still does not reach effective consensus. Therefore, the pathological diagnosis classification of phylliform tumors is relatively lacking in objectivity. The existing molecular markers are also of limited value in diagnosing and predicting tumor biological behavior. The leaf tumor is easy to relapse, and the borderline and malignant leaf tumor can also generate blood circulation metastasis. The treatment effect of chemotherapy and radiotherapy on the foliar tumor is not exact, and the treatment method capable of reducing the recurrence rate and the metastasis rate of the foliar tumor is only expanded surgical excision, but even if the treatment method is expanded surgical excision, the local recurrence rate of malignant foliar tumor is still up to 28-67%, the metastasis rate is up to 21-30%, and the death rate is up to 88% in the case of distant metastasis.
CD146 (MCAM), which belongs to the immunoglobulin superfamily, is a cell surface type I transmembrane protein, first discovered and cloned from melanoma by Johnson team in 1987, and is therefore also known as melanoma adhesion molecule (Mel-CAM). It is mainly expressed in neovascular endothelium to promote angiogenesis. And is highly expressed in partial normal and malignant tumor tissues, such as melanoma, triple negative breast cancer, osteosarcoma, etc. High expression of CD146 in melanoma promotes melanoma growth and metastasis. In breast cancer, CD146 is involved in the epithelial-mesenchymal transition (EMT) process, and has the effect of promoting tumor invasion and metastasis. In osteosarcoma, the expression of CD146 promotes tumor development and metastasis. In addition, it has been reported that CD146 is also expressed on mesenchymal stem cells, and participates in differentiation, migration, and the like of mesenchymal stem cells.
Unlike breast cancer with higher incidence, the development and mechanism of breast phylliform tumor are rarely studied at present, and the development of related diagnostic reagents and corresponding medicaments is less due to the lack of specific markers. Searching molecular markers of breast phylliform tumors is particularly important for clinical diagnosis and accurate treatment of the tumors.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a breast phylliform tumor molecular marker CD146 and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a breast foliar tumour molecular marker CD146 comprising a CD146 gene having 80% or more homology to the sequence shown in SEQ ID No.1 or a CD146 protein having 80% or more homology to the sequence shown in SEQ ID No.2 or SEQ ID No. 3.
The invention also provides a primer group for diagnosing the breast phylliform tumor, which is a primer designed based on the breast phylliform tumor molecular marker CD 146.
As a preferred embodiment of the primer set for diagnosis of breast phylliform tumor according to the present invention, the primer set comprises primer CD146-F and primer CD146-R; the nucleotide sequence of the primer CD146-F is shown as SEQ ID NO. 4; the nucleotide sequence of the primer CD146-R is shown as SEQ ID NO. 5.
The invention also provides application of the molecular marker CD146 of the breast phylliform tumor as a molecular marker in preparation of a reagent for diagnosing breast phylliform tumor.
The inventor of the application finds that the expression rule of CD146 in breast phylliform tumors with different histological grades through a large amount of experimental researches: low expression in the tissue beside the breast phylliform tumor and the interstitial cells of benign breast phylliform tumor tissue, medium expression in the interstitial cells of boundary phylliform tumor and high expression in malignant phylliform tumor tissue. In malignant breast lobular tumor tissue, more than 70% of the stromal tumor cells are CD146 positive, whereas in benign tissue, the proportion of CD146 positive stromal cells is less than 25%. In addition, the expression level of CD146 was lower on fibroadenoma parenchymal cells. Therefore, through pathological detection of the expression level of CD146, differential diagnosis, classification and prognosis of the lobular tumor of the breast can be assisted, and the CD146 serving as a molecular marker can be applied to preparation of reagents for diagnosing the lobular tumor of the breast.
As a preferred embodiment of the application of the molecular marker CD146 of the lobular tumor of the breast as a molecular marker in preparing a reagent for diagnosing lobular tumor of the breast, the reagent comprises an immunohistochemical pathological detection, a molecular biological method, a gene chip or a protein chip for detecting the expression level of the molecular marker CD146 of lobular tumor of the breast so as to diagnose lobular tumor of the breast.
The invention also provides application of the molecular marker CD146 of the lobular breast tumor as a molecular marker in preparing a kit for diagnosing lobular breast tumor, monitoring curative effect of lobular breast tumor or prognosis evaluation of lobular breast tumor.
As a preferred embodiment of the application of the molecular marker CD146 of the lobular mammary tumor in preparing a kit for diagnosing lobular mammary tumor, monitoring curative effect of lobular mammary tumor or prognosis evaluation of lobular mammary tumor, the kit comprises the primer group and an antibody for detecting CD146 protein at tissue level.
The invention also provides application of the molecular marker CD146 of the breast phylliform tumor in preparing medicaments for treating breast phylliform tumor.
The inventor of the application finds that aiming at a CD146 target, the growth, migration and malignant progress of the breast phylliform tumor cells can be effectively inhibited by regulating and controlling the expression level of the CD146 or a signal path mediated by the CD146 through inhibitory antibodies or small molecular drugs and the like, and a new thought and a new method are provided for treating the breast phylliform tumor.
As a preferred embodiment of the application of the molecular marker CD146 for breast phylliform tumor in preparing a medicament for treating breast phylliform tumor, the medicament comprises a medicament for inhibiting proliferation of breast phylliform tumor cells, a medicament for inhibiting invasion and metastasis of breast phylliform tumor, a medicament for inhibiting expression of CD146mRNA, a medicament for inhibiting expression of CD146 protein, a medicament for inhibiting activity of CD146 protein or a medicament for inhibiting CD146 signal transduction.
As a preferred embodiment of the use of the molecular marker CD146 for breast phylliform tumor in the preparation of a medicament for treating breast phylliform tumor, the medicament comprises a nucleic acid medicament, an antibody medicament, a small molecule chemical medicament or a cell therapeutic medicament; the antibody drug includes a monoclonal antibody drug or an antibody conjugated drug.
The invention has the beneficial effects that: the invention provides a CD146 as a molecular marker of breast phylliform tumor, the expression of which is closely related to the malignant progress such as growth, metastasis and the like of the phylliform tumor, and the differential diagnosis, grading and prognosis prediction analysis of the breast phylliform tumor can be assisted by detecting the expression level of the CD146 of the phylliform tumor. Aiming at a CD146 target, the expression level of the CD146 or a signal path mediated by the CD146 is regulated and controlled by an inhibitory antibody or a small molecular drug, so that the growth, migration and malignant progress of the breast phylliform tumor cells can be effectively inhibited, and a new thought and a new method are provided for targeted treatment of the breast phylliform tumor.
Drawings
Fig. 1: a: results of expression level of CD146 in breast phylliform tumor cells and tissues; b: results of CD146mRNA expression levels in benign, borderline, and malignant breast phylliform tumor specimens; c: the level of CD146 protein expression in benign and malignant breast phylliform tumor specimens; d: horizontal flow analysis of CD146 expression on the surface of benign and malignant mammary gland leaf tumor cell line.
Fig. 2: a: results of CD146mRNA expression levels in malignant breast lobular tumor cells knocked down the CD146 gene; b: malignant breast lobular tumor cell proliferation with knockdown of CD146 gene; c: clone formation of malignant breast lobular tumor cells with knockdown CD146 gene; d: malignant breast phylliform tumor cell migration and invasion conditions of the knockdown CD146 gene; e: the collagen contraction of malignant breast lobular tumor cells knocked down by CD146 gene.
Fig. 3: a: results of CD146mRNA expression levels in benign breast lobular tumor cells over-expressing CD 146; b: over-expressing CD146 benign breast phylliform tumor cell proliferation; c: clone formation of benign breast lobular tumor cells overexpressing CD 146; d: over-expressing CD146 benign breast phylliform tumor cell migration and invasion; e: over-expressing CD146 benign breast phylliform tumor cell collagen contraction.
Fig. 4: a: influence of CD146 specific antibodies on proliferation of malignant breast lobular tumor cells; b: influence of CD146 specific antibodies on malignant breast phylliform tumor cell migration and invasion; c: effect of CD146 specific antibodies on breast phylliform tumor cell organogenesis.
Fig. 5: a: effect of CD146 specific antibodies on tumor growth in breast phylliform tumor PDX model; b: effect of CD146 specific antibodies on tumor volume of breast phylliform tumor PDX model.
Fig. 6: a: enrichment analysis results of CD146 related signal paths in breast phylliform tumor cells; b: the effect of cell line overexpression or knockdown of CD146 on PI3K/AKT signaling pathway levels; c: results of phosphorylated AKT levels in tumor tissue of PDX model of CD146 antibody injection group; d: effect of CD146 knockdown and overexpression on the level of DCBLD2 and AKT signaling in malignant breast phylliform tumor cell lines; e: effects of proteasome inhibitor MG132 treatment on CD146 knockdown cell DCBLD2 levels.
Detailed Description
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Example 1 analysis of CD146 expression Spectroscopy at the tissue and cellular level of mammary gland phylliform tumor
1. In this example, immunohistochemical staining was performed using antibodies specific for CD146, and CD146 protein levels in fibroadenoma and breast lobular tumor tissues were assessed.
(1) The experimental method comprises the following steps: the pathological sections adopted in the experiment are all from 73 clinically diagnosed breast phylliform tumor cases. The paraffin-embedded tissue immunohistochemical staining was performed as follows: roasting at 60 ℃ to dewax the xylene twice by preheating 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; and (5) sealing the neutral resin, and photographing in a microscopic imaging system.
(2) The experimental results are shown in FIG. 1A. As can be seen from fig. 1A, CD146 is low-expressed in the tissue beside the breast phyllostachys tumor and in the interstitial cells of benign breast phyllostachys tumor tissue, is moderately expressed in the interstitial cells of borderline phyllostachys tumor, and is highly expressed in the malignant phyllostachys tumor tissue. In malignant breast lobular tumor tissue, more than 70% of the stromal tumor cells are CD146 positive, whereas in benign tissue, the proportion of CD146 positive stromal cells is less than 25%. In addition, the expression level of CD146 was lower on fibroadenoma parenchymal cells. From this, it was found that the expression level of CD146 protein or mRNA on mesenchymal cells of leaf tumor tissue correlated positively with the malignancy of leaf tumor.
2. Detection of CD146mRNA expression level in different types of mammary gland phylliform tumor tissue specimens by adopting fluorescent quantitative PCR
(1) The experimental method comprises the following steps: a. 3 benign foliar tumor tissue specimens, 1 borderline foliar tumor specimen, and 8 malignant foliar tumor tissues were selected respectively. b. Extracting tumor tissue total mRNA: grinding tumor tissues, adding Trizol lysate, repeatedly blowing or oscillating to lyse cells, standing at room temperature for 5min, adding 0.2ml chloroform per 1ml Trizol lysate, shaking vigorously for 15s, standing at room temperature for 2-3 min, centrifuging at 4deg.C for 15min at 12000 Xg, collecting upper water phase into new EP tube, and adding equal volume isopropanol to precipitate RNA; centrifuging at 12000 Xg for 10min at 4deg.C; adding 75% ethanol, cleaning, centrifuging at 4deg.C, 7500 Xg for 15min, and discarding supernatant; and (3) airing RNA precipitate in an ultra-static table at room temperature, dissolving in a proper amount of RNase-free water, and measuring the concentration and purity of RNA. c. Reverse transcription synthesis of cDNA: reverse transcription synthesis of cDNA: adding template RNA1 μg,4 μl of reverse transcriptase SuperScript II mix (containing Buffer, dNTP, hiScript II reverse transcriptase, RNase, random primers/Oligo dT) into PCR tube, adding RNase-free water to 20 μl, reacting at 65deg.C for 5min, adding 8 μl of 5 Xbuffer and 2 μl of 0.1MDTT after standing on ice for 5min, mixing, and adding ddH 2 O to 40. Mu.l, 15min at 50℃for 15s at 85℃and 4 ℃. d. 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 was 1. Mu.l of cDNA, 5. Mu.l of SYBR green dye, 0.3. Mu.l of forward primer, 0.3. Mu.l of reverse primer, and ddH 2 O3.4. Mu.l, and the mixture was centrifuged and homogenized. The reaction is carried out for 5min at 95 ℃, 30s at 95 ℃ and 30s at 55-60 ℃ for 40 cycles. e. The relative transcript levels of the genes were detected using GAPDH as an internal control assay. The primer sequences were as follows: GAPDH-Forward, 5'-ATCACCATCTTCCAGGAGCGA-3'; GAPDH-Reverse 5' -CCTTCTCCATGGTGG TGAAGAC-3'. Relative mRNA expression levels were quantitatively analyzed. Statistical data average value is calculated according to the three repeated experiment results, the significance difference is judged by T-test, and P is used<0.05 was defined as statistically different.
(2) The experimental results are shown in FIG. 1B. As can be seen from FIG. 1B, the expression level of CD146mRNA in benign leaf tumor tissue is low, the expression level of borderline tumor tissue is benign and the expression level of CD146mRNA in malignant leaf tumor tissue is at least 2 times that of borderline tumor tissue.
3. Protein immunoblotting (Western blot) experiments detect the expression level of CD146 protein in different types of leaf tumor tissues.
(1) The experimental method comprises the following steps: after SDS-PAGE electrophoresis, membrane transfer and blocking, the gel is subjected to gel imaging analysis after incubation by using a CD146 specific antibody (AA 98) as a primary antibody and further using a horseradish peroxidase-labeled secondary antibody.
(2) The experimental results are shown in FIG. 1C. As can be seen from FIG. 1C, the CD146 protein expression level was high in 6 cases of malignant foliar tumor tissues, whereas the CD146 protein was not expressed or was expressed low in 3 cases of benign foliar tumor tissues.
4. And detecting and separating the leaf tumor tissue by a flow cytometry to obtain the primary cell strain surface marker.
(1) The experimental method comprises the following steps: benign and malignant breast phylliform tumor cells are collected, PE-labeled anti-CD 146 antibodies and isotype controls are incubated, and flow cytometry is used for detection.
(2) The experimental results are shown in FIG. 1D. As can be seen from the expression level histogram of fig. 1D, the expression of CD146 on the surface of malignant foliar tumor cell line was significantly higher than that of benign tumor cell line.
Example 2
The present example analyzes the correlation between the expression level of CD146 in clinical mammary gland phylliform tumor and clinical pathological index.
(1) The experimental method comprises the following steps: the 203 patients diagnosed with the lobular tumor of the breast in example 1 were used as experimental subjects, and the gender, tumor grade, tumor size, local recurrence, metastasis, mitosis and mesenchymal hyperplasia of the 203 patients were counted.
(2) The analysis results are shown in Table 1.
TABLE 1
The results show that the high expression of CD146 (SI > 4) has significant correlation with the malignant classification of phylliform tumor (P < 0.001); in addition, the local recurrence rate of patients with high expression of CD146 (SI.gtoreq.4) is significantly higher than that of patients with low expression of CD146 (SI < 4) (P < 0.01); more patients with high CD146 expression than patients with low CD146 expression (p= 0.0811) were present in the case of metastasis. It can be seen that the high expression of CD146 is closely related to pathological grading and prognosis of lobular tumor of mammary gland.
Example 3 Effect of knockdown of CD146 Gene on proliferation, clonogenic, migration, invasion and collagen contractility of malignant mammary gland leaf tumor cells
1. Effect of knockdown CD146 gene on proliferation and clonogenic formation of malignant breast phylliform tumor cells
(1) The experimental method comprises the following steps: transiently transfecting siRNA (siRNA 1: sense: CCA GCU CCG CGUCUACAA AdTdT, anti: UUU GUA GAC GCG GAG CUG GdT; siRNA2: sense: GGGAGAGAAAUACAUCGAUTT, anti: AUCGAUGUAUUUCUCUCCCTG; siRNA3: sense: GGAACUACUGGUGAACUAUTT; anti: AUAGUUCACCAGUAGUUCCTG) into a malignant breast lobular tumor cell line with Lipo3000 to knock down CD146 expression (as shown in FIG. 2A); cells of a control group with knockdown CD146 expression and without gene knockdown are inoculated, the survival rate of the cells at different time points (1-4 days) is tested by adopting an MTS colorimetric method, and a cell proliferation curve is drawn. The control cells with knockdown CD146 expression and no gene knockdown were inoculated into complete medium in 60mm dishes of 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. 2B and 2C.
2. Effect of knockdown CD146 gene on migration and invasion ability of malignant breast phylliform tumor cells
(1) The experimental method comprises the following steps: knockdown of CD146 gene expression and non-knockdown of control cells at 2 x 10 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 10% fetal bovine serum, three replicate wells being provided per 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. 2D.
3. Effect of knockdown CD146 gene on collagen contractility of malignant breast phylliform tumor cells
(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. 2E.
As can be seen from fig. 2, knocking down CD146 gene reduced proliferation capacity of malignant breast phylliform tumor cells; knocking down the CD146 gene obviously reduces the clone forming ability of malignant breast phylliform tumor cells; the migration and invasion capacity of malignant breast phylliform tumor cells with the CD146 gene knocked down are obviously reduced; knocking down the CD146 gene significantly reduces the collagen contraction capacity of malignant breast phylliform tumor cells. CD146 is highly expressed on malignant breast phylliform tumor cells, plays a role in promoting cell growth, migration, invasion and collagen contraction, and suggests that CD146 can be used as a molecular marker of malignant breast phylliform tumor to promote malignant progress of breast phylliform tumor.
Example 4 Effect of over-expression of CD146 on benign leaf tumor cell proliferation, clonogenic, migration, invasion and collagen contractility
1. Effect of over-expressed CD146 on proliferation of benign breast phylliform tumor cells
(1) The experimental method comprises the following steps: primary benign leaf tumor cells were inoculated into 6-well plates, CD146 overexpression plasmids (1.5 μg/well) were transfected with the infectious agent via, normal complete medium was changed 4-6 hours after transfection, and RNA samples were collected from the cells and assayed for CD146 overexpression levels (as shown in fig. 3A). Cells of the control group which are over-expressed with CD146 and are not treated are inoculated, the survival rate of the cells at different time points (1-4 days) is tested by adopting an MTS colorimetric method, and a cell proliferation curve is drawn. The cells of the control group, which had over-expressed CD146 and had not been treated, were inoculated into complete medium in 60mm dishes of 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. 3B and 3C.
2. Effect of over-expressed CD146 on the migration and invasive capacity of malignant breast phylliform tumor cells
(1) The experimental method comprises the following steps: the cells of the control group, which had over-expressed CD146 and had not been treated, were treated at 2 x 10 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 10% fetal bovine serum, three replicate wells being provided per 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. 3D.
3. Effect of over-expression CD146 on collagen contractility of malignant breast phylliform tumor cells
(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. 3E.
As can be seen from fig. 3, in the breast lobular tumor cell line overexpressing CD146, a significant enhancement in cell proliferation, clonogenic, migratory, invasive and collagen contractile capacity was observed, exhibiting similar biological behavior to malignant lobular tumor cells.
Example 5CD146 specific antibodies inhibit malignant leaf tumor cell proliferation, migration, invasion and organogenesis
1. CD146 specific antibody inhibition of proliferation of malignant leaf tumor cells
(1) The experimental method comprises the following steps: malignant breast lobular tumor cells are inoculated into a 96-well plate, CD146 antibody mAA (150 mug/ml) or isotype control mIgG and PBS are added at the same time, the cells are cultured for 24 hours, 48 hours, 72 hours and 96 hours, the MTS reagent is adopted to detect the activity of the cells, and a cell proliferation curve is drawn.
(2) The experimental results are shown in FIG. 4A. As can be seen from fig. 4A, the CD146 antibody significantly inhibited the growth of malignant breast tumor cells relative to PBS and IgG control, suggesting that CD146 can be a potential drug new target for breast phylliform tumor.
2.CD146 specific antibodies inhibit malignant leaf tumor cell migration and invasion
(1) The experimental method comprises the following steps: malignant leaf tumor cells were treated at 2 x 10 4 Density of wells/wells was inoculated in a Transwell plate (Costar) upper cell serum-free medium or a pre-plated Matrigel thin layer upper culture chamber, the lower layer was a complete medium containing 10% fetal bovine serum, cells were inoculated while adding CD146 antibody mAA for incubation, and malignant leaf tumor cells with mIgG or PBS added were used as controls, and after incubation in a 37 ℃ incubator for 8 hours or 24 hours, cell migration and invasion numbers were statistically analyzed.
(2) The experimental results are shown in fig. 4B. As can be seen from fig. 4B, the CD146 antibody can significantly inhibit migration and invasion of breast phyllostachys tumor cells, suggesting that the development of new anti-metastatic drugs for breast phyllostachys tumor can be performed by using therapeutic antibodies targeting CD 146.
3. CD146 specific antibodies inhibit malignant leaf tumor cell organogenesis
(1) The experimental method comprises the following steps: primary breast foliate tumor cells were isolated from clinical specimens, organoid cultured, incubated with CD146 antibody mAA (150. Mu.g/ml) added to microplates, and organoid formation in the microplates was observed under a microscope for days 0, 7, 14, 21, 28 and 38, respectively, with the addition of mIgG or PBS as a control.
(2) The experimental results are shown in fig. 4C. As can be seen from fig. 4C, the CD146 antibody AA98 has a significant inhibitory effect on primary leaf tumor cell organogenesis.
Example 6CD146 specific antibodies inhibit human malignant lobular tumor xenograft model (PDX) tumor growth
(1) The experimental method comprises the following steps: constructing a mammary gland phylliform tumor PDX model: malignant breast phylliform tumor tissue is taken, sheared into tissue pieces with the diameter of about 1mm, a 3mm incision is made at a position which is about 2cm away from the subcutaneous breast fat pad of the left forelimb armpit of the mouse, and a trocar sends 3-4 small tissue pieces to the subcutaneous fat pad through a subcutaneous tunnel. When the tumor grows to 100m 3 For left and right, tumor-bearing mice were randomly grouped, 6 mice per group were intraperitoneally injected with CD146 antibody (mAA, chAA 98) and control agent (PBS, mIgG, hIgG) at a dose of 250 μg/mouse, twice weekly, 5 consecutive injections. Tumor size was continuously monitored weekly, according to formula v=1/2 (lxw 2 ) The average volume of the transplanted tumors was calculated. Stopping injection when the tumor volume reaches 1.5cm in diameter, and drawing a tumor growth change curve.
(2) The experimental results are shown in fig. 5A and 5B. As can be seen from fig. 5A and 5B, the CD146 antibody-injected group significantly inhibited tumor growth compared to the control group (PBS, mIgG or hig group).
Example 7CD146 promotes malignant progression of breast phylliform tumors by activating the downstream PI3K-Akt signaling pathway
(1) The experimental method comprises the following steps: a. adopting benign breast phylliform tumor cell strains and corresponding CD146 overexpression cells, and carrying out mRNA expression profile chip sequencing and differential gene expression and signal path enrichment analysis on the malignant breast phylliform tumor cell strains and CD146 knockdown expression cells; b. verifying the expression change of related signal proteins at the downstream of a PI3K-Akt signal channel by adopting western blot; c. performing western blot detection on the mouse PDX tumor tissue of the embodiment 5; d. DCBLD2 and AKT signaling changes after CD146 knockdown and overexpression of the malignant breast phylliform tumor cell line; e. effect of proteasome inhibitor MG132 treatment (4 h) on DCBLD2 levels in CD146 knockdown cells.
(2) The experimental results are shown in FIG. 6. From FIG. 6A, both the PI3K-Akt signaling pathway and the cytokine interaction pathway are involved in more altered levels of gene expression; as can be seen from fig. 6B, in CD146 over-expression or malignant breast lobular tumor cells, expression of phosphorylated Akt (p-Akt) was significantly up-regulated, and p-Akt up-regulation indicates that Akt signaling pathway is activated, and activation of Akt signaling pathway is closely related to occurrence, development and metastasis of tumor; as can be seen from fig. 6C, the expression of phosphorylated Akt (p-Akt) in tumor tissue was significantly down-regulated in the CD146 antibodies (mAA, chAA 98) injected group compared to the control group (PBS, mIgG, or hig group); as can be seen from fig. 6D, knocking down the CD146 antibody significantly down-regulates DCBLD2 expression and p-Akt levels, and restoring CD146 expression up-regulates DCBLD2 expression and p-Akt levels; as can be seen from fig. 6E, treatment of CD146 knockdown cell lines with the proteasome inhibitor MG132 inhibited down regulation of DCBLD2 levels, indicating that CD146 inhibited protein degradation of DCBLD2, thereby activating downstream AKT signaling pathways.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> university of Zhongshan Sun Yixian commemorative Hospital, china academy of sciences Biophysical institute
<120> a molecular marker CD146 of breast phylliform tumor and application thereof
<130> 2021.09.30
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 1941
<212> DNA
<213> artificial sequence
<400> 1
atggggcttc ccaggctggt ctgcgccttc ttgctcgccg cctgctgctg ctgtcctcgc 60
gtcgcgggtg tgcccggaga ggctgagcag cctgcgcctg agctggtgga ggtggaagtg 120
ggcagcacag cccttctgaa gtgcggcctc tcccagtccc aaggcaacct cagccatgtc 180
gactggtttt ctgtccacaa ggagaagcgg acgctcatct tccgtgtgcg ccagggccag 240
ggccagagcg aacctgggga gtacgagcag cggctcagcc tccaggacag aggggctact 300
ctggccctga ctcaagtcac cccccaagac gagcgcatct tcttgtgcca gggcaagcgc 360
cctcggtccc aggagtaccg catccagctc cgcgtctaca aagctccgga ggagccaaac 420
atccaggtca accccctggg catccctgtg aacagtaagg agcctgagga ggtcgctacc 480
tgtgtaggga ggaacgggta ccccattcct caagtcatct ggtacaagaa tggccggcct 540
ctgaaggagg agaagaaccg ggtccacatt cagtcgtccc agactgtgga gtcgagtggt 600
ttgtacacct tgcagagtat tctgaaggca cagctggtta aagaagacaa agatgcccag 660
ttttactgtg agctcaacta ccggctgccc agtgggaacc acatgaagga gtccagggaa 720
gtcaccgtcc ctgttttcta cccgacagaa aaagtgtggc tggaagtgga gcccgtggga 780
atgctgaagg aaggggaccg cgtggaaatc aggtgtttgg ctgatggcaa ccctccacca 840
cacttcagca tcagcaagca gaaccccagc accagggagg cagaggaaga gacaaccaac 900
gacaacgggg tcctggtgct ggagcctgcc cggaaggaac acagtgggcg ctatgaatgt 960
cagggcctgg acttggacac catgatatcg ctgctgagtg aaccacagga actactggtg 1020
aactatgtgt ctgacgtccg agtgagtccc gcagcccctg agagacagga aggcagcagc 1080
ctcaccctga cctgtgaggc agagagtagc caggacctcg agttccagtg gctgagagaa 1140
gagacaggcc aggtgctgga aagggggcct gtgcttcagt tgcatgacct gaaacgggag 1200
gcaggaggcg gctatcgctg cgtggcgtct gtgcccagca tacccggcct gaaccgcaca 1260
cagctggtca acgtggccat ttttggcccc ccttggatgg cattcaagga gaggaaggtg 1320
tgggtgaaag agaatatggt gttgaatctg tcttgtgaag cgtcagggca cccccggccc 1380
accatctcct ggaacgtcaa cggcacggca agtgaacaag accaagatcc acagcgagtc 1440
ctgagcaccc tgaatgtcct cgtgaccccg gagctgttgg agacaggtgt tgaatgcacg 1500
gcctccaacg acctgggcaa aaacaccagc atcctcttcc tggagctggt caatttaacc 1560
accctcacac cagactccaa cacaaccact ggcctcagca cttccactgc cagtcctcat 1620
accagagcca acagcacctc cacagagaga aagctgccgg agccggagag ccggggcgtg 1680
gtcatcgtgg ctgtgattgt gtgcatcctg gtcctggcgg tgctgggcgc tgtcctctat 1740
ttcctctata agaagggcaa gctgccgtgc aggcgctcag ggaagcagga gatcacgcta 1800
cccccgtctc gtaagagcga acttgtagtt gaagttaagt cagataagct cccagaagag 1860
atgggcctcc tgcagggcag cagcggtgac aagagggctc cgggagacca gggagagaaa 1920
tacatcgatc tgaggcatta g 1941
<210> 2
<211> 646
<212> PRT
<213> artificial sequence
<400> 2
Met Gly Leu Pro Arg Leu Val Cys Ala Phe Leu Leu Ala Ala Cys Cys
1 5 10 15
Cys Cys Pro Arg Val Ala Gly Val Pro Gly Glu Ala Glu Gln Pro Ala
20 25 30
Pro Glu Leu Val Glu Val Glu Val Gly Ser Thr Ala Leu Leu Lys Cys
35 40 45
Gly Leu Ser Gln Ser Gln Gly Asn Leu Ser His Val Asp Trp Phe Ser
50 55 60
Val His Lys Glu Leu Arg Thr Leu Ile Phe Arg Val Arg Gln Gly Gln
65 70 75 80
Gly Gln Ser Glu Pro Gly Glu Tyr Glu Gln Arg Leu Ser Leu Gln Asp
85 90 95
Arg Gly Ala Thr Leu Ala Leu Thr Gln Val Thr Pro Gln Asp Glu Arg
100 105 110
Ile Phe Leu Cys Gln Gly Lys Arg Pro Arg Ser Gln Glu Tyr Arg Ile
115 120 125
Gln Leu Arg Val Tyr Lys Ala Pro Glu Glu Pro Asn Ile Gln Val Asn
130 135 140
Pro Leu Gly Ile Pro Val Asn Ser Lys Glu Pro Glu Glu Val Ala Thr
145 150 155 160
Cys Val Gly Arg Asn Gly Tyr Pro Ile Pro Gln Val Ile Trp Tyr Lys
165 170 175
Asn Gly Arg Pro Leu Lys Glu Glu Lys Asn Arg Val His Ile Gln Ser
180 185 190
Ser Gln Thr Val Glu Ser Ser Gly Leu Tyr Thr Leu Gln Ser Ile Leu
195 200 205
Lys Ala Gln Leu Val Lys Glu Asp Lys Asp Ala Gln Phe Tyr Cys Glu
210 215 220
Leu Asn Tyr Arg Leu Pro Ser Gly Asn His Met Lys Glu Ser Arg Glu
225 230 235 240
Val Thr Val Pro Val Phe Tyr Pro Thr Glu Lys Val Trp Leu Glu Val
245 250 255
Glu Pro Val Gly Met Leu Lys Glu Gly Asp Arg Val Glu Ile Arg Cys
260 265 270
Leu Ala Asp Gly Asn Pro Pro Pro His Phe Ser Ile Ser Lys Gln Asn
275 280 285
Pro Ser Thr Arg Glu Ala Glu Glu Glu Thr Thr Asn Asp Asn Gly Val
290 295 300
Leu Val Leu Glu Pro Ala Arg Lys Glu His Ser Gly Arg Tyr Glu Cys
305 310 315 320
Gln Gly Leu Asp Leu Asp Thr Met Ile Ser Leu Leu Ser Glu Pro Gln
325 330 335
Glu Leu Leu Val Asn Tyr Val Ser Asp Val Arg Val Ser Pro Ala Ala
340 345 350
Pro Glu Arg Gln Glu Gly Ser Ser Leu Thr Leu Thr Cys Glu Ala Glu
355 360 365
Ser Ser Gln Asp Leu Glu Phe Gln Trp Leu Arg Glu Glu Thr Gly Gln
370 375 380
Val Leu Glu Arg Gly Pro Val Leu Gln Leu His Asp Leu Lys Arg Glu
385 390 395 400
Ala Gly Gly Gly Tyr Arg Cys Val Ala Ser Val Pro Ser Ile Pro Gly
405 410 415
Leu Asn Arg Thr Gln Leu Val Asn Val Ala Ile Phe Gly Pro Pro Trp
420 425 430
Met Ala Phe Lys Glu Arg Lys Val Trp Val Lys Glu Asn Met Val Leu
435 440 445
Asn Leu Ser Cys Glu Ala Ser Gly His Pro Arg Pro Thr Ile Ser Trp
450 455 460
Asn Val Asn Gly Thr Ala Ser Glu Gln Asp Gln Asp Pro Gln Arg Val
465 470 475 480
Leu Ser Thr Leu Asn Val Leu Val Thr Pro Glu Leu Leu Glu Thr Gly
485 490 495
Val Glu Cys Thr Ala Ser Asn Asp Leu Gly Lys Asn Thr Ser Ile Leu
500 505 510
Phe Leu Glu Leu Val Asn Leu Thr Thr Leu Thr Pro Asp Ser Asn Thr
515 520 525
Thr Thr Gly Leu Ser Thr Ser Thr Ala Ser Pro His Thr Arg Ala Asn
530 535 540
Ser Thr Ser Thr Glu Arg Lys Leu Pro Glu Pro Glu Ser Arg Gly Val
545 550 555 560
Val Ile Val Ala Val Ile Val Cys Ile Leu Val Leu Ala Val Leu Gly
565 570 575
Ala Val Leu Tyr Phe Leu Tyr Lys Lys Gly Lys Leu Pro Cys Arg Arg
580 585 590
Ser Gly Lys Gln Glu Ile Thr Leu Pro Pro Ser Arg Lys Asn Glu Leu
595 600 605
Val Val Glu Val Lys Ser Asp Lys Leu Pro Glu Glu Met Gly Leu Leu
610 615 620
Gln Gly Ser Ser Gly Asp Lys Arg Ala Pro Gly Asp Gln Gly Glu Lys
625 630 635 640
Tyr Ile Asp Leu Arg His
645
<210> 3
<211> 604
<212> PRT
<213> artificial sequence
<400> 3
Met Gly Leu Pro Arg Leu Val Cys Ala Phe Leu Leu Ala Ala Cys Cys
1 5 10 15
Cys Cys Pro Arg Val Ala Gly Val Pro Gly Glu Ala Glu Gln Pro Ala
20 25 30
Pro Glu Leu Val Glu Val Glu Val Gly Ser Thr Ala Leu Leu Lys Cys
35 40 45
Gly Leu Ser Gln Ser Gln Gly Asn Leu Ser His Val Asp Trp Phe Ser
50 55 60
Val His Lys Glu Lys Arg Thr Leu Ile Phe Arg Val Arg Gln Gly Gln
65 70 75 80
Gly Gln Ser Glu Pro Gly Glu Tyr Glu Gln Arg Leu Ser Leu Gln Asp
85 90 95
Arg Gly Ala Thr Leu Ala Leu Thr Gln Val Thr Pro Gln Asp Glu Arg
100 105 110
Ile Phe Leu Cys Gln Gly Lys Arg Pro Arg Ser Gln Glu Tyr Arg Ile
115 120 125
Gln Leu Arg Val Tyr Lys Ala Pro Glu Glu Pro Asn Ile Gln Val Asn
130 135 140
Pro Leu Gly Ile Pro Val Asn Ser Lys Glu Pro Glu Glu Val Ala Thr
145 150 155 160
Cys Val Gly Arg Asn Gly Tyr Pro Ile Pro Gln Val Ile Trp Tyr Lys
165 170 175
Asn Gly Arg Pro Leu Lys Glu Glu Lys Asn Arg Val His Ile Gln Ser
180 185 190
Ser Gln Thr Val Glu Ser Ser Gly Leu Tyr Thr Leu Gln Ser Ile Leu
195 200 205
Lys Ala Gln Leu Val Lys Glu Asp Lys Asp Ala Gln Phe Tyr Cys Glu
210 215 220
Leu Asn Tyr Arg Leu Pro Ser Gly Asn His Met Lys Glu Ser Arg Glu
225 230 235 240
Val Thr Val Pro Val Phe Tyr Pro Thr Glu Lys Val Trp Leu Glu Val
245 250 255
Glu Pro Val Gly Met Leu Lys Glu Gly Asp Arg Val Glu Ile Arg Cys
260 265 270
Leu Ala Asp Gly Asn Pro Pro Pro His Phe Ser Ile Ser Lys Gln Asn
275 280 285
Pro Ser Thr Arg Glu Ala Glu Glu Glu Thr Thr Asn Asp Asn Gly Val
290 295 300
Leu Val Leu Glu Pro Ala Arg Lys Glu His Ser Gly Arg Tyr Glu Cys
305 310 315 320
Gln Gly Leu Asp Leu Asp Thr Met Ile Ser Leu Leu Ser Glu Pro Gln
325 330 335
Glu Leu Leu Val Asn Tyr Val Ser Asp Val Arg Val Ser Pro Ala Ala
340 345 350
Pro Glu Arg Gln Glu Gly Ser Ser Leu Thr Leu Thr Cys Glu Ala Glu
355 360 365
Ser Ser Gln Asp Leu Glu Phe Gln Trp Leu Arg Glu Glu Thr Gly Gln
370 375 380
Val Leu Glu Arg Gly Pro Val Leu Gln Leu His Asp Leu Lys Arg Glu
385 390 395 400
Ala Gly Gly Gly Tyr Arg Cys Val Ala Ser Val Pro Ser Ile Pro Gly
405 410 415
Leu Asn Arg Thr Gln Leu Val Asn Val Ala Ile Phe Gly Pro Pro Trp
420 425 430
Met Ala Phe Lys Glu Arg Lys Val Trp Val Lys Glu Asn Met Val Leu
435 440 445
Asn Leu Ser Cys Glu Ala Ser Gly His Pro Arg Pro Thr Ile Ser Trp
450 455 460
Asn Val Asn Gly Thr Ala Ser Glu Gln Asp Gln Asp Pro Gln Arg Val
465 470 475 480
Leu Ser Thr Leu Asn Val Leu Val Thr Pro Glu Leu Leu Glu Thr Gly
485 490 495
Val Glu Cys Thr Ala Ser Asn Asp Leu Gly Lys Asn Thr Ser Ile Leu
500 505 510
Phe Leu Glu Leu Val Asn Leu Thr Thr Leu Thr Pro Asp Ser Asn Thr
515 520 525
Thr Thr Gly Leu Ser Thr Ser Thr Ala Ser Pro His Thr Arg Ala Asn
530 535 540
Ser Thr Ser Thr Glu Arg Lys Leu Pro Glu Pro Glu Ser Arg Gly Val
545 550 555 560
Val Ile Val Ala Val Ile Val Cys Ile Leu Val Leu Ala Val Leu Gly
565 570 575
Ala Val Leu Tyr Phe Leu Tyr Lys Lys Gly Lys Leu Pro Cys Arg Arg
580 585 590
Ser Gly Lys Gln Glu Met Glu Arg Asn Thr Ser Ile
595 600
<210> 4
<211> 20
<212> DNA
<213> artificial sequence
<400> 4
gctgcccagt gggaaccaca 20
<210> 5
<211> 23
<212> DNA
<213> artificial sequence
<400> 5
atcatggtgt ccaagttcca ggc 23

Claims (2)

1. Application of siRNA with knockdown CD146 gene in preparing medicine for treating malignant mammary gland phylliform tumor; the siRNA is siRNA1: CCA GCU CCG CGUCUACAA AdTdT, anti-sense UUU GUA GAC GCG GAG CUG GdTdT, siRNA2: GGGAGAGAAAUACAUCGAUTT, anti-sense AUCGAUGUAUUUCUCUCCCTG or siRNA3: GGAACUACUGGUGAACUAUTT, anti-sense AUAGUUCACCAGUAGUUCCTG.
Application of CD146 specific antibody in preparing medicine for treating malignant breast phylliform tumor; the CD146 specific antibody is mAA98.
CN202111200916.8A 2021-10-15 2021-10-15 Breast phylliform tumor molecular marker CD146 and application thereof Active CN113929764B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111200916.8A CN113929764B (en) 2021-10-15 2021-10-15 Breast phylliform tumor molecular marker CD146 and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111200916.8A CN113929764B (en) 2021-10-15 2021-10-15 Breast phylliform tumor molecular marker CD146 and application thereof

Publications (2)

Publication Number Publication Date
CN113929764A CN113929764A (en) 2022-01-14
CN113929764B true CN113929764B (en) 2023-12-29

Family

ID=79279604

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111200916.8A Active CN113929764B (en) 2021-10-15 2021-10-15 Breast phylliform tumor molecular marker CD146 and application thereof

Country Status (1)

Country Link
CN (1) CN113929764B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116083567A (en) * 2022-09-08 2023-05-09 中山大学孙逸仙纪念医院 Diagnostic reagent for lobular tumor of mammary gland and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103157109A (en) * 2011-12-12 2013-06-19 中国科学院生物物理研究所 CD146 and antibody diagnosis thereof, and application in treating triple negative breast cancer
CN103399144A (en) * 2008-02-25 2013-11-20 雀巢产品技术援助有限公司 Drug selection for breast cancer therapy using antibody-based arrays
CN110967489A (en) * 2018-09-28 2020-04-07 中国科学院生物物理研究所 Application of soluble CD146 as blood brain barrier damage marker in central nervous system diseases

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103399144A (en) * 2008-02-25 2013-11-20 雀巢产品技术援助有限公司 Drug selection for breast cancer therapy using antibody-based arrays
CN103157109A (en) * 2011-12-12 2013-06-19 中国科学院生物物理研究所 CD146 and antibody diagnosis thereof, and application in treating triple negative breast cancer
CN107929732A (en) * 2011-12-12 2018-04-20 中国科学院生物物理研究所 The application of CD146 and its antibody diagnosis and treatment triple negative breast cancer
CN110967489A (en) * 2018-09-28 2020-04-07 中国科学院生物物理研究所 Application of soluble CD146 as blood brain barrier damage marker in central nervous system diseases

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Kinga A. Kocemba-Pilarczyk等.The relationship between expression of VIMENTIN and CD146 genes in breast cancer.Bio-Algorithms and Med-Systems.2021,第第17卷卷(第第1期期),全文. *
樊静等."CD105和CD146在乳腺组织中的表达".海南医学院学报.2011,第第17卷卷(第第12期期),参见摘要，第1620页左栏第1段-右栏第3段. *

Also Published As

Publication number Publication date
CN113929764A (en) 2022-01-14

Similar Documents

Publication Publication Date Title
Zhang et al. Expression of potential cancer stem cell marker ABCG2 is associated with malignant behaviors of hepatocellular carcinoma
CN109797151A (en) The application of Circ-CDH1 inhibitor
CN112501299A (en) Method for predicting recurrence and metastasis of liver cancer and application
Li et al. Loss of periplakin expression is associated with the tumorigenesis of colorectal carcinoma
CN108203732B (en) Application of TRIM24 in glioma diagnosis
CN106834486B (en) Osteosarcoma molecular diagnosis and treatment marker and application thereof
CN113929764B (en) Breast phylliform tumor molecular marker CD146 and application thereof
Tian et al. LncRNA LINC00974 downregulates miR-122 to upregulate RhoA in oral squamous cell carcinoma
Wu et al. Expression and significance of c-kit and epithelial-mesenchymal transition (EMT) molecules in thymic epithelial tumors (TETs)
KR102157895B1 (en) Markers for predicting the response of a patient with colorectal cancer to anti-cancer drugs
CN103103264B (en) Application of SMOC2 gene in preparation of medicine for detecting or treating endometrial cancer and ovarian cancer
CN111040032B (en) Application of bidirectional regulator in preparation of cell aging and tumor diagnosis or regulation preparation
CN105288659A (en) Application of TENM1 gene and its expression product on diagnosis and treatment of papillary adenocarcinoma
Daum et al. ITIH5 shows tumor suppressive properties in cervical cancer cells grown as multicellular tumor spheroids
CN114561470A (en) Triple negative breast cancer molecular marker and application thereof
Ye et al. HER2 and VEGF expression in breast cancer and their correlations
KR101612356B1 (en) Use of HOXB5 for diagnosis and treatment of breast cancer
CN103525941A (en) Application of CTHRC1 genes in preparation of drugs for detecting/treating cervical cancer
US20080096210A1 (en) Cancer suppressing agent
CN113699240B (en) Medical application of NRK in lung cancer treatment and prognosis diagnosis
CN111593124B (en) UGT3A2 gene and application of protein coded by same in auxiliary diagnosis of upper gastrointestinal tumor
WO2024050860A1 (en) Diagnosis reagent for phyllodes tumor and use thereof
JP7250307B2 (en) Method for predicting onset, metastasis or recurrence of uterine cancer
CN108410986B (en) CDH6 promotes osteosarcoma growth and metastasis
Zheng et al. Tumor-associated fibroblasts promote the migration and invasion of Hepatocellular carcinoma cells by regulating THY-1

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant