阅读说明：本技术 Galnt2作为子宫内膜增生或子宫内膜癌诊治标志物的应用 (Application of GALNT2 as endometrial hyperplasia or endometrial cancer diagnosis and treatment marker ) 是由 周雪妍 印晓星 张蓓 徐吟雪 尹弟 于 2020-05-08 设计创作，主要内容包括：本发明公开了GALNT2作为子宫内膜增生或子宫内膜癌诊治标志物的应用。本发明证明相比正常对照,子宫内膜增生患者的子宫内膜组织或子宫内膜癌患者的子宫内膜组织中GALNT2基因表达降低,表明GALNT2可作为诊断子宫内膜增生或子宫内膜癌的分子标志物。体外细胞实验证明,GALNT2表达与子宫细胞癌细胞增殖相关,故GALNT2可作为靶点用于开发临床治疗子宫内膜增生或子宫内膜癌的药物。(The invention discloses application of GALNT2 as a diagnosis and treatment marker for endometrial hyperplasia or endometrial cancer. The present invention demonstrates a reduction in GALNT2 gene expression in endometrial tissue from a patient with endometrial hyperplasia or endometrial tissue from a patient with endometrial cancer compared to a normal control, indicating that GALNT2 can be used as a molecular marker for diagnosing endometrial hyperplasia or endometrial cancer. In vitro cell experiments prove that the GALNT2 expression is related to the proliferation of uterine cell carcinoma cells, so the GALNT2 can be used as a target point for developing a medicament for clinically treating endometrial hyperplasia or endometrial cancer.)

1. The application of the product for detecting GALNT2 gene or GALNT2 protein in preparing tools for diagnosing endometrial hyperplasia and/or endometrial cancer.

2. The use of claim 1, wherein the product for detecting GALNT2 gene or GALNT2 protein comprises a product for detecting the expression level of GALNT2 gene or GALNT2 protein.

3. The use of claim 2, wherein the product is used to detect the expression level of GALNT2 gene or GALNT2 protein in a sample from a subject, wherein a decrease or absence of the expression level of GALNT2 gene or GALNT2 protein in the sample from the subject, as compared to a normal human, diagnoses the subject as a patient with endometrial hyperplasia or endometrial cancer, or diagnoses the subject as at high risk of endometrial hyperplasia or endometrial cancer.

4. The use of any one of claims 1 to 3, wherein the product comprises a nucleic acid capable of binding to the GALNT2 gene or a substance capable of binding to the GALNT2 protein; the nucleic acid is capable of detecting the expression level of GALNT2 gene; the substance is capable of detecting the expression level of GALNT2 protein.

5. The use according to claim 4, wherein the nucleic acid is a primer for specific amplification of GALNT2 gene used in real-time quantitative PCR as shown in SEQ ID No.1 and SEQ ID No. 2.

6. A means for diagnosing endometrial hyperplasia and/or endometrial cancer, comprising means for detecting an expression level of GALNT2 gene or GALNT2 protein in a sample from a subject.

7. The tool of claim 6, comprising a nucleic acid capable of binding to GALNT2 gene or a substance capable of binding to GALNT2 protein; the nucleic acid is capable of detecting the expression level of GALNT2 gene; the substance is capable of detecting the expression level of GALNT2 protein.

8. The kit of claim 8, wherein the nucleic acid is a primer for specific amplification of GALNT2 gene used in real-time quantitative PCR as shown in SEQ ID No.1 and SEQ ID No. 2.

9. A medicament for treating endometrial hyperplasia or endometrial cancer, comprising a substance that promotes the expression of GALNT2 gene; preferably, the agent comprises a GALNT2 gene overexpression vector.

The use of a GALNT2 gene or a GALNT2 protein in the preparation of a medicament for the treatment of endometrial hyperplasia or endometrial cancer.

Technical Field

The invention relates to the field of disease diagnosis and treatment, in particular to application of GALNT2 as a diagnosis and treatment marker of endometrial hyperplasia or endometrial cancer.

Background

Endometrial Cancer (EC) is one of the common malignant tumors in gynecology, the incidence rate of which is rising year by year and the trend of the cancer is younger, and it is generally considered that the continuous stimulation of estrogen without progestogen antagonism causes the proliferation of endometrium and then the canceration. Endometrial Hyperplasia (EH) is a common gynecological endocrine disease, mainly manifested by irregular vaginal bleeding, infertility, and even malignant changes, and the atypical hyperplasia of the endometrium has a certain canceration tendency and is known as a precancerous lesion of endometrial cancer. Endometrial hyperplasia is usually manifested by abnormal uterine bleeding, which often develops into endometrial cancer if not properly treated. Early diagnosis and early treatment of endometrial cancer are critical to improving the prognosis of endometrial cancer patients. The clinical diagnosis of endometrial hyperplasia and endometrial cancer mainly depends on the pathological histology diagnosis, the commonly used method is diagnostic curettage or hysteroscopy curettage, and a certain rate of missed diagnosis exists. Therefore, the method finds a sensitive, accurate and reliable diagnosis and treatment marker, effectively detects the endometrial hyperplasia or endometrial cancer, and has important significance for guiding clinical treatment decisions and improving the prognosis of patients.

In the present application, proteomics technology is used to detect protein mass spectra of uterine tissue, screening and validating differential proteins by bioinformatic and molecular biological analysis. The results indicate that the enzyme N-acetylgalactosamine transferase 2(GALNT2) which regulates the initiation step of mucin O-glycosylation plays an important role in the occurrence and development of endometrial hyperplasia and endometrial cancer, and the expression level of the enzyme is closely related to the degree of cachexia of the disease.

Disclosure of Invention

The invention aims to provide a method for diagnosing endometrial hyperplasia or endometrial cancer by detecting GALNT2 gene or protein expression difference.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of a product for detecting GALNT2 gene or GALNT2 protein in preparation of a tool for diagnosing endometrial hyperplasia and/or endometrial cancer.

Further, the product for detecting the GALNT2 gene or GALNT2 protein comprises a product for detecting the expression level of the GALNT2 gene or GALNT2 protein. The product comprises a nucleic acid capable of binding to GALNT2 gene or a substance (e.g., an antibody) capable of binding to GALNT2 protein. The nucleic acid is capable of detecting the expression level of GALNT2 gene; the substance is capable of detecting the expression level of GALNT2 protein.

The product for detecting GALNT2 gene of the present invention can exert its function based on a known method using a nucleic acid molecule: such as PCR, e.g., Southern hybridization, Northern hybridization, dot hybridization, Fluorescence In Situ Hybridization (FISH), DNA microarray, ASO methods, high throughput sequencing platforms, etc. The product can be used to conduct the assay qualitatively, quantitatively, or semi-quantitatively.

The nucleic acid contained in the above-mentioned products can be obtained by chemical synthesis, or by preparing a gene containing a desired nucleic acid from a biological material and then amplifying it using a primer designed to amplify the desired nucleic acid.

Further, the PCR method is a known method, for example, ARMS (Amplification Refractorymutation System) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method, or the like. The amplified nucleic acid can be detected by using a dot blot hybridization method, a surface plasmon resonance method (SPR method), a PCR-RFLP method, an in situ RT-PCR method, a PCR-SSO (sequence specific oligonucleotide) method, a PCR-SSP method, an AMPFLP (amplifiable fragment length polymorphism) method, an MVR-PCR method, and a PCR-SSCP (single strand conformation polymorphism) method.

The above-mentioned nucleic acids include primers for amplifying the GALNT2 gene, and the primers included in the product can be prepared by chemical synthesis, appropriately designed by referring to known information using a method known to those skilled in the art, and prepared by chemical synthesis.

In a particular embodiment of the invention, the nucleic acid is an amplification primer used in QPCR experiments, the sequence of the primer is shown as SEQ ID NO.1 (forward sequence) and SEQ ID NO.2 (reverse sequence).

The above-mentioned nucleic acids may further include a probe which can be prepared by chemical synthesis, appropriately designed by referring to known information using a method known to those skilled in the art, and prepared by chemical synthesis, or can be prepared by preparing a gene containing a desired nucleic acid sequence from a biological material and amplifying it using a primer designed for amplifying the desired nucleic acid sequence.

The product for detecting GALNT2 protein of the present invention can exert its function based on a known method using an antibody: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, etc. may be included.

The product for detecting GALNT2 protein of the invention includes an antibody or fragment thereof that specifically binds to GALNT2 protein. An antibody or fragment thereof of any structure, size, immunoglobulin class, origin, etc., may be used so long as it binds to the target protein. The antibodies or fragments thereof included in the assay products of the invention may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody (partial fragment) or a peptide containing a portion of an antibody that retains the binding activity of the antibody to an antigen. Antibody fragments may include F (ab')₂Fab', Fab, single chain fv (scfv), disulfide-bonded fv (dsfv) or polymers thereof, dimerized V regions (diabodies), or CDR-containing peptides. The product for detecting GALNT2 protein of the present invention may include an isolated nucleic acid encoding the amino acid sequence of an antibody or encoding a fragment of an antibody, a vector comprising the nucleic acid, and a cell carrying the vector.

Antibodies can be obtained by methods well known to those skilled in the art. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing an animal with an antigen, immune cells are obtained from the immunized animal and myeloma cells are fused to obtain hybridomas. The antibody is then collected from the hybridoma culture. Finally, a monoclonal antibody against GALNT2 protein can be obtained by subjecting the obtained antibody to antigen-specific purification using GALNT2 protein or a part thereof used as an antigen. Polyclonal antibodies can be prepared as follows: an animal is immunized with the same antigen as above, a blood sample is collected from the immunized animal, serum is separated from the blood, and then antigen-specific purification is performed on the serum using the above antigen. The antibody fragment can be obtained by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.

Binding of the label to the antibody or fragment thereof can be carried out by methods generally known in the art. For example, proteins or peptides may be fluorescently labeled as follows: the protein or peptide is washed with phosphate buffer, a dye prepared with DMSO, a buffer, or the like is added, and the solution is mixed and left at room temperature for 10 minutes. In addition, labeling may be carried out using commercially available labeling kits, such as biotin labeling kit, e.g., biotin labeling kit-NH 2, biotin labeling kit-SH (Dojindo laboratories); alkaline phosphatase labeling kits such as alkaline phosphatase labeling kit-NH 2, alkaline phosphatase labeling kit-sh (dojindo laboratories); peroxidase labeling kits such as peroxidase labeling kit-NH 2, peroxidase labeling kit-NH 2(Dojindo Laboratories); phycobiliprotein labeling kits such as phycobiliprotein labeling kit-NH 2, phycobiliprotein labeling kit-SH, B-phycoerythrin labeling kit-NH 2, B-phycoerythrin labeling kit-SH, R-phycoerythrin labeling kit-NH 2, R-phycoerythrin labeling kit SH (dojindo laboratories); fluorescent labeling kits such as fluorescein labeling kit-NH 2, HiLyte Fluor (TM)555 labeling kit-NH 2, HiLyte Fluor (TM)647 labeling kit-NH 2(Dojindo Laboratories); and DyLight 547 and DyLight647(Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody labeling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation), and EZ-marker protein labeling kit (Funakoshi Corporation). For proper labeling, a suitable instrument can be used to detect the labeled antibody or fragment thereof.

Furthermore, the product for detecting the GALNT2 gene or GALNT2 protein can be a reagent for detecting the GALNT2 gene or GALNT2 protein, can also be a kit, a chip, a test paper and the like containing the reagent, and can also be a high-throughput sequencing platform using the reagent.

Detecting the expression level of GALNT2 gene or GALNT2 protein in the sample of the subject by using the aforementioned detection product, wherein the reduction or absence of the expression level of GALNT2 gene or GALNT2 protein in the sample of the subject as compared with normal persons diagnoses the subject as a patient with endometrial hyperplasia or endometrial cancer or diagnoses the subject as having high risk of endometrial hyperplasia or endometrial cancer.

As a sample of the test product according to the invention, a tissue sample or fluid obtained, for example, from a biopsy subject may be used. The sample is not particularly limited as long as it is suitable for the assay of the present invention; for example, it may comprise tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva, or fractions or treated materials thereof.

The present invention also provides a means for diagnosing endometrial hyperplasia or endometrial cancer, said means being capable of detecting the expression level of GALNT2 gene or GALNT2 protein in a sample from a subject. The means comprises a nucleic acid capable of binding to GALNT2 gene or a substance (e.g., an antibody) capable of binding to GALNT2 protein. The nucleic acid is capable of detecting the expression level of GALNT2 gene; the substance is capable of detecting the expression level of GALNT2 protein.

Further, the properties of the nucleic acid and the substance are the same as those described above.

Further, the tool for diagnosing endometrial hyperplasia or endometrial cancer includes but is not limited to a chip, a kit, a test paper, or a high-throughput sequencing platform; the high-throughput sequencing platform is a special tool for diagnosing endometrial hyperplasia or endometrial cancer, and with the development of a high-throughput sequencing technology, the construction of a gene expression profile of a person becomes very convenient work. By comparing the gene expression profiles of patients with diseases and normal people, the abnormality of which gene is related to the disease can be easily analyzed. Therefore, the knowledge that the abnormality of GALNT2 gene is related to endometrial hyperplasia or endometrial cancer in high-throughput sequencing also belongs to the use of GALNT2 gene, and is also within the scope of the present invention.

The number of amino acids recognized by the anti-GALNT 2 antibody or a fragment thereof used in the detection product, the diagnostic tool of the present invention is not particularly limited as long as the antibody can bind to GALNT 2. When the antibody is used as a therapeutic drug, it is preferable that it recognize as many amino acids as possible as long as it inhibits GALNT2 function. The number of amino acids recognized by the antibody or fragment thereof is at least one, more preferably at least three. The immunoglobulin class of the antibody is not limited and may be IgG, IgM, IgA, IgE, IgD or IgY.

Other properties of the anti-GALNT 2 antibody used in the test product and the diagnostic kit of the present invention are as described above.

Further, the subject sample may use a tissue sample or fluid obtained, for example, from a biopsy subject. The sample is not particularly limited as long as it is suitable for the assay of the present invention; for example, it may comprise tissue, blood, plasma, serum, lymph, urine, serosal cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva, or fractions or treated materials thereof.

The present invention also provides a method of diagnosing endometrial hyperplasia or endometrial cancer, comprising the steps of:

(1) obtaining a sample from a subject with endometrial hyperplasia or endometrial cancer;

(2) detecting the expression level of GALNT2 gene or protein in a sample from the subject;

(3) correlating the measured expression level of GALNT2 gene or protein to the presence or absence of disease in the subject.

(4) A reduced or absent level of expression of GALNT2 gene or protein, as compared to a normal control, then the subject is diagnosed with endometrial hyperplasia or endometrial cancer, or the subject is diagnosed with a high risk of future endometrial hyperplasia or endometrial cancer; a reduced or absent expression level of GALNT2 gene or protein as compared to a patient with endometrial hyperplasia, the subject is diagnosed with endometrial cancer.

In the context of the present invention, "diagnosing endometrial cancer" includes both determining whether a subject has endometrial cancer and determining whether a subject is at risk for endometrial cancer.

The information on NCBI of the "GALNT 2 gene" of the present invention is: chromosome 1, NC _000001.11(230057789.. 230282122).

The invention also provides a medicament containing a substance for promoting GALNT2 gene expression.

The invention also provides application of the GALNT2 gene in preparing a medicament for treating endometrial hyperplasia or endometrial cancer.

The invention also provides application of the GALNT2 gene expression product in preparing a medicament for treating endometrial hyperplasia or endometrial cancer.

The invention also provides application of the GALNT2 gene expression promoting substance in preparing a medicine for treating endometrial hyperplasia or endometrial cancer.

The substance promoting the expression of GALNT2 gene of the present invention is not limited as long as it is a drug that can promote the expression or activity of GALNT2 gene or a factor involved in the upstream or downstream pathway of GALNT2 gene and is effective for the treatment of endometrial hyperplasia or endometrial cancer.

In a specific embodiment of the present invention, the substance promoting the expression of GALNT2 gene comprises an overexpression vector of GALNT2 gene.

The medicament of the present invention may be administered alone or together with other medicaments as a medicine. The other drug that can be administered together with the drug of the present invention is not limited as long as it does not impair the effect of the therapeutic or prophylactic drug of the present invention.

The medicine of the present invention may be prepared into various preparation forms. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.

The route of administration of the drug of the present invention is not limited as long as it exerts the desired therapeutic or prophylactic effect, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, transpleural, topical, inhalation, mucosal, cutaneous, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic. In some cases, the administration may be systemic. In some cases topical administration.

The dose of the drug of the present invention is not limited as long as the desired therapeutic effect or prophylactic effect is obtained, and can be appropriately determined depending on the symptoms, sex, age, and the like. The dose of the therapeutic agent or prophylactic agent of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.

The invention also provides a method of treating endometrial hyperplasia or endometrial cancer, comprising promoting GALNT2 gene expression.

The invention has the advantages and beneficial effects that:

the invention discloses a molecular marker for diagnosing endometrial hyperplasia or endometrial cancer, which can be used as a judgment at the early stage of occurrence of endometrial hyperplasia or endometrial cancer, and provides the survival rate of a patient.

The GALNT2 gene-promoting substance can be used as a new therapeutic drug for endometrial hyperplasia or endometrial cancer, and provides a new therapeutic method for clinical treatment of endometrial hyperplasia or endometrial cancer.

Drawings

FIG. 1 shows the result chart of the rat establishment of the endometrial hyperplasia model, wherein A is the index of the uterus of the rat, B is the thickness (mum) of the endometrium of the rat, C is the result chart of the HE staining of the uterine tissue of the rat (original magnification: 40 ×), D is the immunohistochemical staining chart of the PCNA of the endometrial hyperplasia marker of the rat, the data are expressed by Mean + -SEM, n is 6,^*P<0.05, uterine index (%) - (wet uterine weight (g)/rat weight (g) × 100%) compared to group N;

fig. 2 shows a graph of the results of differential expression of GALNT2 in uterine tissues of rats in the N and EH groups, wherein a: mRNA statistics of GALNT2 in rat uterine tissue; b: protein banding pattern of GALNT2 in rat uterine tissue; c: b shows the statistical profile of the protein bands, data are expressed as Mean + -SEM, n is 6,^**P<0.01, compared to group N;

figure 3 is a graph showing the validation of GALNT2 knock-down experiments and the effect of GALNT2 expression on endometrial cancer cell Ishikawa proliferation, wherein a: the GALNT2 protein band diagram in Ishikawa cells after knockdown; b: a is a protein condition histogram; c: proliferation of Ishikawa cells after knockdown of GALNT2, data expressed as Mean ± SEM, n-3,^##P<0.01, compared to MOCK-N group;

figure 4 shows the results of GALNT2 expression in endometrial tissue and serum from clinical samples, where a: representative results of immunohistochemical staining of GALNT2 in endometrial tissue chips (N: N-6, EH: N-12, EC: N-6); b: statistical plots of GALNT2 immunohistochemical staining in endometrial tissue chips (N: 6, EH: 12, EC: N: 6); c: levels of GALNT2 in the serum of healthy humans and patients with endometrial hyperplasia and endometrial cancer (N-group: 17, EH-group: N-17, EC-group: N-15); the data are expressed as Mean + -SEM,^*P<0.05，^**P<0.01, compared to group N.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press, 1989), or according to the manufacturer's recommendations.

The statistical analysis method used in the present invention is as follows: data were statistically analyzed using SPSS 16.0 software, data are presented as Mean + -SEM, two sample comparisons were performed using Independent sample T-Test (Independent-Samples T Test), and multiple group comparisons were performed using One-Way ANOVA (One-Way ANOVA). Assuming that the test level is judged as α ═ 0.05, P <0.05 indicates that the difference is statistically significant. Statistical analysis was performed using GraphPad Prism Software version 5.0(GraphPad Software, CA, USA).