CN111518890A - Application of GALNT2 as endometrial hyperplasia or endometrial cancer diagnosis and treatment marker - Google Patents
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Abstract
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.
Description
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).
Example 1 establishment of endometrial hyperplasia model rats
1. Animals and treatments
Female Sprague-Dawley rats (160-180g) were purchased from SIPPR-BK Lab Animal, Shanghai, China. Rats were kept under controlled temperature, humidity and light conditions. All animal studies were approved by the animal ethics committee of xu university of medical science and have been conducted according to the declaration of helsinki. Intramuscular injection of Estradiol Benzoate (EB) (60. mu.g/100 g) into rats caused endometrial hyperplasia in rats. 16 female SD rats fed with normal diet were randomly divided into 2 groups (normal (N) group and endometrial hyperplasia model (NH) group). N groups of rats were intramuscularly infused with sesame oil every two days for 4 weeks. NH groups rats were intramuscularly injected with EB (60. mu.g/100 g) every two days for 4 weeks. At the end of the experiment, the serum of the animals, the uterine tissue, were collected, frozen at-80 ℃ or fixed overnight with 10% formaldehyde solution, paraffin embedded, for histological and immunohistochemical analysis.
2. Histopathological examination
Rat endometrial tissue was fixed in 10% formaldehyde solution for 24 h. The tissue samples were then wax embedded. Paraffin embedded with uterine tissue was prepared into 4 μm sections. Following deparaffinization and fluid replacement, the uterine sections were mounted on slides and stained with hematoxylin and eosin (H & E) and subjected to routine histological examination under an optical microscope (Olympus VS 120).
3. Endometrial thickness statistics
HE stained sections were taken, 5 non-overlapping fields of endometrial tissue were observed for each section, and statistical analysis was performed by measuring endometrial thickness in rats using computer image analysis software.
4. Immunohistochemical (IHC) staining
The endometrium tissue chip was purchased from Cian Baisida Biotech, Inc., including 6 JianWell-known (N), 12 Endometrial Hyperplasia (EH) patients and 6 Endometrial Cancer (EC) patients. After dewaxing, IHC was performed. The slides were rinsed in Phosphate Buffered Saline (PBS), excess fluid around the tissue was wiped dry with clean filter paper, and 3% H was added to each tissue2O2Incubate at room temperature for 10min to eliminate endogenous peroxidase activity. The sections were placed in a container with citrate buffer, heated in a microwave oven for 5 minutes, then low heated for 15 minutes for antigen retrieval, and then blocked in 2% BSA for 20 minutes. anti-PCNA (Abcam, 1: 100) and anti-GALNT 2(Abcam, 1: 100) antibodies were diluted with primary antibody dilutions in advance to cover the entire tissue, and incubated at 37 ℃ for 2 h. Visualization was performed using Diaminobenzidine (DAB) and hematoxylin staining followed by examination using a digital slide scanning system (olympus VS120 microscope). Linear measurements were obtained using an Image analysis system (Image-Pro Plus 4.0, Media Cybernetics, Silver Spring, MD).
5. Results
To investigate the effect of GALNT2 on endometrial hyperplasia, rats were developed for a model of endometrial hyperplasia (NH).
As shown in fig. 1A, B, the uterine index and endometrial thickness were significantly increased in the NH group rats compared to the N group. HE staining of rat uterine tissue showed (fig. 1C) that the endometrial epithelial cells of N groups of rats were well-arranged without significant inflammatory cell infiltration and interstitial connective tissue proliferation. However, the endometrial epithelial cells of the NH group rats showed disorganization in the arrangement of the cells, a degree of disorder in the arrangement, and enlargement and deep staining of the nuclei, with pathological nuclear divisions clearly visible.
Proliferating Cell Nuclear Antigen (PCNA) is a proliferating cell nuclear antigen that is synthesized or expressed only in proliferating cells. Here, PCNA protein expression in rat uterine tissue was examined by immunohistochemical staining. As shown in fig. 1D, the level of PCNA was significantly increased in uterine tissue of NH group rats compared to N group, and the difference was statistically significant.
Example 2 expression of GALNT2 in uterine tissue of Normal rats and endometrial hyperplasia model rats
1. Quantitative reverse transcriptase PCR (real-time PCR)
Use of
(Invitrogen) Total tissue RNA was extracted and cDNA was synthesized using the PrimeScript RT kit (TaKaRa Bio-technology) according to the manufacturer's instructions. Use of
480 II (Roche, Switzerland) were subjected to quantitative reverse transcriptase PCR analysis to determine mRNA expression by the method described above. The primer sequences are detailed in Table 1.
TABLE 1 qRT-PCR reaction primers
2. Western blot analysis
Uterine tissue was lysed with ice-cold RIPA and insoluble material was removed. Proteins were separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide (SDS) gel, transferred to a Nitrocellulose (NC) membrane, and blocked with 3% BSA for 2h at room temperature. And (3) incubating the membrane and the primary antibody at 4 ℃ overnight, taking the membrane to room temperature for incubation for 30min the next day, adding the corresponding near-infrared fluorescence labeled secondary antibody, and incubating the membrane and the secondary antibody at room temperature in a dark place for 1 h. The main antibodies used were GALNT2(Abcam), gapdh (proteintech). And placing the NC film into a two-color infrared laser imaging system for scanning, storing the scanned Image, performing gray level statistical analysis through Image J statistical software, and expressing the relative expression content of the target protein by the ratio of optical density.
3. Results
Quantitative reverse transcriptase PCR results showed significant differences in mRNA expression of GALNT2 between the N and NH groups. mRNA expression of GALNT2 was significantly reduced in the NH group compared to the N group (fig. 2A). The results of western blot analysis are shown in fig. 2B and 2C, where GALNT2 protein expression was reduced in the NH group compared to the N group, and the differences were statistically significant (P < 0.05).
Example 3 Effect of GALNT2 on the development of endometrial cancer
1. Validation of GALNT2 knockdown experiments
(1) Cell culture and processing
The human endometrial cancer cell line Ishikawa is provided by a cell bank of the chinese academy of sciences (shanghai, china). Ishikawa cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (Clark, USA) and 1% penicillin, 1% streptomycin, 5% CO2And culturing in a constant-temperature incubator at 37 ℃.
(2) Transfection
Knockdown of GALNT2 expression in Ishikawa cells using small interfering rna (sirna). The sequence of GALNT2siRNA is 5'-GGUGAUCACGUUUCACAAUTT-3' (SEQ ID NO.5), and the sequence of Negative Control (NC) siRNA is 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO. 6). All siRNA oligonucleotides were synthesized by GenePharma (shanghai, china). After plating, Ishikawa cells were cultured for an additional 24h and then transfected with gp-transfer-mate transfection reagent (GenePharma), serum-free medium, GALNT2siRNA, or negative siRNA. After 6h of transfection, cells were transferred to complete medium and cell lysates were collected after 48h of culture. GALNT2 expression was detected using the methods described previously.
The results showed that the si-GALNT2-N group (group transfected with siRNA against GALNT2) had reduced GALNT2 expression compared to the MOCK-N group (transfection negative control group), indicating successful knockdown of GALNT2 in Ishikawa cells (FIGS. 3A and 3B).
2. Effect of knocking down GALNT2 Gene on Ishikawa cell proliferation
After transfection of cells with siRNA, culture in complete medium was continued for 48 hours. Cell proliferation was examined by Cell Counting Kit-8(CCK-8) assay (Dojindo, Japan) according to the manufacturer's instructions.
Cell experiments demonstrated that interfering with GALNT2 expression can induce endometrial cancer cell proliferation (fig. 3C).
Example 4 clinical study
1. Clinical study subjects
Patients and healthy women diagnosed and examined at the xu state medical university affiliated hospital from 2017, 2 months to 2018, 10 months, including 32 female patients diagnosed with Endometrial Hyperplasia (EH) by Endometrial curettage (mean age: 49.50 ± 1.59 years), 30 female patients diagnosed with Endometrial Cancer (EC) (mean age: 51.46 ± 1.30 years) and 30 healthy female volunteers (N) (mean age: 50.53 ± 1.43 years) were collected in this study. Blood samples of patients with endometrial hyperplasia and endometrial cancer are obtained from obstetrics and gynecology department, and blood samples of healthy people are obtained from a physical examination center of a hospital affiliated with Xuzhou medical school, and venous blood is collected in fasting state. The serum was centrifuged and separated within 1 hour and stored at-20 ℃ until experimental analysis. The women in this study had not previously used hormone therapy. The study was registered in Chinese clinical trial registry (code Chi-CTR-1800014658) on 26.1.2018 and was conducted according to the declaration of Helsinki. Each subject signed a written informed consent prior to the study.
Expression of GALNT2 in endometrial tissue and serum of patients with endometrial hyperplasia and endometrial cancer was examined using immunohistochemistry and ELISA. As can be seen from fig. 4A and 4B, the expression of GALNT2 showed a significant decrease in endometrial tissue in endometrial hyperplastic patients (EH) and even lower in endometrial cancer patients (EC) compared to normal control (N). In addition, the level of GALNT2 was also significantly down-regulated in the serum of endometrial hyperplasia patients (EH), and this down-regulation was more pronounced in the serum of endometrial cancer patients (fig. 4C).
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Sequence listing
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Application of <120> GALNT2 as endometrial hyperplasia or endometrial cancer diagnosis and treatment marker
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Claims (10)
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.
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