CN113151473A - Method for detecting EphA2DNA methylation in invasive breast cancer and application - Google Patents
Method for detecting EphA2DNA methylation in invasive breast cancer and application Download PDFInfo
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The invention relates to the technical field of biological medicines, and particularly discloses a method for detecting EphA2DNA methylation in invasive breast cancer, which comprises the steps of extracting tissue whole genome DNA, modifying the whole genome DNA in a methylation mode, and carrying out nMS-PCR amplification detection by utilizing a pair of outer primers and two pairs of inner primers. The method for detecting EphA2DNA methylation in invasive breast cancer can detect the level of EphA2DNA methylation in tissues, thereby judging the incidence of invasive breast cancer.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for detecting EphA2DNA methylation in invasive breast cancer and application thereof.
Background
Breast cancer is one of the most serious malignant tumors in women, about 1700 thousands of breast cancer patients are diagnosed globally at present, and about 100 thousands of people die of the breast cancer patients every year. Research shows that the inflammatory reaction is involved in the occurrence and development of breast cancer diseases, and the mammary gland corpuscles can generate inflammatory changes in early stage of the breast diseases, but the specific mechanism is not clear. The breast cancer is one of the most common three cancers including lung cancer and colon cancer, and the incidence rate of the breast cancer is on the rising trend year by year along with the improvement of the clinical screening level of China and the change of the living standard of people in recent years. Although tumor markers can be analyzed through second-generation gene sequencing and early breast cancer can be screened through pathological biopsy, no active and effective breast cancer treatment method exists at present due to the complex typing, various symptoms and poor prognosis of breast cancer. During the formation of breast nodules, the inflammatory response plays an important role; it has been found that, before the onset of early stage breast disease, inflammatory changes occur in breast nodules first, eventually leading to the development of breast cancer.
Erythropoietin human hepatocyte receptor tyrosine kinase A2(EphA2) is a transmembrane protein widely expressed on tumor cells, and mainly acts on signal transduction aspects such as cell growth, inflammatory cell metastasis, cell rejection, adhesion and the like. Mammalian Ephrin receptors are classified into a-class and B-class according to their transmembrane domains, including 9 ephas (EphA1-8 and EphA10) and five EphB receptors (EphB1-4 and EphB 6). Wherein EphA2 is a transmembrane glycoprotein consisting of 976 amino acids originally discovered in 1990; under normal conditions, EphA2 can interact with ephrin a1 ligands on neighboring cells, modulating inflammatory responses and endothelial cell activation. There is increasing evidence that EphA2 plays a role in the progression and progression of tumors such as esophageal, breast, lung and ovarian cancer. The detection of EphA2 expression in breast tissue of a patient can help us to analyze and diagnose the progression and pathological type of breast cancer. However, the condition of invasive breast cancer cannot be accurately diagnosed merely by detecting the expression of EphA2, and the specific role of EphA2 in invasive breast cancer cannot be analyzed.
Disclosure of Invention
To solve the above technical problems, the present invention provides a method for detecting EphA2DNA methylation in invasive breast cancer.
The invention provides a method for detecting EphA2DNA methylation in invasive breast cancer, comprising: extracting the whole genome DNA of a tissue to be detected, carrying out methylation modification on the whole genome DNA, carrying out nMS-PCR amplification by using a pair of outer primers, then carrying out amplification by using two pairs of inner primers by using the obtained product as a template, and obtaining the methylation level through calculation.
Furthermore, the upstream primer of the pair of outer primers is shown as SEQ ID NO.1, and the downstream primer is shown as SEQ ID NO. 2.
Further, the two pairs of inner primers comprise methylated primers and unmethylated primers;
the sequence of an upstream primer of the methylation primer is shown as SEQ ID NO.3, and the sequence of a downstream primer of the methylation primer is shown as SEQ ID NO. 4;
the upstream primer sequence of the non-methylated primer is shown as SEQ ID NO.5, and the downstream primer sequence of the non-methylated primer is shown as SEQ ID NO. 6.
Further, the methylation level is calculated by:
methylation level ═ methylation OD value/(methylation OD value + non-methylation OD value).
The invention also provides application of the EphA2 gene in preparation of a medicament for treating breast cancer.
The invention also provides application of the expression product of the EphA2 gene in preparing a medicament for treating breast cancer.
Further, the breast cancer is invasive breast cancer.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a method for detecting EphA2DNA methylation in invasive breast cancer, so as to judge the disease development condition of invasive breast cancer, and compared with a normal group and a paracarcinoma group, the DNA methylation level of an EphA2 promoter region in a breast cancer group is obviously reduced.
2. The invention provides a method for detecting EphA2DNA methylation in invasive breast cancer, lays a foundation for researching related mechanisms, and finds that DNMT3a may participate in the process of DNA hypomethylation of an EphA2 promoter region, so that the level of interference DNMT3a can be utilized to control the DNA hypomethylation level of the EphA2 promoter region.
3. The invention can diagnose the progress and pathological type of the invasive breast cancer by detecting the methylation level of EphA2DNA in the invasive breast cancer, the methylation level of EphA2DNA is in negative correlation with the expression level of EphA2 protein, and the invasive breast cancer can be treated by improving the methylation level of EphA2 DNA.
4. In order to improve the accuracy of diagnosis of invasive breast cancer in clinic, the DNA methylation level of the EphA2 promoter region is detected while the EphA2 protein expression level is detected, the specific action of EphA2 in invasive breast cancer can be analyzed, and a new theoretical basis can be provided for diagnosis of invasive breast cancer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a diagram showing the expression of EphA2 in normal breast tissue, paraneoplastic tissue and breast cancer tissue by Western blot detection in the present invention, wherein 1 is normal breast tissue, 2 is paraneoplastic tissue and 3 is breast cancer tissue;
wherein, FIG. 1A is a photograph of the expression of EphA2 in normal breast tissue, paraneoplastic tissue and breast cancer tissue;
FIG. 1B is a statistical plot of the expression levels of EphA2 in normal breast, paracancerous and breast cancer tissues in accordance with the invention;
FIG. 2 is a graph showing the expression of EphA2 in paraffin sections of a para-carcinoma tissue and a breast cancer tissue detected by immunofluorescence in the present invention, wherein 2 is the para-carcinoma tissue, and 3 is the breast cancer tissue;
wherein, figure 2A is an immunofluorescence assay of EphA2 in paracancerous and breast cancer tissues;
FIG. 2B is a statistical plot of the expression levels of EphA2 in paracarcinoma and breast cancer tissues;
FIG. 3 is a graph showing the level of methylation of the DNA of the promoter region EphA2 in normal breast tissue, paracarcinoma tissue and breast cancer tissue according to the present invention using nested methylation-specific PCR (nMS-PCR), wherein 1 is normal breast tissue, 2 is paracarcinoma tissue and 3 is breast cancer tissue;
wherein FIG. 3A is a gel diagram of nested methylation specific PCR (nMS-PCR) assays for DNA methylation of the EphA2 promoter region in normal breast, paracancerous and breast cancer tissues;
FIG. 3B is a graph showing the DNA methylation levels of the promoter region of EphA 2;
FIG. 4 is a diagram showing the expression of DNMT3a in normal breast tissue, paraneoplastic tissue and breast cancer tissue by Western blot detection, wherein 1 is normal breast tissue, 2 is paraneoplastic tissue, and 3 is breast cancer tissue;
wherein, fig. 4A shows a glue pattern of DNMT3a expression in normal breast tissue, paracancerous tissue and breast cancer tissue;
fig. 4B shows a statistical graph of the expression levels of DNMT3a in normal breast tissue, paracancerous tissue, and breast cancer tissue.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Method for detecting EphA2DNA methylation in invasive breast cancer and application
1. Subject: 42 specimens of breast cancer, tissues beside the cancer and normal breast tissue of the general hospital tumor hospital of Ningxia medical university are collected. The pathological type is invasive breast cancer, the experiment is approved by ethical committee of hospital, and informed consent of patients is obtained
2. Instrument materials and reagents
The main apparatus is as follows: clean bench (antai); model 5415D micro bench centrifuge (Eppendorf); precision balance model BS110S (Sartorius); vertical electrophoresis and Model 680 full-automatic enzyme labeling machine (Bio-Rad); homogenizers (MP Biomedicals); laser confocal microscopy (Zeiss);
the main reagents are as follows: genomic DNA extraction kit (tiangen); protein extraction kits and protein quantification kits (kaiky bio ltd); DNA methylation modification kit (ZYMO); anti-EphA 2 antibody (Affinity); anti-DNA methyltransferase 3a (DNA methyltransferase 3a, DNMT3a) antibody (abcam); anti-beta-actin human monoclonal antibodies (sequoia ponticello bio); horseradish peroxidase-labeled goat anti-mouse IgG and horseradish peroxidase-labeled goat anti-rabbit IgG (boaosen biotechnology limited); the primers were synthesized by Shanghai Bioengineering Co., Ltd.
3. Method of producing a composite material
(1) Western blot detection of protein expression of EphA2 and DNMT3a
Extracting the whole protein of each group of mammary tissues according to the specification of the whole protein extraction kit, and adding a certain proportion of sample buffer solution to boil and denature for 5min after quantification by a BCA method; taking 40 mu g of total protein from each group, carrying out SDS-PAGE gel electrophoresis for 80V, then electrically transferring to a PVDF membrane, and sealing for 2h by 5% skimmed milk; incubated overnight at 4 ℃ with anti-EphA 2 and DNMT3a antibodies at the corresponding dilution ratios, incubated for 2h at room temperature with secondary antibodies at a dilution ratio of 1:5000, and washed 3 times for 10min each in PBST. Scanning by a gel image analysis imaging system, taking beta-actin as an internal reference, and calculating the ratio of the gray values of EphA2 and DNMT3a to the gray values of the beta-actin internal reference for semi-quantitative detection.
(2) Expression of EphA2 by immunofluorescence staining
Each group of slices was dewaxed, dehydrated, fixed with 4% paraformaldehyde for 30min, and washed three times with PBS for five minutes each time. Acting with 10% volume fraction hydrogen peroxide for 10 min; incubating with 0.3% Triton X-100 for 10min, washing with PBS, adding goat serum, and sealing for 1 hr. Then adding anti-EphA 2 antibody, and incubating overnight at 4 ℃; adding a fluorescent secondary antibody after washing by PBS, incubating for 2h at room temperature in a dark place, dyeing cell nuclei by DAPI for 10min, and adding an anti-fluorescence quencher for sealing; and (4) observing and photographing under a laser confocal microscope, and analyzing the fluorescence expression condition of the target area by using image J.
(3) Nested methylation specific PCR (nMS-PCR) for detecting DNA methylation level of EphA2 promoter region
Extracting the whole genome DNA of normal tissues, tissues beside the cancer and breast cancer tissues according to the DNA extraction kit specification, and carrying out methylation modification on the whole genome DNA. nMS-PCR method detects changes in the degree of DNA methylation in the promoter region of EphA 2. Aiming at EphA2 sequences, designing a pair of outer primers and two pairs of inner primers, wherein the upstream primer sequence of the outer primers is shown as SEQ ID NO.1, and the downstream primer sequence of the outer primers is shown as SEQ ID NO. 2;
the two pairs of inner primers are methylated primers and unmethylated primers respectively, wherein the sequence of an upstream primer of the methylated primer is shown as SEQ ID NO.3, and the sequence of a downstream primer of the methylated primer is shown as SEQ ID NO. 4; the sequence of the upstream primer of the non-methylated primer is shown as SEQ ID NO.5, and the sequence of the downstream primer of the non-methylated primer is shown as SEQ ID NO. 6;
SEQ ID NO.1：5'-GGGGGATGTTAATAGTTATAATGTG-3'
SEQ ID NO.2：5'-CTCCTACCAATACCAAAAACAAAAC-3'
SEQ ID NO.3：5'-GGTGTTTTAGGTTTGG TGATTC-3'
SEQ ID NO.4：5'-TATTAACATCCCCCTTCTTACGAT-3'
SEQ ID NO.5：5'-GGTG TTTTAGGTTTGGTGATTAGTT-3'
SEQ ID NO.6：5'-CTATTAACATCCCCCTTCTTACAAT-3'
the reaction system of the nMS-PCR is as follows: PCR MIX 12.5. mu. L, H2O 7. mu.L, upstream and downstream primers 1. mu.L each, modified DNA 3.5. mu.L, total 25. mu.L;
the reaction conditions for the outer primer amplification are as follows: 5min at 95 ℃; 30s at 95 ℃, 30s at 63 ℃ and 30s at 72 ℃, and 20 cycles, wherein each cycle is reduced to 0.5-53 ℃ for 7min at 72 ℃; the methylated and unmethylated inner primers were amplified separately using the PCR product of the outer primer as a template, and the reaction conditions were the same as for the outer primer.
The product was then electrophoresed on a 2% agarose gel and the methylated bands were analyzed by imaging with a gel image analyzer and calculated as follows:
methylation level ═ methylation OD value/(methylation OD value + non-methylation OD value)
(4) Statistical treatment
The experimental results are all measured data, the experimental data are subjected to statistical analysis by using Prism 6.0 statistical software, the experimental results of the measured data are expressed by mean +/-standard deviation, t test is adopted for comparison between two groups, one-factor variance analysis is adopted between three groups, and P <0.05 shows that the difference has statistical significance.
4. Results
(1) Protein expression of EphA2 in various groups of breast cancer patients
EphA2 protein expression in each group was detected by immunofluorescence staining and Western blot, and statistical analysis was performed. The results are shown in FIG. 2: the fluorescence expression level of EphA2 in breast cancer tissues is obviously increased compared with that in paracarcinoma tissues (P < 0.05); as shown in fig. 1, the expression level of EphA2 protein was increased in breast cancer tissues compared to normal breast tissues and paraneoplastic tissues (P <0.01 ).
(2) Changes in EphA2 promoter region DNA methylation in breast cancer tissue
To further investigate the specific mechanism of increased EphA2 expression in breast cancer, nMS-PCR was used to detect the levels of EphA2DNA methylation in each group, and the results are shown in fig. 3, where the levels of EphA2 promoter DNA methylation in the breast cancer group were reduced (P <0.05) compared to the normal and paracancerous groups, and the differences were statistically significant.
(3) Expression of DNA methylase DNMT3a in breast cancer
The expression level of DNMT3a protein in each group was detected by Western blot, and the results are shown in FIG. 4: compared with normal tissues and tissues beside the cancer, the expression of DNMT3a protein in the breast cancer group is reduced (P <0.01 and P <0.05), and the difference has statistical significance.
Taken together, EphA2 expression was upregulated in invasive breast cancer tissues; DNMT3a may be involved in the process of DNA hypomethylation of the EphA2 promoter region, the progress and pathological type of invasive breast cancer can be diagnosed by detecting the methylation level of EphA2DNA in the invasive breast cancer, the methylation level of EphA2DNA is in negative correlation with the expression level of EphA2 protein, and the invasive breast cancer can be treated by increasing the methylation level of EphA2 DNA.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
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Claims (7)
1. A method for detecting EphA2DNA methylation in invasive breast cancer, comprising: extracting the whole genome DNA of a tissue to be detected, carrying out methylation modification on the whole genome DNA, carrying out nMS-PCR amplification by using a pair of outer primers, then carrying out amplification by using two pairs of inner primers respectively by using the obtained products as templates to obtain products, and obtaining the methylation level through calculation.
2. The method of claim 1, wherein the pair of outer primers comprises an upstream primer as set forth in SEQ ID No.1 and a downstream primer as set forth in SEQ ID No. 2.
3. The method of claim 2, wherein the two pairs of inner primers comprise a methylated primer and an unmethylated primer;
the sequence of an upstream primer of the methylation primer is shown as SEQ ID NO.3, and the sequence of a downstream primer of the methylation primer is shown as SEQ ID NO. 4;
the upstream primer sequence of the non-methylated primer is shown as SEQ ID NO.5, and the downstream primer sequence of the non-methylated primer is shown as SEQ ID NO. 6.
4. The method of claim 3, wherein said methylation level is calculated by:
methylation level ═ methylation OD value/(methylation OD value + non-methylation OD value).
5. Use of the EphA2 gene of claim 1 for the manufacture of a medicament for the treatment of breast cancer.
6. Use of the expression product of the EphA2 gene of claim 5 in the manufacture of a medicament for the treatment of breast cancer.
7. Use of the expression product of the EphA2 gene of claim 6 in the preparation of a medicament for the treatment of a breast cancer, wherein the breast cancer is invasive.
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| CN114622012A (en) * | 2022-04-21 | 2022-06-14 | 宁夏医科大学 | EphA2 gene and application of methylation level detection reagent thereof |
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