CN114622012A - EphA2 gene and application of methylation level detection reagent thereof - Google Patents

Abstract

The invention belongs to the technical field of medicine, and particularly relates to the application of EphA2 gene and a methylation level detection reagent thereof. The application is specifically the application of the EphA2 gene methylation level detection reagent in the preparation of products for diagnosing diseases caused by islet beta cell apoptosis, and the application of the EphA2 gene for screening potential substances for the treatment of type 2 diabetes. The results of the present invention confirm that the EphA2 gene Hcy is a key gene that causes dysglycemia, and EphA2 DNA hypermethylation is involved in the regulation of homocysteine-induced apoptosis of pancreatic islet β cells in Cbs ^+/- mice, which can be used as early clinical diagnosis and disease development. The evaluation provides a new class of markers, and also provides new therapeutic ideas for the clinical treatment of type 2 diabetes mellitus with insufficient insulin secretion caused by pancreatic β-cell dysfunction, which has potential value.

Description

Application of EphA2 gene and its methylation level detection reagent

technical field

The invention belongs to the technical field of medicine, and specifically relates to the application of EphA2 gene and a methylation level detection reagent thereof.

Background technique

Diabetes mellitus is a general term for heterogeneous metabolic disorders, the main manifestation of which is chronic hyperglycemia. The pathogenesis is mainly due to the destruction of pancreatic β cells, including insulin resistance, progressive insulin secretion insufficiency, or both. It has become a group of metabolic diseases that seriously endanger human health due to its insidious onset, high morbidity and many comorbidities. Worldwide, 415 million people currently have diabetes, and an estimated 193 million people have undiagnosed diabetes. Among them, type 2 diabetes accounts for more than 90% of diabetic patients, often causing microvascular and macrovascular complications, causing profound psychological and physical distress to patients and caregivers, and placing a huge burden on the health care system. So far, despite the collection and analysis of large amounts of data on diabetes, the actual molecular mechanisms underlying the development of type 2 diabetes (T2DM) remain unknown. Based on this, it is urgent to find a molecular marker for the diagnosis of islet β-cell apoptosis induced by homocysteine, and to further explore the clinical prevention and treatment strategy of type 2 diabetes, which has great and far-reaching effects on improving human health and optimizing population quality. meaning.

Homocysteine (Hcy) is a sulfur-containing non-protein amino acid formed during the metabolism of the essential amino acid methionine, which can induce important biological functions such as apoptosis, autophagy, and pyroptosis. According to epidemiological surveys, Hcy levels are elevated in obese patients with insulin resistance and impaired insulin secretion, and even in non-diabetic insulin-resistant populations. It can be seen that Hcy is closely related to abnormal blood sugar. Research evidence suggests that overproduction of proinflammatory cytokines contributes to DM, and that locally produced IL-1β in pancreatic islets leads to islet β-cell dysfunction that reduces insulin production, suggesting that islet β-cell apoptosis may be central to its dysfunction link. At the same time, studies have also shown that bcl-2 is a recognized cellular regulatory target, which can antagonize bax, thereby reducing the amount of free bax. The protein expression of bcl-2 decreases, which increases the translocation of bax to the mitochondrial membrane, enhances the permeability of mitochondria, releases cytochrome-c (Cyt-c), and activates the online reaction of caspase-3. apoptosis. In this study, we cultured Cbs+/- mouse islet β cell line (Min6) in vitro, and detected apoptosis-related indicators by immunofluorescence and Western blot. Compared with the control group, we also found that the expression of bcl-2 protein in Hcy-intervention cells was also found. decreased, the protein expressions of bax and caspase-3 increased significantly, and the ratio of bax/bcl-2 increased, suggesting that the dysfunction of islet β cells caused by Hcy is an important mechanism of blood glucose elevation.

EphA2 is a major member of erythropoietin-producing hepatocyte (Eph) receptors, and Eph receptor tyrosine kinases and their ephrin ligands play important roles in both physiological and pathological processes. Eph receptors and their ligands are divided into two subfamilies, of which Eph receptors are the largest subfamily of receptor tyrosine kinases (RTKs). Studies have shown that EphA2 is overexpressed in breast cancer, cervical cancer and melanoma. So far, there is no correlation study between EphA2 and pancreatic β-cell apoptosis and type 2 diabetes.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a molecular marker for the diagnosis of islet beta cell apoptosis caused by homocysteine and its application, and to provide a theoretical basis for the clinical diagnosis of type 2 diabetes.

In a first aspect, the present invention provides the application of an EphA2 gene methylation level detection reagent in the preparation of a product for diagnosing a disease caused by islet β-cell apoptosis. The sequence of the EphA2 gene is shown in SEQ ID NO.1.

Further, the β-cell apoptosis is induced by homocysteine.

Still further, the disease includes type 2 diabetes.

Furthermore, the methylation level of EphA2 gene was elevated in pancreatic beta cells of patients with type 2 diabetes.

Further, the reagents include nMS-PCR detection reagents.

Further, the nMS-PCR detection reagents include methylation primers for the EphA2 gene, the sequences of which are shown in SEQ ID NO. 6-7.

In the second aspect, the present invention provides the application of the EphA2 gene for screening potential substances for the treatment of type 2 diabetes.

Further, the substance is a substance that downregulates the methylation level of the EphA2 gene.

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

The present invention finds that the protein expression level and mRNA expression level of EphA2 in the islet beta cells mediated by homocysteine are significantly lower than those in the control group, which may suggest that EphA2 may negatively regulate the apoptosis of islet beta cells in the occurrence and development of diabetes. play a role. DNA methylation is the most characteristic phenomenon of epigenetics, which can cause changes in chromatin structure, DNA stability, DNA conformation and stability, and the interaction between DNA and proteins, thereby regulating gene expression.

In the present invention, we found that the EphA2 DNA methylation level was significantly increased in the homocysteine group, which suggested that the decreased expression level of EphA2 was closely related to the increased DNA methylation level of the EphA2 promoter region. DNMT1 is the main enzyme that maintains methylation patterns after DNA replication and plays an important regulatory role in a variety of diseases. The present invention finds that the expression of DNMT1 in the Hcy group is increased, suggesting that DNMT1 may play a significant role in promoting EphA2 DNA methylation, confirming that EphA2 hypermethylation expression is closely related to changes in blood glucose levels and islet β-cell apoptosis.

In summary, it can be seen that EphA2 is a key gene for Hcy-induced dysglycemia, and DNA hypermethylation in the EphA2 promoter region may be the key mechanism of homocysteine-induced apoptosis of pancreatic islet β cells. It provides a new experimental basis and research direction for the occurrence of diabetes caused by pancreatic islet cell secretion dysfunction. It can also be used as a new marker for early clinical diagnosis and assessment of disease development, and is also useful for clinical treatment of insulin caused by pancreatic islet β-cell dysfunction. Type 2 diabetes mellitus with insufficient secretion provides new therapeutic ideas and has potential value.

Description of drawings

Figure 1 shows the effect of Hcy on the apoptosis-related indicators of pancreatic β-cells. A: The expression of bax and bcl-2 protein in each group was detected by immunofluorescence; B: The expression of caspase-3 protein in each group was detected by immunofluorescence; C: The expression of bax/bcl-2 in each group was detected by Western blot Protein expression; D: Western blot test the protein expression of caspase-3 in each group. *P<0.05 compared to the control group.

Figure 2 shows the effect of homocysteine on apoptosis of pancreatic beta cells. A: Flow cytometry was used to detect the apoptosis of islet β cells in each group; B: Statistical chart of apoptosis rate, compared with the control group, *P<0.05.

Figure 3 shows the expression of EphA2 in islet beta cells. A: q-RT PCR was used to detect the mRNA expression of EphA2 in islet β cells; B: Western blot was used to detect the expression of EphA2 protein in each group. *P<0.05 compared to the control group.

Figure 4 shows the detection of EphA2 DNA methylation level by nMS-PCR. A: map of CPG island in the promoter region of EphA2 gene; B: methylation level of EphA2 gene detected by agarose gel electrophoresis; C: statistical map of EphA2 DNA methylation level; compared with the control group, *P<0.05.

Figure 5 shows the expression of DNMT1 and DNMT3a in each group of islet β cells. A: Western blot detection of DNMT1 protein expression in each group B: Western blot detection of DNMT3a protein expression in each group. *P<0.05 compared to the control group.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

1 Materials and methods

1.1 Materials: mouse pancreatic islet beta cell line (min6).

1.2 Main reagents and instruments

Main experimental kits and instruments: fetal bovine serum, RPMI-1640 medium (Gibco, USA); trypsin digestion solution (Biyuntian Institute of Biotechnology); homocysteine (Sigma, USA); genomic DNA Extraction kit, total RNA extraction kit (Beijing, Tiangen); protein extraction kit, protein quantification kit (Nanjing, KGI); reverse transcription and qRT-PCR kit (U.S., Thermo Fisher); DNA A Sylation kit (ZYMO, USA); EphA2 antibody, Bax antibody, Caspase-3 antibody, Bcl-2 antibody (Affinity, China); β-actin antibody (Zhongshan Jinqiao, China); primers provided by Shanghai Sangon Bioengineering Co., Ltd.; CO ₂ incubator (Germany, Heraeus); ultra-clean workbench (Suzhou, Antai); 5415D micro-tabletop centrifuge (Germany, Eppendorf); BS110S precision balance (Germany, Sartorius); fluorescence quantitative PCR instrument (Shanghai, Fengling); electrophoresis apparatus, electroporation apparatus, gel imager (Bio-rad, USA); laser confocal microscope (Zeiss).

2. Experimental method

1.2.1 Cell culture and treatment The islet β cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and placed in an incubator at 37°C and 5% CO ₂ . When the cell density reached 80% of the total volume, the cells were passaged at a ratio of 1:3, once every two days, and the third passage of cells were replaced with a control group with a concentration of 0 μmol/L homocysteine and a homocysteine concentration of 120 μmol/L. For the homocysteine group of cysteine, after 48 hours of intervention in the cells, the cells were collected, and the relevant indicators were detected.

1.2.2 Immunofluorescence staining to detect the expression of bcl-2, bax and caspase-3

The β-cells in the control group and the homocysteine group were fixed with 4% formaldehyde for 30 min respectively, washed with PBS for 3 times for 5 min each, incubated under 10% hydrogen peroxide for 10 min, washed with PBS for three times, and then blocked with goat serum for 60 min. bcl-2, bax, caspase-3 primary antibodies, incubate overnight at 4°C, remove the cells, let stand at room temperature for 30 minutes, add fluorescent secondary antibodies after washing with PBS, incubate at 37°C for 1 h in the dark, rinse with PBS and place in the dark DAPI was added to stain cell nuclei under light conditions for 5 min, rinsed with PBS again, and then mounted with fluorescent quencher dropwise. The stained cells were observed under a laser confocal microscope and photographed.

1.2.3 Detection of apoptosis rate by flow cytometry

Islet β cells were divided into two groups: (1) blank control group; (2) homocysteine intervention group; after 48 hours of homocysteine intervention of islet β cells, the cells were harvested, washed twice with PBS, and treated with PBS without Cells were collected by trypsinization with 0.25% EDTA, transferred to a 1.5 centrifuge tube, centrifuged with a centrifuge (4° C., 10000 rpm, 5 min) to discard the waste liquid, and 1 ml of PBS was added, and the centrifugation was repeated twice. Under dark conditions, the binding solution was added to each centrifuge tube at a ratio of 1:9, then 5 μL of 7-AAD was added, 5 μL of PE was gently mixed, and it was still for 15 minutes. After blowing evenly, the cells were filtered with a filter membrane, and the apoptosis rate was detected by flow cytometry immediately. Streaming data was processed using NovoExpress software.

1.2.4 Real-time fluorescence quantitative PCR to detect the expression of EphA2 mRNA

The total RNA of β cells in each group was extracted according to the instructions of the RNA extraction kit, and the sequence of EphA2 was queried through the GenBank database and primers were designed. EphA2: upstream: 5'-GCACAGGGAAAGG AAGTTGTT-3', shown in SEQ ID NO.2; downstream: 5'-CATGTAGATAGGCATGTCGTCC-3', shown in SEQ ID NO.3. PCR amplification program: 95°C for 30s, 95°C for 5s, 55.1°C for ^34s , a total of 40 cycles, and an internal reference (GAPDH) control group was set at the same time.

1.2.5 Western blot detection of protein expression of bax, bcl-2, caspase-3, EphA2, DNMT1, DNMT3a

According to the instructions of the whole protein extraction kit, the whole protein of β cells in the control group and the homocysteine group was extracted. After quantification by BCA method, a certain proportion of protein loading buffer was added to boil for denaturation for 5 minutes, and 20 μg of total protein was taken from each group for SDS-PAGE. Gel electrophoresis for 80v, then electrotransfer to PVDF membrane, block with 5% skim milk for 2h, wash the membrane with PBST 3 times/10min, and dilute bax, bcl-2, caspase-3, EphA2, DNMT1, DNMT3a and β according to corresponding proportions. -actin antibody, shake overnight at 4°C; use horseradish peroxidase-labeled secondary antibody, diluted 1:5000, incubate at room temperature for 2h, wash the membrane 3 times/10min with PBST; image analysis on a gel imaging analyzer , β-actin was used as the internal reference, the ratios of bax, bcl-2, caspase-3, EphA2, DNMT1 and DNMT3a to the gray value of the β-actin internal reference were calculated and analyzed statistically.

1.4.6 Nested methylation specific PCR (nMS-PCR) to detect the DNA methylation level of EphA2 promoter region

The whole genome DNA of the control group and the homocysteine intervention group was extracted according to the instructions of the DNA extraction kit, and the whole genome was modified by DNA methylation. The changes of DNA methylation in the promoter region of EphA2 were detected by nMS-PCR. For the EphA2 sequence, one pair of outer primers and two pairs of inner primers were designed online (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi) (outer primer: upstream: 5'-GGGGGATGTTAATAGTTATAATGTG-3', SEQ ID NO.4; Downstream: 5'-CTCCTACCAATACCAAAAACAAAAC-3', SEQ ID NO.5; Methylation primer: Upstream: 5'-GGTGTTTTAGGTTTGG TGATTC-3', SEQ ID NO.6; Downstream 5'-TATTAACATCCCCCTTCTTACGAT-3', shown in SEQ ID NO.7; Unmethylated primer: upstream: 5'-GGTG TTTTAGGTTTGGTGATTAGTT-3', shown in SEQ ID NO.8; downstream: 5'-CTATTAACATCCCCCTTCTTACAAT-3', shown in SEQ ID NO. 9). (Reaction system: PCR MIX 12.5 μL, H ₂ O 7 μL, upstream and downstream primers 1 μL each, modified DNA 3.5 μL, a total of 25 μL) The reaction conditions for outer primer amplification are: 95°C for 5 min; 95°C for 30s, 63°C for 30s , 72 ℃ for 30s, 20 cycles, each cycle drops 0.5 ℃ to 53 ℃, 72 ℃ 7min. The PCR products of the outer primers were used as templates, and the methylated and unmethylated inner primers were amplified respectively, and the reaction conditions were the same as those of the outer primers. Then the product was electrophoresed on a 1% agarose gel, and the methylated bands were imaged and analyzed by a gel imaging analyzer, and the calculation was performed according to the following formula: % methylation=M/(M+U)×100 %, M is the methylated OD value, and U is the unmethylated OD value.

3 Statistical processing and analysis

表示，两组间比较采用t检验，P<0.05 表示差异有统计学意义。The experimental results are all measurement data, the experimental data are statistically analyzed using Prism 6.0 statistical software, and the measurement data experimental results are expressed as mean ± standard deviation

The t-test was used for comparison between the two groups, and P<0.05 indicated that the difference was statistically significant.

4 results

4.1 The effect of Hcy on the apoptosis-related indicators of pancreatic β-cells

The apoptosis of islet β cells was detected by immunofluorescence staining and Westen Blot method. The results showed that: Compared with the control group, the fluorescence intensity and protein level of islet β-cell-related apoptosis protein bax in the homocysteine group were significantly increased, indicating that the expression of bax in the homocysteine group was increased; Compared with the control group, the fluorescence intensity and protein level of bcl-2 decreased, indicating that the expression of bcl-2 was decreased. Compared with the control group, the fluorescence intensity and protein level of caspase-3 in the homocysteine group increased, indicating that the expression of caspase-3 increased. It is shown that homocysteine can increase the level of pancreatic β-cell apoptosis, as shown in Figure 1.

4.2 Flow cytometry to detect the effect of homocysteine on apoptosis of pancreatic β cells

Cell apoptosis was detected by flow cytometry. The results showed that compared with the control group, the apoptosis rate of the homocysteine group was significantly increased. It showed that the apoptosis rate of islet β cells could be increased after the intervention of homocysteine, as shown in Figure 2.

4.3 The effect of homocysteine on the expression level of EphA2

In order to prove the role of EphA2 in homocysteine-induced apoptosis of islet β cells, the expression levels of EphA2 mRNA and protein in islet β cells were detected by qRT-PCR and Western blot, respectively. The results showed that compared with the control group, the expression of EphA2 mRNA in pancreatic β cells in the homocysteine group was decreased, and the expression of EphA2-related proteins in pancreatic β cells in the homocysteine group was also significantly decreased, as shown in Figure 3.

4.4 Effect of homocysteine group on EphA2 DNA methylation level

In order to further explore the mechanism by which homocysteine reduces the expression of EphA2, nMs-PCR was used to detect the DNA methylation level of EphA2 in pancreatic β cells. Compared with the control group, the DNA methylation level of EphA2 in the homocysteine group was rise, see Figure 4.

4.5 Expression of DNMT1 and DNMT3a in the apoptosis of pancreatic β cells induced by homocysteine

Western blot was used to detect the protein expression levels of DNMT1 and DNMT3a in islet β cells. The results showed that compared with the control group, the expression level of DNMT1 protein in islet β cells in the homocysteine group increased, and the difference was statistically significant. However, the expression of DNMT3a was not statistically significant, see Figure 5.

Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be construed to include the preferred embodiment and all changes and modifications that fall within the scope of the present invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention. Thus, provided that these modifications and variations of the present invention fall within the scope of the claims of the present invention and technical equivalents thereof, the present invention is also intended to include these modifications and variations.

sequence listing

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Claims (8)

1. The application of the reagent for detecting the methylation level of the EphA2 gene in the preparation of products for diagnosing diseases caused by islet beta cell apoptosis is characterized in that the sequence of the EphA2 gene is shown as SEQ ID NO. 1.
2. 2. The use of claim 1, wherein said β -cell apoptosis is induced by homocysteine.
3. 3. The use according to claim 2, wherein the disease comprises type 2 diabetes.
4. 4. The use of claim 3, wherein the methylation level of the EphA2 gene is elevated in islet beta cells of the type 2 diabetic patient.
5. 5. The use of claim 4, wherein said reagents comprise nMS-PCR detection reagents.
6. 6. The use of claim 5, wherein said nMS-PCR detection reagent comprises a methylated primer for the EphA2 gene having the sequence shown in SEQ ID nos. 6-7.
7. 7. The use of the EphA2 gene of claim 1 for screening for potential agents for the treatment of type 2 diabetes.
8. 8. The use of claim 7 wherein the agent down-regulates the methylation level of the EphA2 gene.

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