CN114622012A - EphA2 gene and application of methylation level detection reagent thereof - Google Patents
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Abstract
The invention belongs to the technical field of medicine, and particularly relates to an EphA2 gene and application of a methylation level detection reagent thereof. The application is specifically the application of the detection reagent for the methylation level of the EphA2 gene in the preparation of products for diagnosing diseases caused by islet beta cell apoptosis, and the application of the EphA2 gene in screening potential substances for treating type 2 diabetes. The results of the invention prove that the key gene of the EphA2 gene Hcy causing blood sugar abnormality, wherein EphA2DNA hypermethylation is involved in homocysteine-induced Cbs+/‑The regulation and control of the mouse islet beta cell apoptosis can be used as a new marker for clinical early diagnosis and disease development evaluation, and also provides a new treatment idea for clinically treating type 2 diabetes with insufficient insulin secretion caused by islet beta cell dysfunction, and has potential value.
Description
Technical Field
The invention belongs to the technical field of medicine, and particularly relates to an EphA2 gene and an application of a methylation level detection reagent thereof.
Background
Diabetes is a collective term for heterogeneous metabolic disorders, and its main manifestation is chronic hyperglycemia. The pathogenesis is mainly due to the destruction of islet beta cells, including insulin resistance, insufficient progressive insulin secretion or both. The metabolic diseases seriously endanger the health of human beings are formed by occult onset, high morbidity and more complications. Currently, about 4.15 million people worldwide suffer from diabetes, and it is estimated that about 1.93 million people suffer from undiagnosed diabetes. Among them, type 2 diabetes accounts for more than 90% of diabetes patients, often causes complications of micro-and macrovascular diseases, brings deep psychological and physical pains to patients and nursing staff, and brings huge burden to medical care systems. To date, despite the large amount of data collected and analyzed regarding diabetes, the actual molecular mechanisms underlying development of type 2 diabetes (T2DM) are unknown. Based on this, a molecular marker for diagnosing islet beta cell apoptosis caused by homocysteine is urgently needed to be searched, and the prevention and treatment strategy of type 2 diabetes in clinic is further discussed, which has great and profound significance for improving the health level of human beings and optimizing the population quality.
Homocysteine (Hcy) is a sulfur-containing non-protein amino acid formed in the metabolism process of essential amino acid methionine, and can cause important biological functions of apoptosis, autophagy, scorch and the like of cells. According to epidemiological investigations, Hcy levels are elevated in obese patients accompanied by impaired insulin resistance and insulin secretion, and even in non-diabetic insulin resistant people, and it can be seen that Hcy is closely associated with blood glucose abnormalities. Research evidence suggests that excessive production of proinflammatory cytokines leads to DM and that local production of IL-1 β in the islets leads to islet β cell dysfunction and thus to decreased insulin production capacity, suggesting that islet β cell apoptosis may be a central link in its dysfunction. Meanwhile, the study also shows that bcl-2 is a recognized cell regulation target and can generate antagonism with bax, so that the amount of free bax is reduced. The decrease of the protein content of bcl-2 can increase bax translocation to a mitochondrial membrane, so that the permeability of mitochondria is enhanced, cytochrome c (Cyt-c) is released, the online reaction of caspase-3 is activated, and apoptosis occurs. In the research, a Cbs +/-mouse islet beta cell strain (Min6) is cultured in vitro, immunofluorescence and Western blot are adopted to detect apoptosis related indexes, and compared with a control group, the expression of bcl-2 protein in cells subjected to Hcy intervention is reduced, the protein expression of bax and caspase-3 is obviously increased, the ratio of bax/bcl-2 is increased, and the fact that islet beta cell dysfunction caused by Hcy is an important mechanism for increasing blood sugar is suggested.
EphA2 is a major member of the erythropoietin-producing hepatocyte (Eph) receptor, the Eph receptor tyrosine kinase and ligands for ephrin playing important roles in both physiological and pathological processes. Eph receptors and their ligands fall into two subfamilies, with Eph receptors being the largest subfamily of Receptor Tyrosine Kinases (RTKs). Studies have shown that EphA2 is overexpressed in breast cancer, cervical cancer, and melanoma. No study on EphA2 related to islet beta cell apoptosis and type 2 diabetes is available.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a molecular marker for diagnosing islet beta cell apoptosis caused by homocysteine and application thereof, and provides a theoretical basis for clinical diagnosis of type 2 diabetes.
In a first aspect, the invention provides an application of a reagent for detecting the methylation level of an EphA2 gene in preparing a product for diagnosing diseases caused by islet beta cell apoptosis, wherein the sequence of the EphA2 gene is shown as SEQ ID NO. 1.
Further, the beta cell apoptosis is induced by homocysteine.
Still further, the disease includes type 2 diabetes.
Furthermore, patients with type 2 diabetes have elevated levels of methylation of the EphA2 gene in islet beta cells.
Further, the reagent includes nMS-PCR detection reagent.
Furthermore, the nMS-PCR detection reagent comprises a methylation primer aiming at the EphA2 gene, and the sequence of the methylation primer is shown as SEQ ID NO. 6-7.
In a second aspect, the invention provides the use of the EphA2 gene for screening potential agents for the treatment of type 2 diabetes.
Further, the agent down-regulates the methylation level of EphA2 gene.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers that the protein expression level and the mRNA expression level of EphA2 in the islet beta cells intervened by homocysteine are obviously lower than those of a control group, which can suggest that EphA2 can play a role in the occurrence and development of diabetes by negatively regulating the apoptosis of the islet beta cells. DNA methylation is the most characteristic phenomenon of epigenetics, and it can cause changes in chromatin structure, DNA stability, DNA conformation and stability, and the way DNA interacts with proteins, thus regulating gene expression.
In the present invention, we found that the methylation level of EphA2DNA in the homocysteine group is obviously increased, which suggests that the reduction of the expression level of EphA2 is closely related to the increase of the methylation level of the DNA in the EphA2 promoter region. DNMT1 is a major enzyme that maintains methylation patterns after DNA replication and plays an important regulatory role in a variety of diseases. The invention discovers that the expression of DNMT1 in the Hcy group is increased, and suggests that DNMT1 has obvious effect in promoting the methylation of EphA2DNA, thereby proving that the hypermethylation expression of EphA2 is closely related to the change of blood sugar level and the apoptosis of islet beta cells.
In conclusion, it can be seen that EphA2 is a key gene of Hcy causing blood sugar abnormality, hypermethylation of DNA in the EphA2 promoter region may be a key mechanism of homocysteine causing islet beta cell apoptosis, which provides a new experimental basis and a new research direction for further research on occurrence of diabetes caused by islet cell secretory dysfunction caused by homocysteine, can also be used as a new marker for clinical early diagnosis and disease development evaluation, and provides a new treatment concept for clinical treatment of type 2 diabetes caused by insulin hyposecretion due to islet beta cell dysfunction, and has potential value.
Drawings
FIG. 1 shows the effect of Hcy on apoptosis-related markers of islet beta cells. A: detecting the expression conditions of bax and bcl-2 proteins in each group by adopting immunofluorescence; b: detecting the expression of caspase-3 protein in each group by immunofluorescence; c: western blot is adopted to test the protein expression of bax/bcl-2 in each group; d: western blot assay protein expression of caspase-3 in each group. P <0.05 compared to control group.
FIG. 2 is a graph of the apoptotic effect of homocysteine on islet beta cells. A: detecting the apoptosis condition of each group of islet beta cells by adopting flow cytometry; b: apoptosis rate statistics, P <0.05 compared to control.
Figure 3 is the expression of EphA2 in islet beta cells. A: detecting the expression of mRNA of EphA2 in islet beta cells by using q-RT PCR; b: the expression of EphA2 protein in each group was examined by Western blot. P <0.05 compared to control.
FIG. 4 shows the detection of the methylation level of EphA2DNA by nMS-PCR. A: CPG island map of EphA2 gene promoter region; detecting the methylation level of the EphA2 gene by agarose gel electrophoresis; c: a statistical map of EphA2DNA methylation levels; p <0.05 compared to control.
FIG. 5 shows the expression of DNMT1 and DNMT3a in each group of islet beta cells. A: detecting DNMT1 protein expression B of each group by Western blot: western blot was used to detect DNMT3a protein expression in each group. P <0.05 compared to control.
Detailed Description
The invention is described in detail below with reference to the drawings and specific examples, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art unless otherwise specified, and the materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
1 materials and methods
1.1 materials: mouse islet beta cell line (min 6).
1.2 Primary reagents and instruments
Main kit and instrument of experiment: fetal bovine serum, RPMI-1640 medium (Gibco, usa); trypsin digest (bi yun day biotechnology research institute); homocysteine (U.S. Sigma); genomic DNA extraction kit, total RNA extraction kit (beijing, tiangen); protein extraction kit protein quantification kit (Nanjing, Kaiyi); reverse transcription and qRT-PCR kit (Thermo Fisher, USA); DNA methylation modification kit (ZYMO, usa); EphA2 antibody, Bax antibody, Caspase-3 antibody, Bcl-2 antibody (Chinese Affinity); beta-actin antibodies (Zhongshan bridge, China); the primer is synthesized by Shanghai biological engineering Co., Ltd; CO 22Incubators (Heraeus, germany); clean bench (suzhou, anta); model 5415D micro bench centrifuge (Eppendorf, Germany); precision balances of the BS110S type (Sartorius, germany); fluorescent quantitative PCR instrument (Shanghai, Feng Ling); electrophoresis, electrotransfer, gel imager (Bio-rad, USA); laser confocal microscopy (Zeiss).
2. Experimental methods
1.2.1 cell culture and treatment islet beta cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, placed at 37 ℃ with 5% CO2In an incubator. When the density of cells reached 80% of the total volume, the ratio of 1: 3, carrying out passage in proportion, carrying out passage once in two days, taking a third generation cell, respectively using a control group with the concentration of 0 mu mol/L homocysteine and a homocysteine group with the concentration of 120 mu mol/L homocysteine, interfering the cell for 48 hours, collecting the cell, and carrying out related index detectionAnd (6) measuring.
1.2.2 immunofluorescence staining to detect the expression of bcl-2, bax and caspase-3
Respectively fixing beta cells of a control group and a homocysteine group by using 4% formaldehyde for 30min, washing by using PBS for 3 times, incubating for 10min under 10% hydrogen peroxide for 5min each time, adding goat serum for sealing for 60min after washing by using PBS for three times, adding relative bcl-2, bax and caspase-3 primary antibody, incubating for one night at 4 ℃, taking out the cells, standing for 30min at room temperature, adding a fluorescent secondary antibody after washing by using the PBS, incubating for 1h in a dark place at 37 ℃, adding DAPI (Dairy amplified peptide) for staining cell nuclei for 5min under the dark place after washing by using the PBS, dripping a fluorescent quencher for sealing after washing again, and observing and photographing the stained cells under a laser confocal microscope.
1.2.3 flow cytometry detection of apoptosis Rate
Islet beta cells are divided into two groups: (1) blank control group; (2) a homocysteine intervention group; after islet beta cell homocysteine is intervened for 48h, the cells are collected, washed by PBS for 2 times, digested by 0.25% pancreatin without EDTA and collected, transferred into a 1.5 centrifuge tube, centrifuged by a centrifuge (4 ℃, 10000rpm, 5min), the waste liquid is poured out, 1ml of PBS is added, and the centrifugation is carried out uniformly by blowing, and the steps are repeated twice. Adding the binding solution into each centrifuge tube at a ratio of 1:9 in dark condition, adding 5 μ L7-AAD and 5 μ L PE, stirring, and standing for 15 min. After being evenly blown, the mixture is filtered by a filter membrane, and the apoptosis rate is immediately detected by a flow cytometer. Streaming data is handled using NovoExpress software.
1.2.4 real-time fluorescent quantitative PCR detection of EphA2 mRNA expression
Total RNA of beta cells in each group is extracted according to the instruction of an RNA extraction kit, and the sequence of EphA2 is inquired through a GenBank database and a primer is designed. EphA 2: upstream: 5'-GCACAGGGAAAGG AAGTTGTT-3', SEQ ID NO. 2; downstream: 5'-CATGTAGATAGGCATGTCGTCC-3' SEQ ID NO. 3. PCR amplification procedure: at 95 ℃ for 30s, 95 ℃ for 5s, and 55.1 ℃ for 34s, 40 cycles in total, while setting an internal reference (GAPDH) control group, the relative amounts of the genes of interest amplified in the same system were calculated according to equation 2-ΔΔCtAnd (4) calculating.
1.2.5 Western blot detection of protein expression of bax, bcl-2, caspase-3, EphA2, DNMT1, DNMT3a
Extracting beta cell holoprotein of a control group and a homocysteine group according to the instruction of a holoprotein extraction kit, adding a certain proportion of protein loading buffer solution after quantification by a BCA method, boiling and denaturing for 5min, taking 20 mu g of total protein in each group, carrying out SDS-PAGE gel electrophoresis for 80v, then electrically transferring to a PVDF membrane, sealing for 2h by using 5% skimmed milk, washing the membrane for 3 times/10 min by using PBST, respectively diluting bax, bcl-2, caspase-3, EphA2, DNMT1, DNMT3a and beta-actin antibodies according to corresponding proportions, and shaking overnight at 4 ℃; using horseradish peroxidase-labeled secondary antibodies, as described in 1: diluting with 5000, incubating at room temperature for 2h, and washing membrane with PBST for 3 times/10 min; and (3) performing imaging analysis on a gel imaging analyzer by taking beta-actin as an internal reference, calculating the ratio of the gray values of bax, bcl-2, caspase-3, EphA2, DNMT1 and DNMT3a to the internal reference value of the beta-actin, and performing statistical analysis.
1.4.6 Nested methylation specific PCR (nMS-PCR) detection of the DNA methylation level of the EphA2 promoter region
And extracting the whole genome DNA of the cells of the control group and the homocysteine intervention group according to the instruction of the DNA extraction kit, and carrying out DNA methylation modification on the whole genome. nMS-PCR method detects changes in the degree of methylation of the DNA of the promoter region of EphA 2. One pair of outer primers and two pairs of inner primers were designed for the EphA2 sequence in-line (http:// www.urogene.org/cgi-bin/methprimer. cgi) (outer primer: upstream: 5'-GGGGGATGTTAATAGTTATAATGTG-3', SEQ ID NO. 4; downstream: 5'-CTCCTACCAATACCAAAAACAAAAC-3', SEQ ID NO. 5; methylated primer: upstream: 5'-GGTGTTTTAGGTTTGG TGATTC-3', SEQ ID NO. 6; downstream 5'-TATTAACATCCCCCTTCTTACGAT-3', SEQ ID NO. 7; non-methylated primer: upstream: 5'-GGTG TTTTAGGTTTGGTGATTAGTT-3', SEQ ID NO. 8; downstream: 5'-CTATTAACATCCCCCTTCTTACAAT-3', SEQ ID NO. 9). (reaction System: PCR MIX 12.5. mu. L, H2O7. mu.L, 1. mu.L each of the upstream and downstream primers, 3.5. mu.L of modified DNA, and 25. mu.L in total) of the external primer amplification reaction conditions were: 5min at 95 ℃; 30s at 95 ℃, 30s at 63 ℃ and 30s at 72 ℃, and 20 cycles of cooling to 0.5-53 ℃ and 7min at 72 ℃ in each cycle. PCR product using outer primers as templatePlate, amplification of methylated and unmethylated inner primers, respectively, the reaction conditions being the same as for the outer primers. The products were then electrophoresed on a 1% agarose gel and the methylated bands were analyzed by imaging with a gel image analyzer, calculated as follows: the% methylation is M/(M + U). times.100%, M is the methylated OD value, and U is the unmethylated OD value.
3 statistical processing and analysis
The experimental results are all measured data, the experimental data are subjected to statistical analysis by using Prism 6.0 statistical software, and the experimental results of the measured data are calculated as mean +/-standard deviation
Showing that the comparison between the two groups adopts t test, P<0.05 indicates that the difference is statistically significant.
4 results
4.1 Effect of Hcy on pancreatic islet beta cell apoptosis-related indices
And (3) detecting the apoptosis condition of the islet beta cells by adopting immunofluorescence staining and a Westen Blot method. The results show that: compared with a control group, the fluorescence intensity and the protein level expression of the islet beta cell related apoptosis protein bax of the homocysteine group are obviously increased, and the increase of the expression of the bax of the homocysteine group is prompted; the fluorescence intensity and protein level of the bcl-2 group of homocysteine are reduced, which indicates that the expression of bcl-2 is reduced; the fluorescence intensity and protein level expression of caspase-3 in the homocysteine group were increased compared to the control group, suggesting that caspase-3 expression was increased. Indicating that homocystine can increase the level of islet beta cell apoptosis, see figure 1.
4.2 flow cytometry detection of the apoptotic Effect of homocysteine on islet beta cells
And detecting the apoptosis condition by adopting a flow cytometry method. The results show that: compared with a control group, the apoptosis rate of the homocysteine group is obviously increased. Indicating that homocysteine dried after the pretreatment can increase islet beta cell apoptosis rate, see figure 2.
4.3 Effect of homocysteine on EphA2 expression levels
To demonstrate the role of EphA2 in homocysteine-induced islet beta cell apoptosis, the expression levels of EphA2 mRNA and protein in islet beta cells were examined using qRT-PCR and Western blot, respectively. The results show that: compared with the control group, the expression of the mRNA of the islet beta cell of the homocysteine group EphA2 is reduced, and the expression of the protein related to the islet beta cell of the homocysteine group EphA2 is also obviously reduced, as shown in FIG. 3.
4.4 Effect of homocysteine group on the methylation level of EphA2DNA
To further explore the mechanism by which homocysteine reduces EphA2 expression, the DNA methylation level of EphA2 in islet beta cells was measured using nMs-PCR, and the DNA methylation level of EphA2 in the homocysteine group was increased compared to the control group, as shown in fig. 4.
4.5 expression of DNMT1 and DNMT3a in homocysteine-induced islet beta cell apoptosis
And detecting the expression levels of DNMT1 and DNMT3a proteins in the pancreatic beta cells by using Western blot. The results show that: compared with a control group, the expression level of the islet beta cell DNMT1 protein in the homocysteine group is increased, and the difference has statistical significance. While DNMT3a expression was not statistically significant, see fig. 5.
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 the preferred embodiment and all changes and modifications that 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 intended to include such modifications and variations.
Sequence listing
<110> Ningxia medical university
<120> EphA2 gene and application of methylation level detection reagent thereof
<160> 9
<170> SIPOSequenceListing 1.0
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tctcacccaa cttccattaa ggacttggag caggagggta gaagttgtct ctgtcggcgg 60
gcgggcagga ttggggcacc gagaccggcg tgcggacagc agggatcgcg gggagcgagg 120
ggtgcggcat ggagctccgg gcagtcggtt tctgcctggc gctgctgtgg ggttgcgcgc 180
tggcggccgc ggcggcacag ggaaaggaag cctgttctcc aggattcttc aagtctgagg 240
catctgagag cccttgcctg gagtgtccag agcataccct gccatccaca gagggtgcca 300
cctcctgcca gtgtgaagaa ggctatttca gggcacctga ggacccactg tccatgtctt 360
gcacacgtcc accctctgcc cccaactacc tcacggccat tggcatgggt gccaaagtag 420
aactgcgttg gacagctccc aaggacactg gtggccgcca ggacattgtc tacagtgtca 480
cttgcgaaca gtgctggcca gagtctggcg agtgtgggcc ctgtgaggcg agcgtgcgct 540
attcagaacc tcctcacgcc ctgacccgca cgagtgtgac agtcagtgac ctggagcccc 600
acatgaacta taccttcgct gtcgaagcac gcaatggtgt ctcaggcctg gtgactagcc 660
gaagcttccg gactgccagc gtcagtatta accaaacaga gccccccaaa gtgaggctgg 720
aggaccgaag caccacctcc ctgagtgtca cctggagcat cccggtgtca cagcagagcc 780
gtgtgtggaa gtacgaagtc acctaccgca agaaggggga tgccaacagc tataatgtgc 840
gccgcacgga aggcttctcc gtgaccctgg atgaccttgc tccggatacc acgtacctgg 900
tgcaggtgca ggcgctgacg caggagggcc agggagccgg cagcaaagtg cacgagttcc 960
agacactgtc cacggaagga tctgccaaca tggcggtgat cggcggtgtg gctgtaggtg 1020
ttgttttgct tctggtactg gcaggagttg gcctcttcat ccatcgaagg aggaggaacc 1080
tgcgggctcg ccagtcctct gaggatgtcc gtttttccaa gtcagaacaa ctaaagcccc 1140
tgaagaccta tgtggatcct cacacttacg aagaccccaa ccaggctgta ctcaagttta 1200
ccaccgagat ccacccatcc tgtgtggcaa ggcagaaggt cattggagca ggagagtttg 1260
gagaggtcta taaagggacg ctgaaggcat cctcggggaa gaaggagata ccggtggcca 1320
tcaagacact gaaagcgggc tacactgaga agcagcgggt ggacttcctg agcgaggcca 1380
gcatcatggg ccagtttagc caccacaata tcatccgcct ggagggcgtg gtctctaaat 1440
acaaacccat gatgattatc acagagtaca tggagaatgg agcgctagac aagttcctta 1500
gggagaagga tggtgagttc agtgtacttc agttggtggg catgctgagg ggtatcgcat 1560
ccggcatgaa gtacctggcc aacatgaact acgtgcaccg ggacctggcc gcccgcaaca 1620
tcctcgtcaa cagcaacctg gtgtgcaagg tgtccgattt tggcctgtcg cgtgtgctgg 1680
aagatgaccc cgaggccacc tacaccacaa gtggcggcaa gatccctatt cgatggacag 1740
ccccagaggc catttcctac cgcaagttca cctcagccag cgatgtgtgg agctacggca 1800
ttgtcatgtg ggaagtgatg acttatggcg aacggcccta ctgggaactg tcaaaccacg 1860
aggtcatgaa agccatcaac gacggcttcc ggctccctac gcccatggac tgcccttcag 1920
ccatttacca gctcatgatg cagtgctggc agcaagagcg ctcccgccga cccaagtttg 1980
ccgacatcgt tagcatcctg gacaagctca tccgagcccc cgactccctc aagacgctgg 2040
ctgactttga tccccgagtg tccatccggc tgcccagcac cagcggctcg gagggagtcc 2100
ccttccgtac ggtgtccgag tggctggaga gcatcaagat gcaacagtac acggaacact 2160
tcatggtggc tggctacacg gccatcgaga aggtggtaca gatgtccaac gaagacatca 2220
aaaggatcgg agtgcgtctt cctggccacc agaagcgtat tgcctacagc ctgctgggac 2280
tcaaggacca ggtcaacaca gtggggattc ctatctgagt ccattggggc cgtgcccaac 2340
aatacttgaa gagccacagt ggtctccctg ccgacctggt gctggcccac tggaacttta 2400
tttatttctg tttcctcgtc tatgcctccc tgaggactct gcagggggct tttgaatgac 2460
accctggcct gagcctggga aacttggatg ctggtcaggg ctctctttcc cctgcaaagg 2520
acccagctaa gcacttagca gtttgccatg gccttcccag catcccctga ggctaaagtt 2580
ccaccaagac cgtcgatatc gacgagggac atttccaaac ggacctcccc atcttcattt 2640
ggcctcctga gaagccacct caggagctga ggctaagcac taagcccagg accatatgac 2700
tagggcactg tacgccccgc ccctagttag agggtaggtt ttggacttgg ctgggtgtgg 2760
tcacagcaat ctcccagtgc cttttacaga cccagggctc tgccctccgc cctcgagggc 2820
cagcttcttg ctttcctagg gccctctcag gatgcttggc tgtgctgagg tttttattaa 2880
atatatattt tatacttgtg gaaagaatga gtgtgtggca gggaccttgc cagggctgga 2940
gacagaggat cccctgcaac agacattccc gggctggggg ctggcggacc tgcaggagac 3000
tttccgccag ccacccagtc tccagcccct ttggacaaat gtcgctgtca gtgttacaga 3060
tttcttttat tgggttgttt ttttgttgta ttttttgaac cttaacttat tatttttttt 3120
atatttattg ttagaaaatg acttatttct gctctggaat aaagttgcag atggttcaaa 3180
ctga 3184
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<400> 2
gcacagggaa aggaagttgt t 21
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<212> DNA
<213> Artificial sequence
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catgtagata ggcatgtcgt cc 22
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<213> Artificial sequence
<400> 4
gggggatgtt aatagttata atgtg 25
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<211> 25
<212> DNA
<213> Artificial sequence
<400> 5
ctcctaccaa taccaaaaac aaaac 25
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<213> Artificial sequence
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ggtgttttag gtttggtgat tc 22
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<211> 24
<212> DNA
<213> Artificial sequence
<400> 7
tattaacatc ccccttctta cgat 24
<210> 8
<211> 25
<212> DNA
<213> Artificial sequence
<400> 8
ggtgttttag gtttggtgat tagtt 25
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Claims (8)
- 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. The use of claim 1, wherein said β -cell apoptosis is induced by homocysteine.
- 3. The use according to claim 2, wherein the disease comprises type 2 diabetes.
- 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. The use of claim 4, wherein said reagents comprise nMS-PCR detection reagents.
- 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. The use of the EphA2 gene of claim 1 for screening for potential agents for the treatment of type 2 diabetes.
- 8. The use of claim 7 wherein the agent down-regulates the methylation level of the EphA2 gene.
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