CN110951866A - Primer for detecting methylation level of GPVI gene promoter region and detection method thereof - Google Patents
Primer for detecting methylation level of GPVI gene promoter region and detection method thereof Download PDFInfo
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Abstract
The invention discloses a primer for detecting methylation level of a GPVI gene promoter region and a detection method thereof, wherein the primer comprises an amplification primer pair and a sequencing primer GPVI-S of the GPVI gene promoter region after bisulfite conversion; the sequence of the upstream primer GPVI-F is 5'-ATTAGGGAGTTTATGGGAGTACGG-3', and the sequence of the downstream primer GPVI-R is: 5 '-Biotin-ATTCCTCAACCCTATCCTAAACTCTAT-3'; the sequence of the sequencing primer GPVI-S is as follows: 5'-AATATAGATTAGGTTTTAGTAGG-3' are provided. According to the application, a pyrosequencing method is adopted for sequencing, the methylation and non-methylation proportion of the CpG island in a target area is efficiently detected, the methylation level of the GPVI promoter region is evaluated, and the methylation level can be used as an early index of myocardial infarction.
Description
Technical Field
The invention relates to the technical field of biological gene detection. In particular to a primer for detecting the methylation level of a GPVI gene promoter region and a detection method thereof.
Background
Excessive platelet activation can lead to unwanted platelet aggregation, ultimately leading to thrombosis and cardiovascular disease. In recent years, many researches show that the coronary heart disease has close relationship with gene epigenetic DNA methylation patterns in the occurrence and development processes. Therefore, the research on the action mechanism of DNA methylation in the pathogenesis of coronary heart disease can provide a new idea for the diagnosis and treatment of coronary heart disease.
Receptors on platelet membranes play a key role in the development of platelets involved in coronary heart disease. Glycoprotein VI (GPVI) is one of many receptors on platelet membranes and is also a hot target for the development of novel antithrombotic drugs. The human GPVI gene is positioned on chromosome 19q13.4-42, the length of the DNA fragment is about 23000bp, the genome structure is composed of 8 exons and 7 introns, and the gene is a type I transmembrane glycoprotein and belongs to a member of immunoglobulin superfamily. GPVI is only expressed on platelets and platelet precursor cells, namely megakaryocytes in bone marrow, is a main receptor of collagen, mediates adhesion of the platelets and the collagen, transmits an adhesion signal from outside to inside to the interior of the platelets through an FcR gamma chain connected with the adhesion signal, starts a series of activities for mediating platelet activation, and generates signal cascade amplification reaction through activation of Syk kinase, gradual activation of SLP76, PI3K, Btk, PLC gamma 2 and PKC, and Ca in cytoplasm2+Elevated concentrations cause cytoskeletal changes, platelet shape changes and granule secretion, and these platelet responses are further enhanced by thromboxane A2 receptor and Adenosine Diphosphate (ADP) receptors P2Y1, P2Y12 signaling, ultimately leading to integrin α IIb β 3 activation, platelet aggregation, and thrombus formationanti-GPVI autoantibodies are detected in the plasma and are confirmed to specifically bind GPVI positive platelets, and soluble GPVI in the plasma is cleared, so that the platelets cannot bind collagen and cannot form thrombus. In addition, knockout of GPVI-Fc dimers is also effective in preventing platelet aggregation, but does not prolong bleeding time, suggesting that reducing GPVI levels may prevent thrombosis, but does not cause pathological bleeding. In addition, studies show that the expression level of GPVI on platelet membranes is increased in patients with cardiovascular diseases, and high levels of GPVI are associated with adverse clinical events and are independent risk factors for myocardial infarction, suggesting that abnormal increase of GPVI can be used as an early indicator of myocardial infarction.
Epigenetics is an important way to regulate genome function, and DNA methylation is one of the most important forms in epigenetic modification. Most mammalian genomic DNA has a small amount of methylated cytosine, mainly concentrated in the 5' non-coding region of the gene, and present in clusters. Cytosine, two nucleotides of CG of DNA, is catalyzed by DNA methyltransferases (DNMTs) to selectively add methyl to form 5-methylcytosine, which is commonly found in 5 '-CG-3' sequences of genes. The DNA methylation status of a CpG site is regulated by both DNA methyltransferases and demethylases and can be inherited by cell division. When the CpG sites are in a methylation state, the conformation of DNA in certain regions can be changed, the structure of the DNA is contracted, the helix of the DNA is deepened, the interaction between a transcription factor or a co-activator and a related regulation region on the DNA is influenced, the initiation of transcription is not facilitated, the expression of a target gene is reduced, and the target gene is expressed in a silent state. Conversely, when the CpG site is in a demethylated modified state, the compact structure of the chromosome will be opened and a loose state will be presented, favoring access of transcription factors or co-activators to the DNA sequence, and thus the modified state is associated with activation of the gene. Through gene sequence analysis, a plurality of CpG islands are found between GPVI 5' regulatory region and promoter region-1576 to +75, and through earlier researches, compared with a healthy group, the methylation level of the GPVI promoter region in a coronary heart disease group is obviously reduced, while the mRNA level of the GPVI is obviously increased, which indicates that GPVI regulates the self gene expression through the methylation of the promoter region. In view of this, it is necessary to establish a method for rapidly detecting the methylation level of the promoter region of the GPVI gene.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a primer capable of rapidly detecting the methylation level of a GPVI gene promoter region and a detection method thereof.
In order to solve the technical problems, the invention provides the following technical scheme:
a primer for detecting the methylation level of a GPVI gene promoter region comprises an amplification primer pair and a sequencing primer GPVI-S (shown as SEQ ID NO: 3) of the GPVI gene promoter region after bisulfite conversion;
the amplification primer pair comprises an upstream primer GPVI-F (shown as SEQ ID NO: 1) and a downstream primer GPVI-R (shown as SEQ ID NO: 2), the sequence of the upstream primer GPVI-F is 5'-ATTAGGGAGTTTATGGGAGTACGG-3', and the sequence of the downstream primer GPVI-R is: 5 '-Biotin-ATTCCTCAACCCTATCCTAAACTCTAT-3';
the sequence of the sequencing primer GPVI-S is as follows: 5'-AATATAGATTAGGTTTTAGTAGG-3' are provided.
A detection method for detecting the methylation level of a GPVI gene promoter region comprises the steps of amplifying the GPVI gene promoter region in human blood after bisulfite conversion by using the amplification primer pair, detecting the methylation ratio of 5 CpG sites in the detected gene region by using the sequencing primer GPVI-S, and determining the expression level of the GPVI gene in the blood by detecting the obtained methylation ratio.
The detection method for detecting the methylation level of the GPVI gene promoter region comprises the following steps:
(1) collecting a blood sample;
(2) separating white blood cells;
(3) extracting genome DNA;
(4) bisulfite conversion;
(5) carrying out PCR amplification reaction on a GPVI gene promoter region;
(6) identifying the PCR amplification product by agarose gel electrophoresis;
(7) pyrosequencing reaction and result analysis.
In the method for detecting the methylation level of the GPVI gene promoter region, in the step (1), 2ml of human venous blood is collected by using an EDTA (ethylene diamine tetraacetic acid) anticoagulation tube.
In the method for detecting the methylation level of the GPVI gene promoter region, in the step (2), the white blood cells in the human venous blood are separated by using a Ficoll density gradient method.
In the method for detecting the methylation level of the GPVI gene promoter region, leukocyte genomic DNA is extracted by using a Biospin whole blood genomic DNA extraction kit in step (3).
In the method for detecting the methylation level of the GPVI gene promoter region, in the step (4), the genome DNA sample in the step (3) is treated by using bisulfite in an EZ DNA methylation kit-gold.
In the step (5), the amplification primer pair of the GPVI gene promoter region after bisulfite conversion comprises an upstream primer GPVI-F and a downstream primer GPVI-R,
the sequence of the upstream primer GPVI-F is 5'-ATTAGGGAGTTTATGGGAGTACGG-3',
the sequence of the downstream primer GPVI-R is 5 '-Biotin-ATTCCTCAACCCTATCCTAAACTCTAT-3'; the size of the amplified target gene fragment is 166 bp;
the PCR amplification reaction system is as follows: a mixed system of 30uL, comprising
0.3 mu L of TaKaRa Taq HS, wherein the concentration of the TaKaRa Taq HS is 5U/mu L;
3 μ L of 10 × PCR Buffer;
2.4. mu.L of dNTP mix at a concentration of 2.5mM,
3. mu.L of bisulfite-treated genomic DNA, wherein the concentration of the bisulfite-treated genomic DNA is 10 to 20 ng. mu.L-1；
1.0 mu L of upstream primer GPVI-F, wherein the concentration of the upstream primer GPVI-F is 10 mu M;
1.0 mu L of downstream primer GPVI-R, the concentration of the downstream primer GPVI-R is 10 mu M;
19.3 μ L of sterile deionized water;
the PCR reaction program is:
(P-1) pre-denaturation at 95 ℃ for 2min,
(P-2) denaturation at 95 ℃ for 10s,
(P-3) annealing at 55 ℃ for 30s,
(P-4) extension at 72 ℃ for 30s,
(P-5) the steps (P-1) to (P-4) are circulated 30 times,
(P-6) final extension at 72 ℃ for 5 min.
In the method for detecting the methylation level of the GPVI gene promoter region, in the step (6), 2% agarose gel electrophoresis is used for identifying the specificity of the PCR amplification product in the step (5), and the size of the target gene fragment is 166 bp.
In the step (7), a sequencing primer GPVI-S is used, and the sequence of the sequencing primer GPVI-S is as follows: 5'-AATATAGATTAGGTTTTAGTAGG-3', sequencing on a Pyro Mark Q24 pyrosequencer of QIAGEN company, analyzing the methylation level by using PyroMark Q24 software to obtain 5 CpG methylation values of the GPVI gene promoter region of the sample,
5 CpG sites of the GPVI gene promoter region are respectively:
the 1 st CpG site is: -211 to-210 CpG islands;
the 2 nd CpG site is: -203 to-202 CpG islands;
the 3 rd CpG site is: -195 to-194 CpG islands;
the 4 th CpG site is: -170 to-169 CpG islands;
the 5 th CpG site is: -168 to-167 CpG islands.
The technical scheme of the invention achieves the following beneficial technical effects:
the application provides a primer for detecting methylation level of a GPVI gene promoter region and a detection method thereof, a pyrosequencing method is adopted for sequencing, the methylation and non-methylation proportion of a CpG island in a target region is efficiently detected, the methylation level of the GPVI promoter region is evaluated, and the primer can be used as an early index of myocardial infarction.
Studies have shown that a lack of GPVI significantly inhibits collagen-induced platelet adhesion, aggregation, and thrombosis. anti-GPVI autoantibodies were detected in the plasma of one ITP patient and demonstrated that their autoantibodies specifically bound GPVI positive platelets, clearing plasma of soluble GPVI, resulting in platelets that were unable to bind collagen and unable to form thrombi. In addition, knockout of GPVI-Fc dimers is also effective in preventing platelet aggregation, but does not prolong bleeding time, suggesting that reducing GPVI levels may prevent thrombosis, but does not cause pathological bleeding. In addition, studies show that the expression level of GPVI on platelet membranes is increased in patients with cardiovascular diseases, and high levels of GPVI are associated with adverse clinical events and are independent risk factors for myocardial infarction, suggesting that abnormal increase of GPVI can be used as an early indicator of myocardial infarction. Epigenetics is an important way to regulate genome function, and DNA methylation is one of the most important forms in epigenetic modification. Through gene sequence analysis, a plurality of CpG islands are found between GPVI 5' regulatory region and promoter region-1576 to +75, but the influence of methylation of the region on GPVI transcriptional regulation is not reported in the literature. Our earlier studies found that the methylation level of the GPVI promoter region was significantly reduced in the coronary heart disease group, while the GPVI mRNA level was significantly increased, compared to the healthy group.
Drawings
FIG. 1 is an electrophoresis detection diagram for detecting amplification fragments of GPVI gene promoter regions by agarose gel electrophoresis in the detection method for detecting methylation levels of GPVI gene promoter regions, wherein M is DL2000DNA Marker, ① is amplification fragments of GPVI gene promoter regions, and the size is 166 bp;
FIG. 2 is a diagram showing the results of detecting the methylation level of 5 CpG sites in the promoter region of the GPVI gene of a CHD patient by pyrosequencing;
FIG. 3 is a diagram showing the results of pyrosequencing for detecting the methylation level of 5 CpG sites in the promoter region of GPVI gene in the healthy group;
FIG. 4 statistical analysis of CHD patients (31 cases) and healthy groups (31 cases) for methylation level of 5 CpG sites in the promoter region of the GPVI gene.
Detailed Description
Apparatus and materials
PCR instrument (Eppendorf, model 5332), cryocentrifuge (Eppendorf, model 5810R), micropipette (Eppendorf), electrophoresis system (six instruments, model DYY-12, Beijing), gel imaging analyzer (BIO-RADChemidoc XRS), pyrosequencing instrument (QIAGEN, Pyro Mark Q24).
Biospin whole blood genomic DNA extraction kit (Hangzhou Bori Co.), EZ DNA Methylation-Goldkit (ZYMO Research Co.), TAE buffer, agarose (Promega Co.), Goldview (Beijing Sorley technologies Co., Ltd.), DNA Taq polymerase (Takara Co.), absolute ethanol was purified for domestic analysis.
Example 1
A primer for detecting the methylation level of a GPVI gene promoter region comprises an amplification primer pair and a sequencing primer GPVI-S (shown as SEQ ID NO: 3) of the GPVI gene promoter region after bisulfite conversion;
the amplification primer pair comprises an upstream primer GPVI-F (shown as SEQ ID NO: 1) and a downstream primer GPVI-R (shown as SEQ ID NO: 2),
the sequence of the upstream primer GPVI-F is 5'-ATTAGGGAGTTTATGGGAGTACGG-3', and the sequence of the downstream primer GPVI-R is: 5 '-Biotin-ATTCCTCAACCCTATCCTAAACTCTAT-3'; the sequence of the sequencing primer GPVI-S is as follows: 5'-AATATAGATTAGGTTTTAGTAGG-3' are provided.
Example 2
A method for detecting the methylation level of a GPVI gene promoter region comprises the steps of amplifying the GPVI gene promoter region in human blood after bisulfite conversion by using the amplification primer pair described in example 1, detecting the methylation ratio of 5 CpG sites in the detected gene region by using the sequencing primer GPVI-S, and determining the expression level of the GPVI gene in the blood by detecting the obtained methylation ratio.
The method specifically comprises the following steps:
(1) using EDTA anticoagulant blood collection tubes to collect 2ml of venous blood of 31 clinical samples CHD and 31 healthy samples CHD;
(2) separating white blood cells in 31 cases of clinical samples CHD collected in the step (1) and 31 cases of blood in a healthy group by a Ficoll method respectively;
(3) extracting the genomic DNA in the white blood cells by a column type extraction method, and extracting the genomic DNA of the white blood cells by using a Biospin whole blood genomic DNA extraction kit;
(4) the DNA sample obtained in step (3) is treated with bisulfite, and the procedure is strictly followed, in this example, with EZ DNA Methylation-Gold Kit from ZYMO Research, cat # D5006, but not limited to this Kit;
(5) and (4) carrying out PCR amplification on the DNA treated by the bisulfite in the step (4).
The PCR amplification reaction system is as follows: a30 uL mixed system was prepared from 0.3. mu.L of TaKaRa TaqHS (5U/. mu.L), 3. mu.L of 10xPCR Buffer, 2.4. mu.L of dNTP mix (2.5 mM each), and 3. mu.L of genomic DNA treated with bisulfite (concentration 10-20 ng. mu.L)-1) 1.0. mu.L of the forward primer (10. mu.M) of example 1, 1.0. mu.L of the reverse primer (10. mu.M) of example 1, 19.3. mu.L of sterile deionized water;
the PCR reaction program is:
(P-1) pre-denaturation at 95 ℃ for 2min,
(P-2) denaturation at 95 ℃ for 10s,
(P-3) annealing at 55 ℃ for 30s,
(P-4) extension at 72 ℃ for 30s,
(P-5) the steps (P-1) to (P-4) are circulated 30 times,
(P-6) final extension at 72 ℃ for 5 min.
(6) As shown in FIG. 1, 2% agarose gel (2g agar/100 mL water) is used for electrophoresis to identify the specificity of the amplified product in step (5), and the size of the amplified fragment should be 166 bp;
(7) the sequencing primer GPVI-S is used, and the sequence of the sequencing primer GPVI-S is as follows: 5'-AATATAGATTAGGTTTTAGTAGG-3', sequencing on a Pyro Mark Q24 pyrosequencer from QIAGEN, operating strictly according to the instructions, methylation level analysis was performed using PyroMark Q24 software, with 5 CpG methylation values in the GPVI gene promoter region of samples from 31 CHD patients and 31 healthy groups, respectively.
(8) Analysis of results
8.1, GPVI gene promoter region 5 CpG sites sequencing as follows:
-250tttatgggag cacgggaaca cagaccaggc cccagcaggcg(underlined cg is the 1 st CpG site))gacaaacg(underlined cg is the 2 nd CpG site) g tgcaacg(underlined cg is the 3 rd CpG site)
-193cca ggctggccag aggagataagcg(underlined cg is the 4 th CpG site)cg(underlined cg is the 5 th CpG site) gctcct tggagcttgt gtgcaagtcactgtactgag
8.2, pyrosequencing the PCR product, and analyzing 5 CpG methylation of GPVI gene promoter regions of CHD patients and healthy groups, wherein the results are as follows:
the sequencing results showed that the methylation level of these 5 CpG sites in the CHD group tended to decrease significantly compared to the healthy group as shown in fig. 2 and 3:
CpG1:91.48±0.4115Vs.87.45±0.6283，P<0.01；
CpG2:84.15±0.6544Vs.76.43±0.9821，P<0.01；
CpG3:86.44±0.5165Vs.81.20±0.8125，P<0.01；
CpG4:88.71±0.4515Vs.85.18±0.6456，P<0.01；
CpG5:71.83±0.3987Vs.66.55±0.6449，P<0.01。
therefore, the primer for detecting the methylation level of the GPVI gene promoter region and the detection method thereof have stable results and strong repeatability, ensure extremely high accuracy, and have simple method and easy operation.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are possible which remain within the scope of the appended claims.
Sequence listing
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101253264A (en) * | 2005-04-28 | 2008-08-27 | 持田制药株式会社 | Monoclonal antibody against platelet membrane glycoprotein VI |
| WO2010141771A1 (en) * | 2009-06-03 | 2010-12-09 | Regado Biosciences, Inc. | Nucleic acid modulators of glycoprotein vi |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101253264A (en) * | 2005-04-28 | 2008-08-27 | 持田制药株式会社 | Monoclonal antibody against platelet membrane glycoprotein VI |
| WO2010141771A1 (en) * | 2009-06-03 | 2010-12-09 | Regado Biosciences, Inc. | Nucleic acid modulators of glycoprotein vi |
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| SACHIKO KANAJI 等: ""Thrombopoietin initiates demethylation-based transcription of GP6 during megakaryocyte differentiation"" * |
| SHUIBO GAO 等: ""Epigenetic modulation of glycoprotein VI gene expression by DNA methylation"" * |
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