CN112322725B - DNA methylation marker of FURIN gene and detection kit thereof - Google Patents
DNA methylation marker of FURIN gene and detection kit thereof Download PDFInfo
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Abstract
The invention discloses a DNA methylation marker of FURIN gene and a detection kit thereof, belonging to the technical field of biological medicine. The DNA methylation marker provided by the invention is a DNA methylation site Chr15:91416118 of a FURIN gene promoter region, and the methylation degree of the DNA methylation marker indicates the onset risk of hypertension of a subject. The DNA methylation marker of the FURIN gene provided by the invention can be used for predicting the onset risk of hypertension, provides a basis for screening hypertension high risk groups, and can also be used as an intervention target for preventing and controlling hypertension. The invention not only helps to explain the molecular mechanism of FURIN deficiency on hypertension, but also provides important epidemiological evidence for the methylation of FURIN gene as a drug target for preventing and controlling hypertension.
Description
Technical Field
The invention relates to a DNA methylation marker of FURIN gene and a detection kit thereof, belonging to the technical field of biological medicine.
Background
Hypertension is an important risk factor for cardiovascular diseases and other cardiovascular and cerebrovascular diseases. Worldwide there are 67% of heart diseases, 54% of strokes caused by hypertension, and 13.5% of deaths due to hypertension. The prevalence rate of global adult hypertension is about 30%, and at present, about 3.3 hundred million of hypertension patients in China account for about 1/3 of the global hypertension patients. Therefore, hypertension is an important public health problem in China, seriously threatens human health, and how to effectively prevent and control hypertension is a major public health challenge in China at present, so that more potential risk factors and intervention targets of hypertension are urgently searched and found.
The natriuretic peptide axis is an important heart endocrine regulation system for the body to respond to external environmental stimulation, and plays an important role in maintaining the water-sodium balance, blood pressure stability and energy metabolism balance of the body. The natriuretic peptide axis is mainly composed of Atrial Natriuretic Peptide (ANP), brain Natriuretic Peptide (BNP), C-type natriuretic peptide (CNP), and their receptors. When blood volume is increased, a large amount of bioactive atrial natriuretic peptide precursor (pro-ANP) and brain natriuretic peptide precursor (pro-BNP) are secreted and released by myocardial cells, and the pro-ANP and the pro-NNP are further activated into active ANP and BNP and are combined with receptors thereof to activate natriuretic peptide axes, so that natriuretic metabolism is promoted, hypovolemia is reduced, blood vessels are expanded, energy metabolism is promoted, and cardiovascular homeostasis is maintained. There is a great deal of epidemiological evidence that the natriuretic peptide axes, in particular ANP and BNP, play an important role in the development of hypertension and its risk factors (obesity, hyperglycemia, atherosclerosis, etc.).
The proteases that convert inactive pro-ANP and pro-BNP into active ANP and BNP are the key to the regulation of the natriuretic peptide axis function. Furin protease is a converting enzyme, can convert pro-BNP into BNP, and participates in the regulation of blood vessels through mechanisms such as diuresis, natriuresis and vasodilation. Both basic research and population research results suggest that furin protease may be a potential intervention target for preventing and controlling hypertension. As a medium for connecting the gene with the environment, DNA methylation regulates the expression and the function of the gene and can be used as a potential drug target spot which can intervene, therefore, as the coding gene of FURIN protease, DNA methylation of the promoter region of the FURIN gene can inhibit the expression of FURIN protease, cause FURIN deficiency and participate in the pathogenesis of hypertension. However, no suitable site has been reported as an intervention target for preventing and controlling hypertension.
Disclosure of Invention
In order to solve the technical problems, the DNA methylation marker of the FURIN gene for predicting the risk of hypertension and the application thereof in predicting the risk of hypertension are provided.
The first purpose of the invention is to provide a DNA methylation marker for predicting the risk of hypertension, wherein the DNA methylation marker is the DNA methylation site Chr15:91416118 of the FURIN gene promoter region, and the methylation degree of the DNA methylation marker indicates the risk of hypertension of a subject.
Further, the methylation level of the DNA methylation site Chr15:91416118 is increased by 5 percent, and the incidence risk of hypertension is increased by 32 percent.
The second purpose of the invention is to provide a primer pair for detecting the DNA methylation marker, wherein the primer pair is an upstream primer and a downstream primer for amplifying a DNA methylation site Chr15: 91416118.
The third purpose of the invention is to provide the application of the DNA methylation marker or the primer pair in preparing a kit for predicting the risk of hypertension.
The fourth purpose of the invention is to provide a kit for predicting the risk of hypertension, wherein the kit is used for detecting the DNA methylation marker of the FURIN gene.
Further, the kit comprises an upstream primer and a downstream primer for amplifying DNA methylation sites Chr15: 91416118.
The fifth purpose of the invention is to provide a method for detecting the DNA methylation marker, which comprises the following steps:
s1, extracting a DNA sample, and carrying out sulfite treatment on the DNA sample;
s2, amplifying the sulfite-treated sample by using a primer pair to amplify a product;
s3, carrying out transcription and enzyme digestion on the amplification product to obtain a transcription and enzyme digestion product;
and S4, detecting the transcription and enzyme digestion products by adopting a flight mass spectrometry method to obtain the methylation degree in the sample sequence.
The invention has the beneficial effects that:
the DNA methylation marker of the FURIN gene can be used for predicting the risk of hypertension, providing a basis for screening hypertension high risk groups and also serving as an intervention target for preventing and controlling hypertension. The invention not only helps to explain the molecular mechanism of FURIN deficiency on hypertension, but also provides important epidemiological evidence for the methylation of FURIN gene as a drug target for preventing and controlling hypertension.
Drawings
FIG. 1 is a schematic diagram of methylation of 1 CpG site in the promoter region of FURIN gene.
Detailed Description
The present invention is further described below in conjunction with the drawings and the embodiments so that those skilled in the art can better understand the present invention and can carry out the present invention, but the embodiments are not to be construed as limiting the present invention.
Example 1:
a population study (including 2312 residents without cardiovascular disease) found that the systolic blood pressure, diastolic blood pressure and mean arterial pressure levels of low levels of furin were all higher than those of high levels of furin; and in the population without hypertension, residents with low levels of furin (the lowest quartile level group) were 1.46 times as at risk for hypertension (P = 0.003) in the high level group (the highest quartile level group). The research result suggests that the FURIN protein deficiency may be the cause of hypertension, however, the molecular mechanism of the FURIN deficiency and the hypertension is not clear, but the research on the molecular mechanism of the FURIN participating in the pathogenesis of the hypertension helps to find the drug target aiming at the FURIN deficiency, thereby promoting the prevention and control of the hypertension. As a vector for connecting genes and the environment, DNA methylation regulates the expression and the function of the genes and can be used as a potential and interventional drug target. Therefore, the invention further researches the role of DNA methylation of FURIN gene in the development of hypertension.
We included 1043 baseline non-hypertensive population in the study cohort and studied the association of DNA methylation of the FURIN gene with the risk of developing hypertension. The whole blood DNA specimen of each study object is extracted, the methylation level of each CpG site in the promoter region of FURIN gene is detected by using a target region sequencing technology, namely, an ENSEMBL database is used for inquiring the promoter region of human FURIN gene (gene number: ENSG 00000140564), the region is Chromosome 15. By selection, the methylation detection is carried out on 1 fragment of the promoter region of the FURIN gene, and the methylation levels of 8 CpG sites are obtained.
Table 1 shows the sequences of the fragments to be detected and the primers:
TABLE 1 DNA methylation detection fragments in promoter region of FURIN gene
The specific detection method comprises the following steps:
first, DNA samples were bisulfite-treated with EZ-96DNA methylation kit (Zymo Research, orange, calif.) to convert all cytosine C that was not methylated in the sample DNA into thymine U. Then, multiplex PCR amplification was performed using the designed primers and the bisulfite-treated sample genome as a template. Specific tag sequences compatible with the illumina platform were then introduced to the ends of the library by PCR amplification using primers with Index sequences. Finally, all sample Index PCR amplification products are equally mixed, and high-throughput sequencing is carried out on an Illumina Hiseq/Miseq platform in a double-end sequencing mode of 2 × 150bp/2 × 250bp, so as to obtain FastQ data. The methylation level at each CpG site is quantified as the number of reads methylated at that site (i.e., the number of reads in which base C is detected)/total number of reads for that site x 100%.
The invention totally detects the methylation level of 8 CpG sites, adjusts the traditional risk factors of age, sex, education degree, smoking, drinking, obesity, blood sugar, blood fat and the like for hypertension, wherein 1 CpG site (Chr 15:91416118, distance TSS:1350 bp) is still obviously associated with hypertension and is positioned in the promoter region of FURIN gene. Every 5% increase in methylation level at this site increases the risk of developing hypertension by 32%. Higher methylation levels mean lower FURIN expression (β = -0.41), i.e. lower serum FURIN protein levels, and it can be seen that the results of this study are consistent with the results of previous studies finding higher blood pressure levels in low serum FURIN populations. Accordingly, this methylation site can be used to predict the risk of developing hypertension.
Based on the above studies, it can be known that: as shown in figure 1, after methylation of the CpG sites located in the promoter region, FURIN gene expression and FURIN protein expression may be inhibited, and further the inhibition of the expression of FURIN protein may be involved in the onset of hypertension, and the inhibition of the expression of FURIN protein may be used as a prediction marker and a potential drug target for the risk of the onset of hypertension. Therefore, the invention constructs a methylation detection kit based on the CpG sites.
The specific detection method comprises the following steps:
(1) whole blood DNA extraction and quality control
a. Agarose gel electrophoresis for genomic DNA integrity: the electrophoresis strip is clearly visible, has no obvious degradation and has no RNA pollution.
Nanodrop 2000 detection of genomic DNA quality: the concentration is more than or equal to 20 ng/mu L, the total amount is more than or equal to 1 mu g, OD260/280= 1.7-2.0, and OD260/230 is more than or equal to 1.8.
(2) Sulfite treatment
The qualified DNA samples were sulfite-treated with EZ-96DNA methylation kit (Zymo Research, orange, calif.) to convert all unmethylated cytosine C in the sample DNA to uracil U.
(3) Multiplex PCR amplification
Then, DNA amplification is carried out on the sulfite-treated specimen by using the designed primer, and an amplification product with a T7 RNA polymerase promoter sequence is obtained.
(4) Cleavage of CpG fragments
The amplified DNA product is then transcribed into RNA fragments using T7 RNA polymerase, and the resulting RNA fragments are cleaved into CpG-bearing small fragments using RNase A.
(5) Flight mass spectrometry
Finally, within each small RNA fragment, the unmethylated CpG end-product was CpA and the methylated CpG end-product was CpG, and the molecular weights of these two end-products were determined using the Agena MassArray flight mass spectrometry system.
(6) Methylation level calculation and hypertension risk prediction
The methylation level at each CpG site was quantified as the product mass CpG/(CpG + CpA). Times.100%. Methylation level of any CpG locus is more than 80% which indicates that the hypertension is high in risk and needs to pay close attention and take preventive treatment measures.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
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Claims (4)
1. A primer pair for detecting a DNA methylation marker is characterized in that the DNA methylation marker isFURINDNA methylation site Chr15:91416118 of gene promoter region, the primer pair is an upstream primer and a downstream primer of amplification DNA methylation site Chr15:91416118, the primer pair is a primer pair with a specific sequence of primer pairs, and the primer pair is a primer pair with a specific sequence of primer pairsFURINThe gene numbers of the genes in the ENSEMBL database are as follows: ENSG00000140564.
2. Use of the primer pair of claim 1 for the preparation of a kit for predicting the risk of developing hypertension.
3. A kit for detecting a DNA methylation marker, the DNA methylation marker beingFURINDNA methylation site of gene promoter region Chr15:91416118, saidFURINThe gene numbers of the genes in the ENSEMBL database are as follows: ENSG00000140564.
4. The kit of claim 3, wherein the kit comprises an upstream primer and a downstream primer for amplifying DNA methylation sites Chr15: 91416118.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315410A (en) * | 2018-04-28 | 2018-07-24 | 南京医科大学 | DNA methylation marker, primer and its application of one group of assessment early abortion risk |
| CN111286534A (en) * | 2019-08-06 | 2020-06-16 | 苏州大学 | CORIN gene DNA methylation marker and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315410A (en) * | 2018-04-28 | 2018-07-24 | 南京医科大学 | DNA methylation marker, primer and its application of one group of assessment early abortion risk |
| CN111286534A (en) * | 2019-08-06 | 2020-06-16 | 苏州大学 | CORIN gene DNA methylation marker and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Furin 基因对高血压心肌梗死细胞增殖, 迁移以及作用机制的研究;李艳等;《临床和实验医学杂志》;20180510;第17卷(第9期);全文 * |
| FURINPromoter Methylation Predicts the Risk of Incident Hypertension: A Prospective Analysis of the Gusu Cohort;Ma S Q et al.;《Cardiology Plus》;20210330;第6卷(第1期);全文 * |
| FURIN基因变异与原发性高血压的关联研究;张菊红等;《第十三次全国心血管病学术会议论文集》;20110623;全文 * |
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