CN114438188A - Use of hypermethylated CDK2AP1 gene as molecular marker for alpha radiation damage prediction - Google Patents
Use of hypermethylated CDK2AP1 gene as molecular marker for alpha radiation damage prediction Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, and relates to application of hypermethylated CDK2AP1 gene as a molecular marker for alpha radiation damage prediction. The methylation level of the hypermethylated CDK2AP1 gene in the promoter region of CDK2AP1 gene is 43.00 +/-27.91%. The hypermethylated CDK2AP1 gene is used as a molecular marker for alpha radiation damage prediction, and early alpha radiation damage prediction can be conveniently, accurately and inexpensively performed.
Description
Technical Field
The invention belongs to the technical field of biological detection, and relates to application of hypermethylated CDK2AP1 gene as a molecular marker for alpha radiation damage prediction.
Background
The radiation carcinogenic effect of alpha particles is a major concern in the field of radiation bioeffect research. The ICRP 31 publication reviews a large number of animal experimental results, and it is believed that the biological effects of alpha radionuclide including plutonium are mainly manifested by lymphopenia, respiratory insufficiency, fibrosis of lung and lymph node, cell degeneration, lung cancer, osteosarcoma, etc., and the decrease of lymphocyte level due to blood irradiation affects the immunological competence of animals, thereby increasing the susceptibility to tumorigenesis.
In the molecular mechanism of alpha radiation carcinogenic injury, besides the change of gene structure, epigenetic change can also be generated, which can cause the expression change of some key genes and influence biological functions, such as promoting the activation of oncogene, inhibiting the inactivation of oncogene, interfering the regulation of cell cycle and the like.
DNA methylation is the major form of epigenetic modification. Cyclin-dependent kinase 2-related protein 1(cyclin-dependent kinase2-associated protein1, CDK2AP1, GenBank accession No.: NC — 000012(123260976..123272240)) is a cyclin-dependent kinase 2(CDK 2). The results of the current research suggest that CDK2AP1 promotes cell cycle arrest and may lead to apoptosis, and has the function of an anti-cancer gene. The hypermethylation of cancer suppressor genes has important clinical significance in the early diagnosis of tumors.
Disclosure of Invention
The invention aims to provide application of hypermethylated CDK2AP1 gene as a molecular marker for alpha radiation damage prediction so as to predict early alpha radiation damage conveniently, accurately and at low cost.
To achieve this object, in a basic embodiment, the present invention provides the use of hypermethylated CDK2AP1 gene as a molecular marker for the prediction of α -radiation damage (i.e. the use of hypermethylated CDK2AP1 gene as a molecular marker for the preparation of a kit for the diagnosis of α -radiation damage), said hypermethylated CDK2AP1 gene having a methylation level of the promoter region of CDK2AP1 gene of 43.00 ± 27.91% (corresponding to a methylation level of 18.50 ± 31.66% in the control group, P < 0.05).
The number of radiation operating personnel in China increases year by year, and the irradiation ways comprise external irradiation and internal irradiation. Alpha internal irradiation generates continuous internal irradiation on a human body, but the health injury effect is unclear, and specific health monitoring indexes are lacked. DNA methylation is one of the most important forms of epigenetic modification. Studies have demonstrated that DNA methylation changes are an important biological basis in the early stages of carcinogenesis. DNA methylation has been used clinically for early diagnosis and prognosis of cancer. In the early molecular biological change of ionizing radiation carcinogenesis, DNA methylation and the regulation mechanism thereof are hot spots for researching the biological effect of ionizing radiation at present.
In a preferred embodiment, the invention provides the use of a hypermethylated CDK2AP1 gene as a molecular marker for the prediction of alpha radiation damage, wherein the methylation site of the CDK2AP1 gene comprises the CG site of CG09084244 of the promoter region.
In a preferred embodiment, the invention provides the use of hypermethylated CDK2AP1 gene as a molecular marker for the prediction of alpha radiation damage, wherein said radiation damage is local or systemic adverse tissue-organ reactions (e.g. nausea, vomiting, anorexia, leukopenia and/or red skin itching, etc.).
The invention has the beneficial effects that the hypermethylated CDK2AP1 gene can be used as the molecular marker for alpha radiation damage prediction, so that early alpha radiation damage prediction can be conveniently, accurately and at low cost.
Detailed Description
The following examples further illustrate the practice of the present invention, but the embodiments of the present invention are not limited to the following examples.
Example 1:
1. the gene chip is used for detecting and analyzing the methylation of the DNA of the peripheral blood of alpha illuminated persons, and the methylation gene with obvious difference is primarily screened
1) Collection of samples
Blood samples of the persons to be irradiated (irradiation dose range within plutonium from 24.0mSv to 2254.0mSv) and the control persons were selected.
2) Peripheral blood genome DNA extraction
Extracting the genomic DNA of the sample. The whole blood genome DNA of the sample is extracted by adopting a blood genome DNA extraction kit of Illumina company according to a product instruction. And detecting and extracting the DNA concentration by using a spectrophotometer, and simultaneously evaluating that the ratio of A260/A280 is between 1.8 and 2.0, so that the quality meets the experiment requirement, and subsequent experiments can be carried out.
3) Sulfite conversion
With sulfite reagent (NaHSO)3In biological engineering (Shanghai) Co., Ltd.) to dissolve the DNA of the sample, and a sulfite reaction component (NaHSO) was added3Biological engineering (Shanghai) Co., Ltd.) in a PCR apparatus, circulation and amplification were initiated (conditions are shown in Table 1), and DNA conversion by sulfite was completed by PCR.
TABLE 1 PCR reaction conditions
| Serial number | Procedure | Temperature of | Time |
| 1 | Denaturation | 95℃ | 5min |
| 2 | Renaturation | 60℃ | 25min |
| 3 | Denaturation of the material | 95℃ | 5min |
| 4 | Renaturation | 60℃ | 85min |
| 5 | Denaturation of the material | 95℃ | 5min |
| 6 | Renaturation | 60℃ | 175min |
| 7 | Holding | 20℃ | —— |
4) DNA methylation chip detection
The methylation level of the whole genome DNA was determined by using Illumina850K methylation chip (850K chip for short, Zymo CA, Illumina).
5) Methylation chip screening differential methylation sites
Values of Δ β: the result is obtained by subtracting the methylation value beta of each CpG site of the control group and the control group, i.e. the difference degree of the methylation of each site between the experimental group and the control group.
② difference Score (Diff Score value): comparing the expression difference of each gene locus between the irradiated group and the control group, wherein the positive value of the difference score indicates that the methylation degree of the irradiated group is higher than that of the control group, and the negative value indicates that the methylation degree of the irradiated group is lower than that of the control group.
③ screening standard of differential methylation gene locus:
the difference score > 13| and Δ β > 0.10| of the experimental group samples (calculation of Δ β value, i.e. the degree of difference in methylation at each site between the experimental group and the control group).
Hypermethylation sites in the promoter region of the CDK2AP1 gene were selected by bioinformatic analysis, as shown in Table 2.
TABLE 2 Primary screening of the promoter region hypermethylated sites of the CDK2AP1 Gene
2. Crowd verification of experimental verification sites by means of pyrosequencing technology
Specific location of the promoter region
1) Sample acquisition and irradiation and chromosome aberration analysis
Peripheral blood was collected from about 30 persons who received irradiation (irradiation dose range within plutonium was 24.0mSv-2254.0mSv) and about 15 persons who controlled the group.
2) Peripheral blood genome DNA extraction
Extracting the genomic DNA of the sample. The whole blood genome DNA extraction is carried out by adopting a blood genome DNA extraction kit of Illumina company according to a product instruction. And detecting and extracting the DNA concentration by using a spectrophotometer, and simultaneously evaluating that the ratio of A260/A280 is between 1.8 and 2.0, so that the quality meets the experiment requirement, and subsequent experiments can be carried out.
3) Pyrophosphoric acid sequencing for detecting methylation degree of CDK2AP1 gene
Design of primer
TABLE 3 CDK2AP1 Gene primer design
② PCR amplification reaction
TABLE 4 PCR reaction System
| Template DNA | Concentration of | Volume of |
| Primer F | 10μM | 2μL |
| Primer R | 10mΜ | 2μL |
| dNTP (hybrid) | 10μM | 2μL |
| Taq buffer (containing MgCl)2) | 10× | 5μL |
| Taq enzyme | 5U/μL | 0.5μL |
| ddH2O | - | 50μL |
TABLE 5 PCR reaction conditions
| Serial number | Procedure | Temperature of | Time |
| 1 | Pre-denaturation | 95℃ | 5min |
| 2 | Denaturation of the material | 94℃ | 30sec |
| 3 | Annealing | 55℃ | 30sec |
| 4 | Extension | 72℃ | 50sec |
| 5 | Cycle 2-4 | 35 cycle | |
| 6 | Prosthetic extensions | 72℃ | 8min |
Sequencing of Pyrophosphoric acid
a) The PCR product was mixed with water to prepare a 38.5. mu.l reaction system.
b) mu.L of reaction binding beads, 38. mu.L of binding buffer and 40. mu.L of PCR product were added to a 96-well PCR reaction plate and mixed well for 10min at room temperature.
c) The vacuum pump was turned on to suck the binding beads and PCR product suspension, and immersed in 70% (v/v) ethanol, 0.2M NaOH and wash buffer for 5s each.
d) After the vacuum pump was turned off, the beads on the probe and the PCR product were placed in 40. mu.l annealing buffer (containing 1.5. mu.l of sequencing primer), denatured at 85 ℃ for 2min, cooled to room temperature, and the primers were annealed to the template for hybridization.
e) The dose was calculated according to Pyrose sequencing software sequence design information, and the substrate mixture, enzyme mixture and four dntps were added in sequence in the reagent compartment.
f) The reagent chamber and the 96-well reaction plate were placed in a Pyrosequencing detector (QIAGEN) for reaction, and the methylation level of each site was automatically analyzed by pyroQ-CpG software. And each primer of each sample has a sequencing peak map, and sites corresponding to each percentage in the sequencing peak map are methylation sites.
4) Statistical analysis
Statistical analysis is carried out on the methylation degree data of the differentially methylated genes by adopting SPSS 22.0 statistical software, a t test (normal) or a Mann-Whitney test (non-normal) is used for comparison between two groups according to whether the data accord with normal distribution, and a Spearman method is used for correlation analysis between the two groups. And analyzing the influence factors of the methylation level by adopting a step-by-step multiple linear regression equation. The statistical test is two-sided, with a test level α of 0.05. The results are shown in Table 6.
TABLE 6 comparison of promoter region methylation levels between groups
Note: p < 0.05
Dose correlation with differential methylation levels was analyzed using a stepwise multiple linear regression equation. The results show that: the exposure to chemical noxious factors has a linear correlation with CDK2AP1 gene methylation levels (P ═ 0.011), i.e., the exposure to chemical noxious factors CDK2AP1 gene methylation levels increased by 24.396% on average; the alpha dose had a linear correlation with CDK2AP1 gene methylation levels, the partial regression coefficient was 19.021(P ═ 0.061), and with a history of plutonium doses, the CDK2AP1 gene methylation levels increased by an average of 19.02%.
TABLE 7 CDK2AP1 Gene methylation level multiple Linear regression analysis
Therefore, the hypermethylated CDK2AP1 gene promoter region was screened as a molecular marker for the prediction of early radiation damage.
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. The foregoing examples or embodiments are merely illustrative of the present invention, which may be embodied in other specific forms or in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims should be construed to be included therein.
Claims (3)
1. Use of the hypermethylated CDK2AP1 gene as a molecular marker for alpha radiation damage prediction, characterized in that: the methylation level of the hypermethylated CDK2AP1 gene in the promoter region of CDK2AP1 gene is 43.00 +/-27.91%.
2. Use according to claim 1, characterized in that: the methylation site of the CDK2AP1 gene included the CG site of CG09084244 of the promoter region.
3. Use according to claim 1, characterized in that: the radiation injury is local or systemic tissue organ adverse reaction.
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Citations (3)
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| US20110287968A1 (en) * | 2009-01-28 | 2011-11-24 | Ait Austrian Institute Of Technology Gmbh | Methylation Assay |
| CN103998622A (en) * | 2011-07-01 | 2014-08-20 | 加利福尼亚大学董事会 | Multigene prognostic assay for lung cancer |
| WO2018008740A1 (en) * | 2016-07-08 | 2018-01-11 | 有限会社ハヌマット | Method for judging onset possibility of large bowel cancer |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110287968A1 (en) * | 2009-01-28 | 2011-11-24 | Ait Austrian Institute Of Technology Gmbh | Methylation Assay |
| CN103998622A (en) * | 2011-07-01 | 2014-08-20 | 加利福尼亚大学董事会 | Multigene prognostic assay for lung cancer |
| WO2018008740A1 (en) * | 2016-07-08 | 2018-01-11 | 有限会社ハヌマット | Method for judging onset possibility of large bowel cancer |
| CN109415771A (en) * | 2016-07-08 | 2019-03-01 | 哈鲁曼有限公司 | The determination method of colorectal cancer initiation potential |
Non-Patent Citations (2)
| Title |
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| WANG等: "Comprehensive analysis of gene expression and DNA methylation for preeclampsia progression" * |
| 何颖;邹立津;高蔓蔓;梁堂钊;邹学农;: "体外长期培养的猪BMSCs发生转化过程中基因表达谱和DNA甲基化谱关联分析" * |
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