CN111172272A - Application of TRIP12 gene as molecular marker for judging susceptibility to radiation damage - Google Patents
Application of TRIP12 gene as molecular marker for judging susceptibility to radiation damage Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, and relates to application of TRIP12 gene as a molecular marker for judging susceptibility to radiation damage. By using the TRIP12 gene as a molecular marker for judging susceptibility to radiation damage, the radiation damage can be detected by whether the site rs13018957 of the TRIP12 gene is mutated with high efficiency and low cost.
Description
Technical Field
The invention belongs to the technical field of biological detection, and relates to application of TRIP12 gene as a molecular marker for judging susceptibility to radiation damage.
Background
At present, about 20 thousands of radiation workers exist in China. Radiation workers are required to receive occupational irradiation for a long time during daily work, and the injury of accumulated irradiation to susceptible people is obvious, so that an effective method is needed for judging whether the personnel meet the post requirements or not so as to reduce occupational injury.
In addition, radiation therapy is one of the main therapeutic approaches to malignancy, and over 50% of cancer patients require radiation therapy. However, even with the same radiation treatment regimen, the degree of radiation damage varies from patient to patient, and the cause of this is mainly related to the individual differences in susceptibility to radiation damage: under the same treatment condition, people susceptible to radiation damage may cause serious adverse reactions, and people not susceptible to radiation damage may have a risk of poor treatment effect.
Therefore, the method can accurately distinguish the person susceptible to radiation damage, is favorable for the post adaptability judgment of the radiation worker, and is also favorable for customizing a clinical personalized treatment scheme or optimizing the treatment scheme.
The TRIP12rs13018957 locus is located on chromosome 2, the gene of the locus is a thyroid hormone receptor interaction factor, and the protein encoded by the gene is E3 ubiquitin protein ligase and participates in the degradation of p19ARF/ARF subtype of tumor suppressor CDKN 2A. The encoded protein also plays a role in DNA damage response by modulating the stability of USP 7.
Susceptibility refers to the different tendency of different individuals to become predisposed to disease under the influence of the external environment due to genetic differences. At present, a great deal of research proves that different individuals have great difference in response to ionizing radiation, and the effect of susceptibility factors of the individuals on the occurrence of radiation damage is not negligible. The problem of susceptibility to radiation damage among individuals of different genetic backgrounds was mentioned earlier in publications ICRP60 and 79, and there is a high-risk subpopulation of people who are highly sensitive to ionizing radiation damage, the presence of which increases the uncertainty of the risk of radiation carcinogenesis. With the support of the eu 5 th framework, the research group found that phenotypic changes with early effects of radiation of more than 70% were attributed to intrinsic differences among individuals. Accordingly, the research group believes that inherent differences in individual susceptibility to radiation can be identified by Single Nucleotide Polymorphism (SNP) analysis methods.
Disclosure of Invention
The invention aims to provide application of TRIP12 gene as a molecular marker for judging susceptibility to radiation damage, so that radiation damage can be detected through high efficiency and low cost of whether mutation at site rs13018957 of TRIP12 gene is high.
To achieve this object, in a basic embodiment, the present invention provides the use of the TRIP12 gene as a molecular marker for susceptibility determination of radiation damage.
In a preferred embodiment, the invention provides the use of the rs13018957 mutation site of the TRIP12 gene as a molecular marker for judging the susceptibility to radiation damage.
In a preferred embodiment, the invention provides the use of TRIP12 gene as a molecular marker for determining susceptibility to radiation damage, wherein the radiation damage is local or systemic tissue-organ adverse reactions (such as nausea, vomiting, anorexia, leukopenia and/or red skin itching, etc.).
The sequence of the TRIP12 gene is shown as SEQ ID NO.1, the mutation site is rs13018957 site (mutation from C to T) of the TRIP12 gene, and the sequence of the mutated TRIP12 gene is shown as SEQ ID NO. 2.
The invention has the beneficial effects that by using the TRIP12 gene as a molecular marker for judging the susceptibility to radiation damage, the radiation damage can be detected by judging whether the site rs13018957 of the TRIP12 gene is mutated with high efficiency and 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. preliminary screening of molecular markers for judging susceptibility to radiation damage by using full exon capture sequencing technology
1) Sample acquisition and irradiation and chromosome aberration analysis
Collecting peripheral blood of healthy adult male of 20-30 years old, and administering 0, 2Gy60And (4) irradiating Co gamma rays. And (3) carrying out chromosome aberration analysis on the 2Gy gamma ray irradiation sample, and dividing the population into a susceptible group, a general group and an insensitive group. After the irradiation, the blood sample is cultured for 52h, and then the chromosome is harvested and sliced. Each sample was analyzed for 200 metaphase phases.
2) Peripheral blood genome DNA extraction
Genomic DNA of 0Gy irradiated samples of susceptible and non-susceptible groups was extracted. The whole blood genome DNA extraction is carried out by adopting a blood genome DNA extraction kit of Beijing Tianzhu Biochemical technology Co., Ltd according to a product specification, and the specific steps are shown in the specification. The quantitative detection A260/280 of the sampled nucleic acid is between 1.70 and 1.90, the quality meets the experiment requirements, and the subsequent experiment can be carried out.
3) Whole exon capture sequencing
Sequencing data is firstly subjected to data filtration to remove low-quality data, and clear Reads are obtained. The sequencing needs to reach the clean Reads rate of more than 90%, the clean base rate of more than 20G, the clean base rate of more than 90%, and the Q20 rate of more than 98%, and the experimental sample meets the requirements.
4) Biological information analysis
Clear Reads were aligned to the reference genome and differential SNP sites were screened. Reference genome version: GRCh37(hg19),ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/human_ g1k_v37.fasta.gz. By utilizing biological information analysis, the site TRIP12rs13018957 which is the difference site between the susceptible group and the less susceptible group is screened out, and the table 1 shows.
TABLE 1 preliminary screening of radiation injury susceptible sites
2. Experimental verification of primary screening sites by matrix-assisted laser desorption ionization mass spectrometry
1) The method utilizes a blood genome DNA extraction kit (non-centrifugal column type; catalog number: DP319) human whole blood genome (derived from 1) of step 1) DNA extraction was performed according to the product instructions.
2) The amplification primer pair of SEQ ID NO.3 and SEQ ID NO.4 is adopted, and a specific reaction system (5 mu l of the reaction system comprises 0.95 mu l H)2O、0.625μl PCR Buffer(10×)、0.325μl MgCl2PCR reaction (25mM), 1. mu.l dNTP (2.5mM), 1. mu.l primer, 0.1. mu.l HotstarTaq (5U/. mu.L)) was performed according to the following reaction program: 15min at 94 ℃; [94 ℃, 20sec, 56 ℃, 30sec ]]45 cycles; 72 ℃ for 4 min. The reaction product was stored at 4 ℃.
3) Using an SAP reaction solution (2. mu.l SAP reaction solution included 1.53. mu.l H)2O, 0.17. mu.l SAP Buffer (10X), 0.3. mu.l SAP enzyme (1U/. mu.L)) were applied to the reaction product of step 2) according to the following procedure: at 37 ℃ for 40 min; 85 ℃ for 5 min. The treated product was stored at 4 ℃.
4) Carrying out extension reaction on the treated product in the step 3) by adopting an extension primer of SEQ ID NO.5,
2 μ l reaction included 0.755 μ l H2O, 0.2. mu.l of iPLex Buffer (10X), 0.2. mu.l of iPLEX termination mix, 0.041. mu.l of iPLex enzyme, 0.804. mu.l of primer.
The reaction procedure is as follows: 30s at 94 ℃; 5 cycles of [94 ℃, 5s, (52 ℃, 5s, 80 ℃, 5s) ]40 cycles; 72 ℃ for 3 min. The extension product was stored at 4 ℃.
5) The extension product from step 4) was purified (6 mg of resin was uniformly covered on 384 well plates and left for 20 min. The 384 well plate containing the extension product of step 4) was centrifuged at 1000rpm for 1min, 25. mu.L of deionized water was added to each well, inverted on the resin plate, and then the resin plate was snapped on the 384 well plate in the inverted position, and the resin was dropped into the 384 well plate by tapping, and the membrane was sealed. The 384 well plate was inverted for 20 minutes with the long axis of the 384 well plate as the axis, centrifuged at 3500rpm for 5 minutes, and then prepared).
6) Detecting the genotype of the gene locus: transferring the sample treated in step 5) to MassARRAYPectroCHIP chip (MassArray)TMNanodispenser, SAMSUNG), was put into a mass spectrometer (massarrycompact System, SEQUENOM), and the results were shown in table 2.
Table 2 experimental verification results
3. Crowd verification of experimental verification sites by matrix-assisted laser desorption ionization mass spectrometry
1) Sample acquisition and irradiation and chromosome aberration analysis
Collecting peripheral blood of healthy adult male of 20-30 years old, and administering 0, 2Gy60And (4) irradiating Co gamma rays. And (3) carrying out chromosome aberration analysis on the 2Gy gamma ray irradiation sample, and dividing the population into a susceptible group, a general group and an insensitive group. After culturing for 52h, the chromosomes are harvested and sliced. Each sample was analyzed for 200 metaphase phases.
2) Peripheral blood genome DNA extraction
Genomic DNA of 0Gy irradiated samples of susceptible and non-susceptible groups was extracted. The whole blood genome DNA extraction is carried out by adopting a blood genome DNA extraction kit of Beijing Tianzhu Biochemical technology Co., Ltd according to a product specification, and the specific steps are shown in the specification. The quantitative detection A260/280 of the sampled nucleic acid is between 1.70 and 1.90, the quality meets the experiment requirements, and the subsequent experiment can be carried out.
3) Mass spectrometry detection
And performing population verification on the sample, and further verifying the site.
4) Statistical analysis
The Hardy-Weinberg equilibrium test is used for testing the rs13018957 locus susceptible group and the non-susceptible group of the TRIP12 gene respectively, wherein P is more than 0.05 in the two groups, which indicates that the sample has population representativeness, and the results are shown in a table 3.
Comparing the difference of locus rs13018957 of TRIP12 gene between susceptible group and non-susceptible group by using one-way anova2＝9.326，P<0.01, the site was significantly different between the two groups, and the results are shown in Table 4.
The differences between the susceptible and non-susceptible TRIP12 gene at locus rs13018957 were compared by logistic regression analysis, and the results are shown in Table 5. The P of TT type, TC type and CC type is less than 0.05, the TC type is 5.5 times of the risk of TT type, and the CC type is 0.583 times of the risk of TT type.
TABLE 3 Hardy-Weinberg equilibrium test results
TABLE 4 results of one-way ANOVA
TABLE 5 results of logistic regression analysis
| Genotype(s) | P | Risks |
| TT | 0.045 | 1 |
| TC | 0.027 | 5.5 |
| CC | 0.257 | 0.583 |
Therefore, the locus rs13018957 of the TRIP12 gene is screened to be used as a molecular marker for judging the susceptibility to radiation damage, and the TC type is a genotype for judging the susceptibility to radiation damage and can be used as an index for judging and detecting the susceptibility of people.
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.
Sequence listing
<110> China institute for radiation protection
Application of <120> TRIP12 gene as molecular marker for judging susceptibility to radiation damage
<130>-
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agcatttgta tataaaactc tgcctgaatt tgatgattat ttttggccat actactttaa 120
attgagagca aagcattttc attgtgaaac cattctatta acataaacat ttttcttaaa 180
tgctcaagtt ttatatttta taaaagctat tatgtgaata taaatagaca aaaaagagct 240
caagatactc ctaacaaatt tttgtgcata ttttttctat tattacttta gaggtttctg 300
aaattacttg cagagatttg tatttgtaaa atgtttctac tattttcatt aacacacctt 360
gaaacagcat gatttttcag aacatccttc agaagttcag catccgcaaa ataaattatc 420
ctaagaattg ctctaaggca cttatgtctg accgcaggtc ctgctgagga actatacact 480
tcataaagaa caccaaataa cgtcttaata aaagacttag ccagttccgg atcctctttc 540
ataagctgtg ctcgagcatc atccttcttt gactctgaat atccacctat tacattaaaa 600
aaaaatatat gtgcaatcct gaagtgacag acttcagaaa cacaataaac aatataaaat 660
actcaagcca gtgtaattct tgaaatgcta tgaaaacttt ttcattatag aacacatgaa 720
cttctatgat atgatcaact aggatttaca aagatgctaa aggcaacatt aagacagaca 780
aaattcagct gtctagaaaa ctagtgaccc attggggcca gctgaaagta acaattcatc 840
accgcactgt acctctttat tttactgata tacattcaat gaaacaatag ggaaatccaa 900
tcctaaccaa ccaaaccagt ggtaagaaac tgagttctag aaatgcacca tatttcacta 960
aaagctttgt aaaatggata aagaaggtaa ttctaccact t 1001
<210>2
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cccactacta tcttcaggtc ttggcttgac agcatggaag caatgtgact aaaaaaagaa 60
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attgagagca aagcattttc attgtgaaac cattctatta acataaacat ttttcttaaa 180
tgctcaagtt ttatatttta taaaagctat tatgtgaata taaatagaca aaaaagagct 240
caagatactc ctaacaaatt tttgtgcata ttttttctat tattacttta gaggtttctg 300
aaattacttg cagagatttg tatttgtaaa atgtttctac tattttcatt aacacacctt 360
gaaacagcat gatttttcag aacatccttc agaagttcag catccgcaaa ataaattatc 420
ctaagaattg ctctaaggca cttatgtctg accgcaggtc ctgctgagga actatacact 480
tcataaagaa caccaaataa tgtcttaata aaagacttag ccagttccgg atcctctttc 540
ataagctgtg ctcgagcatc atccttcttt gactctgaat atccacctat tacattaaaa 600
aaaaatatat gtgcaatcct gaagtgacag acttcagaaa cacaataaac aatataaaat 660
actcaagcca gtgtaattct tgaaatgcta tgaaaacttt ttcattatag aacacatgaa 720
cttctatgat atgatcaact aggatttaca aagatgctaa aggcaacatt aagacagaca 780
aaattcagct gtctagaaaa ctagtgaccc attggggcca gctgaaagta acaattcatc 840
accgcactgt acctctttat tttactgata tacattcaat gaaacaatag ggaaatccaa 900
tcctaaccaa ccaaaccagt ggtaagaaac tgagttctag aaatgcacca tatttcacta 960
aaagctttgt aaaatggata aagaaggtaa ttctaccact t 1001
<210>3
<211>30
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<213> human (Homo sapiens)
<400>3
acgttggatg gtcctgctga ggaactatac 30
<210>4
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<212>DNA
<213> human (Homo sapiens)
<400>4
acgttggatg tgaaagagga tccggaactg 30
<210>5
<211>23
<212>DNA
<213> human (Homo sapiens)
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acttcataaa gaacaccaaa taa 23
Claims (3)
- Use of the TRIP12 gene as a molecular marker for determining susceptibility to radiation damage.
- Use of the rs13018957 mutation site of the TRIP12 gene as a molecular marker for judging susceptibility to radiation damage.
- 3. Use according to claim 1 or 2, characterized in that: the radiation injury is local or systemic tissue organ adverse reaction.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114214324A (en) * | 2021-11-23 | 2022-03-22 | 中国辐射防护研究院 | miR-219a-2-3p and application thereof as molecular marker for early diagnosis of radiation damage |
| CN114214323A (en) * | 2021-11-23 | 2022-03-22 | 中国辐射防护研究院 | hsa-miR-23c and application thereof as molecular marker for early diagnosis of radiation damage |
| CN114480635A (en) * | 2021-11-29 | 2022-05-13 | 中国辐射防护研究院 | Application of hypomethylated PDGFRL gene as molecular marker for alpha radiation damage prediction |
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| CN101258250A (en) * | 2005-07-07 | 2008-09-03 | 拜奥默里克斯股份有限公司 | Method for breast cancer diagnosis |
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| JUANCONG DONG: "Single nucleotide polymorphisms of radiation susceptible genes and radiosensitive miRNA and LncRNA in Chinese population", 《ARADOS MEETING》 * |
| L WANG等: "TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects", 《ONCOGENE》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114214324A (en) * | 2021-11-23 | 2022-03-22 | 中国辐射防护研究院 | miR-219a-2-3p and application thereof as molecular marker for early diagnosis of radiation damage |
| CN114214323A (en) * | 2021-11-23 | 2022-03-22 | 中国辐射防护研究院 | hsa-miR-23c and application thereof as molecular marker for early diagnosis of radiation damage |
| CN114480635A (en) * | 2021-11-29 | 2022-05-13 | 中国辐射防护研究院 | Application of hypomethylated PDGFRL gene as molecular marker for alpha radiation damage prediction |
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