CN1616674A - Method for detecting lung cancer relative CYP2A13 resistance gene and its resistance gene - Google Patents
Method for detecting lung cancer relative CYP2A13 resistance gene and its resistance gene Download PDFInfo
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Abstract
The present invention relates to method of detecting lung cancer relative blastomogen metabolizing enzyme gene CYP2A13 as equipotential resistance gene. The method is a PCR-RFLP process. The present invention also relates to the reagent kit for detecting the resistance gene and its usage. In addition, the C3375T(Arg257Cys) functional polymorphism of blastomogen metabolizing enzyme gene CYP2A13 is linked with the lung cancer risk, so as to identify one new resistance gene, and this provides new genetic consultation content and molecular criterion for preventing lung cancer and intervening smoking behavior.
Description
Technical field
The present invention relates to individual resistance and genetics detection method thereof in the crowd prevention of tumour.Particularly, the present invention relates to resistance allele type and the detection method thereof of the carcinogens metabolic enzyme gene CYP2A13 relevant with the ill risk of lung cancer.The invention further relates to the detection kit of above-mentioned resistant gene type, and the application of this test kit.In addition, the invention still further relates to the application in lung cancer prevention and cigarette smoking intervention and correlated inheritance consulting of above-mentioned detection method and resistant gene.
Background technology
Lung cancer is one of modal malignant tumour, has confirmed that smoking and fume exposure are the most important risk factors of lung cancer.But lung cancer is the environment and heredity results of interaction, has only the part individuality finally to suffer from lung cancer in the smoking population.Therefore, judge lung cancer resistant gene and genetic counseling based on this and cigarette smoking intervention, to crowd's prevention of tumour, reducing the relevant tumor incidence of smoking has important public health meaning.
Tobacco comprises the carcinogenic substance more than 40 kinds, and major part is all by causing dna damage to bring out lung cancer.Nicotine is Nicotine (nicotine) again, is the alkaloid in the tobacco.Nicotine burns and sucks or in moment that flue gas sucks at processing, the cigarette of tobacco, can generate 4-(methyl nitrosamine)-1-(3-pyridyl)-1-butanone (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK), it is an intensive animal carcinogens, can bring out the golden vole of small white mouse, big white mouse and Syria lung cancer takes place
[1]Studies show that, in metabolic activity, NNK can make living animal and the tissue that exsomatizes in DNA (thymus nucleic acid) methylate, this isolates 7-methyl guanine and O-6-methyl guanine and obtains proof from various tissues
[2]Nitrosamines derivative NNK wherein just can become final carcinogens and bring out lung cancer after the aerobicization activation in vivo.Bibliographical information to the highest metabolic enzyme CYP2A13 of NNK oxidation capacity mainly at tissue expressions such as lung and respiratory tracts
[3]There is mononucleotide polymorphic (the single nucleotidepolymorphism of a function in this gene; SNP) site; a base replacement C3375T who is positioned at the 5th exon causes coded amino acid to change (Arg257Cys); and the 257Cys varient enzyme of CYP2A13 is significantly lower to the oxidation activity of substrates such as NNK than wild-type enzyme in experiment in vitro, points out this equipotential gene may play the provide protection that reduces the carcinogens metabolism activation and alleviate its genotoxicity in the expression of carcinogens target spot tissue
[4]Though though the function of the Arg257Cys polymorphism of CYP2A13 has been carried out the zymetology checking in experiment in vitro, investigate its meaning in tumor aetiology, particularly, do not have any bibliographical information so far with the dependency of lung cancer resistance at population level.
That has reported on the other hand, carries out polymerase chain reaction-restriction enzyme length polymorphism analysis (PCR-RFLP) to CYP2A13 C3375T function SNP and analyzes certain defective is arranged
[4]Because (homology of genome nucleotide sequence CYP2A13) is very high for CYP2A6, CYP2A7, when carrying out the DNA cloning of gene fragment, causes the mistake of non-specific amplification and analytical results probably for several members of CYP2A gene subfamily.Therefore, need the amplification condition of gene-specific primer design and PCR be optimized, to guarantee before based on the genetic counseling of colony or community and cigarette smoking intervention, carrying out specificity and all very high high-throughput gene type of accuracy.
Therefore, the objective of the invention is to distinguish the C3375T function SNP of CYP2A13 and the dependency of lung cancer resistance, and the method for this site being carried out the high-throughput gene type with PCR-RFLP is provided.
Summary of the invention
The contriver has directly investigated the C3375T function SNP of carcinogens metabolic enzyme gene CYP2A13 and the dependency of the ill risk of lung cancer.By extensive case control study based on hospital, the a large amount of clinical and laboratory experiments and the statistical study of large sample have been carried out, the individuality of distinguishing T 3375 (Cys 257) allelotype of carrying CYP2A13 is suffered from risk to lung cancer and is shown significant resistance, first at population level, identified the polymorphic dependency with the ill risk of lung cancer of the Arg257Cys function that influences carcinogens NNK oxidation activity of CYP2A13.Thereby the present invention has identified in the carcinogens metabolic enzyme system and has suffered from a related resistance allele type of risk with lung cancer.
The invention provides a kind of method of suffering from the related resistance allele type CYP2A13 C3375T of risk with lung cancer that detects, particularly, this method is the PCR-RFLP method.
The present invention also provides with lung cancer and has suffered from a related resistance allele type CYP2A13 C3375T of risk.
The present invention also provides a kind of and has judged individual method to the lung cancer resistance by detecting carcinogens metabolic enzyme gene CYP2A13 polymorphism.
The present invention also provides the primer that is used to detect carcinogens metabolic enzyme gene CYP2A13 C3375T functional polymorphisms right.
The present invention further provides and a kind ofly detected the test kit of suffering from a related resistance allele type CYP2A13C3375T of risk with lung cancer, and the application of mentioned reagent box in lung cancer crowd prevention and cigarette smoking intervention.
The present invention also provides above-mentioned detection method and resistant gene type in the application in lung cancer crowd prevention and cigarette smoking are intervened.
According to an aspect of the present invention, the invention provides a kind of detection method of suffering from a related resistance allele type CYP2A13 C3375T of risk with lung cancer, this method is the PCR-RFLP method.Specifically, comprised following steps:
A: C3375T place the 5th exon to CYP2A13 designs the gene order Auele Specific Primer, and sample genomic dna is carried out the high specific pcr amplification of warm start and gradient;
B: the PCR product is cut with restriction enzyme HhaI, and analyze the banding pattern of restriction endonuclease digestion product, distinguish the genotype of CYP2A13 C3375T with this with agarose gel electrophoresis;
On the other hand, the present invention also provides with lung cancer and has suffered from a related resistance allele type CYP2A13C3375T of risk.Wherein be specially, the T 3375 of CYP2A13 (Cys 257) allelotype is the lung cancer resistant gene.
On the other hand, the present invention also provides a kind of by detecting carcinogens metabolic enzyme gene CYP2A13 polymorphism judgement crowd or individual method to the lung cancer resistance.Wherein, this method comprises
A: C3375T place the 5th exon to CYP2A13 designs the gene order Auele Specific Primer, and sample genomic dna is carried out the high specific pcr amplification of warm start and gradient;
B: the PCR product is cut with restriction enzyme HhaI, and analyze the banding pattern of restriction endonuclease digestion product, distinguish the genotype of CYP2A13 C3375T with this with agarose gel electrophoresis;
C. when the polypeptide of described gene performance CYP2A13 T3375 or this genes encoding carries Cys 257, show that then this individuality has certain lung cancer resistance.
On the other hand, the gene-specific primer of the C3375T function SNP of detection carcinogens metabolic enzyme gene CYP2A13 is right, and its sequence is: forward primer: see sequence 1 in the sequence table, reverse primer: see sequence 2 in the sequence table.
On the other hand, the present invention also provides a kind of and lung cancer to suffer from the detection kit of the related resistance allele type CYP2A13 C3375T of risk, one or more containers are housed in it, are equipped with in the container in order to detect one or more components of CYP2A13 C3375T resistance allele type.Provide simultaneously with it can be through the audit of government's food and drug regulatory department, relevant medicine or biological products manufacturing, use and the information of sale aspect.For example, in the pcr amplification method of DNA is implemented, the test kit of the CYP2A13 C3375T resistance allele type of direct test sample, the primer that can contain PCR is to (see in the sequence table sequence 1 and see sequence 2 in the sequence table), dNTP is used for one or more of the archaeal dna polymerase of PCR reaction and damping fluid thereof etc.It is known to those skilled in the art that above component only is schematically, the archaeal dna polymerase of the described PCR of being used for reaction can be the TaqDNA polysaccharase, for example, HotStar Taq enzyme Klenow fragment, the Tth archaeal dna polymerase, VENT DNA enzyme etc. can be used in the enzyme of pcr amplification.But consider the homology of CYP2A gene family member genome sequence height, for guaranteeing specific amplification, except the optimization of primer and reaction conditions, preferred HotStar Taq enzyme.In the rflp analysis of PCR product, this test kit can contain HhaI and damping fluid thereof, the HhaI restriction enzyme mapping of this gene fragment etc.
And the explanation using method is as follows:
1) pcr amplification
To sample gene group DNA (its concrete preparation method is referring to embodiment), use primer provided by the invention, carry out pcr amplification according to following reaction system and condition: 25-μ l reaction system is: 20～100ng genomic dna, 0.2 μ M primer, 0.2mM dNTP, 1.5mM MgCl
2, 1.0unit HotStarTaq
TMArchaeal dna polymerase and 1 * damping fluid (QIAGEN, Chatsworth, CA).Carry out the amplification in 2 stages after 95 ℃ of pre-sex change of 15min, its sex change and extension term harmonization are 94 ℃ of 30s and 72 ℃ of 50s, in 13 circulations of fs, annealing temperature is since 0.5 ℃ of 63 ℃ of each cycle down, and the annealing temperature of subordinate phase amplification then is fixed on 57.5 ℃.72 ℃ of last extension times are 7min.Specific 375bpPCR product is identified with 1% agarose gel electrophoresis.
2) rflp analysis
To the PCR product of identifying through agarose gel electrophoresis, carry out the cutting digestion of restriction enzyme according to following reaction system and condition: the reaction system of 10 μ l is: 5 μ l PCR product D NA, 2 unit limit restriction endonuclease Hha I, 10 * damping fluid, 1 μ l, and bi-distilled water.Behind the restriction enzyme digestion reaction terminating, sample is carried out 1.5% agarose gel electrophoresis, judge thereby carry out genotype.Wherein, the foundation that genotype is distinguished is, the PCR product of 375bp with Hha I digestion with restriction enzyme after, C/C wild-type homozygote is cut into two bar segment, length is respectively 217 and 158 Nucleotide; The C/T heterozygote is after digestion, and three fragments of this of different lengths all occur; Band of 375bp then only appears in anomaly homozygote T/T after digestion because there not being restriction enzyme site.
The present invention also provide the resistance allele of the carcinogens metabolic enzyme gene CYP2A13 relevant and detection method thereof with the ill risk of lung cancer in lung cancer crowd's prevention and the application in the behavior intervention of smoking.For example; because the present invention has confirmed CYP2A13 and the provide protection that the relevant allelotrope (T3375 or Cys257) of the oxidation activity reduction of carcinogens NNK etc. is had remarkable resistance to the ill risk of lung cancer; and the resistant gene type is frequency and outer showing to spend all lower allelotrope (embodiment 2) in the crowd; therefore; in the design and enforcement of the behavior intervention of the crowd of lung cancer prevention and smoking; just need analyze, whether carry the T3375 of CYP2A13 (Cys257) lung cancer resistance allele to distinguish this individual subject to C3375T (Arg257Cys) genotype of the genomic CYP2A13 of individual subject that seeked advice from.To CYP2A13 wild-type homozygote (C3375C, the performance lung cancer susceptibility) individuality, then can carry out the intervention of science to cigarette smoking, comprise that especially suggestion reduces cigarette consumption (comprising second hand smoking) as far as possible, consume the cigarette of low nicotine content as far as possible, can reduce the lung cancer risk of these Susceptible population so pointedly.
Description of drawings
Fig. 1. the electrophoretogram of restriction enzyme Hha I (available from Promega company) digestion 375bp pcr amplification product.Wherein, swimming lane 1 (right-to-left): standard molecular weight object of reference DL2 000 (available from TaKaRa company); Swimming lane 2,5,7 and 8: contain the result of C/C wild-type homozygote PCR product, the PCR product is cut into two bar segment that length is respectively 217bp and 158bp; Swimming lane 4 and 6: contain the result of C/T heterozygote PCR product, three bar segment that length is respectively 217bp, 158bp and 375bp occurred; Swimming lane 2: contain the result of T/T anomaly homozygote PCR product, the band of 375bp only occurs.
Embodiment:
Embodiment 1.
It is right that this embodiment has designed and synthesized a cover primer, and on the right basis of this primer, designed a kind of method of suffering from the related resistance allele type CYP2A13 C3375T functional polymorphisms of risk with lung cancer that detects.
1. the extraction of genomic dna:
Take the Miller improvement salting-out process of this area routine to extract the peripheral blood leucocyte genomic dna.
1) gets Trisodium Citrate or EDTA-Na
2Anticoagulated whole blood 5ml (as far as possible without anticoagulant heparin) places 50ml to cover centrifuge tube;
2) add 3-5 times of volume cold distilled water, put upside down mixing repeatedly, placed 5 minutes in the ice, centrifugal 20 minutes of 4 ℃ of 2000rpm;
3) slowly topple over supernatant, precipitation adds the 0.1%Triton-X100 (volume is the same) of precooling, and soft mixing precipitation is as above centrifugal, abandons supernatant;
4) (50Mm Tris-Cl-10mM EDTA pH8.0), thoroughly smashes precipitation to precipitation adding 4ml lysate, and adding 10%SDS then is 0.5% to final concentration, mixing;
5) adding Proteinase K (10mg/ml) is 200 μ g/ml to final concentration, mixing, 55 ℃ of 3 hours or 37 ℃ of digested overnight (is good with digested overnight);
6) add 6M NaCl 1.2ml, thermal agitation 20 seconds, 2, centrifugal 10 minutes of 200rpm is transferred to another with supernatant and covers centrifuge tube;
7) add 2 times of volume precooling dehydrated alcohols, put upside down mixing repeatedly, choose DNA to another Eppendorf pipe to cotton-shaped DNA precipitation occurring;
8) precipitate 2 times with 75% pre-cooled ethanol washing DNA;
9) with 0.5ml TE (10mM Tris-Cl-EDTA, pH8.0) or dissolved in distilled water DNA precipitation, electrophoresis detection dna fragmentation size, or survey 260/280nm OD ratio.
2.PCR amplification:
Use primer provided by the invention, carry out pcr amplification according to following reaction system and condition: 25-μ l reaction system is: 20～100ng genomic dna, 0.2 μ M primer, 0.2mM dNTP, 1.5mM MgCl
2, 1.0unit HotStarTaq
TMArchaeal dna polymerase and 1 * damping fluid (QIAGEN, Chatsworth, CA).Carry out the amplification in 2 stages after 95 ℃ of pre-sex change of 15min, its sex change and extension term harmonization are 94 ℃ of 30s and 72 ℃ of 50s, in 13 circulations of fs, annealing temperature is since 0.5 ℃ of 63 ℃ of each cycle down, and the annealing temperature of subordinate phase amplification then is fixed on 57.5 ℃.72 ℃ of last extension times are 7min.Specific 375bp PCR product carries out the electrophoresis evaluation with 1% sepharose.
3.RFLP analyze:
Carry out the cutting digestion of restriction enzyme according to following reaction system and condition: the reaction system of 10 μ l is: 5 μ l PCR product D NA, 2 unit limit restriction endonuclease Hha I (Promega), 10 * damping fluid, 1 μ l, and bi-distilled water.37 ℃ of digestion are spent the night, and stop endonuclease reaction with bromjophenol blue load sample damping fluid, and reaction product is separated with 1.5% agarose gel electrophoresis, judge thereby carry out genotype.Wherein, the foundation that genotype is distinguished is, the PCR product of 375bp with Hha I digestion with restriction enzyme after, C/C wild-type homozygote is cut into two bar segment, length is respectively 217 and 158 Nucleotide; The C/T heterozygote is after digestion, and three fragments of this of different lengths all occur; Band of 375bp then only appears in anomaly homozygote T/T after digestion because there not being restriction enzyme site.
Embodiment 2.
The method that this embodiment sets up with embodiment 1 is analyzed the genotypic distribution of CYP2A13 C3375T in the healthy individual of 724 routine patients with lung cancer and 791 contrasts.
1. subjects: among this embodiment at 724 routine patients and 791 example contrasts be Chinese northern Han nationality individuality, do not have directly under kinship.The case group all is the primary lung cancer patient who makes a definite diagnosis through histopathology that Cancer Hospital of Chinese Academy of Medical Sciences is accepted for medical treatment in the period of 1997-2001, before the art without radiation and anticancer chemotherapy.The extensive nutrition-epidemiology investigation that the normal control crowd of no tumour medical history and clinical sign comes comfortable Beijing area to carry out, the frequency of the sex of control group and age and case group is complementary.In collection organization's sample or blood sample, each research object is all signed Informed Consent Form and is provided detailed social demography's data and data such as smoking history, every day smoking capacity, smoking initial sum smoking cessation age.
2. analyze the genotypic distribution of CYP2A13 C3375T: in the healthy individual of 724 routine patients with lung cancer and 791 contrasts.CYP2A13 C3375T genotype is shown in Table 1 in the case of experiment and the frequency distribution among the contrast crowd: C3375T is respectively 11.7% and 16.4% in the frequency of case group and control group; Because the frequency of anomaly homozygote (T/T) in the crowd is very low, be used as one group and compare when T/T homozygote and the merger of C/T heterozygote being risen with wild homozygote (C/C) according to dominant models, the result shows two groups lung cancer morbidity risk significant difference: the ill risk of carrying the individuality (genotype is C/T or T/T) of resistance allele type T has only 0.62 times of wild-type (C/C) individuality, and the fiducial interval of its statistics 95% is 0.45 to 0.87 times.Proofread and correct OR in the table and be with CYP2A13 C/C genotype and be reference (promptly this genotypic odds ratio is 1) and age-sex and smoking state are carried out correction analysis obtain, T/T wherein and C/T are one group according to the merger of dominant inheritance pattern and compare with reference group, represent the significance of adding up with 95% fiducial interval.Just, use method of the present invention, resolved the C3375T function polymorphic site of CYP2A13 and the dependency of lung cancer resistance.
CYP2A13 C3375T control group case group
Genotype (N=791) (N=724)
N(％) N(％)
C/C 652(82.4) 632(87.3)
C/T 130(16.4) 85(11.7)
T/T 9(1.2) 7(1.0)
-/T:C/T or T/T 139 (17.6) 92 (12.7)
Proofread and correct OR (95%CI): with wild-type C/C be reference carry T 0.62
Allelic-/the ill risk (odds ratio) of T is (0.45-0.87)
Among table 1: embodiment 2 cases and the contrast crowd the genotypic distribution of CYP2413 C3375T and with the dependency of lung cancer risk
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, but the equivalent form of value doing to change or revise drop on equally in the application's letter of authorization institute restricted portion.
Reference
1.Hecht，S.S.Biochemistry，biology，and?carcinogenicity?of?tobacco-specific?nitrosamines.Chem.Res.Toxicol.，11：559-603，1997.
2.Hecht，S.S.Tobacco?smoke?carcinogens?and?lung?cancer.J.Natl.Cancer?Inst.，91：1194-1210，1999.
3.Su，T.，Bao，Z.，Zhang，Q.-Y.，Smith，T.J.，Hong?J.-Y.，and?Ding，X.Human?cytochrome?P450CYP2A13：predominant?expression?in?the?respiratory?tract?and?its?high?efficiency?metabolicactivation?of?a?tobacco-specific?carcinogen，4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.Cancer?Res.，60：5074-5079，2000.
4.Zhang，X.，Su，T.，Zhang，Q.Y.，Gu，J.，Caggana，M.，Li，H.，and?Ding，X.Geneticpolymorphisms?of?the?human?CYP2A13?gene：identification?of?single-nucleotidepolymorphisms?and?functional?characterization?of?an?Arg257Cys?variant.J.Pharmacol.Exp.Ther.，302：416-423，2002.
Sequence table
＜110〉Tumar Inst of Tumoor Hospital, Chinese Academy of Medical Sciences of Institute of Radiation Medicine, Academy of Military Medical Sciences, PLA
＜120〉method and the resistant gene thereof of the CYP2A13 resistant gene that a kind of detection of lung cancer is relevant
<130>
<140>200310113359.1
<141>2003-11-12
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<170>PatentIn?version?3.2
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<212>DNA
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<400>1
taactccgtt?ccttccttgc?t 21
<210>2
<211>21
<212>DNA
<213>
<400>2
taatttgaat?gggcctgtgt?c 21
Claims (6)
1. the method for CYP2A13 T3375 (Cys 257) resistance allele that a detection of lung cancer is relevant, this method is PCR-RFLP, i.e. polymerase chain reaction-restriction enzyme length polymorphism analysis is characterized in that comprising following aspect:
A: to the gene order of the 5th exon of the CYP2A13 that comprises C3375T (Arg257Cys) function polymorphic site, the primer of design gene specific, its sequence is: forward primer: see sequence 1 in the sequence table, reverse primer: see sequence 2 in the sequence table.
B: the 5th exon to the CYP2A13 that comprises C3375T (Arg257Cys) function polymorphic site, with designed primer, by the polymerase chain reaction of warm start and gradient, carry out DNA cloning with gene-specific primer.
2. the method for CYP2A13 T3375 (Cys 257) resistance allele that detection of lung cancer as claimed in claim 1 is relevant, the primer of C3375T (Arg257Cys) function polymorphic site that it is characterized in that detecting carcinogens metabolic enzyme gene CYP2A13 is right, its sequence is: forward primer: see sequence 1 in the sequence table, reverse primer: see sequence 2 in the sequence table.
3. judge the method for individual lung cancer resistance by detecting carcinogens metabolic enzyme gene CYP2A13 polymorphism for one kind, it is characterized in that when described gene shows CYP2A13 T3375, showing that then this individuality has certain lung cancer resistance.
4. judge the method for individual lung cancer resistance by detecting carcinogens metabolic enzyme gene CYP2A13 polymorphism for one kind, it is characterized in that when the polypeptide of described genes encoding carries Cys 257, show that then this individuality has certain lung cancer resistance.
5. suffer from a kind of detection kit of a related resistance allele type CYP2A13 C3375T of risk with lung cancer, it comprises:: the amplification gene-specific primer, dNTP, be used for one or more of the archaeal dna polymerase of PCR reaction and damping fluid thereof, be used for one or more and restriction enzyme mapping thereof of the restriction enzyme Hha I of RFLP and corresponding damping fluid.
6. as each described method or test kit among the claim 1-5, it is in crowd's prevention and the behavior intervention of smoking and the application in the genetic counseling of lung cancer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102168061A (en) * | 2010-12-21 | 2011-08-31 | 南京医科大学 | Human lung cell BEAS-2B/CYP2A13, and preparation method and application thereof |
| CN102911855A (en) * | 2012-10-19 | 2013-02-06 | 天津金思德生物技术有限公司 | Warm start method of PCR (Polymerase Chain Reaction) instrument and PCR instrument with warm start function |
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| JP3888807B2 (en) * | 1999-08-06 | 2007-03-07 | 凸版印刷株式会社 | Gene detection method, detection apparatus, and detection chip |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168061A (en) * | 2010-12-21 | 2011-08-31 | 南京医科大学 | Human lung cell BEAS-2B/CYP2A13, and preparation method and application thereof |
| CN102168061B (en) * | 2010-12-21 | 2013-05-22 | 南京医科大学 | Human lung cell BEAS-2B/CYP2A13, and preparation method and application thereof |
| CN102911855A (en) * | 2012-10-19 | 2013-02-06 | 天津金思德生物技术有限公司 | Warm start method of PCR (Polymerase Chain Reaction) instrument and PCR instrument with warm start function |
| CN102911855B (en) * | 2012-10-19 | 2016-01-13 | 天津金思德生物技术有限公司 | A kind of PCR instrument hot start method and there is the PCR instrument of warm start function |
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