CN102796820B - Hepatocellular carcinoma-related klotho gene single-nucleotide polymorphism and its construction method and use - Google Patents
Hepatocellular carcinoma-related klotho gene single-nucleotide polymorphism and its construction method and use Download PDFInfo
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- CN102796820B CN102796820B CN2012102997487A CN201210299748A CN102796820B CN 102796820 B CN102796820 B CN 102796820B CN 2012102997487 A CN2012102997487 A CN 2012102997487A CN 201210299748 A CN201210299748 A CN 201210299748A CN 102796820 B CN102796820 B CN 102796820B
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Abstract
The invention provides hepatocellular carcinoma-related klotho gene single-nucleotide polymorphism and its construction method and use. The hepatocellular carcinoma-related klotho gene single-nucleotide polymorphism and its construction method have the advantages that 1, through a detection method provided by the invention, polymorphism of a single nucleotide located in an exon 4 area of a human klotho gene can be detected fast and simply and it is diagnosed that if an individual has susceptibility of hepatocellular carcinoma so that the hepatocellular carcinoma-susceptible population screening and hepatocellular carcinoma prevention are promoted; 2, a T allele point of rs648202 is used as a drug design target point for drug screening so that an active molecule adjusting a klotho gene expression level is selected and development of a novel antitumor drug is promoted; 3, through primer sequences and HaeIII endonuclease provided by the invention, simple, efficient and specific detection of rs648202 polymorphism is realized; and 4, according to hepatocellular carcinoma-related klotho gene single-nucleotide polymorphism, a hepatocellular carcinoma genetic diagnosis kit can be constructed.
Description
Technical field
The invention provides its construction process of klotho gene mononucleotide polymorphism and application thereof that a kind of primary hepatocellular carcinoma (hcc) is relevant, belong to the genetically engineered biological technical field.
Background technology
Primary hepatocellular carcinoma (hcc) (is called for short liver cancer, HCC) be one of modal malignant tumour in world wide, the data presentation of in December, 2007 ACS (ACS) issue: increase 66.7 ten thousand examples liver cancer year, dead 59.8 ten thousand examples, wherein approximately 50% new trouble case and death in China, be that liver cancer is larger to China people's health threat, become China second tumour killer, not only make the patient suffering can't bear, survival rate is low, and brings serious economy and psychological burden to family, society.
Single nucleotide polymorphism (SNP) refers on genomic level the polymorphism that the variation by the single core thuja acid causes.It is modal a kind of in the heritable variation of the mankind, accounts for more than 90% of all known polymorphisms.SNP is as the early stage sudden change of genetic stability, closely related with disease.Apparently higher than non-patient, point out this SNP disease-related therewith when the frequency of occurrences of a kind of SNP in the patient, by analyzing and researching both haplotype and linkage disequilibrium, the Disease-causing gene of any the unknown in genome can be located.The effect of SNP performance in the Disease-causing gene location mainly comprises: the one, finding the SNP that causes a disease, the appearance of this SNP can cause gene expression amount and (or) variation of protein expressioning product structure, thereby cause the generation of certain disease or make individual to certain disease-susceptible humans; The 2nd, SNP is as a kind of genetic marker, chain with disease or phenotype.
Klotho finds the gene relevant with aging in 1997, is positioned at human chromosomal 13q12, and length is about 50kb, 5 exons and 4 introns, consists of.The expression deletion of Klotho gene in mouse causes its various phenotypes that the mankind aging occurs being similar to, as arteriosclerosis, osteoporosis, pulmonary emphysema, the lost of life, skin atrophy, infertility, ataxia etc.In recent years, increasing research discovery klotho gene can be regulated IGF, Wnt, the signal paths such as TGF-β l, FGF, participate in the oxidative stress process, and relevant with the immune deficiency of body, the vital role of the similar cancer suppressor gene of performance in the carcinogenesis of human of malignant tumour.Therefore can utilize that klotho is used as that tumour occurs, the mark of classification and as target spot for antineoplastic treatment.
Domestic and foreign literature and patent to prior art are retrieved, the relevant report of so far there are no any klotho gene pleiomorphism and hepatocellular carcinoma susceptibility relation.
Summary of the invention
The object of the invention is to fill up the blank of prior art, its construction process of klotho gene mononucleotide polymorphism and application thereof that a kind of primary hepatocellular carcinoma (hcc) is relevant are provided.
The invention provides a kind of method that detects the klotho gene mononucleotide polymorphism that primary hepatocellular carcinoma (hcc) is relevant, adopt the PCR-RFLP method, comprise the following steps:
(1) take the people's complete genome DNA to be measured that comprises the klotho gene is template, and pcr amplification obtains the DNA sequence dna that comprises the rs648202 mononucleotide polymorphic in klotho gene extron 4th district, i.e. sequence shown in the 2190th to the 2352nd in SEQ ID No.1,
Wherein, the PCR primer sequence is as follows:
Upstream primer is F:5 '-ATAGCCTTGCAGGCTGATTG-3 '
Downstream primer is R:5 '-TTCTTTGGTTCAGCCAGTCC-3 '
(2) pcr amplification product is carried out after enzyme is cut carrying out the rs648202 single nucleotide polymorphism typing by agarose gel electrophoresis through restriction enzyme Hae III, the DNA fragmentation length after CC genotype enzyme is cut is still 163bp; CT genotype enzyme is cut the rear DNA fragmentation that produces 163bp, 100bp, tri-kinds of length of 63bp; TT genotype enzyme is cut the DNA fragmentation of rear generation 100bp, bis-kinds of length of 63bp.
The present invention also provides a kind of isolating nucleic acid, have sequence shown in the 2190th to the 2352nd in SEQ ID No.1, and the base of the 2255th in SEQ ID No.1 is T.
The present invention also provides a kind of Auele Specific Primer that detects the klotho gene mononucleotide polymorphism that primary hepatocellular carcinoma (hcc) is relevant, upstream primer is F:5 '-ATAGCCTTGCAGGCTGATTG-3 ', and downstream primer is R:5 '-TTCTTTGGTTCAGCCAGTCC-3 '.
The present invention also provides a kind of diagnostic kit of primary hepatocyte hepatocarcinoma susceptibility, and it comprises:
(1) primer of specific amplification klotho gene mononucleotide polymorphism rs648202, wherein, upstream primer is F:5 '-ATAGCCTTGCAGGCTGATTG-3 ', downstream primer is R:5 '-TTCTTTGGTTCAGCCAGTCC-3 ';
(2) detect amplified production and compare the reagent that whether exists variation required with normal klotho gene.
Utilize the nucleotide sequence of the pleomorphism site relevant to primary hepatocyte hepatocarcinoma provided by the present invention, can build the test kit that primary hepatocyte hepatocarcinoma Susceptible population is carried out to genetic screening, target spot using it as medicinal design, find out the bioactive molecule with adjusting klotho genetic expression, thereby promote the discovery of new antitumor drug.
The present invention has following technique effect:
(1) method that the present invention sets up can detect the genotype that is positioned at human chromosomal 13q12 zone klotho gene extron 4 district mononucleotide polymorphism site rs648202 quickly and easily, judge by detecting T allelic having or not whether individuality has susceptibility to primary hepatocyte hepatocarcinoma, thereby be conducive to the examination of primary hepatocyte hepatocarcinoma Susceptible population and the prevention of primary hepatocyte hepatocarcinoma;
(2) utilize the T loci of rs648202 to carry out the screening of medicine as the medicinal design target spot, filter out the bioactive molecule that can regulate the klotho gene expression dose, promote the discovery of new antitumor drug;
(3) utilize primer sequence provided by the invention and Hae III endonuclease to detect easy, efficiently, specifically rs648202 polymorphic;
(4) utilize the klotho gene mononucleotide polymorphism that primary hepatocellular carcinoma (hcc) provided by the invention is relevant, build the test kit that primary hepatocyte hepatocarcinoma is carried out to the heredity diagnosis;
(5) because klotho has participated in malignant proliferation, apoptosis, pathologic process that invasion and attack are relevant to cellular activities such as transfers, so the present invention provides experience and application foundation to the relation of further investigated klotho and other diseases from now on.
The accompanying drawing explanation
Figure l is for being used Auele Specific Primer to carry out the agarose gel electrophoresis figure after pcr amplification, Hae III enzyme are cut to klotho gene extron 4th district.M:DNA molecular weight standard wherein; The l:CT genotype; The 2:CC genotype; The 3:TT genotype; 4: blank.
Embodiment
Further illustrate the present invention below in conjunction with specific embodiment.Should be noted, following examples, only for the present invention is described, limit the scope of the invention and be not used in.
(1) collect blood sample and DNA extraction
Collect sporadic primary hepatocyte hepatocarcinoma clinical samples 212 examples from The Second Affiliated Hospital of Nanjing Medical University, all patients all make a definite diagnosis through histopathology, male 150 examples wherein, female's 62 examples.Patient's mean age is 62 years old; Control group 288 examples, the mean age is 61 years old.The age of case group and control group, sex form there was no significant difference (P>0.05).Above result show two groups aspect age, sex equilibrium comparable.All research objects
All signaturesInformed Consent Form.
Use ordinary method to isolate white corpuscle from the above-mentioned blood sample of collecting, and adopt classical
PhenolAfter-chloroform method extracting white corpuscle genomic dna, the sample unification is diluted to 0.1 μ g/ μ l, as DNA profiling, remaining DNA is positioned over-20 ℃ of preservations.
(2) design and synthesize nucleotide primer, primer sequence is as follows,
Upstream primer is F:5 '-ATAGCCTTGCAGGCTGATTG-3 ',
Downstream primer is R:5 '-TTCTTTGGTTCAGCCAGTCC-3 '.
Carry out polymerase chain reaction (PCR)
Carry out the PCR reaction system: total amount is 20 μ l
Reaction conditions is set in the pcr amplification instrument:
What obtain klotho gene extron 4 district 163bp after final amplification comprises the polymorphic DNA sequence dna of rs648202 mononucleotide, i.e. sequence shown in the 2190th to the 2352nd in SEQ ID No.1.
(3) DNA sequence dna amplification obtained carries out the single nucleotide polymorphism somatotype
To the DNA fragmentation product obtained in step (2), utilize restriction endonuclease to carry out the restriction fragment length polymorphism somatotype.
Concrete grammar is: the restriction enzyme HaeIII and the corresponding 10 * enzyme that add 5U in 5 μ l pcr amplification products
Cut reactionDamping fluid 1.0 μ l, add water to 10 μ l, after mixing, under 37 ℃, digests 6 hours.
Getting enzyme cutsProduct 10 μ l add 1.0 μ l
The loading bufferingLiquid, fully mix, the well that adds 3% sepharose, do reference with DNA Marker, the electrophoresis working fluid is 1 * TBE, and sample is moved to positive pole by negative pole, constant voltage 80V approximately will coagulate the chamber plate and be placed on ultraviolet transilluminator silica glass platform and detected after 40 minutes, observe each swimming lane and fluorescence band whether occurs, gel images analyser enzyme analysis is cut the genotype type of product, takes pictures and records enzyme and cut result.As shown in Figure 1, the DNA fragmentation length of CC genotype enzyme after cutting is still 163bp; CT genotype enzyme is cut the rear DNA fragmentation that produces 163bp, 100bp, tri-kinds of length of 63bp; TT genotype enzyme is cut the DNA fragmentation of rear generation 100bp, bis-kinds of length of 63bp.
(4) correlation analysis between Klotho coding region rs648202 mononucleotide polymorphic and primary hepatocyte hepatocarcinoma genetic predisposition
Statistical method: adopt SPSS11.0 software building database.Allelotrope and genotypic frequency to the klotho gene of case and contrast
The distribution state property andDemographic variables, smoking and the variable such as drink adopt χ
2Whether check, exist genotype or allelic distribution bias to judge selected crowd, thereby determine its representativeness to whole crowd.Obtain odds ratio (OR) and 95% credibility interval (CI), all OR with single argument logistic regression analysis
Value is through sexWith the age, correct.Statistical study SAS9.0 software, P<0.05 is for statistics has significant difference, and all checks are two-tailed test.
Statistics is as follows:
1. the genetic equilibrium of Klotho gene rs648202 loci gene type frequency check
Klotho gene rs648202 loci gene type frequency is carried out to the check of Hardy-Weniberg genetic equilibrium, χ
2=2.272, P=0.132, difference not statistically significant between this locus gene frequency observed value and expected value, show that this genotype frequency all meets the Hardy-Weniberg ratio, sample has colony's representativeness.
2. the rs648202 polymorphic site genotype between primary hepatocyte hepatocarcinoma patient group and normal healthy controls group and loci distribution frequency are as shown in table 1 below:
The genotype that table 1Klotho gene rs648202 is polymorphic and gene frequency and with the relation of liver cancer
CC, CT and TT genotype frequency are respectively 57.1%37.7% and 5.2% in case, in contrast, are respectively 68.7%, 27.1% and 4.2%, and genotypic distributional difference has significant statistical significance (P=0.026).GA and AA merging are compared with GG, and both still have significant statistical significance (P=0.007).The Logistic regression analysis shows, with carrying the CC wild-type, compares, and the genotypic individual danger that liver cancer occurs of CT of carrying variation significantly raises, and increased value is 59.6%, and the danger of carrying CT+TT mutator gene type person increases by 60.4%.
These results suggest, klotho gene extron 4 district rs648202 are significant sites to the primary hepatocyte hepatocarcinoma susceptible.When this site is carried T allelotrope the genotype in this site is T/T or C/T, the individuality that its individual danger that liver cancer occurs is C/C apparently higher than genotype, show that the genotypic individuality of C/T or T/T is to the primary hepatocyte hepatocarcinoma susceptible, and the genotypic individuality of C/C is to primary hepatocyte hepatocarcinoma susceptible not comparatively.
Organization Applicant
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<110 > OrganizationName: The Second Affiliated Hospital of Nanjing Medical University
Individual Applicant
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<110 > LastName: Huang
<110 > FirstName: daybreak
<110> MiddleInitial :
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Application Project
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<120 > Title: its construction process of klotho gene mononucleotide polymorphism that a kind of primary hepatocellular carcinoma (hcc) is relevant and
Application
<130> AppFileReference :
<140> CurrentAppNumber :
<141> CurrentFilingDate : ____-__-__
Sequence
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<213> OrganismName :
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gcgcgcagac ctgggcccgt ttctcgcggc ctcctgcccc cgaggccgcg ggcctcttcc 180
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cgctgcgcga gctcggggtc actcactacc gcttctccat ctcgtgggcg cgagtgctcc 480
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tttcctggat tgaccttgaa tttaaccatc ctcaaatatt tattgtggaa aatggctggt 1260
ttgtctcagg gaccaccaag agagatgatg ccaaatatat gtattacctc aaaaagttca 1320
tcatggaaac cttaaaagcc atcaagctgg atggggtgga tgtcatcggg tataccgcat 1380
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actgggccct gattctccct ctgggtaacc agtcccaggt gaaccacacc atcctgcagt 1860
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ggcagcctat ggccccgaac caaggactgc cgcgcctcct ggccaggcag ggcgcctggg 1980
agaaccccta cactgccctg gcctttgcag agtatgcccg actgtgcttt caagagctcg 2040
gccatcacgt caagctttgg ataacgatga atgagccgta tacaaggaat atgacataca 2100
gtgctggcca caaccttctg aaggcccatg ccctggcttg gcatgtgtac aatgaaaagt 2160
ttaggcatgc tcagaatggg aaaatatcca tagccttgca ggctgattgg atagaacctg 2220
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Claims (1)
1. the purposes of a species-specific primer on the diagnostic kit for preparing the primary hepatocellular carcinoma (hcc) susceptibility, it is characterized in that, described Auele Specific Primer, upstream primer is F:5'-ATAGCCTTGCAGGCTGATTG-3', downstream primer is R:5'-TTCTTTGGTTCAGCCAGTCC-3', described diagnostic kit, for detection of the genotype in klotho gene rs648202 site, detects and adopts the PCR-RFLP method, comprises the following steps:
(1) the people's complete genome DNA to be measured that comprises the klotho gene of take is template, carry out with above-mentioned Auele Specific Primer the DNA sequence dna that comprises the rs648202 mononucleotide polymorphic that pcr amplification obtains klotho gene extron 4th district, i.e. sequence shown in the 2190th to the 2352nd in SEQ ID No.1;
(2) pcr amplification product is carried out after enzyme is cut carrying out the rs648202 single nucleotide polymorphism typing by agarose gel electrophoresis through restriction enzyme Hae III.
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| CN102961739A (en) * | 2012-12-12 | 2013-03-13 | 黄曙 | Application of KLOTHO protein |
| CN107312829A (en) * | 2017-04-24 | 2017-11-03 | 首都医科大学附属北京佑安医院 | The mononucleotide polymorphism site related from different recurrence possibility liver cancer and its application |
| CN107312830B (en) * | 2017-04-24 | 2021-04-13 | 首都医科大学附属北京佑安医院 | Single nucleotide polymorphism sites related to liver cancers with different degrees of malignancy and application thereof |
| CN106957921B (en) * | 2017-05-18 | 2020-12-29 | 中国水产科学研究院珠江水产研究所 | SNP locus suitable for klotho gene of micropterus salmoides fed with artificial compound feed and application of SNP locus in breeding |
| CN107557461B (en) * | 2017-10-20 | 2021-02-19 | 武汉赛云博生物科技有限公司 | Detection method of nucleic acid mass spectrum for early screening of liver cancer susceptibility genes |
| CN108333355B (en) * | 2018-02-01 | 2020-05-26 | 黄曙 | Application of KLOTHO protein in preparation of reagent for diagnosing gastrointestinal stromal tumor risk |
| CN112915192B (en) * | 2021-03-05 | 2022-10-25 | 南方医科大学南方医院 | Application of KP-1 in preparation of medicine for treating chronic liver diseases |
| CN113444730A (en) * | 2021-03-17 | 2021-09-28 | 昆明市延安医院 | Screening and constructing method of primary hepatocyte klotho gene transduction stem cells |
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| WO2008135993A1 (en) * | 2007-05-08 | 2008-11-13 | Tel Hashomer Medical Research Infrastructure And Services Ltd. | Klotho protein and related compounds for the treatment and diagnosis of cancer |
| KR101766627B1 (en) * | 2008-03-19 | 2017-08-08 | 차이나 신테틱 러버 코포레이션 | Methods and agents for the diagnosis and treatment of hepatocellular carcinoma |
| CN101608217A (en) * | 2008-06-20 | 2009-12-23 | 上海主健生物工程有限公司 | Liver cancer genetic test kit |
| WO2010064247A1 (en) * | 2008-12-03 | 2010-06-10 | Tel Hashomer Medical Research Infrastructure And Services Ltd. | Methods and kits for determining predisposition to cancer |
| EP2388336A1 (en) * | 2010-05-19 | 2011-11-23 | Signature Diagnostics AG | Method and kits for diagnosing colorectal cancer |
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