CN101041854A - Polymorphism sites of primary liver cancer correlative coding small molecule RNA and usage thereof - Google Patents
Polymorphism sites of primary liver cancer correlative coding small molecule RNA and usage thereof Download PDFInfo
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Abstract
The invention discloses a nonencode small molecule ribonucleic acid hsa-mir-146a correlated with primary liver cancer and polymorphism site, which is characterized by the following: providing a method to analyze mononucleotide polymorphism in has-mir-146a fore-body, check polymorphism and relate to work efficiency of ripe body; possessing usage in aspect of pre-treat primary liver cancer.
Description
Technical field
The present invention relates to a kind of non-coding small molecule RNA hsa-miR-146a relevant and the mononucleotide polymorphism site on the precursor thereof with primary hepatocarcinoma, the invention still further relates to a kind of this pleomorphism site of detection, analyze the method for itself and this microRNA working (machining) efficiency dependency, and on the hsa-miR-146a precursor this polymorphism in the purposes of examining in advance aspect the primary hepatocarcinoma.
Background technology
Malignant tumour has become one of human main causes of death, and wherein primary hepatocarcinoma sickness rate in all tumours is listed as the 6th, and mortality ratio is listed as the 3rd.Liver cancer is in second (Ceng Yixin, oncology (second edition) in the various cancers especially in China's city scope.The People's Health Publisher, Beijing, 2003,5-6).Malignant tumour be a process that multifactor multistep is rapid, wherein the pathogenesis of inherited genetic factors has play a part important.Single nucleotide polymorphism (SNP) is meant in a certain species, the sequence of the single Nucleotide that specific position exists on genome changes, its frequency is higher in human genome, be widely used for as genetic marker (Collins, F.S., et al.A DNA polymorphism discovery resource for research on human genetic variation, GenomeRes, 1998,8 (12), 1229-31).More and more evidences shows, the SNP that exists in albumen coded sequence and the upstream and downstream regulating and controlling sequence thereof in the protein coding gene is when playing the important regulating and controlling effect to expression of gene, function etc., also influenced the generation that comprises diseases such as malignant tumour, development and prognosis.The report of such the existing a lot of at present SNP and the dependency of cancer is auxiliary diagnosis and the treatment that mark carries out disease with the SNP site, will be the important references index that realizes the disease personalized treatment.
Microrna (microRNA) is the noncoding Yeast Nucleic Acid of small molecule that extensively is present in the various living species, and its length is about 20-22 base.It is coded protein or polypeptide not, but by combining with the specificity of the 3 ' UTR (3 ' end non-translational region) of the mRNA (messenger RNA(mRNA)) of target gene, promotes its degraded or suppresses its translation process and realize regulation and control to genetic expression.Many evidences show that microRNA has participated in the regulate several biological processes of cell, unusual morbidity and the deterioration that directly affects malignant tumour that himself is regulated and control.Since microRNA and tumorigenic substantial connection, the SNP site that exists in their genome sequence, the expression or the course of processing of regulating them probably, thus relevant with the morbidity of cancer.
Through domestic and foreign literature retrieval to prior art, so far do not see the research report that pleomorphism site and disease in any microRNA molecular sequences are arranged, do not have pleomorphism site and various diseases in the hsa-miR-146a molecule in the microRNA disclosed in this invention family yet, comprise the relevant report of primary hepatocarcinoma.
Summary of the invention
The object of the invention be to disclose a mononucleotide polymorphism site on the non-coding small molecule RNA hsa-miR-146a precursor and this small molecules working (machining) efficiency, with the dependency of primary hepatocarcinoma susceptible degree, with and in the purposes of examining in advance aspect the primary hepatocarcinoma.
At first, the invention provides the method for single nucleotide polymorphism on a kind of hsa-miR-146a of detection molecular precursor.
Concrete steps are as follows:
(a) single nucleotide polymorphism of determining to exist in the non-coding small molecule RNA hsa-miR-146a precursor (numbering rs2910164), i.e. the 460th Nucleotide (being labeled as N) in the SEQ ID NO:1 sequence, two kinds of polymorphisms are respectively G or C;
(b) adopt the polymerase chain reaction, specific amplification comprises the genomic DNA fragment of hsa-miR-146a precursor sequence;
(c) pleomorphism site in the dna fragmentation that includes the hsa-miR-146a precursor that amplification is obtained with restriction fragment length polymorphism (RFLP) method carries out gene type.
SEQ ID NO：1
1 CCCCTACAGA TTAGTTTTTG TTTTGACAGG GTCTCTCTCT GTGGCCCAGA
51 CTGGAGTGCA GTGGTGCAAT CATAGCTCAC TGCAACCTCC AATTCCCAGG
101 CTCAAGCGAT CCTCCCACCA CAGGCCATCA TGCATGGCTC ATTTTTTATT
151 TTTAGTAGAG ACAAATTCTC CATGTTGCCC AGGCTAGTCC TGAACTCCTG
201 GGCTCAAGAG ATCCACCCAC ATCAGCCTTC CAGACTGCTG GCCTGGTCTC
251 CTCCAGATGT TTATAACTCA TGAGTGCCAG GACTAGACCT GGTACTAGGA
301 AGCAGCTGCA TTGGATTTAC CAGGCTTTTC ACTCTTGTAT TTTACAGGGC
351 TGGGACAGGC CTGGACTGCA AGGAGGGGTC TTTGCACCAT CTCTGAAAAG
401 CCGATGTGTA TCCTCAGCTT TGAGAACTGA ATTCCATGGG TTGTGTCAGT
451 GTCAGACCTN TGAAATTCAG TTCTTCAGCT GGGATATCTC TGTCATCGTG
501 GGCTTGAGGA CCTGGAGAGA GTAGATCCTG AAGAACTTTT TCAGTCTGCT
551 GAAGAGCTTG GAAGACTGGA GACAGAAGGC AGAGTCTCAG GCTCTGAAGG
601 TATAAGGAGT GTGAGTTCCT GTGAGAAACA CTCATTTGAT TGTGAAAAGA
651 CTTGAATTCT ATGCTAAGCA GGGTTCCAAG TAGCTAAATG AATGATCTCA
701 GCAAGTCTCT CTTGCTGCTG CTGCTACTCG TTTACATTTA TTGATTACTT
751 ACGATGATTC AGGTACTGTT GTAAGTGCTT TACATGCTGT TATACGAGAC
801 TCTTGGGAGA AATCACTTTA ATGAAGCTTG AGACACATGG CATTGCCATG
851 CAATGATTTT TCCCCCCTCT TCACGGGATC AGAGGGAACT AATAGAATG
The present invention provides specific amplification hsa-miR-146a molecular dna segmental nucleotide primer simultaneously, and length is 20-22 base.
Amplimer is as follows:
Forward primer: 5 '-GGAGGGGTCTTTGCACCATC-3 ' (SEQ ID NO:2)
Reverse primer: 5 '-TGCCTTCTGTCTCCAGTCTTCC-3 ' (SEQ ID NO:3)
Secondly, the invention provides the described pleomorphism site of a kind of detection to the technological method of hsa-miR-146a precursor (the 401-499 sequence of sequence shown in the SEQ IDNO:1) to the influence of ripe body (the 422-441 sequence of sequence shown in the SEQ ID NO:1) working (machining) efficiency, concrete steps are as follows:
(a) make up microRNA expression vector respectively, and distinguish transfection pattern cell with it with the two kinds of pleomorphism sites (genotype is respectively G or C) that as above identify;
(b) the pattern cell behind the collection transfection expression carrier utilizes the ribonucleotide in the RNA extractive technique acquisition cell;
(c) adopt Northern blot technology, detect the precursor of hsa-miR-146a in the ribonucleotide that obtains and the expression level of ripe body molecule.
The present invention provides specificity to clone that hsa-miR-146a expresses segmental nucleotide primer in the described expression vector simultaneously, and specificity is in conjunction with the oligonucleotide probe of hsa-miR-146a precursor and ripe body.Clone's primer is as follows:
Forward primer: 5 '-ATGCTCGAGCCTGGTCTCCTCCAGATGTT-3 ' (SEQ ID NO:4)
Reverse primer: 5 '-ATGTCTAGATTCTCACAGGAACTCACACTCC-3 ' (SEQ IDNO:5)
Probe: 5 '-AACCCATGGAATTCAGTTCTCA-3 ' (SEQ ID NO:6)
At last, the invention provides a kind of pre-diagnosis reagent box that detects primary hepatocarcinoma.Comprise:
(1) it is right that specific amplification includes the primer of genomic fragment of hsa-miR-146a precursor sequence;
(2) detect the required restriction enzyme of polymorphic site rs2910164 genotype in the amplified production.
The invention has the beneficial effects as follows: the test kit of mononucleotide polymorphism site sequence described in the analysis hsa-miR-146a precursor sequence provided by the invention, can be applicable to auxiliary diagnosis to primary hepatocarcinoma patient, individuality is passed judgment on the susceptibility of primary hepatocarcinoma, thereby helped prevention, the early diagnosis and therapy of primary hepatocarcinoma; Can be used as the potential target spot of medicine, screening has regulates the molecule of pharmaceutical that hsa-miR-146a expresses, and promotes the discovery of new medicines resistant to liver cancer.
Description of drawings
Fig. 1: pleomorphism site is to the influence of hsa-miR-146a precursor to ripe body working (machining) efficiency
Embodiment
Embodiment 1: the collection of blood sample and the extraction of genomic dna
Collect 671 parts of sporadic Patients with Primary samples from Guangdong Province Tumor Hospital Attached to Zhongshan Univ., 46.8 ± 12.2 years old mean age (standard deviation), wherein 65 examples are the women.Healthy population from Guangdong Province's consanguinity-less relation contrasts 655 examples, 45.8 ± 13.7 years old mean age (standard deviation), wherein women's sample 64 examples equally.
After from the above-mentioned blood preparation of collecting, extracting genomic dna with conventional phenol chloroform method, the unified dilution of sample is 10ng/ul.
Embodiment 2: the somatotype of mononucleotide polymorphism site
The present invention adopts the single nucleotide polymorphism technology (PCR-SSCP) based on the polymerase chain reaction, and the polymorphic site rs2910164 (its allelotrope point is to being G/C) that exists in the hsa-miR-146a molecular precursor has been carried out gene type in case group and control group.
PCR is reflected on the MJ PTC-200 instrument and carries out.
Amplimer is as follows:
Forward primer: 5 '-GGAGGGGTCTTTGCACCATC-3 ' (SEQ ID NO:2)
Reverse primer: 5 '-TGCCTTCTGTCTCCAGTCTTCC-3 ' (SEQ ID NO:3)
PCR reaction reagent and program:
The amplified reaction that it is 20ul that each sample carries out a cumulative volume comprises the genomic dna diluent 20ng of preparation, 10 * Taq Buffer 2ul, 5mM dNTP 0.8ul, 25mM MgCl in the system
21.6ul the Taq archaeal dna polymerase of 0.25 unit and each 0.5ul of above-mentioned primer (being diluted to 10uM) add ddH at last
2O joins cumulative volume and is 20ul.Be reflected at 94 ℃ of pre-sex change after 2 minutes, carry out 35 94 ℃ of sex change 30 seconds, annealed 30 seconds for 55 ℃, 72 ℃ of amplification cycles of extending 30 seconds are carried out 72 ℃ of benefits of 2 minutes at last and are put down.
The length that amplification is obtained with restriction fragment length polymorphism (RFLP) method is that the pleomorphism site of the dna fragmentation that includes the hsa-miR-146a precursor of 210bp (being arranged in the 372-581 sequence of sequence shown in the SEQ ID NO:1) carries out gene type.Concrete grammar is: get the above-mentioned amplification PCR reaction product of 3ul, add the enzyme cutting buffering liquid of 1ul, the restriction enzyme MamI of 1 unit adds ddH
2O to cumulative volume be 10ul, and spend the night 37 ℃ of digestion.After this, electrophoresis on 12% denaturing polyacrylamide gel is with carrying out gene type assay after the argentation development: after enzyme is cut processing, if the rs2910164 loci gene type is when isozygotying G, 82nt to occur, the band of 77nt and 51nt in the test sample; If genotype is when isozygotying C, 82nt to occur, 51nt, the band of 45nt and 32nt; If when genotype is heterozygosis GC, 82nt then occurs, 77nt, 51nt, the band of 45nt and 32nt.
Embodiment 3: detect this pleomorphism site to the influence of hsa-miR-146a precursor to ripe body working (machining) efficiency
The present invention is based on cell model and detect the external hsa-miR-16a of the carrying out molecule working (machining) efficiency detection of employing, the technology path is as follows:
(1) based on the pCDNA3.0 carrier framework of business-like invitrogen company, sample with two kinds of homozygous genotypes is a template respectively, the genomic dna sequence that wherein contains the hsa-miR-146a precursor is cloned in this skeleton, is configured to the G of this microRNA or two expression vectors of C multiformity;
Clone's primer is as follows:
Forward primer:
5’-ATGCTCGAGCCTGGTCTCCTCCAGATGTT-3’(SEQ ID NO：4)
Reverse primer:
5’-ATGTCTAGATTCTCACAGGAACTCACACTCC-3’(SEQ ID NO：5)
(2) with the microRNA expression vector of two kinds of multiformities making up in the step 1 and empty carrier pCDNA3.0 with conventional calcium phosphate transfection method, import separately the 293T cell (obtaining) from American Type Culture Collecti, continued culturing cell 24 hours after the transfection;
(3) the 293T cell after the transfection and total RNA of non-transfected cells in the collection step 2, behind electrophoresis on 15% the denaturing polyacrylamide, change film and isotope probe hybridization according to the method for the Northern blot of routine, developing by the phosphorus screen detects the expression level of hsa-miR-146a molecular precursor and ripe body in the different RNA sample.Used specific hybridization probe is: 5 '-AACCCATGGA ATTCAGTTCTCA-3 ' (SEQ ID NO:6);
(4) the expression level ratio of ripe body and precursor in the cell sample after the transfection of two kinds of multiformity expression vectors of comparison, promptly the hsa-miR-146a molecular precursor is to the working (machining) efficiency of ripe body.
The result as shown in Figure 1, the precursor of this molecule was significantly improved during for C with respect to loci to the working (machining) efficiency of ripe body when loci was G.
The correlation analysis of mononucleotide polymorphic site and primary hepatocarcinoma in the embodiment 4:hsa-miR-146a molecular precursor
Statistical method: utilizing GDA (http://hydrodictyon.eeb.uconn.edu/people/plewis/software.php) to simulate number of times is 10000 times chi-square analysis, calculates the Hardy-Weinberg balance of healthy population control group sample.Utilization SPSS specialty statistics software, select for use chi square test that the genotype distribution frequency of polymorphic site in patient's group and the control group sample is carried out statistical analysis, fiducial interval is set position 95% (being 95%CI), statistical significant difference level set is p＜0.05, calculates Odds Ratio (OR) value simultaneously.
Statistics:
The rs2910164 polymorphic site genotype and the loci distribution frequency of primary hepatocarcinoma patient group and normal healthy controls group sample are as shown in the table:
| Grouping | Total number of samples | Genotype number (frequency %) | Loci number (frequency %) | |||
| CC | GC | GG | C | G | ||
| Patient | 671 | 226(33.7) | 339(50.5) | 106(15.8) | 791(58.9) | 551(41.1) |
| Contrast | 665 | 259(38.9) | 327(49.2) | 79(11.9) | 845(63.5) | 485(36.5) |
| P-value | 0.041 | 0.015 | ||||
| OR(95％CI) | 1.21(1.04＜OR＜1.42) | |||||
According to last table distribution frequency, the genotype of utilizing the computing of GDA software to draw the control group sample distributes and meets the Hardy-Weinberg balance.
From last table as seen, be positioned at the G loci of the mononucleotide polymorphism site rs2910164 on the hsa-miR-146a precursor, distribution frequency significance (P=0.015) in Patients with Primary colony is a significant site of primary hepatocarcinoma susceptible greater than its distribution frequency in normal healthy controls cohort body.The pleomorphism site that this is relevant with the primary hepatocarcinoma susceptibility, be positioned at the stem position on the loop-stem structure of hsa-miR-146a precursor molecule, this is polymorphic to have changed the complementary pairing situation of base on the stem and (if this site is the G multiformity, then can form the distinctive G:U pairing of RNA with the U base on another arm; If this site is the C multiformity, then should pairing destroyed), thus the complementary integrity of stem changed.This influences the secondary structure of RNA probably, thereby influence the working (machining) efficiency of its precursor to ripe body, shown in embodiment 3, the difference in this equipotential site has been proved the generation that can influence its ripe body, and this has also supported above-mentioned statistical conclusion from a functional angle.
Embodiment 5: detection kit
Preparation detects the test kit of primary hepatocarcinoma susceptibility, and it is right wherein to contain following oligonucleotide primer, is used for including from the genome sample specific amplification of detected object the genomic fragment of hsa-miR-146a precursor sequence.
Forward primer: 5 '-GGAGGGGTCTTTGCACCATC-3 ' (SEQ ID NO:2)
Reverse primer: 5 '-TGCCTTCTGTCTCCAGTCTTCC-3 ' (SEQ ID NO:3)
This test kit also comprises restriction enzyme MamI, is used for enzyme and cuts the PCR product that above-mentioned primer obtains amplification.Enzyme is cut product carry out developing behind 15% denaturing polyacrylamide gel electrophoresis, can detect the G-C allelotype of rs2910164 pleomorphism site in the sample easily.
The present invention has the illustration of practicality:
(1) method that the present invention set up can be used for the G-C allelotype of the mononucleotide polymorphism site rs2910164 on analyst's the hsa-miR-146a molecular precursor sequence, be applied to the auxiliary diagnosis of primary hepatocarcinoma and whether individuality is had the primary hepatocarcinoma susceptibility judge, thereby help the prevention and the treatment of primary hepatocarcinoma;
(2) can utilize the method for this micromolecular non-encoding ribonucleic acid working (machining) efficiency of research that the present invention introduces, the active drug that screening is a target with above-mentioned rs2910164 polymorphic site, searching has the bioactive molecule of regulating the processing of hsa-miR-146a molecule, expressing, and promotes the discovery of new medicines resistant to liver cancer;
(3) utilize the small molecules non-encoding ribonucleic acid of primary hepatocarcinoma dependency provided by the invention and the sequence and the methods of genotyping of pleomorphism site thereof, make up the detection kit of primary hepatocarcinoma being carried out the molecular genetics diagnosis;
(4) because the hsa-miR-146a molecule has also participated in other and the relevant pathologic processes of cellular activity imbalance such as propagation, apoptosis probably, comprise the tumour of other types etc.Therefore the present invention provides experience and application foundation to the relation of furtheing investigate hsa-miR-146a molecule and other diseases from now on;
(5) in the precursor sequence of other similar small molecules non-encoding ribonucleic acids, also has similar mononucleotide polymorphism site, these sites may have regulating effect to corresponding micromolecular processing and expression equally, thereby influence its biological function even influenced the generation of various diseases equally.The pleomorphism site that exists on this micromolecular non-encoding ribonucleic acid and the report of disease-related are not arranged at present both at home and abroad yet, and therefore, the present invention has also announced the most valuable clue, experience and utilisation technology to the research in this field from now on.
Sequence table
＜110〉Zhongshan University
＜120〉a kind of pleomorphism site of primary liver cancer correlative coding small molecule RNA and application thereof
<160>6
<210>1
<211>899
<212>DNA
＜213〉human (human)
<220>
<221>misc_feature
<222>(460)
＜223〉n=g or c
<400>1
cccctacaga ttagtttttg ttttgacagg gtctctctct gtggcccaga ctggagtgca 60
gtggtgcaat catagctcac tgcaacctcc aattcccagg ctcaagcgat cctcccacca 120
caggccatca tgcatggctc attttttatt tttagtagag acaaattctc catgttgccc 180
aggctagtcc tgaactcctg ggctcaagag atccacccac atcagccttc cagactgctg 240
gcctggtctc ctccagatgt ttataactca tgagtgccag gactagacct ggtactagga 300
agcagctgca ttggatttac caggcttttc actcttgtat tttacagggc tgggacaggc 360
ctggactgca aggaggggtc tttgcaccat ctctgaaaag ccgatgtgta tcctcagctt 420
tgagaactga attccatggg ttgtgtcagt gtcagacctn tgaaattcag ttcttcagct 480
gggatatctc tgtcatcgtg ggcttgagga cctggagaga gtagatcctg aagaactttt 540
tcagtctgct gaagagcttg gaagactgga gacagaaggc agagtctcag gctctgaagg 600
tataaggagt gtgagttcct gtgagaaaca ctcatttgat tgtgaaaaga cttgaattct 660
atgctaagca gggttccaag tagctaaatg aatgatctca gcaagtctct cttgctgctg 720
ctgctactcg tttacattta ttgattactt acgatgattc aggtactgtt gtaagtgctt 780
tacatgctgt tatacgagac tcttgggaga aatcacttta atgaagcttg agacacatgg 840
cattgccatg caatgatttt tcccccctct tcacgggatc agagggaact aatagaatg 899
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>2
ggaggggtct ttgcaccatc 20
<210>3
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>3
tgccttctgt ctccagtctt cc 22
<210>4
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>4
atgctcgagc ctggtctcct ccagatgtt 29
<210>5
<211>31
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>5
atgtctagat tctcacagga actcacactc c 31
<210>6
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial sequence description: primer
<400>6
aacccatgga attcagttct ca 22
Claims (9)
1, a kind of method that detects the single nucleotide polymorphism of hsa-mir-146a molecule, it is characterized in that: it may further comprise the steps:
(1) determines the mononucleotide polymorphic site rs2910164 that on the has-mir-146a molecular precursor, exists, i.e. the Nucleotide of the 460th of SEQ ID NO:1;
(2) adopt the polymerase chain reaction, specific amplification comprises the genomic DNA fragment of has-mir-146a precursor sequence;
(3) pleomorphism site in the dna fragmentation that includes the has-mir-146a precursor that amplification is obtained with restriction fragment length polymorphism (RFLP) method carries out gene type.
2, the method for the single nucleotide polymorphism of detection hsa-mir-146a molecule according to claim 1, it is characterized in that: the sequence of the primer that adopt step (2) polymerase chain reaction is respectively SEQ ID NO:2, sequence shown in the SEQ ID NO:3.
3, the method for the single nucleotide polymorphism of detection hsa-mir-146a molecule according to claim 1, it is characterized in that: the concrete grammar of step (3) is: with restriction enzyme MamI the PCR product of step step (2) is carried out enzyme and cut, the length of the dna fragmentation after cutting according to enzyme is then determined the polymorphic site genotype.
4, a kind of single nucleotide polymorphism that detects the hsa-mir-146a molecule may further comprise the steps the method for its precursor to ripe body working (machining) efficiency influence:
(1) structure has the microRNA expression vector that two kinds of pleomorphism site genotype of mirror are respectively G or C respectively, and distinguishes transfection pattern cell with it;
(2) the pattern cell behind the collection transfection expression carrier utilizes the ribonucleotide in the RNA extractive technique acquisition cell;
(3) adopt Northern blot technology, detect the precursor of has-mir-146a in the ribonucleotide that obtains and the expression level of ripe body molecule.
5, the single nucleotide polymorphism of detection hsa-mir-146a molecule according to claim 4 is to the method for its precursor to ripe body working (machining) efficiency influence, it is characterized in that: step (1) is with SEQ ID NO:4, sequence shown in the SEQID NO:5 is that primer is right, two kinds of genotypic gene DNA fragments that mononucleotide polymorphic site rs2910164 are respectively G or C and comprise the has-mir-146a precursor sequence are cloned into respectively on the pCDNA3.0 carrier, are configured to the G of this microRNA or two expression vectors of C multiformity.
6, the single nucleotide polymorphism of detection hsa-mir-146a molecule according to claim 4 is to the method for its precursor to ripe body working (machining) efficiency influence, it is characterized in that: step (3) is specially: utilize the method for conventional Northern Blot to change film and isotope probe hybridization, hybridization probe is a sequence shown in the SEQ ID NO:6, and developing by phosphorus screen then detects the expression level of hsa-mir-146a molecular precursor and ripe body in the different RNA sample.
7, a kind of diagnostic kit of primary hepatocarcinoma is characterized in that, it comprises:
(1) it is right that specific amplification includes the primer of genomic fragment of hsa-mir-146a precursor sequence;
(2) detect the required restriction enzyme of polymorphic site rs2910164 genotype in the amplified production.
By the diagnostic kit of the described primary hepatocarcinoma of claim 7, it is characterized in that 8, specific amplification includes the primer of genomic fragment of hsa-mir-146a precursor sequence to having SEQ ID NO:2, sequence shown in the SEQID NO:3.
9, by the diagnostic kit of the described primary hepatocarcinoma of claim 7, it is characterized in that described restriction enzyme is restriction enzyme MamI.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333524B (en) * | 2008-06-25 | 2010-06-02 | 中山大学 | Small molecule noncoding RNA gene hsa-mir-101 and antineoplastic use thereof |
| CN102985558A (en) * | 2008-11-13 | 2013-03-20 | 复旦大学 | Compositions and methods for micro-RNA expession profiling of hepatocellular cancer |
| CN104372004A (en) * | 2014-12-04 | 2015-02-25 | 广东医学院 | Detection method and application of single-nucleotide polymorphic sites of miR-27a gene associated with myocardial infarction susceptibility |
| CN104450703A (en) * | 2014-04-11 | 2015-03-25 | 中国人民解放军军事医学科学院基础医学研究所 | Kit and method for detecting serum of liver cancer patient by taking miR-146a as marker |
| CN111826443A (en) * | 2020-07-03 | 2020-10-27 | 清华大学深圳国际研究生院 | Application of serum exosome micro RNAs and liver cancer detection kit |
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2006
- 2006-09-15 CN CNB2006101221702A patent/CN100547081C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333524B (en) * | 2008-06-25 | 2010-06-02 | 中山大学 | Small molecule noncoding RNA gene hsa-mir-101 and antineoplastic use thereof |
| CN102985558A (en) * | 2008-11-13 | 2013-03-20 | 复旦大学 | Compositions and methods for micro-RNA expession profiling of hepatocellular cancer |
| CN102985558B (en) * | 2008-11-13 | 2015-08-19 | 复旦大学 | For composition and the method for the micro-RNA expression spectrum analysis of hepatocellular carcinoma |
| CN104450703A (en) * | 2014-04-11 | 2015-03-25 | 中国人民解放军军事医学科学院基础医学研究所 | Kit and method for detecting serum of liver cancer patient by taking miR-146a as marker |
| CN104372004A (en) * | 2014-12-04 | 2015-02-25 | 广东医学院 | Detection method and application of single-nucleotide polymorphic sites of miR-27a gene associated with myocardial infarction susceptibility |
| CN111826443A (en) * | 2020-07-03 | 2020-10-27 | 清华大学深圳国际研究生院 | Application of serum exosome micro RNAs and liver cancer detection kit |
| CN111826443B (en) * | 2020-07-03 | 2022-06-21 | 清华大学深圳国际研究生院 | Application of serum exosome micro RNAs and liver cancer detection kit |
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| CN100547081C (en) | 2009-10-07 |
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