CN106011278B - Detection reagent for PIEZO2 gene in human gastrointestinal tumor - Google Patents
Detection reagent for PIEZO2 gene in human gastrointestinal tumor Download PDFInfo
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- CN106011278B CN106011278B CN201610564373.0A CN201610564373A CN106011278B CN 106011278 B CN106011278 B CN 106011278B CN 201610564373 A CN201610564373 A CN 201610564373A CN 106011278 B CN106011278 B CN 106011278B
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- 238000001514 detection method Methods 0.000 title claims abstract description 29
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 17
- 208000002699 Digestive System Neoplasms Diseases 0.000 title claims abstract description 11
- 101100243956 Homo sapiens PIEZO2 gene Proteins 0.000 title description 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 27
- 238000007069 methylation reaction Methods 0.000 claims abstract description 18
- 230000011987 methylation Effects 0.000 claims abstract description 16
- 206010017758 gastric cancer Diseases 0.000 claims abstract description 14
- 208000005718 Stomach Neoplasms Diseases 0.000 claims abstract description 13
- 201000011549 stomach cancer Diseases 0.000 claims abstract description 13
- 239000002773 nucleotide Substances 0.000 claims abstract description 10
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 10
- 206010009944 Colon cancer Diseases 0.000 claims abstract description 8
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 7
- 208000029742 colonic neoplasm Diseases 0.000 claims description 6
- 230000002496 gastric effect Effects 0.000 abstract description 12
- 208000001333 Colorectal Neoplasms Diseases 0.000 abstract description 7
- 206010028980 Neoplasm Diseases 0.000 abstract description 6
- 238000000034 method Methods 0.000 abstract description 6
- 201000009030 Carcinoma Diseases 0.000 abstract description 2
- 230000002349 favourable effect Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 5
- 208000029565 malignant colon neoplasm Diseases 0.000 description 5
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000012188 paraffin wax Substances 0.000 description 4
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 3
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 2
- 230000007067 DNA methylation Effects 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 210000002249 digestive system Anatomy 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- YNDXUCZADRHECN-JNQJZLCISA-N triamcinolone acetonide Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@H]3OC(C)(C)O[C@@]3(C(=O)CO)[C@@]1(C)C[C@@H]2O YNDXUCZADRHECN-JNQJZLCISA-N 0.000 description 2
- 102100024326 Contactin-1 Human genes 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 101710113436 GTPase KRas Proteins 0.000 description 1
- 101000909520 Homo sapiens Contactin-1 Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical class NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001839 endoscopy Methods 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000006607 hypermethylation Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000011880 melting curve analysis Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
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Abstract
The invention discloses a method for treating human gastrointestinal tumorPIEZO2The gene detection reagent is a pair of specific primers, the nucleotide sequence of an upstream primer is GGAGTTAGGC GGGAGTATAG TAC, and the nucleotide sequence of a downstream primer is: TTCTTCAAAA ATAACTAATA CCGAA, respectively; the detection reagent aims at human gastrointestinal tumorsPIEZO2The gene(s) is (are),PIEZO2the relationship between gene and tumor is not clear at present, but we find that in gastrointestinal tumor, the canceration tissue and the tissue beside the canceration tissuePIEZO2Gene detection in fluorescence quantitative methylationPIEZO2The degree of gene methylation is different, and the tissues become cancerousPIEZO2The gene methylation degree is obviously higher than that of tissues beside carcinoma, and the detection reagent has the advantages of flexible and quick detection, simplicity, convenience, strong pertinence, accuracy, reliability, high efficiency, economy and economy, and is favorable for early discovery and timely treatment of colorectal cancer and gastric cancer.
Description
Technical Field
The invention relates to a detection reagent for digestive system tumor, in particular to human gastrointestinal tumorPIEZO2A gene detection reagent.
Background
The digestive system tumors are mainly malignant gastric cancer and colorectal cancer, about half of all digestive system malignant tumor death cases die of gastric cancer, and the incidence rate of colorectal cancer in recent years has a remarkable rising trend. The methods currently used for diagnosing malignant tumors in the digestive system mainly include digestive endoscopy, imaging examination, biochemical and immunological examination, and genetic diagnosis. In immunological examination, the markers of gastric cancer mainly include CA199, CEA, CA242, CA125, AFP, NES, etc., and the markers of colorectal cancer mainly include CA19-9, CEA, P53, K-ras, etc. Gene diagnosis is an emerging detection technology in recent years, and has the advantages of rapidness, specificity and sensitivity, for example, the invention patent with the publication number of CN102787174 discloses a detection reagent for CPG (cancer suppressor gene) apparent mutation related to gastrointestinal tumors, wherein 25 pairs of primers are used for sequencing a promoter region of the CPG gene, and hypermethylation of the promoter region of the gene can cause the expression of the cancer suppressor gene to be silent, so that tumors are generated, and the gastrointestinal tumors are confirmed. However, the invention needs 25 pairs of primers for PCR detection of gastrointestinal tumor DNA, has long detection time and complex detection process, and delays the time for diagnosis and treatment of patients.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a drug for treating human gastrointestinal tumorPIEZO2The gene detection reagent has the advantages of flexible and quick detection, simplicity, convenience and strong pertinence.
The technical scheme adopted by the invention for solving the technical problems is as follows: in human gastrointestinal tumorsPIEZO2The gene detection reagent is a pair of specific primers, and the nucleotide sequences of the specific primers are as follows:
the nucleotide sequence of the upstream primer is as follows: 5'-GGAGTTAGGC GGGAGTATAG TAC-3' the flow of the air in the air conditioner,
the nucleotide sequence of the downstream primer is as follows: 5'-TTCTTCAAAA ATAACTAATA CCGAA-3' are provided.
Compared with the prior art, the invention has the advantages that the invention is applied to human gastrointestinal tumorsPIEZO2The gene detection reagent is a pair of specific primers, the nucleotide sequence of an upstream primer is GGAGTTAGGC GGGAGTATAG TAC, and the nucleotide sequence of a downstream primer is: TTCTTCAAAA ATAACTAATA CCGAA, respectively; the detection reagent aims at human gastrointestinal tumorsPIEZO2The gene(s) is (are),PIEZO2the relationship between gene and tumor is not clear at present, but we find that in gastrointestinal tumor, the canceration tissue and the tissue beside the canceration tissuePIEZO2Gene detection in fluorescence quantitative methylationPIEZO2The degree of gene methylation is different, and the tissues become cancerousPIEZO2The degree of gene methylation is obviously higher than that of paracancer groupThe detection reagent has the advantages of flexible and quick detection, simplicity, convenience, strong pertinence, accuracy, reliability, high efficiency, economy and economy, and is favorable for early discovery and timely treatment of colorectal cancer and gastric cancer.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
In human gastrointestinal tumorsPIEZO2The gene detection reagent is a pair of specific primers, and the nucleotide sequence of an upstream primer is as follows: 5'-GGAGTTAGGC GGGAGTATAG TAC-3', the nucleotide sequence of the downstream primer is: 5'-TTCTTCAAAA ATAACTAATA CCGAA-3' are provided. Is directed toPIEZO2The fluorescent quantitative methylation specific amplification upstream primer and the fluorescent quantitative methylation specific amplification downstream primer designed in the promoter region of the gene are obtained by mass screening, testing and confirmation, and the primers are synthesized by Shanghai Shenggong bioengineering GmbH.
Example 2
The case detection is carried out by using the pair of primers in the example 1, and the specific detection process is as follows:
1. DNA extraction: we collected 5 paraffin samples of stomach cancer and corresponding paraffin samples of paracarcinoma tissues from Ningbo third people hospital, extracted DNA samples using QIAamp DNA FFPE Tissue Kit (available from Qiagen, Hilden, Germany), and we collected 5 paraffin samples of colorectal cancer and corresponding paraffin samples of paracarcinoma tissues from Ningbo third people hospital, extracted the same, and obtained DNA samples requiring a ratio (A260/A280) of absorbance (A) of DNA of about 1.80.
2. DNA sample methylation: using EZ DNA methylation kit (available from Zymo, USA), bisulfite conversion is performed on sample DNA, and after treating the DNA sample with bisulfite, methylated cytosine (C) base is kept unchanged while unmethylated C is converted to uracil (U), and then PCR reaction is performed by fluorescence quantification, sequencingPIEZO2The methylation rate of the gene.
3. And (3) PCR reaction: 1.6ul of DNA sample was added to 10ul of SYBRGreen mixture (available from Roche, USA), 6.8ul of DNA and RNase free water is added, and the above pair is addedPIEZO2Specific amplification primers of the gene are used for carrying out fluorescent quantitative PCR methylation amplification; the fluorescent quantitative methylation procedure used was: firstly, denaturation at 95 ℃ for 10 min; then carrying out 45 cycles of annealing reaction at 95 ℃ for 20s, 59 ℃ for 30s and 72 ℃ for 30 s; after the reaction is finished, the reaction is carried out at 95 ℃ for 1min, at 59 ℃ for 30s and at 95 ℃ for 30s, and then the melting curve analysis is carried out to judge the specificity of the product.
4. After completion, use 2-△CTMethod of analyzing samplesPIEZO2Degree of gene methylation, wherein △ CT = CT CNTN1 -CT ACTB -CTPositive control，ACTBDetection of (housekeeping gene, purchased from shanghai bio-engineering gmbh) and positive control (methylated standard DNA, purchased from Zymo corporation) in the same steps 2 and 3, data were analyzed by SPSS 16.0, and we found that: there were significant differences in DNA methylation levels between colorectal and paraneoplastic tissues, and between gastric carcinoma and paraneoplastic tissues as shown in table 1.
Table 1: comparison table of methylation rates of cancer tissue genes and paracarcinoma tissue genes
| Name (I) | Disorders of the disease | Methylation rate of cancer tissue gene | Gene methylation rate of para-carcinoma tissue |
| Flower of beautiful Fang | Stomach cancer | 0.400535 | 0.063813 |
| Pay to the sky | Stomach cancer | 0.289172 | 0.044811 |
| Xingxing' e | Stomach cancer | 0.368567 | 0.017948 |
| Chapter Anshan | Stomach cancer | 0.332171 | 0.051474 |
| Wangxiao yu | Stomach cancer | 0.211686 | 0.007239 |
| Zhuwangchu | Cancer of colon | 0.316439 | 0.031250 |
| Dongqing law | Cancer of colon | 0.447513 | 0.070316 |
| Shao Weibo | Cancer of colon | 0.233258 | 0.097396 |
| King friend officer | Cancer of colon | 0.316439 | 0.031250 |
| Bingqingsheng wine | Cancer of colon | 0.554785 | 0.044194 |
As can be seen from Table 1, in patients with gastric and colorectal cancer disorders, the treatment is performed byPIEZO2The methylation rate of the canceration tissue is obviously higher than that of the tissue beside the cancer by the detection of the gene detection reagent.
<110> subsidiary hospital of Ningbo university medical college
<120>In human gastrointestinal tumorsPIEZO2Gene detection reagent
<160>2
<170>PatentIn version 3.1
<210>1
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223>In amplifying gastrointestinal tumorsPIEZO2Upstream primer sequence of gene
<400>1
GGAGTTAGGC GGGAGTATAG TAC 23
<210>2
<211>25
<212>DNA
<213>In amplifying gastrointestinal tumorsPIEZO2Downstream primer sequence of gene
<400>2
TTCTTCAAAA ATAACTAATA CCGAA 25
Claims (1)
1. Detecting in tissue samplesPIEZO2The application of the specific primer pair with gene methylation level in the preparation of the human gastrointestinal tumor detection reagent is characterized in that the human gastrointestinal tumor is gastric cancer or colon cancer, and the primer pair is as follows: the nucleotide sequence of the upstream primer is as follows: GGAGTTAGGC GGGAGTATAG TAC, the nucleotide sequence of the downstream primer is: TTCTTCAAAAATAACTAATA CCGAA are provided.
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| CN201610564373.0A CN106011278B (en) | 2016-07-18 | 2016-07-18 | Detection reagent for PIEZO2 gene in human gastrointestinal tumor |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610564373.0A CN106011278B (en) | 2016-07-18 | 2016-07-18 | Detection reagent for PIEZO2 gene in human gastrointestinal tumor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018232735A1 (en) * | 2017-06-23 | 2018-12-27 | Tsinghua University | Use of piezo regulator in preparation of a medicament |
| CN111751532B (en) * | 2020-07-20 | 2022-07-12 | 河南省医药科学研究院 | Application of PIEZO1 protein as esophageal cancer marker |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103249878A (en) * | 2010-08-23 | 2013-08-14 | Irm有限责任公司，特拉华有限责任公司 | Mechanically-activated cation channels |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6017704A (en) * | 1996-06-03 | 2000-01-25 | The Johns Hopkins University School Of Medicine | Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103249878A (en) * | 2010-08-23 | 2013-08-14 | Irm有限责任公司，特拉华有限责任公司 | Mechanically-activated cation channels |
Non-Patent Citations (3)
| Title |
|---|
| "Piezo2 protein: A novel regulator of tumor angiogenesis and hyperpermeability";Hong Yang等;《Oncotarget》;20160617;第7卷(第28期);第44630-44643页 * |
| "PIEZO2蛋白的生物信息学分析及其在肠道中的表达";白涛等;《重庆医学》;20160503;第45卷(第15期);摘要，正文1.2.2小节及2.4-2.5节，图4 * |
| "Promoter hypermethylation of PIEZO2 is a risk factor and potential clinical biomarker for laryngeal squamous cell carcinoma";Lixin Cheng等;《Int J Clin Exp Pathol》;20171215;第10卷(第12期);第11635-11643页 * |
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