CN105368945A - Methylation marker for early detection of cancer and detection method thereof - Google Patents
Methylation marker for early detection of cancer and detection method thereof Download PDFInfo
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Abstract
The invention relates to a methylation marker for early detection of cancer and a detection method thereof. The methylation marker comprises PCDHGB6, HOXA9, MGMT and microRNA-126 genes. The methylation marker has the advantages of quickness, high throughput, high sensitivity and high specificity when being adopted for combined detection, and the detection method is suitable for early detection of cancer, can be used for late detection of lung cancer and has a good application prospect.
Description
Technical field
The invention belongs to lung cancer detection field, particularly a kind of detection of early lung cancer mark and detection method thereof of methylating.
Background technology
Lung cancer, also known as primary bronchogenic carcinoma, is that its mortality ratio occupies first of all kinds of malignant tumour to one of maximum malignant tumour of human health risk.The World Health Organization predicts, will have more than 1,000,000 new cases every year to China in 2025, thus greatly compromises the life security of China people and healthy.Research is thought, 5 years survival rates of total 5 years survival rates of lung cancer only 10%-16%, I phase patient can reach 65-75%, and the total 5 years survival rates of II ~ IV phase patient then drop to 5% from 40%.Due to the pathogenic factor of lung cancer and pathogenesis very complicated, and clinical manifestation has individual difference, be developed to late period when most of patients is gone to a doctor, therefore early discovery, early diagnosis, early treatment have great significance to extending lung cancer malignant tumor patient lifetime and reducing mortality ratio.
The generation of lung cancer and development are very complicated multifactor, processes that multistage regulation and control are abnormal, are the coefficient results of genetics and epigenetics.Genetics regulation and control refer to the change of nucleotide sequence, comprise the sudden change of base, disappearance, insertion, restructuring etc.; And epigenetic regulation affects Gene Transcription in vitro mainly through the mode such as DNA methylation and histone modification.DNA methylation refers under the effect of dnmt rna, the methyl (-CH3) of S-adenosylmethionine is covalently bound on 5 carbon atoms of cytosine(Cyt) (C) base of DNA molecular, form 5-methylcytosine (5mC), but do not change the sequence of DNA.Research in recent years shows, generation development and the CPG island, cancer suppressor gene promoter region of the lung cancer expression of tumor suppressor gene inactivation caused that methylates is relevant, and it may occur in the early stage of lung cancer, is the molecular indexes of a very potential early diagnosis lung cancer.Therefore, the methylation state of the multiple gene promoter of joint-detection, significant to the aspect such as diagnosis, treatment, Index for diagnosis of lung cancer.
At present, methylated detection method mainly contains methylation status of PTEN promoter, bisulfite sequencing and methyl fluorescent method.But these methods exist the shortcomings such as complex operation, false positive rate is high, cost is higher, limit its widespread use in clinical labororatory.High resolving power solubility curve technology [WojdaczTK, the DobrovicA.Methylation-sensitivehighresolutionmelting (MS-HRM): anewapproachforsensitiveandhigh-throughputassessmentofme thylation.NucleicAcidsRes.2007 of development in recent years; 35 (6): e41], [MigheliF, StoccoroA, CoppedeF, etal.ComparisonstudyofMS-HRMandpyrosequencingtechniquesf orquantificationofAPCandCDKN2Agenemethylation.PLoSOne.20 13; 8 (1): e52501] be pcr amplification and solubility curve analysis are combined, the minor sequence difference due to object fragment causes the change of PCR solvent temperature, thus can detect transgenation, gene type, gene methylation etc.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of detection of early lung cancer mark and detection method thereof of methylating, the feature such as joint-detection has fast, high-throughput, sensitive and specificity are good that adopts this mark to carry out, detection method is not only applicable to the early stage detection of lung cancer, also may be used for the detection of advanced lung cancer, have a good application prospect.
A kind of detection of early lung cancer mark that methylates of the present invention, described in the mark that methylates comprise PCDHGB6, HOXA9, MGMT and microRNA-126.
Described PCDHGB6 upstream primer sequence is as shown in SEQIDNO.1, and downstream primer sequence is as shown in SEQIDNO.2;
Described HOXA9 upstream primer sequence is as shown in SEQIDNO.3, and downstream primer sequence is as shown in SEQIDNO.4;
Described MGMT upstream primer sequence is as shown in SEQIDNO.5, and downstream primer sequence is as shown in SEQIDNO.6;
Described microRNA-126 upstream primer sequence is as shown in SEQIDNO.7, and downstream primer sequence is as shown in SEQIDNO.8.
SEQ1：5-AATTTGAGGGGGATGTATATTT-3(SEQIDNO.1)；
SEQ2：5-AAAATCCCAAACCAAAAACT-3(SEQIDNO.2)；
SEQ3：5-GAGTTGTGGTTGTTTTTTTTTG-3(SEQIDNO.3)；
SEQ4：5-ACCTTTCAAAACTCCTTCCTC-3(SEQIDNO.4)；
SEQ5：5-GCGTTTCGGATATGTTGGGATAGT-3(SEQIDNO.5)；
SEQ6：5-AACGACCCAAACACTCACCAA-3(SEQIDNO.6)；
SEQ7：5-TGGGTTGGTTTTTGTTAGG-3(SEQIDNO.7)；
SEQ8：5-TAACCCTCACCTACTCCACAA-3(SEQIDNO.8)。
The described mark composition test kit that methylates, wherein test kit also comprises: 1 × master and 25mMMgCl
2.
Described test kit also comprises: positive control and negative control.
A kind of detection of early lung cancer detection method of the mark that methylates of the present invention, comprises the steps:
(1) from tissue to be checked, extract DNA, and respectively moditied processing carried out to sample DNA, standard substance DNA, the DNA (at least 20ng) after conversion with after water dissolution, as the template of PCR;
(2) pair of primers, Master, MgCl of gene is added according to PCR system ratio
2, make often pair of primer, MgCl
2final concentration be respectively 1uM, 4mM; Carry out pcr amplification subsequently, the melting temperature difference according to the standard substance DNA of the different ratio that methylates does typical curve, then according to the PCR result of sample, obtains the relative methylation level of sample with typical curve.
Pcr amplification condition in described step (2) is: 95 degree of sex change, 10min; 45cycles, 95 DEG C of 10s, the 10s from 62 DEG C to 51 DEG C, 72 DEG C of 20s; 95 DEG C of 1min, 40 DEG C of 1min, 65 DEG C are warming up to 94 DEG C.
The technology that the present invention adopts: the primer designing a pair specific DNA methylation assay in the promoter region of gene, then removes amplification sulfuration sample DNA with this primer, determines the methylation level of sample to be tested according to the relative fluorescence of pcr amplification result.
beneficial effect
Compared with the method for lung cancer diagnosis of routine, the feature such as joint-detection has fast, high-throughput, sensitive and specificity are good that adopts mark of the present invention to carry out, detection method is not only applicable to the early stage detection of lung cancer, also may be used for the detection of advanced lung cancer, the method can make lung cancer patient early discovery, early treatment, prolongation lifetime, thus improve the quality of life of tumour patient, have a good application prospect.
Accompanying drawing explanation
Fig. 1 is the typical curve that four genes are drawn with the PCR result that standard substance DNA is template; Wherein, A is HOXA9, B be PCDHGB6, C be MGMT, D is miR-126;
Fig. 2 is four genes (PCDHGB6, HOXA9, MGMT and microRNA-126) in the DNA methylation assay distribution of results of cancerous lung tissue and far-end healthy tissues.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
One group for the specific primer design of pulmonary cancer diagnosis with the mark that methylates:
According to the disclosed mankind's whole genome sequence of NCBI (US National Biotechnology Information center), use PrimerPremier3.0 and MethylPrimerExpressv1.0 design, synthesized by Shanghai Sheng Gong company limited.Upstream primer and the downstream primer of described PCDHGB6 gene are respectively shown in SEQIDNO.1 and SEQIDNO.2; Upstream primer and the downstream primer of described HOXA9 gene are respectively shown in SEQIDNO.3 and SEQIDNO.4; The upstream primer of described mgmt gene and downstream primer are respectively shown in SEQIDNO.5 and SEQIDNO.6; Upstream primer and the downstream primer of described MicroRNA-126 gene are respectively shown in SEQIDNO.7 and SEQIDNO.8.
Embodiment 2
Test kit of the present invention is in the application of the methylation level of detection people lung cancer:
The Master that the present invention adopts is purchased from Roche company limited; DNA extraction kit is, QIAmpDNAMiniKit (QIAGEN); Conversion reagent box is, EZDNAMethylationkit (ZymoResearch, Orange, CA, USA); Other reagent are domestic analytical reagent.
Biomaterial of the present invention is all from Shanghai City Zhong Shan hospital.
Method:
One, biological specimen:
The tumor tissues of 54 routine patients with lung cancer and pairing healthy tissues.
Two, the extraction of tissue DNA and conversion:
The operating scissors got after about 25mg cancerous lung tissue or healthy tissues autoclaving sterilization shred and join in the centrifuge tube of 1.5ml, use DNA extraction kit to extract the DNA of sample to be tested, then transform extraction DNA with conversion reagent box.
Three, PCR flow process
The 1 PCR instrument device used for Roche LightCycler480, Master and magnesium ion be also the reagent of Roche, reaction system is 20ul;
The preparation of 2PCR reaction system and condition are as shown in following table 1 and table 2:
The preparation of table 1, PCR reaction system
| Component | Volume |
| Master | 10ul |
| 25mM MgCl 2 | 3.2ul |
| Aqua sterilisa | 5.8ul |
| Template | 1ul |
Table 2, PCR response procedures:
Four, interpretation of result
1, according to the Comparative dissolution curve difference after the standard substance DNA cloning that methylates of different ratios, draws the typical curve (see accompanying drawing 1) of each gene.
2 calculate methylated ratio in unknown sample according to the typical curve set up, and data analysis adopts Fisher, and exact method analysis, P<0.05 is that difference has statistical significance.
The distribution situation that methylates (see accompanying drawing 2) of 4 genes of 3 cancerous lung tissues and far-end healthy tissues.
Embodiment 3
Performance curve analyzes (ROC tracing analysis):
Build ROC curve and compare 4 methylate the marks differentiation tumor tissues of lung cancer patient and the diagnosis capabilitys of healthy tissues.The ROC area under curve of 4 marks is as follows respectively: PCDHGB6,0.796; HOXA9,0.694; MGMT, 0.594; Mir-126,0.658.Under the cutoff value of the best, the Sensitivity and Specificity of gene is as follows: PCDHGB6, and 66.7% and 90.7%; HOXA9,42.6% and 96.3%; MGMT, 25.9% and 94.4%; Mir-126,38.9% and 90.7%.The AUC that these 4 marks join together to detect reaches 0.891, and Sensitivity and Specificity is respectively 85.2% and 81.5% (see table 3).These results show, 4 markers in detecting lung cancer that methylate have higher sensitivity and specificity.
Table 3 methylates the ROC tracing analysis of mark
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive.In addition should be understood that those skilled in the art can do various change, amendment to the present invention after having read above-mentioned teachings of the present invention, even equivalence is changed, but all will fall within the scope of protection of the present invention.
Claims (6)
1. the detection of early lung cancer mark that methylates, is characterized in that: described in the mark that methylates comprise PCDHGB6, HOXA9, MGMT and microRNA-126.
2. a kind of detection of early lung cancer mark that methylates according to claim 1, is characterized in that: described PCDHGB6 upstream primer sequence is as shown in SEQIDNO.1, and downstream primer sequence is as shown in SEQIDNO.2;
Described HOXA9 upstream primer sequence is as shown in SEQIDNO.3, and downstream primer sequence is as shown in SEQIDNO.4;
Described MGMT upstream primer sequence is as shown in SEQIDNO.5, and downstream primer sequence is as shown in SEQIDNO.6;
Described microRNA-126 upstream primer sequence is as shown in SEQIDNO.7, and downstream primer sequence is as shown in SEQIDNO.8.
3. a kind of detection of early lung cancer mark that methylates according to claim 1, is characterized in that: described in methylate mark composition test kit, wherein test kit also comprises: 1 × master and 25mMMgCl
2.
4. a kind of detection of early lung cancer mark that methylates according to claim 3, is characterized in that: described test kit also comprises: positive control and negative control.
5. a detection of early lung cancer as claimed in claim 1 detection method for the mark that methylates, comprises the steps:
(1) from tissue to be checked, extract DNA, and respectively moditied processing is carried out to sample DNA, standard substance DNA, after the DNA water dissolution after conversion, as the template of PCR;
(2) pair of primers, Master, MgCl of gene is added according to PCR system ratio
2, make often pair of primer, MgCl
2final concentration be respectively 1uM, 4mM; Carry out pcr amplification subsequently, the melting temperature difference according to the standard substance DNA of the different ratio that methylates does typical curve, then according to the PCR result of sample, obtains the relative methylation level of sample with typical curve.
6. a kind of detection of early lung cancer detection method of the mark that methylates according to claim 5, is characterized in that: the pcr amplification condition in described step (2) is: 95 degree of sex change, 10min; 45cycles, 95 DEG C of 10s, the 10s from 62 DEG C to 51 DEG C, 72 DEG C of 20s; 95 DEG C of 1min, 40 DEG C of 1min, 65 DEG C are warming up to 94 DEG C.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107475369A (en) * | 2017-07-07 | 2017-12-15 | 南方医科大学 | HOXA9 genes are preparing the application in treating cutaneous squamous cell carcinoma medicine |
| CN108342477A (en) * | 2017-01-24 | 2018-07-31 | 北京艾克伦医疗科技有限公司 | Detection kit based on multiple gene diagnosis patients with lung cancer |
| CN110567906A (en) * | 2019-09-12 | 2019-12-13 | 深圳大学 | Method for representing RNA methylation modification and application |
| WO2019242754A1 (en) * | 2018-06-22 | 2019-12-26 | 深圳市圣必智科技开发有限公司 | Method for detecting methylation of multiplex gene for non-small cell lung cancer |
| CN110964812A (en) * | 2018-09-29 | 2020-04-07 | 广州市康立明生物科技有限责任公司 | Application of HOXA9 methylation detection reagent in preparation of lung cancer diagnosis reagent |
| CN110964810A (en) * | 2018-09-29 | 2020-04-07 | 广州市康立明生物科技有限责任公司 | Application of HOXA7 and HOXA9 methylation detection reagent in preparation of lung cancer diagnostic reagent |
| CN110964811A (en) * | 2018-09-29 | 2020-04-07 | 广州市康立明生物科技有限责任公司 | HOXA9 methylation detection reagent |
| CN111630186A (en) * | 2018-01-23 | 2020-09-04 | 北京艾克伦医疗科技有限公司 | Methods and kits for identifying lung cancer status |
| CN111676286A (en) * | 2020-05-29 | 2020-09-18 | 武汉爱基百客生物科技有限公司 | Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application |
| CN117625795A (en) * | 2024-01-25 | 2024-03-01 | 北京迈基诺基因科技股份有限公司 | Probe set, kit and detection system for methylation detection of lung cancer and application |
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| CN108342477A (en) * | 2017-01-24 | 2018-07-31 | 北京艾克伦医疗科技有限公司 | Detection kit based on multiple gene diagnosis patients with lung cancer |
| CN107475369B (en) * | 2017-07-07 | 2021-05-04 | 南方医科大学 | Application of HOXA9 gene in preparation of medicine for treating skin squamous cell carcinoma |
| CN107475369A (en) * | 2017-07-07 | 2017-12-15 | 南方医科大学 | HOXA9 genes are preparing the application in treating cutaneous squamous cell carcinoma medicine |
| CN111630186A (en) * | 2018-01-23 | 2020-09-04 | 北京艾克伦医疗科技有限公司 | Methods and kits for identifying lung cancer status |
| WO2019242754A1 (en) * | 2018-06-22 | 2019-12-26 | 深圳市圣必智科技开发有限公司 | Method for detecting methylation of multiplex gene for non-small cell lung cancer |
| CN110964810A (en) * | 2018-09-29 | 2020-04-07 | 广州市康立明生物科技有限责任公司 | Application of HOXA7 and HOXA9 methylation detection reagent in preparation of lung cancer diagnostic reagent |
| CN110964811A (en) * | 2018-09-29 | 2020-04-07 | 广州市康立明生物科技有限责任公司 | HOXA9 methylation detection reagent |
| CN110964812A (en) * | 2018-09-29 | 2020-04-07 | 广州市康立明生物科技有限责任公司 | Application of HOXA9 methylation detection reagent in preparation of lung cancer diagnosis reagent |
| CN110964812B (en) * | 2018-09-29 | 2021-10-22 | 广州康立明生物科技股份有限公司 | Application of HOXA9 methylation detection reagent in preparation of lung cancer diagnosis reagent |
| CN110964810B (en) * | 2018-09-29 | 2022-04-29 | 广州康立明生物科技股份有限公司 | Application of HOXA7 and HOXA9 methylation detection reagent in preparation of lung cancer diagnostic reagent |
| CN110964811B (en) * | 2018-09-29 | 2022-04-29 | 广州康立明生物科技股份有限公司 | HOXA9 methylation detection reagent |
| CN110567906A (en) * | 2019-09-12 | 2019-12-13 | 深圳大学 | Method for representing RNA methylation modification and application |
| CN111676286A (en) * | 2020-05-29 | 2020-09-18 | 武汉爱基百客生物科技有限公司 | Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application |
| CN111676286B (en) * | 2020-05-29 | 2023-04-14 | 武汉爱基百客生物科技有限公司 | Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application |
| CN117625795A (en) * | 2024-01-25 | 2024-03-01 | 北京迈基诺基因科技股份有限公司 | Probe set, kit and detection system for methylation detection of lung cancer and application |
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