CN107988366B - Kit for diagnosing and evaluating breast cancer, detection of methylated HOXA4/DPP6 gene and application thereof - Google Patents
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Abstract
The invention relates to a kit for diagnosing and evaluating breast cancer, detection of a methylated HOXA4/DPP6 gene and application thereof, in particular to methylation detection of a HOXA4/DPP6 gene and a HOXA4/DPP6 gene, application thereof in preparation of medicines for diagnosing, detecting and screening breast cancer and a kit for the application. The research of the invention finds that the methylation degree of the HOXA4/DPP6 gene in the tumor tissue of a breast cancer patient is obviously higher than that of the normal tissue beside the tumor tissue, and the methylation level is closely related to the tumor progression.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a methylated HOXA4/DPP6 gene and application thereof, and more particularly relates to a kit for diagnosing and evaluating breast cancer, methylation detection of HOXA4/DPP6 gene and HOXA4/DPP6 gene and application thereof in breast cancer.
Background
According to data of Cancer Statistics in China tumor registration center, the number of breast cancers in urban areas (34.3/10 ten thousand women) is 2 times (17.0/10 ten thousand women) of the rural areas, the number of developed coastal urban diseases of socioeconomic efficiency is the highest, the number of breast cancers in Guangzhou is 46.6/10 ten thousand women, the average age of breast cancers in China is diagnosed to be 45-55 years, the average age of breast cancers in China is younger than that of Western women, the average increase of breast cancers in China is 3.1%, the growth rate of breast cancers in each region of China is 2007-2007 is 1.2-2.2, the growth rate of breast cancers in China is increased by 3.1%, the growth rate of breast cancers in each region of China is increased by 1.2-2, the growth rate of breast cancers in China is increased by 3.1%, the growth rate of breast cancers in each year of 1988 is increased by one year, the health of old women is increased by 1.2 times, and the growth rate of breast cancers in China is increased by the same as the growth rate of breast cancers in China.
At present, no mature female breast cancer screening scheme exists in China, and ACS (acute coronary syndrome) guidelines are referred to as recommended schemes of breast cancer diagnosis and treatment guidelines and specifications (2013 edition) of the Chinese anticancer Association. However, because the peak of the breast cancer incidence age of women in China is earlier than that of the United states, the breast molybdenum target radiography (MAM) screening initial age is recommended to be earlier than that of the United states by 5 years in China, and because of the difference of the breast texture, the breast volume and the economic condition of women in Asia, the breast ultrasound without radioactivity becomes an important screening method for the breast cancer in China. At present, the clinical rapid auxiliary diagnosis means is molybdenum target X-ray film detection, and micro-calcification is an important early imaging symptom. However, the microcalcifications are very small and are mixed in the background of a large number of images, which makes the visual recognition difficult and requires a high experience for the doctor. Therefore, in recent years, people have paid more attention to the computer-aided diagnosis technology of molybdenum-palladium images. However, because high-resolution images are huge and individual differences are large, even though images and artificial intelligence algorithms are continuously developed in recent years, the early diagnosis of breast cancer based on the imaging still is restricted by the problems of slow calculation, low accuracy, easy occurrence of experience errors and the like. Therefore, molecular diagnosis is a very essential detection means. At present, the diagnosis and molecular typing of breast cancer mainly rely on invasive means to obtain tissue samples, such as needle biopsy, surgical excision focus, intraoperative frozen consultation samples and the like, and have the defects of needing assistance of imaging and the like, relatively high detection cost, risk and incapability of implementing tracking and monitoring of the disease condition. Therefore, the search for a rapid and effective breast cancer detection molecular diagnosis method has important clinical significance.
DNA methylation is one of the earliest discovered ways of modifying gene appearance, and methylation in eukaryotes occurs only in cytosine, i.e., cytosine at the 5 '-end of dinucleotides is converted into 5' -methylcytosine by DNA methyltransferases (DNMTs). In the prior art, related researches are still lacked, and more researches are urgently needed to reveal methylation genes related to breast cancer and clarify the relationship of the methylation sites with the breast cancer.
The invention screens a plurality of candidate genes which are hypermethylated in a case group and methylation sites thereof by carrying out high-throughput methylation sequencing analysis on tumor tissues of a plurality of breast cancer patients and normal tissues beside the tumor tissues for a long time. And verified by a plurality of experiments, the technology of the application is obtained.
Disclosure of Invention
In view of the above, the present invention provides a kit for diagnosing and evaluating breast cancer, detection of methylated HOXA4/DPP6 gene and application thereof, so as to solve the above-mentioned deficiencies in the prior art.
The purpose of the invention is realized by the following technical scheme:
a kit for the diagnostic assessment of breast cancer, which is a kit with a methylated HOXA4 gene and/or a methylated DPP6 gene as a biomarker.
Further, the kit includes primers designed to be specific for the biomarkers.
Further, the kit also comprises a TRIzol reagent, sodium citrate, a CT conversion agent, an M-binding buffer solution, an M-washing solution, an M-desulfonation buffer solution, bisulfite and Zymo Taq premix solution.
Further, the sodium citrate is 0.1M sodium citrate with 10% ethanol.
Further, the primers in the kit are primers comprising a CpG amplifying the CpG of the 5 'UTR or the CpG of the exon 1 region of the methylated HOXA4 gene and/or the methylated DPP6 gene and a CpG amplifying the CpG of the 5' UTR or the CpG of the exon 1 region of the unmethylated HOXA4 gene and/or the unmethylated DPP6 gene.
Further, the primers comprise a primer of a methylated HOXA4 gene and/or a primer of a methylated DPP6 gene;
the primers of the methylated HOXA4 gene comprise a methylated HOXA4 specific upstream primer and a methylated HOXA4 specific downstream primer, and the sequence of the methylated HOXA4 specific upstream primer is as follows: 5' -TTATATTGGAGGAGAGTTTAAACGG; the sequence of the methylated HOXA4 specific downstream primer is: 5' -TCTAAAACCAAATCTTCACCTAACG;
the primer of the methylated DPP6 gene comprises a methylated DPP6 specific upstream primer and a methylated DPP6 specific downstream primer, and the sequence of the methylated DPP6 specific upstream primer is as follows: 5' -ATTTGGTTTTGAAAGGTAAGGTTAC; the sequence of the methylated DPP6 specific downstream primer is as follows: 5' -ATTTACTCGTCGATATCGACGTT.
Further, the primers also comprise a primer of an unmethylated HOXA4 gene and/or a primer of an unmethylated DPP6 gene;
the primers of the unmethylated HOXA4 gene comprise an unmethylated HOXA4 specific upstream primer and an unmethylated HOXA4 specific downstream primer, and the sequence of the unmethylated HOXA4 specific upstream primer is as follows: 5' -TTATATTGGAGGAGAGTTTAAATGG; the sequence of the unmethylated HOXA4 specific downstream primer is: 5' -TAAAACCAAATCTTCACCTAACATT;
the primers of the unmethylated DPP6 gene comprise an unmethylated DPP6 specific upstream primer and an unmethylated DPP6 specific downstream primer, and the sequence of the unmethylated DPP6 specific upstream primer is as follows: 5' -TTTGGTTTTGAAAGGTAAGGTTATG; the sequence of the non-methylated DPP6 specific downstream primer is as follows: 5' -CAATTTACTCATCAATATCAACATT.
Further, the primers include a primer of a methylated HOXA4 gene, a primer of a methylated DPP6 gene, a primer of an unmethylated HOXA4 gene, and a primer of an unmethylated DPP6 gene.
The detection method is characterized in that a methylated HOXA4/DPP6 gene is used as a target point, and the methylation degree of the HOXA4/DPP6 gene is detected by a methylation chip technology method.
The application of the methylated HOXA4/DPP6 gene and the application of the methylated HOXA4 gene and/or the methylated DPP6 gene in breast cancer assessment, diagnosis, detection or screening medicines.
The invention has at least the following beneficial effects:
according to the present application, the expression level of the methylated HOXA4/DPP6 gene in the tumor tissue is greatly improved, and a large number of case studies by the applicant show that the methylation rate of the HOXA4/DPP6 gene in the tumor tissue of all tumor patients is significantly higher than that of the normal tissue, the methylation rate is over 55%, the methylation rate of the normal tissue beside the tumor is extremely low, the methylation rate of the normal tissue beside the tumor of more than 90% of the patients is negative, the positive rate is extremely low, the methylation rate of the positive patients is mostly about 10%, and the value is far lower than that of the tumor tissue, so that the methylated HOXA4/DPP6 gene can be effectively applied to the breast cancer and applied to the medicines for evaluation, diagnosis, detection or screening. The application provides a kit for evaluating, diagnosing or detecting breast cancer, the kit takes a methylated HOXA4/DPP6 gene as a biomarker, and specially develops a plurality of specific primers for detecting the methylated HOXA4/DPP6 gene, the specificity is good, the accuracy rate is high, and the final accuracy rate of the primers is extremely high and can reach more than 99% through hundreds of tests, comparisons and the like; the kit can be used for efficiently detecting the methylated HOXA4/DPP6 genes in tumor tissues and normal tissues. In a word, the application provides a new breast cancer diagnosis and treatment target, has important clinical application value, and has extremely important significance in the field of cancers such as breast cancer.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent from the following description of the embodiments of the present invention with reference to the accompanying drawings, in which:
FIG. 1 is a schematic structural diagram showing the degree of methylation of the HOXA4/DPP6 gene in tumor tissue and normal tissue according to the present invention;
FIG. 2 is a schematic structural diagram showing the expression levels of HOXA4/DPP6 genes in tumor tissues and normal tissues in the examples of the present invention.
Detailed Description
The present invention will be described below based on examples, but the present invention is not limited to only these examples.
Example 1
The application of a methylated HOXA4/DPP6 gene, wherein the accession numbers of the HOXA4 gene and the DPP6 gene in a DNA sequence database are shown in Table 1; methylation of the HOXA4/DPP6 gene is the conversion of cytosine at the 5 '-end of CpG dinucleotide to 5' -methylcytosine. The research of the inventor finds that the methylated HOXA4/DPP6 gene in breast cancer tumor is greatly improved, wherein the methylation rate of HOXA4 gene in tumor tissue reaches 80.58%, and the methylation rate of DPP6 gene is 46.9%, which is obviously improved compared with the methylated HOXA4/DPP6 gene in normal tissue. Therefore, the methylated HOXA4/DPP6 gene can be applied to medicines for evaluation, diagnosis, detection or screening of breast cancer.
Table 1: accession number of HOXA4/DPP6 Gene
| Gene symbol | Genbank accession number |
| HOXA4 | NM_001039350 |
| DPP6 | NM_001039350 |
Example 2
A kit for the diagnostic assessment of breast cancer, which is a kit with a methylated HOXA4 gene and/or a methylated DPP6 gene as biomarker, preferably with a methylated HOXA4/DPP6 gene as biomarker.
The kit comprises specific primers designed for the biomarkers, TRIzol reagent (Purchase, L ife Tech Inc, USA), 0.1M sodium citrate containing 10% ethanol, and CT conversion agent (from EZ DNAmethlation)TMKit (ZYMO RESEARCH, cat # D5002)), M-binding buffer, M-Wash, M-desulfonation buffer, bisulfite solution, Zymo Taq premix.
The primer is a primer for amplifying CpG including CpG of 5 'UTR of methylated HOXA4/DPP6 gene or CpG of exon 1 region to detect CpG methylation, preferably simultaneously including CpG of 5' UTR of unmethylated HOXA4/DPP6 gene or CpG of exon 1 region.
Specifically, the primers include a primer of a methylated HOXA4 gene and a primer of a methylated DPP6 gene. The primers of the methylated HOXA4 gene comprise a methylated HOXA4 specific upstream primer and a methylated HOXA4 specific downstream primer, and the sequence of the methylated HOXA4 specific upstream primer is as follows: 5' -TTATATTGGAGGAGAGTTTAAACGG; the sequence of the methylated HOXA4 specific downstream primer is: 5' -TCTAAAACCAAATCTTCACCTAACG. The primer of the methylated DPP6 gene comprises a methylated DPP6 specific upstream primer and a methylated DPP6 specific downstream primer, and the sequence of the methylated DPP6 specific upstream primer is as follows: 5' -ATTTGGTTTTGAAAGGTAAGGTTAC; the sequence of the methylated DPP6 specific downstream primer is as follows: 5' -ATTTACTCGTCGATATCGACGTT.
The primers also comprise a primer of the unmethylated HOXA4 gene and a primer of the unmethylated DPP6 gene; the primers of the unmethylated HOXA4 gene comprise an unmethylated HOXA4 specific upstream primer and an unmethylated HOXA4 specific downstream primer, and the sequence of the unmethylated HOXA4 specific upstream primer is as follows: 5' -TTATATTGGAGGAGAGTTTAAATGG; the sequence of the unmethylated HOXA4 specific downstream primer is: 5' -TAAAACCAAATCTTCACCTAACATT. The primers of the unmethylated DPP6 gene comprise an unmethylated DPP6 specific upstream primer and an unmethylated DPP6 specific downstream primer, and the sequence of the unmethylated DPP6 specific upstream primer is as follows: 5' -TTTGGTTTTGAAAGGTAAGGTTATG; the sequence of the non-methylated DPP6 specific downstream primer is as follows: 5' -CAATTTACTCATCAATATCAACATT.
Example 3
The detection method is to detect the methylation degree of the HOXA4/DPP6 gene by taking the methylated HOXA4/DPP6 gene as a target point, adopting an illumina methylation chip (the product number is WG-314-. See example 4 for a specific method.
Example 4: collection and sequencing of samples
9 breast cancer patients and 11 scattered collected patients in other places were collected at 2015.05-2016.04 time in Shenzhen, Baoan region maternal-child healthcare institute, and tumor tissues and normal tissues beside the tumor tissues were taken and stored in liquid nitrogen tank rapidly. After sample collection, the whole genome methylation sequencing is carried out on the tumor tissue and normal tissues beside the tumor tissue, and a primary analysis result is provided. The sequencing results of all cases collected showed that the candidate gene HOXA4/DPP6 was significantly more methylated in the tumor tissue of the patients than in the normal group, and the results of the mean values are shown in FIG. 1, which is consistent with the results of the present invention.
Example 5: expression of HOXA4/DPP6 Gene in Normal tissue alongside Breast tumor tissue and control tumor tissue
Firstly, experimental materials:
1. selecting tumor tissues of 3 breast cancer patients and normal tissues beside the tumor tissues, wherein case groups all accord with breast cancer diagnosis standards and are numbered in groups; the experimental group is tumor tissue of breast patient, the control group is normal tissue beside tumor tissue, and fresh tissue with size of 5mm is taken3And storing at-80 deg.C.
2. Primary screening of nucleic acid markers: transcriptome sequencing is carried out on 3 tumor tissues and normal tissues beside the tumor tissues, and the sequencing result is further subjected to bioinformatics analysis, so that the result shows that the expression levels of HOXA4 and DPP6 genes are obviously reduced, and the result is shown in figure 2, and the comparison between an experimental group and a control group in figure 2 shows that the expression level in the normal tissues is more than 10 times higher than that in the tumor tissues for the HOXA4 gene; for the DPP6 gene, the expression level in normal tissues is more than 5 times higher than that in tumor normal tissues. The transcriptome results were further verified by real-time fluorescent quantitative PCR.
3. Respectively extracting RNA in tissues of an experimental group and a control group by using Trizol reagent (a purchase manufacturer: L ife Tech Inc, USA), and measuring the concentration and the purity of the RNA, specifically, (1) adding 1m L Trizol to 0.5g of tissue of each sample, violently shaking for 1min, storing the mixture at room temperature for 10min, (2) adding 0.2ml of chloroform, violently shaking for 1min, fully mixing the mixture, and storing the mixture at room temperature for 3min to 5min, (3) centrifuging the mixture at 12000rpm for 15min at 4 ℃ and then absorbing 70% of an upper aqueous phase into another new centrifuge tube, taking out protein substances between the two aqueous phases, transferring the mixture into the new tube, adding equal volume of isopropanol and 1 to 2ul of glycogen, fully inverting the mixture, storing the mixture at-20 ℃ for more than 6 hours, keeping the centrifuge tube in a vertical state, (4) adding 75% of DEPC (diethyl DEPC) to 4 ℃, fully washing the mixture at 12000rpm for 15min, and carefully removing supernatant after high-speed separation at 4 ℃, adding cold ethanol (DEPC) at an equal volume of the same volume of the mixture with Trizol, fully dissolving the precipitate, adding DEPC, drying the precipitate at a temperature, and then fully washing the precipitate at 10 rpm, and then fully dissolving the precipitate at 10 ℃ and then adding the precipitate at 10 rpm, and then directly performing the concentration measurement by using an ultraviolet light, (5) and performing the precipitation procedure (5) and the concentration measurement.
4. Reverse transcription into cDNA, the mRNA is reverse transcribed to obtain cDNA by a conventional method, a 25ul reaction system is adopted, and 1ug of total RNA is taken from each sample as template RNA. The obtained cDNA was stored in a freezer at-20 ℃ for further use.
5. Real-time quantitative fluorescent PCR (Real-time PCR)
Primers for HOXA4/DPP6 gene were designed, and the sequence listing of nucleic acid primers for these two mRNAs is shown in Table 2.
TABLE 2 mRNA primer sequences of HOXA4 Gene and DPP6 Gene
(1) Fluorescent quantitative PCR was performed on HOXA4 and DPP6 according to the following reaction systems:
(2) fluorescent quantitative PCR reaction conditions:
94 ℃ below zero: 30 s; 94 ℃ below zero: 5s, 60 ℃: 34s, plate 40 cycles; dissolution curve analysis: the temperature is 60-95 ℃.
(3) Results of the experiment
The inflection point of the real-time fluorescence quantitative PCR amplification curve is clear, the overall parallelism of the amplification curve is good, the amplification efficiency of each reaction tube is similar, the dissolution curves of the sample amplification products are all single peaks, and the condition that only one amplification product is used is specific amplification is shown; according to the relative quantitative formula of real-time fluorescent quantitative PCR, the expression level of the HOXA4 gene in the breast cancer tissue is about 0.07 times of that of the control group, and the expression level of the DPP6 gene in the breast cancer tissue is about 0.15 times of that of the control group, and the results verify the results of the integrated analysis of the expression data of the high-throughput transcriptome and the low expression of the HOXA4 and DPP6 genes in the breast cancer patient tissue.
Example 6: detection of methylation degree of HOXA4/DPP6 gene
1. DNA extraction
Selecting tumor tissue and normal tissue beside the tumor tissue of 3 breast cancer patients, wherein the case groups all accord with the breast cancer diagnosis standard, the tumor tissue of the breast cancer patients is used as an experimental group, the normal tissue beside the tumor tissue is used as a control group, and the fresh tissue with the size of 5mm is taken3And storing in liquid nitrogen at-80 deg.C.
Rapidly taking out the tissue from liquid nitrogen, and grinding into powder; placing the tissue into a homogenizer with a TRIzol reagent added in advance by using a curette, and homogenizing for a plurality of minutes; transferring the homogenized liquid into a centrifugal tube without RNA enzyme, adding chloroform, and centrifuging at 4 ℃ for layering; putting the middle-layer solid phase into a fresh centrifugal tube, adding 0.3ml of absolute ethyl alcohol (calculated according to 1ml of Trizol), fully and uniformly mixing, standing for 3 minutes at room temperature, and centrifugally precipitating DNA at 2000g at 4 ℃; collecting supernatant, washing DNA precipitate with 0.1M sodium citrate containing 10% ethanol, standing at room temperature for 30min, and centrifuging at 4 deg.C for 2000g to precipitate DNA; collecting the supernatant, repeatedly washing and centrifuging once, and finally collecting the precipitate; washing the DNA precipitate with 75% ethanol, adding 2ml 75% ethanol according to 1ml Trizol, standing at room temperature for 10-20min, centrifuging at 4 deg.C for 2000g to precipitate DNA; after natural air drying, the precipitate is fully dissolved by 30-50ul double distilled water, and the integrity of DNA is detected by agarose electrophoresis and frozen at-20 ℃ for standby.
2. Bisulphite modification
Bisulfite deaminates unmethylated cytosines in DNA to uracil, while methylated cytosines remain unchanged, reflecting the methylation status of individual cytosines. Using EZ DNA MethylationTMThe specific steps of Kit (ZYMO RESEARCH, cat # D5002) are as follows:
adding 130ul of CT transforming agent into 20ul of genomic DNA solution, recording as a mixed solution, incubating the mixed solution at 98 ℃ for 10min, incubating at 64 ℃ for 2.5h, and incubating at 4 ℃ for 20 h; 150ul of this mixture and 600ul of M-binding buffer (from EZDDNA Methylation) were addedTMKit) to Zymo-SpinTMIC column (from EZ DNA Methylation)TMKit), centrifuged at full speed for 30s, the filtrate was discarded, and 100ul of M-wash (from EZ DNA Methylation) was addedTMKit) to a filter column; after washing the column, 200ul of M-desulfonation buffer (from EZ DNA Methylation) was added to the filtration columnTMKit), standing at room temperature for 15min, centrifuging at full speed, and washing the column twice with M-washing solution; finally 10ul of M-eluent (from EZ DNAmethation) was usedTMKit) the DNA on the adsorption column was dissolved and the modified DNA was collected by centrifugation.
3. CpG of 5' UTR of HOXA4/DPP6 gene or CpG of exon 1 region to detect detection of CpG hypermethylation.
Specific primer sequences are shown in table 3:
TABLE 3 specific primers for HOXA4/DPP6 Gene
(1) MSP method detection is adopted, and the total reaction volume is 20 ul: 1ul of bisulfite treated DNA is used as a template, 1ul of each of the 8 upstream and downstream specific primers is used, 10ul of 2xZymo Taq premixed, and 7ul of deionized water is used. The reaction parameters were as follows: pre-denaturation at 95 ℃ for 5 min; 40 cycles were set as follows, denaturation at 95 ℃ for 30 s; annealing at 62 ℃ for 30s, and extending at 72 ℃ for 30 s; finally, extension is carried out for 10min at 72 ℃. The PCR amplification product was detected by 1.5% agarose gel electrophoresis.
(2) Results of the experiment
The existence of the amplification product of the non-methylation specific primer, and the non-existence of the amplification product of the methylation specific primer is non-methylation; the presence of amplification products of the non-methylation specific primers is indicative of methylation. And (3) displaying an electrophoresis result: among 9 breast cancer tissues, 5 HOXA4/DPP6 genes with positive methylation rate were 55.56%, and among 9 breast cancer tissues, only 1 of normal tissues beside the control breast cancer tissues with positive methylation rate was 11.11%.
In the present invention, DNA methylation typically suppresses gene expression, and demethylation induces reactivation and expression of the gene. The DNA modification mode realizes the regulation and control of gene expression on the premise of not changing gene sequences. Alterations in DNA methylation levels and patterns are an important factor in tumorigenesis. These changes include the hypermethylated and genomic DNA hypomethylated states locally at CpG islands. In normal cells, CpG islands located in the promoter region of the cancer suppressor gene are in a low-level or unmethylated state, at which time the cancer suppressor gene is in a normal open state, and the cancer suppressor gene is continuously expressed to suppress tumor occurrence. In tumor cells, CpG islands in the region are highly methylated, chromatin conformation is changed, and the expression of cancer suppressor genes is closed, so that the cells enter a cell cycle, apoptosis is lost, DNA repair defects, angiogenesis, cell adhesion function loss and the like are caused, and finally, tumorigenesis is caused.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A kit for the diagnostic assessment of breast cancer, characterized by: the primer group in the kit consists of a primer for amplifying a methylated HOXA4 gene, a primer for amplifying a methylated DPP6 gene, a primer for an unmethylated HOXA4 gene and a primer for an unmethylated DPP6 gene;
wherein the sequence of the amplified methylated HOXA4 specific upstream primer is as follows: 5' -TTATATTGGAGGAGAGTTTAAACGG; the sequence of the amplified methylated HOXA4 specific downstream primer is as follows: 5' -TCTAAAACCAAATCTTCACCTAACG;
wherein the sequence of the amplified methylated DPP6 specific upstream primer is as follows: 5' -ATTTGGTTTTGAAAGGTAAGGTTAC; the sequence of the amplified methylated DPP6 specific downstream primer is as follows: 5' -ATTTACTCGTCGATATCGACGTT;
wherein the sequence of the unmethylated HOXA4 specific upstream primer is as follows: 5' -TTATATTGGAGGAGAGTTTAAATGG; the sequence of the unmethylated HOXA4 specific downstream primer is: 5' -TAAAACCAAATCTTCACCTAACATT;
wherein the sequence of the non-methylated DPP6 specific upstream primer is as follows: 5' -TTTGGTTTTGAAAGGTAAGGTTATG; the sequence of the non-methylated DPP6 specific downstream primer is as follows: 5' -CAATTTACTCATCAATATCAACATT.
2. The kit for the diagnostic evaluation of breast cancer according to claim 1, characterized in that: the kit also comprises a TRIzol reagent, sodium citrate, a CT conversion agent, an M-binding buffer solution, an M-washing solution, an M-desulfonation buffer solution, bisulfite and a Zymo Taq premix solution.
3. The kit for the diagnostic evaluation of breast cancer according to claim 2, characterized in that: the sodium citrate is 0.1M sodium citrate containing 10% ethanol.
4. Use of the primer set of claim 1 in the preparation of a product for breast cancer assessment, diagnosis and detection.
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| WO2009108917A3 (en) * | 2008-02-29 | 2010-07-01 | Oncomethylome Sciences, S.A. | Markers for improved detection of breast cancer |
| CN102181520A (en) * | 2011-03-01 | 2011-09-14 | 杭州博谱医药科技有限公司 | Application of methylated SLC19A3 (solute carrier family 19 member 13) gene |
| CN106498076A (en) * | 2010-05-11 | 2017-03-15 | 威拉赛特公司 | For diagnosing the method and composition of symptom |
-
2017
- 2017-12-22 CN CN201711406093.8A patent/CN107988366B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009108917A3 (en) * | 2008-02-29 | 2010-07-01 | Oncomethylome Sciences, S.A. | Markers for improved detection of breast cancer |
| CN106498076A (en) * | 2010-05-11 | 2017-03-15 | 威拉赛特公司 | For diagnosing the method and composition of symptom |
| CN102181520A (en) * | 2011-03-01 | 2011-09-14 | 杭州博谱医药科技有限公司 | Application of methylated SLC19A3 (solute carrier family 19 member 13) gene |
Non-Patent Citations (4)
| Title |
|---|
| Common gene pathways and families altered by DNA methylation in breast and prostate cancers;Tanya K Day and Tina Bianco Miotto;《Endocrine-Related Cancer》;20130701;第20卷(第5期);R215–R232 * |
| DNA methylation data for identifification of epigenetic targets of resveratrol in triple negative breast cancer cells;Rubiceli Medina-Aguilar等;《Data in Brief 》;20170209;第11卷;169–182 * |
| Microarray‐based analysis of whole‐genome DNA methylation profiling in early detection of breast cancer;Shao‐Ying Li等;《J Cell Biochem》;20180911;第120卷;658-670 * |
| 乳腺癌HOXA4 基因甲基化的检测及其临床意义;李少英等;《中华乳腺病杂志( 电子版) 》;20190831;第13卷(第4期);225-233 * |
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