CN107904309A - Methylated genes composition and the purposes for preparing diagnosis three negative type breast cancers Bone tumour kits of indication - Google Patents

Methylated genes composition and the purposes for preparing diagnosis three negative type breast cancers Bone tumour kits of indication Download PDF

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CN107904309A
CN107904309A CN201711200315.0A CN201711200315A CN107904309A CN 107904309 A CN107904309 A CN 107904309A CN 201711200315 A CN201711200315 A CN 201711200315A CN 107904309 A CN107904309 A CN 107904309A
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methylates
bone
breast cancer
methylate
indication
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CN107904309B (en
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薛守海
李宜健
童强
刘敏涛
胡雨祝
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Jiaxing Maiwei Metabolic Biotechnology Co., Ltd.
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薛守海
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    • 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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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Abstract

The invention discloses the purposes of methylated genes composition and preparation diagnosis three negative type breast cancers Bone tumour kits of indication.Present invention discover that, the serum PITX1 and AMOT that methylates that methylates can combine for diagnosing indication LuminalA types Bone of Breast Cancer transfer, the serum PTPN1 and SLIT2 that methylates that methylates can combine for diagnosing indication LuminalB types Bone of Breast Cancer transfer, serum methylates MYLK2, the EFEMP1 and SOSTDC1 that methylates that methylates can combine for diagnosing indication Her 2 overexpression type Bone of Breast Cancer transfers, serum methylate MYLK3 and methylate SCARA5 can combine for diagnose indication three negative type breast cancers Bone tumours, accuracy is up to more than 90%, testing cost is low, non-invasi, it is convenient and efficient.

Description

Methylated genes composition and preparation diagnosis indication three negative type breast cancers Bone tumours examination The purposes of agent box
Technical field
The invention belongs to biochemical field, is related to diagnosis composition and diagnostic kit, and in particular to one kind methylates Gene diagnosis composition and the application in terms of the diagnostic kit for diagnosing the Bone of Breast Cancer transfer of indication different molecular hypotype is prepared.
Background technology
Breast cancer is one of malignant tumour for threatening women life and health, there are about ten thousand people of 40-45 every year and dies of breast cancer (ginseng Examine document：Different molecular hypotype Bone of Breast Cancer shifts the Clinical symptoms and prognostic analysis of patient, XI AN JIAOTONG UNIVERSITY Subject Index doctor Learn version, in September, 2017 the 5th phase of volume 38).Breast cancer is very easy to that DISTANT METASTASES IN occurs, and bone is the most common distant place of breast cancer Metastasis site, is bone tissue (bibliography more than the starting metastasis site of 50% patient：Genes associated with breast cancer metastatic to bone,J Clin Oncol,2006；Implications of Bone-Only Metastases in Breast Cancer:Favorable Preference with Excellent Outcomes of Hormone Receptor Positive Breast Cancer,Cancer Res Treat,2011).According to estrogen receptor (estrogen receptor, ER), progesterone receptor (progesterone receptor, PR), human epidermal growth factor receptor Breast cancer, can be divided into by the expression of body -2 (human epidermal growth factor receptor-2, HER-2) 4 hypotypes, are respectively：Luminal A types, Luminal Type Bs, Her-2 overexpressions type and triple negative breast cancer (bibliography： Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications,PNAS,2001).Research shows that prognosis and its molecule of Bone of Breast Cancer transfer divide Closely related (the bibliography such as type, clinical stages, lymph node status：Prevalence and risk factors of bone metastasis and skeletal related events in patients with primary breast cancer in Japan,Int J Clin Onco,2014).The specific molecular biology of different molecular hypotype breast cancer and clinical pathology are special Sign, determines the difference of its therapeutic modality and prognosis.There is also difference for the gene phenotype of different molecular hypotype Bone of Breast Cancer transfer.
Early diagnosis Bone of Breast Cancer transfer is to save the key of patient vitals.At present, radionuclide bone scan (ECT), CT scan (CT), nuclear magnetic resonance (MRI), Positron emission computed tomography (PET-CT) and bone tissue Biopsy is to find and make a definite diagnosis the goldstandard of Bone of Breast Cancer transfer.But there are different deficiencies, such as Laboratory Fee for these methods With height, intervention diagnosis adds the burden of patient.Which increase the pressure of patient with breast cancer's Bone tumour conventional detection.
DNA methylation refers to one methyl base of covalent bond on No. 5 carbon atoms of cytimidine of genome CpG dinucleotides Group, it is primarily involved in the expression regulation of gene.Methylating for many genes is proved to closely related with various clinical diseases, some Even it can be determined that the independent factor that disease is caused a disease.The abnormal disease such as with tumour of DNA methylation is closely related.Therefore, first Base gene can be used as the marker of tumor cells diagnosis, while also can be as the target (bibliography of molecular therapy： Value of the DNA methylation marker in molecule Clinics and Practices, molecule Clinics and Practices magazine in September, 2009 volume 1 the 3rd Phase).
Research shows that the several genes expression of breast cancer primary tumo(u)r is necessary to Bone tumour occurs, it is thus regarded that special The transfer for determining organ is that multiple-factor is coefficient as a result, the conclusion is prompted, and the transspecific of bone is bone in primary tumo(u)r The selection of different phenotype tumour cells and bone source sex factor induction as a result, different molecular hypotype Bone of Breast Cancer transfer methylate Gene is expected to become the diagnosis marker (bibliography of Bone of Breast Cancer transfer：Kang Y,Siegel PM,Shu W,et al.A multigenic program mediating breast cancer metastasis to bone.Cancer Cell,2003)。
Applicant is intended to research and compares the base that methylates in generation Bone tumour and the blood serum of patients with human breast carcinoma that Bone tumour does not occur The difference of cause, finds, verification may be used as diagnosing, indicating the methylated genes mark that different molecular hypotype Bone of Breast Cancer shifts Thing, with provide it is a kind of can the kit that shifts of quick diagnosis, indication different molecular hypotype Bone of Breast Cancer and method by blood.
The content of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of methylated genes diagnosis composition, prepares Into a kind of testing cost is low, non-invasi, convenient and efficient diagnosis indication different molecular hypotype Bone of Breast Cancer transfer diagnostic reagent Box.
The above-mentioned purpose of the present invention is achieved by following technical solution：
First,Luminal A types Bone of Breast Cancer shifts
A kind of methylated genes diagnosis composition, by methylating, the PITX1 and AMOT that methylates is formed.
Above-mentioned diagnosis composition is in terms of the diagnostic kit for diagnosing the Bone of Breast Cancer transfer of indication Luminal A types is prepared Using.
A kind of diagnostic kit for being used to diagnose indication Luminal A types Bone of Breast Cancer transfer, including methylate PITX1 and Methylate the PCR amplification primer of AMOT.
Preferably, in the diagnostic kit, the PCR amplification sense primer such as Sequence NO.1 for the PITX1 that methylates Shown, PCR amplification anti-sense primer is as shown in Sequence NO.2.
Preferably, in the diagnostic kit, the PCR amplification sense primer such as Sequence NO.4 for the AMOT that methylates Shown, PCR amplification anti-sense primer is as shown in Sequence NO.5.
Preferably, the pyrosequencing that methylate PITX1 and the AMOT that methylates are further included in the diagnostic kit draws Thing.
Preferably, in the diagnostic kit, the Pyrosequencing primer such as Sequence NO.3 for the PITX1 that methylates It is shown.
Preferably, in the diagnostic kit, the Pyrosequencing primer such as Sequence NO.6 institutes for the AMOT that methylates Show.
Preferably, in the diagnostic kit, PCR amplification and enzyme and reagent needed for pyrosequencing are further included.
A kind of method for diagnosing the Bone of Breast Cancer transfer of indication Luminal A types, includes the following steps：
Step S1, gathers Luminal A type patient with breast cancer's limosis vein bloods, serum is centrifuged out after natural coagulation；
Step S2, extracts serum STb gene, through in PCR amplification, the modification of DNA sulphite and pyrosequencing measure STb gene Methylate PITX1 and the index that methylates of AMOT of methylating, and uses X successively1、X2Represent；
Step S3, by X1、X2Value substitutes into dualistic logistic regression equation Y=1/ [1+EXP (1.499X1+2.302X2- 0.258) Y value] is obtained, Y value is less than 0.238 and indicates that Bone tumour occurs for the patient with breast cancer, and bone does not occur more than 0.238 indication turns Move.
2nd, Luminal Type Bs Bone of Breast Cancer shifts
A kind of methylated genes diagnosis composition, by methylating, the PTPN1 and SLIT2 that methylates is formed.
Above-mentioned diagnosis composition is in terms of the diagnostic kit for diagnosing the Bone of Breast Cancer transfer of indication Luminal Type Bs is prepared Using.
A kind of diagnostic kit for being used to diagnose indication Luminal Type Bs Bone of Breast Cancer transfer, including methylate PTPN1 and Methylate the PCR amplification primer of SLIT2.
Preferably, in the diagnostic kit, the PCR amplification sense primer such as Sequence NO.7 for the PTPN1 that methylates Shown, PCR amplification anti-sense primer is as shown in Sequence NO.8.
Preferably, in the diagnostic kit, the PCR amplification sense primer such as Sequence for the SLIT2 that methylates Shown in NO.10, PCR amplification anti-sense primer is as shown in Sequence NO.11.
Preferably, in the diagnostic kit, the pyrosequencing of methylate PTPN1 and the SLIT2 that methylates are further included Primer.
Preferably, in the diagnostic kit, the Pyrosequencing primer such as Sequence NO.9 for the PTPN1 that methylates It is shown.
Preferably, in the diagnostic kit, the Pyrosequencing primer such as Sequence NO.12 for the SLIT2 that methylates It is shown.
Preferably, in the diagnostic kit, PCR amplification and enzyme and reagent needed for pyrosequencing are further included.
A kind of method for diagnosing the Bone of Breast Cancer transfer of indication Luminal Type Bs, includes the following steps：
Step S1, gathers Luminal Type B patient with breast cancer's limosis vein bloods, serum is centrifuged out after natural coagulation；
Step S2, extracts serum STb gene, through in PCR amplification, the modification of DNA sulphite and pyrosequencing measure STb gene Methylate PTPN1 and the index that methylates of SLIT2 of methylating, and uses X successively1、X2Represent；
Step S3, by X1、X2Value substitutes into dualistic logistic regression equation Y=1/ [1+EXP (2.016X1+1.898X2- 0.455) Y value] is obtained, Y value is less than 0.310 and indicates that Bone tumour occurs for the patient with breast cancer, and bone does not occur more than 0.310 indication turns Move.
3rd,Her-2 overexpression types Bone of Breast Cancer shifts
A kind of methylated genes composition, is made of the MYLK2 that methylates, the EFEMP1 and SOSTDC1 that methylates that methylates.
Above-mentioned diagnosis composition is in terms of the diagnostic kit for diagnosing the Bone of Breast Cancer transfer of indication Her-2 overexpression types is prepared Application.
A kind of diagnostic kit for being used to diagnose the Bone of Breast Cancer transfer of indication Her-2 overexpression types, including methylate The PCR amplification primer and Pyrosequencing primer of MYLK2, methylate EFEMP1 and the SOSTDC1 that methylates.
Preferably, in the diagnostic kit, the PCR amplification sense primer such as Sequence for the MYLK2 that methylates Shown in NO.13, PCR amplification anti-sense primer is as shown in Sequence NO.14, Pyrosequencing primer such as Sequence NO.15 It is shown.
Preferably, in the diagnostic kit, the PCR amplification sense primer such as Sequence for the EFEMP1 that methylates Shown in NO.16, PCR amplification anti-sense primer is as shown in Sequence NO.17, Pyrosequencing primer such as Sequence NO.18 It is shown.
Preferably, in the diagnostic kit, the PCR amplification sense primer such as Sequence for the SOSTDC1 that methylates Shown in NO.19, PCR amplification anti-sense primer is as shown in Sequence NO.20, Pyrosequencing primer such as Sequence NO.21 It is shown.
Preferably, in the diagnostic kit, PCR amplification and enzyme and reagent needed for pyrosequencing are further included.
A kind of method for diagnosing the Bone of Breast Cancer transfer of indication Her-2 overexpression types, includes the following steps：
Step S1, gathers Her-2 overexpression type patient with breast cancer's limosis vein bloods, bleeding is centrifuged after natural coagulation Clearly；
Step S2, extracts serum STb gene, through in PCR amplification, the modification of DNA sulphite and pyrosequencing measure STb gene Methylate MYLK2, the index that methylates of methylate EFEMP1 and the SOSTDC1 that methylates, and is followed successively by X1、X2、X3；
Step S3, by X1、X2、X3Substitute into equation Y=1/ [1+EXP (1.342X1+1.401X2+1.345X3- 2.035)] To Y value, Y value is less than 0.308 and indicates that Bone tumour occurs for the patient with breast cancer, and Bone tumour does not occur more than 0.308 indication.
4th,Triple negative breast cancer Bone tumour
A kind of methylated genes diagnosis composition, by methylating, the MYLK3 and SCARA5 that methylates is formed.
Above-mentioned diagnosis composition answering in terms of the diagnostic kit for diagnosing three negative type breast cancers Bone tumours of indication is prepared With.
A kind of diagnostic kit for being used to diagnose three negative type breast cancers Bone tumours of indication, including methylate MYLK3 and first The PCR amplification primer of base SCARA5.
Preferably, in the diagnostic kit, the PCR amplification sense primer such as Sequence for the MYLK3 that methylates Shown in NO.22, PCR amplification anti-sense primer is as shown in Sequence NO.23.
Preferably, in the diagnostic kit, the PCR amplification sense primer such as Sequence for the SCARA5 that methylates Shown in NO.25, PCR amplification anti-sense primer is as shown in Sequence NO.26.
Preferably, the pyrosequencing that methylate MYLK3 and the SCARA5 that methylates are further included in the diagnostic kit draws Thing.
Preferably, methylate the Pyrosequencing primer such as Sequence NO.24 institutes of MYLK3 in the diagnostic kit Show.
Preferably, methylate the Pyrosequencing primer such as Sequence NO.27 institutes of SCARA5 in the diagnostic kit Show.
Preferably, in the diagnostic kit, PCR amplification and enzyme and reagent needed for pyrosequencing are further included.
A kind of method for diagnosing three negative type breast cancers Bone tumours of indication, includes the following steps：
Step S1, gathers three negative type breast cancers patient's limosis vein bloods, serum is centrifuged out after natural coagulation；
Step S2, extracts serum STb gene, through in PCR amplification, the modification of DNA sulphite and pyrosequencing measure STb gene Methylate MYLK3 and the index that methylates of SCARA5 of methylating, and uses X successively1、X2Represent；
Step S3, by X1、X2Value substitutes into dualistic logistic regression equation Y=1/ [1+EXP (1.775X1+1.236X2- 0.398) Y value] is obtained, Y value is less than 0.366 and indicates that Bone tumour occurs for the patient with breast cancer, and bone does not occur more than 0.366 indication turns Move.
It is a discovery of the invention that serum methylates, the PITX1 and AMOT that methylates can combine for diagnosing indication Luminal A types Breast cancer whether Bone tumour, individual authentication concentrate diagnosis indication rate of accuracy reached more than 90%；Serum methylates PTPN1 and methyl Change SLIT2 can combine for diagnose indication Luminal Type Bs breast cancer whether Bone tumour, concentrate diagnosis indication in individual authentication Rate of accuracy reached more than 90%；Methylate MYLK2, the EFEMP1 and SOSTDC1 that methylates that methylates of serum can combine for diagnosing Indicate Her-2 overexpression types breast cancer whether Bone tumour, concentrate diagnosis indication rate of accuracy reached more than 90% in individual authentication；Serum Methylate MYLK3 and methylate SCARA5 can combine for diagnose indication three negative type breast cancers whether Bone tumour, in independence Diagnosis indication rate of accuracy reached more than 90% is concentrated in verification.Indication different molecular hypotype breast is diagnosed using above-mentioned serum methylated genes The accuracy of gland cancer Bone tumour is high, and testing cost is low, non-invasi, convenient and efficient, greatly reduces patient suffering and burden.
Brief description of the drawings
Fig. 1 combines for the PITX1 and AMOT that methylates that methylates in test set and is used to diagnose differentiation Luminal A type breast cancer The ROC curve with the transfer of Luminal A types Bone of Breast Cancer is not shifted；
Fig. 2 is to verify that concentration methylates PITX1 and the AMOT joints that methylate for diagnosis differentiation Luminal A type breast cancer The accuracy rate with the transfer of Luminal A types Bone of Breast Cancer is not shifted；
Fig. 3 combines for the PTPN1 and SLIT2 that methylates that methylates in test set and is used to diagnose differentiation Luminal Type B mammary gland Cancer does not shift the ROC curve with the transfer of Luminal Type Bs Bone of Breast Cancer；
Fig. 4 is to verify that concentration methylates PTPN1 and the SLIT2 joints that methylate for diagnosis differentiation Luminal Type B mammary gland Cancer does not shift the accuracy rate with the transfer of Luminal Type Bs Bone of Breast Cancer；
Fig. 5 is that MYLK2, the EFEMP1 and SOSTDC1 that the methylates joints that methylate are methylated in test set for diagnosing differentiation Her-2 overexpression type breast cancer does not shift the ROC curve with the transfer of Her-2 overexpression types Bone of Breast Cancer；
Fig. 6 is that verification concentration methylates MYLK2, the EFEMP1 and SOSTDC1 that the methylates joints that methylate for diagnosing differentiation Her-2 overexpression type breast cancer does not shift the accuracy rate with the transfer of Her-2 overexpression types Bone of Breast Cancer；
Fig. 7 combines for the MYLK3 and SCARA5 that methylates that methylates in test set and is used to diagnose three negative type breast cancers of differentiation The ROC curve with three negative type breast cancers Bone tumours is not shifted；
Fig. 8 for verification concentration methylate MYLK3 and methylate SCARA5 Combining diagnosis distinguish three negative type breast cancers do not turn Move the accuracy rate with three negative type breast cancers Bone tumours.
Embodiment
The specific guarantor for introducing essentiality content of the present invention, but the present invention not being limited with this with reference to the accompanying drawings and examples Protect scope.
All breast cancer samples of this project are taken from September, 2014 to 2017 Nian9Yue Lai Hospital Attached to Nantong Univ. or south Tong Shi First People's Hospital or Nanjing drum tower hospital inspection are diagnosed as the patient of other malignant tumours of breast cancer and nonjoinder.It is all Sample is divided into Luminal A types, Luminal Type Bs, Her-2 overexpressions type and three negative types according to immunohistochemistry detection, various Molecular isoform is according to whether transfer is divided into the non-transfer group of breast cancer and Bone of Breast Cancer transfer group, and each case of Bone of Breast Cancer transfer group It is starting DISTANT METASTASES IN position to belong to bone.The non-transfer group of breast cancer and Bone of Breast Cancer transfer group pass through radionuclide bone scan (ECT), CT scan (CT), nuclear magnetic resonance (MRI), Positron emission computed tomography (PET-CT) And/or the inspection such as tissue biopsy confirms.The non-transfer group of breast cancer and Bone tumour group patient age compare without bright in each molecular isoform Significant difference is different, is comparable.Each group sample is finally half-and-half divided into test set at random and verification collects.
All sample packet information and sample number are as shown in the table after the diagnosis of above-mentioned goldstandard：
The collection of serum specimen：Patient limosis vein blood 5.0mL is gathered, centrifuged after natural coagulation (4000r/min, 2860 × g) serum is isolated after 7min, -80 DEG C of preservations are placed in, for detecting target methylated genes in serum.
Embodiment 1：Luminal A types Bone of Breast Cancer shifts
First, experiment sample and experimental method
1st, experiment sample
The non-transfer group of breast cancer, the test set of Luminal A type Bone of Breast Cancer transfer groups and verification in Luminal A types Collection.
2nd, serum Genome DNA extraction
Serum CRP extraction is carried out according to DNA Blood Midi Kit specifications, and every part of sample uses 0.8mL serum.Carry The DNA purity UV spectrophotometer measurings taken, absorbance A 260/A280 ratios carry out subsequent operation between 1.7-2.0. DNA content is calculated, -70 DEG C save backup.
3rd, the modification of DNA sulphite and pyrosequencing detection
DNA sulphite is modified：
1 μ g DNA are taken, the modification that methylates is carried out to genomic DNA according to DNA Methylation-Gold kit specifications Afterwards, save backup for -70 DEG C.Polymerase chain reaction：Using PCR to PITX1 and AMOT gene promoter methylations area in sample Domain is expanded.Reaction system includes 2 μ l, 10 × PCR buffer, 0.25U/ μ l Hot star of sulphite processing rear pattern plate Taq enzyme, 0.5mmol/L dNTP, each 1 μ l of upstream and downstream primer, 50 μ l of cumulative volume.Blank control is used as using distilled water.
Pyrosequencing detects：
(1) 45 μ l pcr amplification products are taken respectively into 96 Plate Low sample preparation plates A of PSQ, it is each to add 45 μ l knots Close buffer solution and 8 μ l are coated with the magnetic bead of streptavidin, 43 DEG C of vibration 25min.The magnetic bead for combining PCR product is transferred to denaturation In the plate B of buffer solution, double-stranded DNA is set fully to be denatured.Transfer is combined with the magnetic beads of single stranded PCR products to 150 μ l annealing buffers Plate C vortex oscillations washing 3min.
(2) sequencing primer hybridizes：The magnetic bead for combining single stranded PCR products is transferred in 50 μ l renaturation buffers, adds 10 μ l Sequencing primer, 75 DEG C of hybridization 7min.
(3) PSQ96 pyrosequencings instrument and sequencing reaction kit (Pyro Gold Reagents) are used, is detected respectively The base frequency of methylation sites in PITX1 and AMOT promoter regions.
Wherein PCR amplification primer and sequencing primer are as follows：
PITX1
Upstream 5 '-GGAAGGTATTTAGTATAGGTGAGTTTGA-3 '
Downstream 5 '-AAACCTTAATATTCACTACACTTTATC-3 '
5 '-GTGTTTATTTTGGATTGTTTAATT-3 ' are sequenced
AMOT
Upstream 5 '-TGAGTTAATATGAAAGAAGATAGTA-3 '
Downstream 5 '-TGATCTCTACATCTCAACTAATATAC-3 '
5 '-GTAGGTTTATTTAGGTT-3 ' are sequenced
Each methylation sites are analyzed by using pyrosequencing instrument allelic frequency analysis function.According to Equation below calculates the index that methylates of each gene promoter region, which can reflect the methyl of the gene promoter region Change degree：
4th, statistical procedures
Data are analyzed using SPSS 19.0.Measurement data is represented with mean value ± deviation, is examined using t, enumeration data It is expressed as a percentage, using χ2Examine, it is statistically significant for difference with P ＜ 0.05, and ROC curve is established, under calculated curve Area (area under the curve, AUC) and 95% credibility interval.Selection variables are returned with Logistic, are established back Return equation, produce one group of new variables Y.ROC curve analysis is carried out to new variables and each single index.
2nd, experimental result
1st, Luminal A types breast cancer does not shift and Bone tumour group methylates the journey that methylates of PITX1 and the AMOT that methylates Degree
In test set, the index that methylates of methylate in each sample PITX1 and the AMOT that methylates are measured respectively.With The non-transfer group of Luminal A type breast cancer is compared, and methylate PITX1 and first in Luminal A type Bone of Breast Cancer transfer group samples The index that methylates of base AMOT significantly raises, Bone tumour group methylate PITX1 and methylate AMOT methylate index difference Methylate (3.5 ± 0.4) times, (3.3 ± 0.5) times of index for non-transfer group.
2nd, methylate the PITX1 and AMOT that methylates the index that methylates be individually used for diagnosis distinguish Luminal A type mammary gland Cancer does not shift the ROC curve analysis with the transfer of Luminal A types Bone of Breast Cancer
The principle of ROC curve evaluation assessment：
The Basic Evaluation index of diagnostic test has susceptibility, specificity etc., comprehensive evaluation index have Youden indexes, ROC, AUC etc..Evaluation for diagnostic test, it is necessary first to the true group of sample to be tested is known by goldstandard.For down payment mark Accurately fixed disease group (equivalent to the Bone of Breast Cancer transfer group in the present invention) and healthy group are (equivalent to the breast cancer in the present invention Non- transfer group), the result detected using diagnostic test can be divided into following situation：
Positive (True Positive, TP)；Diagnostic test test positive (consistent with goldstandard result)；
Negative (True Negative, TN)；Diagnostic test is detected as negative (consistent with goldstandard result)；
False positive (False Positive, FP)：Diagnostic test test positive (inconsistent with goldstandard result)；
False negative (False Negative, FN)：Diagnostic test is detected as negative (inconsistent with goldstandard result).
It can be represented with following table：
Susceptibility=A/ (A+C) of diagnostic test；Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically Property can draw diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.High represent of susceptibility examines disease example Break few for negative number, rate of missed diagnosis is low；The number that healthy example is diagnosed as the positive by specific high representative is few, and misdiagnosis rate is low.
The curve that ROC curve is based on above-mentioned susceptibility and specificity is drawn out.With possible diagnosis in diagnostic test Dividing value calculates corresponding susceptibility and specificity as diagnostic points according to above table.Then, using susceptibility as ordinate, 1- specificity is abscissa, the susceptibility of each point during each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point Smoothed curve is obtained, which is ROC curve.Diagnostic points are set more much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, the size of its area under the curve AUC shows The size of diagnostic test accuracy.Intrinsic accuracy indexs of the area AUC as diagnostic test Authentic Assessment under ROC curve It has been be commonly recognized that, when AUC is 0.5, i.e., without diagnostic significance；AUC represents that accuracy rate of diagnosis is relatively low at 0.5~0.7；AUC exists When 0.7~0.9, represent that diagnostic accuracy is medium；During AUC ＞ 0.9, represent that diagnosis has higher accuracy.
The index that methylates that methylate PITX1 and the AMOT that methylates are drawn in SPSS19.0 is individually used for diagnosis differentiation Luminal A types breast cancer does not shift the ROC curve with the transfer of Luminal A types Bone of Breast Cancer, AUC is respectively 0.715, 0.707, there is medium accuracy.
3rd, methylate the PITX1 and AMOT that methylates the index Combining diagnosis model that methylates structure and for diagnose distinguish Luminal A types breast cancer does not shift the ROC curve analysis with the transfer of Luminal A types Bone of Breast Cancer
Methylate PITX1 using in test set sample and the index that methylates of AMOT of methylating (sets X as independent variable1=first The index that methylates of base PITX1, X2The index that the methylates of=AMOT that methylates), with group (i.e. according to the goldstandard sample category In Bone tumour group still non-transfer group) dependent variable is used as, to methylating PITX1 and AMOT is methylated in Luminal A type mammary gland Cancer does not shift the index that methylates shifted with Luminal A types Bone of Breast Cancer in sample and carries out dualistic logistic regression, obtains binary Logistic regression equation：Y=1/ [1+EXP (1.499X1+2.302X2-0.258)]；
The index that methylates of methylate in each sample PITX1 and the AMOT that methylates are substituted into the dualistic logistic regression side again Journey, you can the regressand value Y of each sample is obtained, using possible regressand value Y as diagnostic points, meter sensitivity and specificity, according to This draws ROC curve (as shown in Figure 1), and AUC 0.935, has higher accuracy.Calculated and tieed up according to the coordinate of ROC curve Mounting index=specificity+sensitivity -1, corresponding Y value distinguishes Luminal A types for that can carry out diagnosis when tieing up mounting index maximum The optimal cut-off values 0.238 (i.e. diagnostic threshold) of the non-transfer group of breast cancer and Bone tumour group.
4th, verification concentrates the index Combining diagnosis that methylates for verifying the PITX1 and AMOT that methylates that methylates to distinguish Luminal A types breast cancer does not shift the order of accuarcy with the transfer of Luminal A types Bone of Breast Cancer
Concentrated in verification, the above-mentioned recurrence mould of index substitution that methylates for the PITX1 and AMOT that methylates that each sample is methylated Type, the regressand value Y, Y for obtaining each sample are predicted as the transfer of Luminal A types Bone of Breast Cancer less than diagnostic threshold 0.238, are higher than The Luminal A type breast cancer that is predicted as of diagnostic threshold 0.238 does not shift, and accuracy is 95.5% (105/110), such as Fig. 2 institutes Show.
Embodiment 2：Luminal Type Bs Bone of Breast Cancer shifts
First, experiment sample and experimental method
1st, experiment sample
The non-transfer group of breast cancer, the test set of Luminal Type B Bone of Breast Cancer transfer groups and verification in Luminal Type Bs Collection.
2nd, serum Genome DNA extraction
Serum CRP extraction is carried out according to DNA Blood Midi Kit specifications, and every part of sample uses 0.8mL serum.Carry The DNA purity UV spectrophotometer measurings taken, absorbance A 260/A280 ratios carry out subsequent operation between 1.7-2.0. DNA content is calculated, -70 DEG C save backup.
3rd, the modification of DNA sulphite and pyrosequencing detection
DNA sulphite is modified：
1 μ gg DNA are taken, genomic DNA methylate repairing according to DNA Methylation-Gold kit specifications After decorations, -70 DEG C save backup.Polymerase chain reaction：Using PCR to PTPN1 and SLIT2 gene promoter methylations in sample Region is expanded.Reaction system includes 2 μ l, 10 × PCR buffer, 0.25U/ μ l Hot of sulphite processing rear pattern plate Star Taq enzymes, 0.5mmol/L dNTP, each 1 μ l of upstream and downstream primer, 50 μ l of cumulative volume.Blank control is used as using distilled water.
Pyrosequencing detects：
(1) 45 μ l pcr amplification products are taken respectively into 96 Plate Low sample preparation plates A of PSQ, it is each to add 45 μ l knots Close buffer solution and 8 μ l are coated with the magnetic bead of streptavidin, 43 DEG C of vibration 25min.The magnetic bead for combining PCR product is transferred to denaturation In the plate B of buffer solution, double-stranded DNA is set fully to be denatured.Transfer is combined with the magnetic beads of single stranded PCR products to 150 μ l annealing buffers Plate C vortex oscillations washing 3min.
(2) sequencing primer hybridizes：The magnetic bead for combining single stranded PCR products is transferred in 50 μ l renaturation buffers, adds 10 μ l Sequencing primer, 75 DEG C of hybridization 7min.
(3) PSQ96 pyrosequencings instrument and sequencing reaction kit (Pyro Gold Reagents) are used, is detected respectively The base frequency of methylation sites in PTPN1 and SLIT2 promoter regions.
Wherein PCR amplification primer and sequencing primer are as follows：
PTPN1
Upstream 5 '-AGCGGGTTAGAGGGTAGATGT-3 '
Downstream 5 '-TAGGTTTCTCCTCTCCCACATAT-3 '
5 '-TTTCCATTCATCCTAA-3 ' are sequenced
SLIT2
Upstream 5 '-TGAAGTTTTATTAGGTTGTGGAGGAGTA-3 '
Downstream 5 '-ATACCAAATATCCTATCCTTATCTTC-3 '
5 '-GTTTAAGGTTTATGATA-3 ' are sequenced
Each methylation sites are analyzed by using pyrosequencing instrument allelic frequency analysis function.According to Equation below calculates the index that methylates of each gene promoter region, which can reflect the methyl of the gene promoter region Change degree：
4th, statistical procedures
Data are analyzed using SPSS 19.0.Measurement data is represented with mean value ± deviation, is examined using t, enumeration data It is expressed as a percentage, using χ2Examine, it is statistically significant for difference with P ＜ 0.05, and ROC curve is established, under calculated curve Area (area under the curve, AUC) and 95% credibility interval.Selection variables are returned with Logistic, are established back Return equation, produce one group of new variables Y.ROC curve analysis is carried out to new variables and each single index.
2nd, experimental result
1st, Luminal Type Bs breast cancer does not shift and Bone tumour group methylates the journey that methylates of PTPN1 and the SLIT2 that methylates Degree
In test set, the index that methylates of methylate in each sample PTPN1 and the SLIT2 that methylates are measured respectively.With The non-transfer group of Luminal Type B breast cancer is compared, and methylate PTPN1 and first in Luminal Type B Bone of Breast Cancer transfer group samples The index that methylates of base SLIT2 significantly raises, and Bone tumour group methylates the index point of methylating of PTPN1 and the SLIT2 that methylates Not Wei non-transfer group methylate (2.9 ± 0.5) times, (3.4 ± 0.5) times of index.
2nd, methylate the PTPN1 and SLIT2 that methylates the index that methylates be individually used for diagnosis distinguish Luminal Type B mammary gland Cancer does not shift the ROC curve analysis with the transfer of Luminal Type Bs Bone of Breast Cancer
The principle of ROC curve evaluation assessment：
The Basic Evaluation index of diagnostic test has susceptibility, specificity etc., comprehensive evaluation index have Youden indexes, ROC, AUC etc..Evaluation for diagnostic test, it is necessary first to the true group of sample to be tested is known by goldstandard.For down payment mark Accurately fixed disease group (equivalent to the Bone of Breast Cancer transfer group in the present invention) and healthy group are (equivalent to the breast cancer in the present invention Non- transfer group), the result detected using diagnostic test can be divided into following situation：
Positive (True Positive, TP)；Diagnostic test test positive (consistent with goldstandard result)；
Negative (True Negative, TN)；Diagnostic test is detected as negative (consistent with goldstandard result)；
False positive (False Positive, FP)：Diagnostic test test positive (inconsistent with goldstandard result)；
False negative (False Negative, FN)：Diagnostic test is detected as negative (inconsistent with goldstandard result).
It can be represented with following table：
Susceptibility=A/ (A+C) of diagnostic test；Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically Property can draw diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.High represent of susceptibility examines disease example Break few for negative number, rate of missed diagnosis is low；The number that healthy example is diagnosed as the positive by specific high representative is few, and misdiagnosis rate is low.
The curve that ROC curve is based on above-mentioned susceptibility and specificity is drawn out.With possible diagnosis in diagnostic test Dividing value calculates corresponding susceptibility and specificity as diagnostic points according to above table.Then, using susceptibility as ordinate, 1- specificity is abscissa, the susceptibility of each point during each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point Smoothed curve is obtained, which is ROC curve.Diagnostic points are set more much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, the size of its area under the curve AUC shows The size of diagnostic test accuracy.Intrinsic accuracy indexs of the area AUC as diagnostic test Authentic Assessment under ROC curve It has been be commonly recognized that, when AUC is 0.5, i.e., without diagnostic significance；AUC represents that accuracy rate of diagnosis is relatively low at 0.5~0.7；AUC exists When 0.7~0.9, represent that diagnostic accuracy is medium；During AUC ＞ 0.9, represent that diagnosis has higher accuracy.
The index that methylates that methylate PTPN1 and the SLIT2 that methylates are drawn in SPSS 19.0 is individually used for diagnosis differentiation Luminal Type Bs breast cancer does not shift the ROC curve with the transfer of Luminal Type Bs Bone of Breast Cancer, AUC is respectively 0.723, 0.741, there is medium accuracy.
3rd, methylate the PTPN1 and SLIT2 that methylates the index Combining diagnosis model that methylates structure and for diagnostic region Luminal Type Bs breast cancer is divided not shift the ROC curve analysis with the transfer of Luminal Type Bs Bone of Breast Cancer
Methylate PTPN1 using in test set sample and the index that methylates of SLIT2 of methylating (sets X as independent variable1=first The index that methylates of base PTPN1, X2The index that the methylates of=SLIT2 that methylates), with group (i.e. according to the goldstandard sample Belong to Bone tumour group still non-transfer group) dependent variable is used as, to the PTPN1 and SLIT2 that methylates that methylates in Luminal Type Bs breast Gland cancer does not shift the index that methylates shifted with Luminal Type Bs Bone of Breast Cancer in sample and carries out dualistic logistic regression, obtains two Metalogic regression equation：Y=1/ [1+EXP (2.016X1+1.898X2-0.455)]；
The index that methylates of methylate in each sample PTPN1 and the SLIT2 that methylates are substituted into the dualistic logistic regression side again Journey, you can the regressand value Y of each sample is obtained, using possible regressand value Y as diagnostic points, meter sensitivity and specificity, according to This draws ROC curve (as shown in Figure 3), and AUC 0.942, has higher accuracy.Calculated and tieed up according to the coordinate of ROC curve Mounting index=specificity+sensitivity -1, corresponding Y value distinguishes Luminal Type Bs for that can carry out diagnosis when tieing up mounting index maximum The optimal cut-off values 0.310 (i.e. diagnostic threshold) of the non-transfer group of breast cancer and Bone tumour group.
4th, verification concentrates the index Combining diagnosis that methylates for verifying the PTPN1 and SLIT2 that methylates that methylates to distinguish Luminal Type B breast cancer does not shift the order of accuarcy with the transfer of Luminal Type Bs Bone of Breast Cancer
Concentrated in verification, the above-mentioned recurrence mould of index substitution that methylates for the PTPN1 and SLIT2 that methylates that each sample is methylated Type, the regressand value Y, Y for obtaining each sample are predicted as the transfer of Luminal Type Bs Bone of Breast Cancer less than diagnostic threshold 0.310, are higher than The Luminal Type B breast cancer that is predicted as of diagnostic threshold 0.310 does not shift, and accuracy is 93.7% (59/63), as shown in Figure 4.
Embodiment 3：Her-2 overexpression types Bone of Breast Cancer shifts
First, experiment sample and experimental method
1st, experiment sample
The non-transfer group of breast cancer in Her-2 overexpression types, Her-2 overexpression type Bone of Breast Cancer transfer groups test set and test Card collection.
2nd, serum Genome DNA extraction
Serum CRP extraction is carried out according to DNA Blood Midi Kit specifications, and every part of sample uses 0.8mL serum.Carry The DNA purity UV spectrophotometer measurings taken, absorbance A 260/A280 ratios carry out subsequent operation between 1.7-2.0. DNA content is calculated, -70 DEG C save backup.
3rd, the modification of DNA sulphite and pyrosequencing detection
DNA sulphite is modified：
1 μ gg DNA are taken, genomic DNA methylate repairing according to DNA Methylation-Gold kit specifications After decorations, -70 DEG C save backup.Polymerase chain reaction：MYLK2, EFEMP1 and SOSTDC1 gene in sample are opened using PCR The mover region that methylates is expanded.Reaction system includes sulphite processing 2 μ l, 10 × PCR buffer of rear pattern plate, 0.25U/ μ l Hot star Taq enzymes, 0.5mmol/L dNTP, each 1 μ l of upstream and downstream primer, 50 μ l of cumulative volume.
Pyrosequencing detects：
(1) 45 μ l pcr amplification products are taken respectively into 96 Plate Low sample preparation plates A of PSQ, it is each to add 45 μ l knots Close buffer solution and 8 μ l are coated with the magnetic bead of streptavidin, 43 DEG C of vibration 25min.The magnetic bead for combining PCR product is transferred to denaturation In the plate B of buffer solution, double-stranded DNA is set fully to be denatured.Transfer is combined with the magnetic beads of single stranded PCR products to 150 μ l annealing buffers Plate C vortex oscillations washing 3min.
(2) sequencing primer hybridizes：The magnetic bead for combining single stranded PCR products is transferred in 50 μ l renaturation buffers, adds 10 μ l Sequencing primer, 75 DEG C of hybridization 7min.
(3) PSQ96 pyrosequencings instrument and sequencing reaction kit (Pyro Gold Reagents) are used, is detected respectively The base frequency of methylation sites in MYLK2, EFEMP1 and SOSTDC1 promoter region.
Wherein PCR amplification primer and sequencing primer are as follows：
MYLK2
Upstream 5 '-GAGGGAAAGGATATGGTTGATT-3 '
Downstream 5 '-AACTCCACTCCATTCTCCC-3 '
5 '-AGTAAGTTATTTATTTGTTATTTG-3 ' are sequenced
EFEMP1
Upstream 5 '-GGTTTAGGTGGGGAGTATGATAG-3 '
Downstream 5 '-ACCAACAACCCAACTTTAACATAACC-3 '
5 '-TAATGAGGGGTTGAG-3 ' are sequenced
SOSTDC1
Upstream 5 '-GTAAAGGAGAAAGTTTGGTATATGG-3 '
Downstream 5 '-CAAAACTATACAAAAGTATCTCTCTCAAT-3 '
5 '-ATAATTTAATTGTTAGAGTTGAATA-3 ' are sequenced
Each methylation sites are analyzed by using pyrosequencing instrument allelic frequency analysis function.According to Equation below calculates the index that methylates of each gene promoter region, which can reflect the methyl of the gene promoter region Change degree：
4th, statistical procedures
Data are analyzed using SPSS 19.0.Measurement data is represented with mean value ± deviation, is examined using t, enumeration data It is expressed as a percentage, using χ2Examine, it is statistically significant for difference with P ＜ 0.05, and ROC curve is established, under calculated curve Area (area under the curve, AUC) and 95% credibility interval.Selection variables are returned with Logistic, are established back Return equation, produce one group of new variables Y.ROC curve analysis is carried out to new variables and each single index.
2nd, experimental result
1st, Her-2 overexpressions type breast cancer do not shift and Bone tumour group methylate MYLK2, methylating EFEMP1 and methylates The methylation of SOSTDC1
In test set, the MYLK2 that methylates in each sample is measured respectively, methylating EFEMP1 and methylates SOSTDC1's Methylate index.Compared with the non-transfer group of Her-2 overexpression type breast cancer, Her-2 overexpression type Bone of Breast Cancer transfer group samples In methylate MYLK2, the index that methylates for the EFEMP1 and SOSTDC1 that methylates that methylates significantly raise, respectively non-transfer group Methylate (3.7 ± 0.6) times, (2.6 ± 0.4), (3.1 ± 0.5) times of index.
2nd, methylate MYLK2, the index that methylates for the EFEMP1 and SOSTDC1 that methylates that methylates be individually used for diagnosis distinguish Her-2 overexpression type breast cancer does not shift the ROC curve analysis with the transfer of Her-2 overexpression types Bone of Breast Cancer
The principle of ROC curve evaluation assessment：
The Basic Evaluation index of diagnostic test has susceptibility, specificity etc., comprehensive evaluation index have Youden indexes, ROC, AUC etc..Evaluation for diagnostic test, it is necessary first to the true group of sample to be tested is known by goldstandard.For down payment mark Accurately fixed disease group (equivalent to the Bone of Breast Cancer transfer group in the present invention) and healthy group are (equivalent to the breast cancer in the present invention Non- transfer group), the result detected using diagnostic test can be divided into following situation：
Positive (True Positive, TP)；Diagnostic test test positive (consistent with goldstandard result)；
Negative (True Negative, TN)；Diagnostic test is detected as negative (consistent with goldstandard result)；
False positive (False Positive, FP)：Diagnostic test test positive (inconsistent with goldstandard result)；
False negative (False Negative, FN)：Diagnostic test is detected as negative (inconsistent with goldstandard result).
It can be represented with following table：
Susceptibility=A/ (A+C) of diagnostic test；Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically Property can draw diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.High represent of susceptibility examines disease example Break few for negative number, rate of missed diagnosis is low；The number that healthy example is diagnosed as the positive by specific high representative is few, and misdiagnosis rate is low.
The curve that ROC curve is based on above-mentioned susceptibility and specificity is drawn out.With possible diagnosis in diagnostic test Dividing value calculates corresponding susceptibility and specificity as diagnostic points according to above table.Then, using susceptibility as ordinate, 1- specificity is abscissa, the susceptibility of each point during each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point Smoothed curve is obtained, which is ROC curve.Diagnostic points are set more much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, the size of its area under the curve AUC shows The size of diagnostic test accuracy.Intrinsic accuracy indexs of the area AUC as diagnostic test Authentic Assessment under ROC curve It has been be commonly recognized that, when AUC is 0.5, i.e., without diagnostic significance；AUC represents that accuracy rate of diagnosis is relatively low at 0.5~0.7；AUC exists When 0.7~0.9, represent that diagnostic accuracy is medium；During AUC ＞ 0.9, represent that diagnosis has higher accuracy.
Methylate MYLK2, the index that methylates of methylate EFEMP1 and the SOSTDC1 that methylates are drawn in SPSS 19.0 It is individually used for diagnosis differentiation Her-2 overexpression type breast cancer and does not shift the ROC songs shifted with Her-2 overexpression types Bone of Breast Cancer Line, AUC are respectively 0.794,0.688,0.738, have relatively low or medium accuracy.
3rd, methylate MYLK2, the EFEMP1 and SOSTDC1 that methylates that methylates the index Combining diagnosis model that methylates structure Build and do not shift the ROC curve shifted with Her-2 overexpression types Bone of Breast Cancer for diagnosing differentiation Her-2 overexpression type breast cancer Analysis
Methylated using in test set sample MYLK2, the EFEMP1 and SOSTDC1 that methylates that methylates methylate index as Independent variable (sets X1The index that the methylates of=MYLK2 that methylates, X2The index that the methylates of=EFEMP1 that methylates, X3=methylate The index that methylates of SOSTDC1), using group (i.e. the sample belongs to Bone tumour group still non-transfer group according to goldstandard) as should Variable, the MYLK2 that methylates, the EFEMP1 and SOSTDC1 that methylates that methylates are not shifted in Her-2 overexpression type breast cancer and The index that methylates in Her-2 overexpression types Bone of Breast Cancer transfer sample carries out dualistic logistic regression, obtains dualistic logistic regression Equation：Y=1/ [1+EXP (1.342X1+1.401X2+1.345X3-2.035)]；
The index that methylates of the MYLK2 that methylates in each sample, the EFEMP1 and SOSTDC1 that methylates that methylates are substituted into again should Dualistic logistic regression equation, you can obtain the regressand value Y of each sample, using possible regressand value Y as diagnostic points, calculate sensitive Degree and specificity, draw ROC curve (as shown in Figure 5), AUC 0.950, has higher accuracy accordingly.According to ROC curve Coordinate calculate dimension mounting index=specificity+sensitivity -1, corresponding Y value is can carry out diagnosis differentiation when tieing up mounting index maximum The optimal cut-off values 0.308 (diagnostic threshold) of the non-transfer group of Her-2 overexpression type breast cancer and Bone tumour group.
4th, the index joint that methylates for verifying the MYLK2 that methylates, the EFEMP1 and SOSTDC1 that methylates that methylates is concentrated in verification Diagnosis distinguishes Her-2 overexpression type breast cancer and does not shift the order of accuarcy shifted with Her-2 overexpression types Bone of Breast Cancer
Concentrated in verification, the finger that methylates of each sample is methylated MYLK2, the EFEMP1 and SOSTDC1 that methylates that methylates Number substitutes into above-mentioned regression model, and the regressand value Y, Y for obtaining each sample are predicted as Her-2 overexpressions less than diagnostic threshold 0.308 Type Bone of Breast Cancer shifts, and the Her-2 overexpression type breast cancer that is predicted as higher than diagnostic threshold 0.308 does not shift, and accuracy is 96.4% (54/56), as shown in Figure 6.
Embodiment 4：Three negative type breast cancers Bone tumours
First, experiment sample and experimental method
1st, experiment sample
The non-transfer group of breast cancer, the test set of three negative type breast cancers Bone tumour groups and verification collection in three negative types.
2nd, serum Genome DNA extraction
Serum CRP extraction is carried out according to DNA Blood Midi Kit specifications, and every part of sample uses 0.8mL serum.Carry The DNA purity UV spectrophotometer measurings taken, absorbance A 260/A280 ratios carry out subsequent operation between 1.7-2.0. DNA content is calculated, -70 DEG C save backup.
3rd, the modification of DNA sulphite and pyrosequencing detection
DNA sulphite is modified：
1 μ gg DNA are taken, genomic DNA methylate repairing according to DNA Methylation-Gold kit specifications After decorations, -70 DEG C save backup.Polymerase chain reaction：Using PCR to MYLK3 and SCARA5 gene promoters methyl in sample Change region to be expanded.Reaction system includes 2 μ l, 10 × PCR buffer, 0.25U/ μ l Hot of sulphite processing rear pattern plate Star Taq enzymes, 0.5mmol/L dNTP, each 1 μ l of upstream and downstream primer, 50 μ l of cumulative volume.Blank control is used as using distilled water.
Pyrosequencing detects：
(1) 45 μ l pcr amplification products are taken respectively into 96 Plate Low sample preparation plates A of PSQ, it is each to add 45 μ l knots Close buffer solution and 8 μ l are coated with the magnetic bead of streptavidin, 43 DEG C of vibration 25min.The magnetic bead for combining PCR product is transferred to denaturation In the plate B of buffer solution, double-stranded DNA is set fully to be denatured.Transfer is combined with the magnetic beads of single stranded PCR products to 150 μ l annealing buffers Plate C vortex oscillations washing 3min.
(2) sequencing primer hybridizes：The magnetic bead for combining single stranded PCR products is transferred in 50 μ l renaturation buffers, adds 10 μ l Sequencing primer, 75 DEG C of hybridization 7min.
(3) PSQ96 pyrosequencings instrument and sequencing reaction kit (Pyro Gold Reagents) are used, is detected respectively The base frequency of methylation sites in MYLK3 and SCARA5 promoter regions.
Wherein PCR amplification primer and sequencing primer are as follows：
MYLK3
Upstream 5 '-TAGGGGAGGTTAAGAAAGTGTA-3 '
Downstream 5 '-AACTCCTTATCAATTCCTAACATACAAT-3 '
5 '-GGAGTAATGATGTAATGTGTAT-3 ' are sequenced
SCARA5
Upstream 5 '-AGGAATTAGGTAAGGTATGTTAGTA-3 '
Downstream 5 '-AAAACTCCAACCTATTCCAACCATACCTAC-3 '
5 '-GTTTTAAGTTTTGGTGTTTGATAT-3 ' are sequenced
Each methylation sites are analyzed by using pyrosequencing instrument allelic frequency analysis function.According to Equation below calculates the index that methylates of each gene promoter region, which can reflect the methyl of the gene promoter region Change degree：
4th, statistical procedures
Data are analyzed using SPSS 19.0.Measurement data is represented with mean value ± deviation, is examined using t, enumeration data It is expressed as a percentage, using χ2Examine, it is statistically significant for difference with P ＜ 0.05, and ROC curve is established, under calculated curve Area (area under the curve, AUC) and 95% credibility interval.Selection variables are returned with Logistic, are established back Return equation, produce one group of new variables Y.ROC curve analysis is carried out to new variables and each single index.
2nd, experimental result
1st, three negative type breast cancers do not shift and Bone tumour group methylates the methylation of MYLK3 and the SCARA5 that methylates
In test set, the index that methylates of methylate in each sample MYLK3 and the SCARA5 that methylates are measured respectively.With three The non-transfer group of negative type breast cancers is compared, and methylating in three negative type breast cancers Bone tumour group samples MYLK3 and methylates The index that methylates of SCARA5 significantly raises, Bone tumour group methylate MYLK3 and methylate SCARA5 methylate index difference Methylate (2.1 ± 0.3) times, (3.6 ± 0.7) times of index for non-transfer group.
2nd, methylate the MYLK3 and SCARA5 that methylates the index that methylates be individually used for diagnosis distinguish three negative type breast cancers Do not shift and analyzed with the ROC curve of three negative type breast cancers Bone tumours
The principle of ROC curve evaluation assessment：
The Basic Evaluation index of diagnostic test has susceptibility, specificity etc., comprehensive evaluation index have Youden indexes, ROC, AUC etc..Evaluation for diagnostic test, it is necessary first to the true group of sample to be tested is known by goldstandard.For down payment mark Accurately fixed disease group (equivalent to the Bone of Breast Cancer transfer group in the present invention) and healthy group are (equivalent to the breast cancer in the present invention Non- transfer group), the result detected using diagnostic test can be divided into following situation：
Positive (True Positive, TP)；Diagnostic test test positive (consistent with goldstandard result)；
Negative (True Negative, TN)；Diagnostic test is detected as negative (consistent with goldstandard result)；
False positive (False Positive, FP)：Diagnostic test test positive (inconsistent with goldstandard result)；
False negative (False Negative, FN)：Diagnostic test is detected as negative (inconsistent with goldstandard result).
It can be represented with following table：
Susceptibility=A/ (A+C) of diagnostic test；Specificity=D/ (B+D) of diagnostic test.By susceptibility and specifically Property can draw diagnosis sensitivity level and specific degree of the diagnostic test relative to goldstandard.High represent of susceptibility examines disease example Break few for negative number, rate of missed diagnosis is low；The number that healthy example is diagnosed as the positive by specific high representative is few, and misdiagnosis rate is low.
The curve that ROC curve is based on above-mentioned susceptibility and specificity is drawn out.With possible diagnosis in diagnostic test Dividing value calculates corresponding susceptibility and specificity as diagnostic points according to above table.Then, using susceptibility as ordinate, 1- specificity is abscissa, the susceptibility of each point during each diagnostic points and specificity point is marked in coordinate diagram, connection coordinate point Smoothed curve is obtained, which is ROC curve.Diagnostic points are set more much closeer, and obtained ROC curve is more smooth.
ROC curve is using each testing result as possible diagnosis dividing value, the size of its area under the curve AUC shows The size of diagnostic test accuracy.Intrinsic accuracy indexs of the area AUC as diagnostic test Authentic Assessment under ROC curve It has been be commonly recognized that, when AUC is 0.5, i.e., without diagnostic significance；AUC represents that accuracy rate of diagnosis is relatively low at 0.5~0.7；AUC exists When 0.7~0.9, represent that diagnostic accuracy is medium；During AUC ＞ 0.9, represent that diagnosis has higher accuracy.
The index that methylates that methylate MYLK3 and the SCARA5 that methylates are drawn in SPSS 19.0 is individually used for diagnostic region Three negative type breast cancers are divided not shift the ROC curve with three negative type breast cancers Bone tumours, AUC is respectively 0.644,0.809, tool There is relatively low or medium accuracy.
3rd, methylate the MYLK3 and SCARA5 that methylates the index Combining diagnosis model that methylates structure and for diagnostic region Point three negative type breast cancers do not shift and the analysis of the ROC curve of three negative type breast cancers Bone tumours
Methylate MYLK3 using in test set sample and the index that methylates of SCARA5 of methylating (sets X as independent variable1= Methylate the index that methylates of MYLK3, X2The index that the methylates of=SCARA5 that methylates), with group (i.e. according to the goldstandard sample Originally Bone tumour group still non-transfer group is belonged to) dependent variable is used as, to the MYLK3 and SCARA5 that methylates that methylates in three negative types breast Gland cancer does not shift carries out dualistic logistic regression with the index that methylates in three negative type breast cancers Bone tumour samples, obtains binary and patrols Collect regression equation：Y=1/ [1+EXP (1.775X1+1.236X2-0.398)]；
The index that methylates of methylate in each sample MYLK3 and the SCARA5 that methylates are substituted into the dualistic logistic regression side again Journey, you can the regressand value Y of each sample is obtained, using possible regressand value Y as diagnostic points, meter sensitivity and specificity, according to This draws ROC curve (as shown in Figure 7), and AUC 0.954, has higher accuracy.Calculated and tieed up according to the coordinate of ROC curve Mounting index=specificity+sensitivity -1, corresponding Y value distinguishes three negative type mammary gland for that can carry out diagnosis when tieing up mounting index maximum The optimal cut-off values 0.366 (i.e. diagnostic threshold) of the non-transfer group of cancer and Bone tumour group.
4th, verification concentrates the index Combining diagnosis that methylates for verifying the MYLK3 and SCARA5 that methylates that methylates to distinguish three feminine genders Type breast cancer does not shift the order of accuarcy with three negative type breast cancers Bone tumours
Concentrated in verification, the above-mentioned recurrence of index substitution that methylates for the MYLK3 and SCARA5 that methylates that each sample is methylated Model, the regressand value Y, Y for obtaining each sample are predicted as three negative type breast cancers Bone tumours less than diagnostic threshold 0.366, are higher than Three negative type breast cancers that are predicted as of diagnostic threshold 0.366 do not shift, and accuracy is 94.6% (53/56), as shown in Figure 8.
Embodiment 5：The diagnostic kit of diagnosis indication different subtype Bone of Breast Cancer transfer
1st, Luminal A types Bone of Breast Cancer transfer diagnosis indication kit
Include the PCR amplification primer of methylate PITX1 and the AMOT that methylates：Methylate the PCR amplification sense primer of PITX1 As shown in Sequence NO.1, PCR amplification anti-sense primer is as shown in Sequence NO.2；Methylate in the PCR amplification of AMOT Primer is swum as shown in Sequence NO.4, PCR amplification anti-sense primer is as shown in Sequence NO.5；Further include and methylate The Pyrosequencing primer of PITX1 and the AMOT that methylates, the Pyrosequencing primer such as Sequence NO.3 for the PITX1 that methylates Shown, the Pyrosequencing primer for the AMOT that methylates is as shown in Sequence NO.6.
Further include PCR amplification and enzyme and reagent needed for pyrosequencing.
2nd, Luminal Type Bs Bone of Breast Cancer transfer diagnosis indication kit
Include the PCR amplification primer of methylate PTPN1 and the SLIT2 that methylates；The methylate PCR amplification upstream of PTPN1 is drawn Thing is as shown in Sequence NO.7, and PCR amplification anti-sense primer is as shown in Sequence NO.8；Methylate the PCR amplification of SLIT2 Sense primer is as shown in Sequence NO.10, and PCR amplification anti-sense primer is as shown in Sequence NO.11；Further include and methylate The Pyrosequencing primer of PTPN1 and the SLIT2 that methylates, the Pyrosequencing primer such as Sequence NO.9 for the PTPN1 that methylates Shown, the Pyrosequencing primer for the SLIT2 that methylates is as shown in Sequence NO.12.
Further include PCR amplification and enzyme and reagent needed for pyrosequencing.
3rd, Her-2 overexpressions type Bone of Breast Cancer transfer diagnosis indication kit
PCR amplification primer and pyrosequencing including the MYLK2 that methylates, methylate EFEMP1 and the SOSTDC1 that methylates Primer；Methylate MYLK2 PCR amplification sense primer as shown in Sequence NO.13, PCR amplification anti-sense primer is such as Shown in Sequence NO.14, Pyrosequencing primer is as shown in Sequence NO.15；Methylate in the PCR amplification of EFEMP1 Primer is swum as shown in Sequence NO.16, PCR amplification anti-sense primer is as shown in Sequence NO.17, Pyrosequencing primer Methylate as shown in Sequence NO.18 SOSTDC1 PCR amplification sense primer as shown in Sequence NO.19, PCR expand Increase anti-sense primer as shown in Sequence NO.20, Pyrosequencing primer is as shown in Sequence NO.21.
Further include PCR amplification and enzyme and reagent needed for pyrosequencing.
4th, three negative type breast cancers Bone tumours diagnosis indication kit
Include the PCR amplification primer of methylate MYLK3 and the SCARA5 that methylates；The methylate PCR amplification upstream of MYLK3 is drawn Thing is as shown in Sequence NO.22, and PCR amplification anti-sense primer is as shown in Sequence NO.23；Methylate the PCR of SCARA5 Sense primer is expanded as shown in Sequence NO.25, PCR amplification anti-sense primer is as shown in Sequence NO.26；Further include first The Pyrosequencing primer of base MYLK3 and the SCARA5 that methylates, the Pyrosequencing primer such as Sequence for the MYLK3 that methylates The Pyrosequencing primer of NO.24, the SCARA5 that methylates such as Sequence NO.27.
Further include PCR amplification and enzyme and reagent needed for pyrosequencing.
In summary, it is a discovery of the invention that serum methylates, PITX1 and the AMOT that methylates can combine for diagnosing indication Luminal A types breast cancer whether Bone tumour, individual authentication concentrate diagnosis indication rate of accuracy reached more than 90%；Serum methylates The PTPN1 and SLIT2 that methylates can combine for diagnose indication Luminal Type Bs breast cancer whether Bone tumour, in individual authentication Concentrate diagnosis indication rate of accuracy reached more than 90%；Serum methylate MYLK2, methylate the EFEMP1 and SOSTDC1 that methylates can be with Joint be used to diagnosing indication Her-2 overexpression types breast cancer whether Bone tumour, concentrate diagnosis indication rate of accuracy reached in individual authentication More than 90%；Serum methylate MYLK3 and methylate SCARA5 can combine for diagnose indication three negative type breast cancers whether Bone tumour, diagnosis indication rate of accuracy reached more than 90% is concentrated in individual authentication.Diagnosed and indicated using above-mentioned serum methylated genes Not only accuracy is high for the transfer of different molecular hypotype Bone of Breast Cancer, but also testing cost is low, non-invasi, convenient and efficient, very big drop Low patient suffering and burden.
The effect of above-described embodiment is the essentiality content for specifically introducing the present invention, but those skilled in the art should know Protection scope of the present invention, should not be confined to the specific embodiment by road.
Sequence table
<110>Xue Shouhai
<120>Methylated genes composition and the purposes for preparing diagnosis three negative type breast cancers Bone tumour kits of indication
<160> 29
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ggaaggtatt tagtataggt gagtttga 28
<210> 2
<211> 27
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
aaaccttaat attcactaca ctttatc 27
<210> 3
<211> 24
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gtgtttattt tggattgttt aatt 24
<210> 4
<211> 25
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tgagttaata tgaaagaaga tagta 25
<210> 5
<211> 26
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
tgatctctac atctcaacta atatac 26
<210> 6
<211> 17
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gtaggtttat ttaggtt 17
<210> 7
<211> 21
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
agcgggttag agggtagatg t 21
<210> 8
<211> 23
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
taggtttctc ctctcccaca tat 23
<210> 9
<211> 16
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
tttccattca tcctaa 16
<210> 10
<211> 28
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
tgaagtttta ttaggttgtg gaggagta 28
<210> 11
<211> 26
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
ataccaaata tcctatcctt atcttc 26
<210> 12
<211> 17
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
gtttaaggtt tatgata 17
<210> 13
<211> 22
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
gagggaaagg atatggttga tt 22
<210> 14
<211> 19
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
aactccactc cattctccc 19
<210> 15
<211> 24
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
agtaagttat ttatttgtta tttg 24
<210> 16
<211> 23
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
ggtttaggtg gggagtatga tag 23
<210> 17
<211> 26
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
accaacaacc caactttaac ataacc 26
<210> 18
<211> 15
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
taatgagggg ttgag 15
<210> 19
<211> 25
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
gtaaaggaga aagtttggta tatgg 25
<210> 20
<211> 29
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
caaaactata caaaagtatc tctctcaat 29
<210> 21
<211> 25
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
ataatttaat tgttagagtt gaata 25
<210> 22
<211> 3
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
<210> 23
<211> 22
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
taggggaggt taagaaagtg ta 22
<210> 24
<211> 28
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
aactccttat caattcctaa catacaat 28
<210> 25
<211> 22
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 25
ggagtaatga tgtaatgtgt at 22
<210> 26
<211> 3
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 26
<210> 27
<211> 25
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 27
aggaattagg taaggtatgt tagta 25
<210> 28
<211> 30
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 28
aaaactccaa cctattccaa ccatacctac 30
<210> 29
<211> 24
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 29
gttttaagtt ttggtgtttg atat 24

Claims (10)

1. a kind of methylated genes diagnosis composition, it is characterised in that the MYLK3 and SCARA5 that methylates is formed by methylating.
2. the diagnosis composition described in claim 1 is preparing the diagnostic kit of diagnosis three negative type breast cancers Bone tumours of indication The application of aspect.
A kind of 3. diagnostic kit for being used to diagnose three negative type breast cancers Bone tumours of indication, it is characterised in that：Including methylating The PCR amplification primer of MYLK3 and the SCARA5 that methylates.
4. diagnostic kit according to claim 3, it is characterised in that：Methylate MYLK3 PCR amplification sense primer such as Shown in Sequence NO.22, PCR amplification anti-sense primer is as shown in Sequence NO.23.
5. diagnostic kit according to claim 3, it is characterised in that：Methylate the PCR amplification sense primer of SCARA5 As shown in Sequence NO.25, PCR amplification anti-sense primer is as shown in Sequence NO.26.
6. diagnostic kit according to claim 3, it is characterised in that：Further include to methylate and MYLK3 and methylate The Pyrosequencing primer of SCARA5.
7. diagnostic kit according to claim 6, it is characterised in that：The pyrosequencing of the MYLK3 that methylates draws Thing is as shown in Sequence NO.24.
8. diagnostic kit according to claim 6, it is characterised in that：The pyrosequencing of the SCARA5 that methylates draws Thing is as shown in Sequence NO.27.
9. according to any diagnostic kits of claim 3-8, it is characterised in that：Further include PCR amplification and pyrophosphoric acid is surveyed Enzyme and reagent needed for sequence.
A kind of 10. method for diagnosing three negative type breast cancers Bone tumours of indication, it is characterised in that include the following steps：
Step S1, gathers three negative type breast cancers patient's limosis vein bloods, serum is centrifuged out after natural coagulation；
Step S2, extracts serum STb gene, through methyl in PCR amplification, the modification of DNA sulphite and pyrosequencing measure STb gene Change the index that methylates of MYLK3 and the SCARA5 that methylates, use X successively1、X2Represent；
Step S3, by X1、X2Value substitutes into dualistic logistic regression equation Y=1/ [1+EXP (1.775X1+1.236X2- 0.398)] obtain Y value, Y value are less than 0.366 and indicate that Bone tumour occurs for the patient with breast cancer, and Bone tumour does not occur more than 0.366 indication.
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