CN108588226A - Detect the miRNA combination of breast cancer patients with brain transfer and the kit containing the combination - Google Patents
Detect the miRNA combination of breast cancer patients with brain transfer and the kit containing the combination Download PDFInfo
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Abstract
The invention discloses the miRNA combination of detection breast cancer patients with brain transfer and containing the kit of the combination, the miRNA combination includes one or more of miR 10b and miR 200a, 122 miR, miR 21, miR 181c.The kit includes forward and reverse primer, cerebrospinal fluid RNA extraction systems, reverse transcription reagents, PCR amplification reagent and the cDNA mixing positive reference substances of forward and reverse primer of above-mentioned miRNA combination, outer ginseng U6 RNA.The present invention is new breast cancer patients with brain metastatic marker object with one group of miRNAs, will improve single labelled object and is difficult to overcome low specificity and muting sensitivity caused by Tumor Heterogeneity, effectively improves breast cancer patients prognosis effect appraisal, to guiding clinical treatment guide.Above-mentioned miRNA combination is prepared into the kit or biochip detected for Prognosis in Breast Cancer using fluorescent quantitation/digital pcr method by the present invention, breast cancer patients prognosis effect appraisal can be effectively improved to guiding clinical treatment guide to avoid due to low difference caused by Tumor Heterogeneity and low sensitivity.
Description
Technical field
The invention belongs to Medical Molecular Biology technical fields, and in particular to the miRNA combination of detection breast cancer patients with brain transfer
And the kit containing the combination.
Background technology
Breast cancer is the first malignant tumour of modern female morbidity, and its death rate is occupied for a long time in all tumor mortalities of women
First five position.Breast cancer incidence is still gradually rising in worldwide, it has been reported that by the end of 2015, incidence
Account for about the 27% of women whole malignant tumour.Breast cancer is the cancer for being easier to occur brain metastes, accounts for brain metastases second, about
10% ~ 16%.The incidence of China's breast cancer is also increased with the growth rate of 3%-4%, higher than global similar disease average level.According to
Report, the patient with breast cancer of about 25%-40% may recur or shift.Wherein Symptomatic metastatic encephaloma accounts for all mammary gland
The 6%-16% of metastasis of cancer patient.Currently used is pathological diagnosis, although pathological diagnosis specificity is high, has wound
Property, it is not easy for patients to accept, and there are certain difficulty for materials.In addition, breast cancer patients with brain transfer is invisible extremely strong, in early days without any
Sign has missed golden hour window when being made a definite diagnosis by pathology, influence the prognosis of patient, causes the survival of patients time notable
Shorten.In addition, computed tomography（CT）, magnetic resonance imaging（MRI）Equal Imaging Technologies, which are diagnosed, to be also difficult to find to dislike
Property tumour brain metastes early stage minimal disease, and patient by economic condition restrict etc. factors fail to receive continuous imageological examination.
Since 2016, molecule parting had just gradually been applied to the diagnosis of nervous system neoplasm, in WHO central nerve neuromas in 2016
The genetic test that the relevant molecular genetics of multinomial nervous system neoplasm changes is proposed in classification.Therefore, it is badly in need of better mammary gland
Cancer brain metastes risk assessment means solve the above problems.
In recent years, many scholars are dedicated to including some genetic markers, Serological markers and cytological marker etc. to carry out
The assessment of tumour individual relapse and metastasis risk, but since the specificity and sensibility of these indexs are different, with RNA conducts
The appearance of tumor markers prompts during tumor development, and RNA is appeared earlier than receptor, albumen etc., and this is to seeking
Look for the marker of effective prediction malignant tumour brain metastes particularly important.
Microrna（miRNA）It is a kind of small nuclear rna, is about 22 nucleotide, not coding protein, but can be with courier
RNA（mRNA）In conjunction with and playing an important role in gene regulation.MiRNA almost participates in the movable links of regulating cell.Greatly
Quantity research shows that miRNA plays a part of promotion sensitivity gene or tumor suppressor gene in tumour, and can adjust multiple target genes simultaneously,
Participate in the occurrence and development of tumour.MiRNA not only in the tissue, also in body fluid（Blood, urine, cerebrospinal fluid）In be stabilized.Brain
Spinal fluid is directly connected with maincenter, participates in the metabolism of brain and spinal cord, when lesion occurs for central nervous system, miRNA can be from disease
Become tissue and is released into cerebrospinal fluid.Therefore there are the miRNA of a large amount of brains and myeloid tissue source in cerebrospinal fluid, and expression, which changes, to fill
Divide the lesion situation of reflection maincenter.Currently, although the cytological analysis of cerebrospinal fluid also can be shown that, there are cancer cell, CSF's is thin
Born of the same parents' sensitivity for analysis is relatively low, is non-quantitation, technically challenging.
In view of the present situation waited low to breast cancer patients with brain transfer detection sensitivity at present.In existing patent, application No. is
The patent of CN200680036598.3 discloses miR-10b in the cancer cell for detecting subject by using micro probe array method,
Tens kinds of miRNA combinations such as miR-21, miR-122 determine Prognosis in Breast Cancer situation, but are not described the miRNA groups of the detection
It whether closes with brain metastes correlation, is only to determine whether patient has the risk to suffer from breast cancer；Application No. is CN201680076769.4
Patent disclose by detecting miR-181c in blood, predict cancer brain metastes, but with the deep development of cancer research, by
Exist in Tumor Heterogeneity, depend merely on a certain marker and go to diagnose the illness, influence factor is too many, is not so good as the value of Combining diagnosis
Greatly；In addition, it has been reported that miR-181c in serum, the miR-21 malignant change that participates in tumour are related to transfer, and application number：
CN201510090768.7, which discloses miR-21, can indicate early diagnosing mammary cancer, but for breast cancer patients with brain transfer, due to blood
MiRNA gene expression abundances in slurry/serum are generally relatively low, and source is unknown, can not accurate judgement disease using serum or blood plasma
People whether there is brain metastes.
Invention content
It is an object of the invention to overcome the above problem, provide that a kind of preparation method is simple, accuracy is high, applicability is wide
Detect the miRNA combination of breast cancer patients with brain transfer and the kit containing the combination.
Breast cancer patients with brain transfer is studied in detail in inventor, and early period is examined using upper Haikang at biological miRNA chip of expression spectrum
Surveyed 10 breast cancer patients with brain transfer patients, 10 therewith age-sex match the table of the non-diverting crowd's cerebrospinal fluid miRNA of breast cancer
Up to situation, a series of candidate miRNA of differential expression multiples at 2.5 times or more is filtered out, further in large sample breast cancer patients with brain
Transfer case compares the expression for verifying candidate miRNA in crowd by RT-qPCR methods with non-brain metastes, finds 5 miRNA
It is proportionate, therefore the expression by detecting miRNA in patient with breast cancer's cerebrospinal fluid, can predict with breast cancer patients with brain transfer
The risk of patient with breast cancer's brain metastes is ready to carry out corresponding prevention and remedy measures.
To achieve the above object, the present invention provides the miRNA combinations of detection breast cancer patients with brain transfer, including miR-10b points
One or more of son and miR-200a, miR-122, miR-21, miR-181c molecule, the sequence of the miRNA molecule
Number and sequence is shown in Table the expression trend of 1, miRNA and outer ginseng gene is shown in Table 2.
Kit for detecting breast cancer patients with brain transfer, including the miRNA combination that above-mentioned detection breast cancer patients with brain shifts is just
Reverse primer, forward and reverse primer of outer ginseng U6 RNA, cerebrospinal fluid RNA extraction systems, reverse transcription reagents, PCR amplification reagent and
CDNA mixing positive reference substances.
Preferably, nucleotide sequence such as SEQ ID NO.1, the SEQ of forward and reverse primer of the miRNA and outer ginseng U6 RNA
ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID
NO.8, SEQ ID NO.9, SEQ ID NO.10, shown in SEQ ID NO.11 and SEQ ID NO.12（It is shown in Table 4）.
Preferably, the preparation method of the cDNA mixing positive reference substance is：50 breast cancer patients with brain are shifted into crowd's brain ridge
After the total serum IgE mixing of liquid sample extraction, cDNA mixing positive reference substances, the feasibility as detection primer series are obtained after reverse transcription
Positive quality control product.
Preferably, the cerebrospinal fluid RNA extraction systems include lysate, HiBind RNA columns, RNA wash
Buffer I, RNA wash buffer II and collecting pipe.
Preferably, the reverse transcription reagents are 5 × iScript Reaction Mix, iScript Reverse
Transcriptase and RNase free dH2O。
Preferably, the PCR amplification reagent is 5 × FastStart Universal SYBR Green Master
(ROX) and RNase free dH2O。
When being detected breast cancer patients with brain transfer with the kit of the present invention, miR-10b molecules are detected first, then again to other
MiRNA molecule is detected, and whether above-mentioned detection breast cancer three kinds of the miRNA differential expression situations point of brain metastes patient occurs：
（1）One or more of miR-10b molecules and miR-200a, miR-122, miR-21, miR-181c molecule are positive
Expression, illustrates that brain metastes occurs in patient at this time;（2）MiR-10b feminine genders express, but miR-200a, miR-122, miR-21,
Two or more equal positive expressions in miR181c molecules, illustrate that brain metastes occurs in patient at this time;（3）miR-10b、
The negative expression of miR-200a, miR-122, miR-21, miR-181c molecule, illustrates that patient does not occur brain metastes at this time, it is proposed that
Periodic review.
Preferably, its operation instructions is also contained in kit of the present invention.
The beneficial effects of the invention are as follows：
(1) one group screened from cerebrospinal fluid is verified in breast cancer patients with brain transfer and non-brain metastes patient by chip and RT-qPCR
Between differential expression miRNA, the miRNA combination filtered out can assess patient with breast cancer's cancer cell brain metastes proliferation and
Invasive ability, further guiding clinical treatment.
(2) one group of miRNAs will be filtered out as the molecular marked compound of breast cancer detection prognosis, prepared by using glimmering
Light is quantitative/digital pcr method comes the kit detected for Prognosis in Breast Cancer or biochip, can to avoid due to tumour it is heterogeneous
Property caused by low difference and low sensitivity, breast cancer patients prognosis effect appraisal can be effectively improved and referred to guiding clinical treatment
South.The detection error that breast cancer tumour heterogeneity can be avoided to bring improves the accuracy of diagnosis.
(3) use one group of miRNAs new breast cancer patients with brain metastatic marker object, the marker institute for improving single is difficult
To overcome low difference and low sensitivity caused by Tumor Heterogeneity, can effectively improve breast cancer patients prognosis effect appraisal from
And guiding clinical treatment guide.
Description of the drawings
Fig. 1 is the ROC (Receiver of cerebrospinal fluid miR-10b independent Diagnosis of Breast cancer brain metastes and non-brain metastes
Operation Characterization) curve；
Fig. 2 is the ROC curve of cerebrospinal fluid miR-200a independent Diagnosis of Breast cancer brain metastes and non-brain metastes；
Fig. 3 is the ROC curve of cerebrospinal fluid miR-122 independent Diagnosis of Breast cancer brain metastes and non-brain metastes；
Fig. 4 is the ROC curve of cerebrospinal fluid miR-21 independent Diagnosis of Breast cancer brain metastes and non-brain metastes；
Fig. 5 is the ROC curve of cerebrospinal fluid miR-181c independent Diagnosis of Breast cancer brain metastes and non-brain metastes；
Fig. 6 is the ROC curve of five miRNA Combining diagnosis breast cancer patients with brain transfers and non-brain metastes.
Specific implementation mode
Below by specific embodiment, the invention will be further described.
Embodiment 1
Accuracy Verification of the miRNA small molecule markers combination to indication breast cancer patients with brain transfer
One, material and method
1. general information
All research objects are in November, 2016 in March, 2018 inpatient（Han nationality Chinese）, totally 116.According to
Whether there is or not brain metastes to be divided into two groups by seminar patient：（1）Breast cancer patients with brain transfer group（Seminar）：48, age 25-71 Sui；It receives
Enter standard：1）Patient with breast cancer is diagnosed as through iconography and pathological examination and without other systems tumour；2）Cerebrospinal fluid is collected
It is preceding not undergo surgery and chemicotherapy；Exclusion criteria：1）：Exclude other systems tumour, diabetes, hypertension and cardiovascular and cerebrovascular disease
Disease；2）It excludes to merge autoimmune disease and severe cardiac, lung, Liver and kidney function obstacle person；3）It excludes not connect before cerebrospinal fluid is collected
It is performed the operation and chemotherapy radiotherapy.（2）Non- brain metastes group（Control group）：68, age 25-75 Sui.Control group includes through pathological tissue
Learn or cytology be diagnosed as breast cancer but non-diverting patient and Non-cancerous patient with nervous system disease include Parkinson, epilepsy,
The Non-cancerous patient with nervous system disease such as migraine, myelitis, dementia, neuropathy, Benign Cerebellar lesion.Collection crowd's faces
Bed data and CSF sample.Seminar and control group no difference of science of statistics (the P ＞ 0.05) between gender, age.
2. key instrument equipment
Vertical pressure steam sterilizer（LDZM-60KCS, Shenan Medical Appliances Factory, Shanghai）, low speed centrifuge（The good literary instrument in Anhui
Co., Ltd）, desk centrifuge (German Eppendorf companies), water-bath（Guo Hua Co., Ltds）, PCR instrument（Bio-Rad）,
Fluorescence quantitative PCR instrument（Bio-Rad-CFX-96）, nucleic acid-protein detector（Thermo Science companies of the U.S.）, electrophoresis apparatus
（DYY-BC types, Beijing Liuyi Instrument Factory）, scroll machine（Shanghai Kang Hua biochemical instrument Manufacturing Co., Ltd）.
3. cerebrospinal fluid acquires
Breast cancer patients with brain transfer patient carries out CSF sample acquisition after making a definite diagnosis, control group carries out CSF sample acquisition after entering group.
Acquisition method is as follows, and waist leaves and takes cerebrospinal fluid 2ml after wearing, and is put into RNase free EP pipes；After leaving and taking CSF sample 6h it
Interior micro- centrifuge (3000 revs/min) 10min removes cells in sample and fragment, extracts all clear liquids in upper layer point
It is not put into RNase free EP pipes, each pipe at least puts 200ul, preserves to -80 DEG C of refrigerators.In the feelings using glassware
Under condition, hot air sterilization please be use, or in 0.1% DEPC solution, 12h is impregnated at 37 DEG C, it is wet then to carry out high temperature and pressure again
Heat sterilization（121 DEG C, 30min）.
4. cerebrospinal fluid Total RNAs extraction
（1）Pretreatment：The clear area of less RNA enzyme at room temperature, thaws spinal fluid samples；
（2）The cerebrospinal fluid of 100 μ L is added in the centrifuge tube of 1.5 ml, 1 ml lysates are added（RNA-Solv-
Reagent）, and 2-3min is stood at room temperature.
（3）Separation phase：200 μ L chloroforms are added in every 1 ml RNA-Solv-Reagent, cover tightly pipe lid, vortex 15s is set
In 10 min on ice, 4 DEG C, 12000 rpm centrifugations 15min.
（4）HiBind RNA columns are placed in 2 ml centrifuge tubes, transfer every time is no more than the mixed liquor of 700 ul extremely
In HiBind RNA columns, room temperature, 10000 rpm centrifuge 1min.
（5）Collection liquid in pipe is abandoned, 80% ethyl alcohol of 1 ml is added in centrifugal column column, at room temperature 7500rpm centrifugations 5
Min abandons collection liquid in pipe.
（6）Repeat step（4）, until all mixed liquors are centrifuged and are attached on HiBind RNA columns.
（7）HiBind RNA columns are placed in a 2 new ml collecting pipes, in addition 300 μ L RNA wash
Buffer I, room temperature, 10000 rpm centrifuge 1min, discard efflux.
（8）Pillar is washed once with 500 μ L RNA wash buffer II, room temperature, 10000rpm centrifuge 1min, abandon
Efflux.
（9）Repeat step（8）Once, centrifuge and then be placed in a new empty collecting pipe, at room temperature with maximum (top) speed (>=
2min 12000rpm) is centrifuged, the HiBind matrix made is completely dried.
（10）The dissolving of RNA：Centrifugal column is placed on to the collecting pipe of a 1.5 new ml, the center along centrifugal column film adds
Enter the RNase free dH of 15-30 μ L2O elutes pillar.
5. sample rna quality control
RNA concentration mensurations：RNA gears are selected on spectrophotometer, are returned to zero with 100 μ L DEPC water, and select dilution appropriate
Concentration, record measure the data obtained（The OD value of RNA concentration, A260 and A280, A260/A280, A260/A230 etc.）, with
Just experiment can be monitored with RNA mass.It should repeatedly measure, be averaged, OD（A260/A280）It is worth the trouble in 1.8-2.0
Person can be used for carrying out next step experiment；OD values illustrate that there are organic matters, OD values to illustrate that RNA has more than 2.0 in extract less than 1.8
Degradation.
6. the integrity detection of total serum IgE
1% denaturing formaldehyde agarose gel electrophoresis detaches the rRNA (rRNA) in total serum IgE, maximum two band 28s
The brightness of RNA and 18s RNA is substantially than being 2:When 1, illustrates that the integrality of RNA is preferable, do not degrade.
7. miRNA reverse transcriptions
The synthesis of first chain cDNA：Reverse transcription reaction system is shown in Table 5.It is sequentially added in the PCR pipe of no RNA enzyme each in table 5
Component, after sample mixes well, centrifugation makes liquid as tube bottom, and first step reaction, 65 DEG C of 5 min, 4 DEG C of drops are carried out in PCR instrument
Temperature, 42 DEG C of 60 min, 70 DEG C of 15 min, 4 DEG C of preservations.It is -20 DEG C long-term to protect after first chain cDNA of synthesis takes out
It deposits spare.
5 reverse transcription reaction system of table
8, Q-PCR analyzes the differential expression of correlation miRNA
U6 genes do internal reference, carry out PCR amplification detection template quality, and reaction system is shown in Table 6.Q-PCR reaction detection instrument is Bio-
The CFX96 systems of Rad companies, reaction condition are：94 DEG C of pre-degeneration 5 min, 94 DEG C of denaturation 15 s, 55 DEG C of annealing 30 s, 72 DEG C
Extend 30 s, totally 40 cycles, 72 DEG C of 10 min of overall elongation, solubility curve is between 65 ~ 95 DEG C.Q-PCR uses relative quantification
Method, gene expression analysis software are that Bio-Rad CFX manager 2.0 in triplicate per a sample utilize 2-△△CtMethod into
Row analysis.
6 Q-PCR reaction systems of table (10 μ L)
9, statistical analysis
Analyzed and mapped using Prism5.0 statistical softwares, between two groups the comparison of mean examined using t, enumeration data
Compare and uses Chi-square Test.The expression of two groups of cerebrospinal fluid miRNAs is indicated using mean ± standard deviation, two groups of data
Carry out F inspections.The diagnosis capability of miRNA is analyzed with Receiver operating curve (ROC) and area under a curve (AUC).
Logistic regression models are established to predict that 5 kinds of miRNA shift Combining diagnosis ability to breast cancer patients with brain.
Two, result
Breast cancer patients with brain transfer group（48）And control group（68）5 miRNAs in Cerebrospinal Fluid in Patients sample（miR-10b、miR-
200a、miR-122、miR-21、 miR-181c）In differential expression.
1, the relative expression quantity of each group miR-10b, miR-200a, miR-122, miR-21, miR-181c
Group difference it was found that, 5 kinds of tumour brain metastes marker levels are significantly higher than control group (P ＜ in seminar's cerebrospinal fluid
0.01), miR-10b raises 1-3 times in seminar's sample, and miR-200a raises 3-5 times, and miR-122 raises 2-5 times, miR-
181c raises 3-6 times, and miR-21 up-regulations are the most notable, raises 10-15 times.Homogeneity test of variance（F is examined）And P values（It is shown in Table 3）.
The expression of 5 miRNAs compares in 3 two groups of cerebrospinal fluid of table（x±s）
2, the ROC curve of miRNA combination is analyzed in cerebrospinal fluid
In ROC curve analysis, area is bigger under ROC curve, and diagnostic value is bigger.When AUC is 0.5, i.e., without diagnostic significance；
AUC indicates that accuracy rate of diagnosis is relatively low at 0.5~0.7；AUC indicates that diagnostic accuracy is medium at 0.7~0.9；AUC ＞
When 0.9, indicate that diagnosis has higher accuracy.
Based on miR-10b, miR-200a, miR-122, miR-181c, miR-21 in seminar and control group cerebrospinal fluid
Expression,（ROC）Tracing analysis is respectively：(a) area under the curve of miR-10b（AUC）It is 0.9332（Standard is mistaken for
0.02560,95%CI, 0.8830-0.9834）；(b) AUC of miR-200a is 0.8454（Standard is mistaken for 0.03875,95%CI,
0.7694-0.9214）；（c）The AUC of miR-122 is 0.8776（Standard is mistaken for 0.03535,95%CI, 0.8083-
0.9469）；（d）The AUC of miR-21 is 0.9152（Standard is mistaken for 0.02869,95%CI, 0.8590-0.9714）；（e）miR-
The AUC of 181c is 0.9648（Standard is mistaken for 0.014,95%CI, 0.9360-0.9936）（See Fig. 1-5）.
Further verification proof joint miR-10b, miR-200a for concentrating in independent sample, miR-122, miR-21,
Accuracy of the miR-181c for Diagnosis of Breast cancer brain metastes is up to 90% or more, has higher clinical value（See Fig. 6）.
Three, conclusion
When the kit of the present invention is detected breast cancer patients with brain transfer, miR-10b molecules are detected first, then again to other
MiRNA molecule is detected, and whether above-mentioned detection breast cancer three kinds of the miRNA differential expression situations point of brain metastes patient occurs：
（1）One or more of miR-10b molecules and miR-200a, miR-122, miR-21, miR-181c molecule are positive
Expression, illustrates that brain metastes occurs in patient at this time；（2）MiR-10b feminine genders express, but miR-200a, miR-122, miR-21,
Two or more equal positive expressions in miR181c molecules, illustrate that brain metastes occurs in patient at this time.（3）miR-10b、
The negative expression of miR-200a, miR-122, miR-21, miR-181c molecule, illustrates that patient does not occur brain metastes at this time, it is proposed that
Periodic review.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention god.
1 miRNA sequence of table
The expression trend and reference gene of 2 miRNA of table
4 miRNA qPCR primer sequences of table
Claims (8)
1. detecting the miRNA combination of breast cancer patients with brain transfer, which is characterized in that including miR-10b molecules and miR-200a,
One or more of miR-122, miR-21, miR-181c molecule.
2. the kit for detecting breast cancer patients with brain transfer, which is characterized in that turn including detecting breast cancer patients with brain described in claim 1
Forward and reverse primer of the miRNA combination of shifting, forward and reverse primer of outer ginseng U6 RNA, cerebrospinal fluid RNA extraction systems, reverse transcription examination
Agent, PCR amplification reagent and cDNA mixing positive reference substances.
3. being used to detect the kit of breast cancer patients with brain transfer according to claim 2, which is characterized in that the miRNA and outer
Join nucleotide sequence such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, the SEQ ID of forward and reverse primer of U6 RNA
NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID
Shown in NO.10, SEQ ID NO.11 and SEQ ID NO.12.
4. being used to detect the kit of breast cancer patients with brain transfer according to claim 2, which is characterized in that the cDNA mixing pair
Preparation method according to product is：After the total serum IgE mixing that breast cancer patients with brain is shifted to Cerebrospinal Fluid in Patients sample extraction, reverse transcription simultaneously dilutes,
Obtain cDNA mixing positive reference substances.
5. being used to detect the kit of breast cancer patients with brain transfer according to claim 2, which is characterized in that the cerebrospinal fluid RNA
Extraction system includes lysate, HiBind RNA columns, RNA wash buffer I, RNA wash buffer II and receipts
Collector.
6. being used to detect the kit of breast cancer patients with brain transfer according to claim 2, which is characterized in that the reverse transcription reagents
For 5 × iScript Reaction Mix, iScript Reverse Transcriptase and RNase free dH2O。
7. being used to detect the kit of breast cancer patients with brain transfer according to claim 2, which is characterized in that the PCR amplification examination
Agent is 5 × FastStart Universal SYBR Green Master (ROX) and RNase free dH2O。
8. the miRNA combination of detection breast cancer patients with brain transfer according to claim 1, which is characterized in that above-mentioned detection breast cancer
Whether the miRNA differential expression situation point three kind of brain metastes patient is occurred：（1）MiR-10b molecules and miR-200a, miR-
122, the equal positive expression of one or more of miR-21, miR-181c molecule illustrates that brain metastes occurs in patient at this time;（2）
MiR-10b feminine genders are expressed, but two or more equal sun in miR-200a, miR-122, miR-21, miR181c molecule
Property expression, illustrate that brain metastes occurs in patient at this time;（3）MiR-10b, miR-200a, miR-122, miR-21, miR-181c points
Sub negative expression, illustrates that patient does not occur brain metastes, it is proposed that periodic review at this time.
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| CN110607366A (en) * | 2019-06-18 | 2019-12-24 | 中山大学孙逸仙纪念医院 | Kit for predicting hormone receptor positive breast cancer preoperative chemotherapy sensitivity |
| CN113866415A (en) * | 2021-10-13 | 2021-12-31 | 戴勇 | Diagnostic and prognostic markers for brain metastasis of breast cancer |
| CN113881769A (en) * | 2021-06-03 | 2022-01-04 | 南京世和基因生物技术股份有限公司 | Application of cerebrospinal fluid cfDNA in diagnosis and treatment of breast cancer metastasis |
| WO2022036869A1 (en) * | 2020-08-17 | 2022-02-24 | 中山大学孙逸仙纪念医院 | Method and system for predicting pathological complete remission probability of breast cancer after neoadjuvant chemotherapy |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012068288A2 (en) * | 2010-11-16 | 2012-05-24 | The Brigham And Women's Hospital, Inc. | Diagnosing and monitoring cns malignancies using microrna |
| CN103866018A (en) * | 2005-08-01 | 2014-06-18 | 俄亥俄州立大学研究基金会 | MicroRNA-Based Methods and Compositions For the Diagnosis, Prognosis and Treatment of Breast Cancer |
| EP2940151A1 (en) * | 2014-05-02 | 2015-11-04 | Ruprecht-Karls-Universität Heidelberg | Circulating miRNAs as early detection marker and prognostic marker |
| CN107250378A (en) * | 2015-02-24 | 2017-10-13 | 株式会社遗传科技 | Diagnosis, prevention and the treatment method of the brain metastes of cancer and for the drug delivery system by blood-brain barrier |
-
2018
- 2018-06-22 CN CN201810647475.8A patent/CN108588226A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103866018A (en) * | 2005-08-01 | 2014-06-18 | 俄亥俄州立大学研究基金会 | MicroRNA-Based Methods and Compositions For the Diagnosis, Prognosis and Treatment of Breast Cancer |
| WO2012068288A2 (en) * | 2010-11-16 | 2012-05-24 | The Brigham And Women's Hospital, Inc. | Diagnosing and monitoring cns malignancies using microrna |
| EP2940151A1 (en) * | 2014-05-02 | 2015-11-04 | Ruprecht-Karls-Universität Heidelberg | Circulating miRNAs as early detection marker and prognostic marker |
| CN107250378A (en) * | 2015-02-24 | 2017-10-13 | 株式会社遗传科技 | Diagnosis, prevention and the treatment method of the brain metastes of cancer and for the drug delivery system by blood-brain barrier |
Non-Patent Citations (2)
| Title |
|---|
| MIRANDA Y. FONG ET AL.: ""Breast cancer-secreted miR-122 reprograms glucose metabolism in pre-metastatic niche to promote metastasis"", 《NATURE CELL BIOLOGY》 * |
| 祝敏捷等: ""血清miR-21和miR-10b在早期宫颈癌患者中的表达及临床意义"", 《现代中西医结合杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110184351A (en) * | 2019-05-29 | 2019-08-30 | 南通普惠精准医疗科技有限公司 | The application of detection kit |
| CN110607366A (en) * | 2019-06-18 | 2019-12-24 | 中山大学孙逸仙纪念医院 | Kit for predicting hormone receptor positive breast cancer preoperative chemotherapy sensitivity |
| WO2022036869A1 (en) * | 2020-08-17 | 2022-02-24 | 中山大学孙逸仙纪念医院 | Method and system for predicting pathological complete remission probability of breast cancer after neoadjuvant chemotherapy |
| CN113881769A (en) * | 2021-06-03 | 2022-01-04 | 南京世和基因生物技术股份有限公司 | Application of cerebrospinal fluid cfDNA in diagnosis and treatment of breast cancer metastasis |
| CN113881769B (en) * | 2021-06-03 | 2023-08-18 | 南京世和基因生物技术股份有限公司 | Application of cerebrospinal fluid cfDNA in diagnosis and treatment of breast cancer metastasis |
| CN113866415A (en) * | 2021-10-13 | 2021-12-31 | 戴勇 | Diagnostic and prognostic markers for brain metastasis of breast cancer |
| CN113866415B (en) * | 2021-10-13 | 2024-02-06 | 戴勇 | Diagnostic and prognostic markers for breast cancer brain metastasis |
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