CN110452982A - Breast cancer circulating tumor cell miRNA and EMT markers in detecting kit and its application - Google Patents
Breast cancer circulating tumor cell miRNA and EMT markers in detecting kit and its application Download PDFInfo
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Abstract
The present invention relates to a kind of metastatic breast cancer prognosis risk assessment mRNA detection primer groups, including nucleotide sequence, kit, mathematical model as shown in NO.1~6 SEQ ID, and its application in metastatic breast cancer risk assessment.The present invention is using miR-106b, EMT marker E-cadherin and Vimentim in Peripheral Circulation tumour cell as combination molecule marker, exploitation applied to metastatic breast cancer CTC Research of predicting markers molecular detection kit, with real time fluorescence quantifying PCR method, the prognosis evaluation of metastatic breast cancer may be implemented, screening has the patient of recurrence high risk, individualized clinical treatment is instructed, and improves the accuracy for the treatment of.Detection kit detects quick and convenient, detection sensitivity, specificity detection needs that are high, at low cost, can satisfy logarithm tumour patient big absolutely, and application range is extremely wide, and prediction accuracy is high according to clinical verification.
Description
Technical field
The invention belongs to nucleic acid detection technique fields, specifically, the present invention relates to detection breast cancer Peripheral Circulation is swollen
The application of miRNA and EMT marker in oncocyte.In addition, the invention further relates to reagent used in the application and
Kit, and the mould based on circulating tumor cell miRNA and EMT marker joint assessment metastatic breast cancer prognostic risk
Type.
Background technique
Breast cancer is one of the most common malignant tumors in women, and disease incidence is in rise year by year trend.Global female in 2018
Property breast cancer new cases 2,088,849, accounts for the 25% of women whole malignant tumour.Women with breast cancer death in 2018
62,667, women's health is seriously endangered.Although clinically the detection of breast cancer and treatment means are constantly progressive, breast cancer
DISTANT METASTASES IN is still difficult to cure, and is the maximum reason for leading to tumour associated death.
Peripheral Circulation tumour cell (circulating tumor cells, CTCs) is the seed of metastases, CTCs
It is the key cells group of breast cancer DISTANT METASTASES IN.During metastases, single tumor cell or small tumour cell group
Block is discharged from primary tumo(u)r lesion or transfer stove to split away off into blood circulation, and is diffused by the circulatory system of whole body
The tissue and organ of distant place.And circulating tumor cell just refers to the tumour cell propagated in the circulatory system in vivo.Major part is invaded
The tumour cell for entering the circulatory system can be dead in a short time, and only only a few has the CTCs of height metastasis tendency and vigor can
Overcome and lose nest pressure, survived in the form of unicellular or cell mass, and under given conditions can at a distance in organ again
Secondary plantation simultaneously continues proliferation, differentiation, ultimately forms clinical visible dominant transfer stove and recurrence stove, and in secondary metastatic tumor
Cancer cell can still be again introduced into circulation, form more CTCs and metastatic tumor.
In recent years, CTCs has been obtained for significant progress in the research of clinical application, more and more researches show that its
Important prognosis information can be provided for kinds of tumors.Since infantile tumour (diameter of tumor < 1mm) may occur in which tumour cell
Hematogenous spread, CTCs are commonly used for the early diagnosis of tumour and the monitoring of relapse and metastasis as the bellwether of liquid biopsy.At present
Frequently with CTCs quantity as prediction patient with breast cancer's prognosis index.Clinical research confirmation, CTCs in 7.5ml before chemotherapy
Quantity be 10.1 months, and the metastatic breast cancer patients overall survival of CTCs<5 is substantially better than the patient greater than CTCs>=5 phase
(vs.10.1 months 18 months).
Current research suggests that CTCs can promote the progress and transfer of tumour through a variety of ways, it among these include: conduct
The cell origin of metastases starting promotes the transfer of tumour；The primary lesion for extravasating into tumour again by blood circulation promotes
The growth of tumour；The growth and transfer for influencing and vivo immunization cell being utilized to promote tumour, such as the relevant macrophage of tumour and
Myeloid cell.Therefore, CTCs may not be merely the transfer initiator cell source of tumour DISTANT METASTASES IN, while be also to promote tumour
One very important factor of transfer.
The characteristic of tumour cell itself is to determine the key factor of CTCs transfer clone.Current research suggests that tumour
The transfer of CTCs is the process of a multi-step, comprising: Tumor cell dissemination overcomes and loses nest pressure, distant organs infiltration, is immunized
Tolerance adapts to microenvironment before shifting, repopulating cell survival and forms transfer stove.Wherein, the tumour cell success sent out is at a distance
The process for shifting initial tumor transforming growth in tissue or organ is referred to as the formation for shifting clone.In this of CTCs transfer clone
During a, in addition to the influence of patient's body environment, its own characteristic of tumour cell is final to determine to send out distal organs
Or can the tumour cell in tissue form the key factor of apparent metastatic lesion in metastasis site.And not all CTCs is
It is likely to form transfer stove, only there is stemness (stem-like), Epithelial and stromal to convert (epithelial-mesenchymal
Transition, EMT), the CTCs of the features such as cluster and organotropism is likely to ultimately form transfer stove.
EMT conversion is current to the effect of growth and metastasis of tumours, and oneself is largely studied.EMT means epithelial cell
Polarity is lost to be migrated and invasive ability.CTCs obtains interstitial phenotype by EMT, and it is de- from epithelial tissue dissociation to be conducive to it
It falls, while increased cell elasticity and transfer ability, helps to seep blood circulation in it.Few tumour cells express epithelium simultaneously
And mesenchymal, but CTCs is but more interstitial type, interstitial type CTCs can be detected in single CTC and CTC groups.
Vimentin can be used as the marker of EMT type CTCs.Rahman etc. is from pulmonary metastasis cell and Serum of Patients with Lung Cancer
The excretion body in source induces CTCs Vimentin, promotes the EMT of CTCs.Bourcy etc. confirms that the CTCs of the EMT positive can be with
Promote the formation of transfer clone.The study found that EMT can promote cell aggregation and blood coagulation, EMT- group with the induced tissue factor, the latter
The transfer ability of EMT positive CTCs can be promoted by knitting factor adjustment axis.To sum up described in research, the EMT characteristic of CTCs is its generation
The key factor of DISTANT METASTASES IN.
EMT is considered as the critical event during cancer metastasis, unconventionality expression and invasion including EMT relevant molecule
The acquisition of interstitial phenotype.The variation that there is also EMT on morphology and phenotype in breast cancer CTCs.The CTCs that EMT occurs loses
Contact and polarity, epithelium related gene expression downward of the cell with cell, and mesenchyma gene expression is raised.E-cadherin
(cadherin) and the core protein that Vimentin (vimentin) is that epithelial adherence connects, multiple studies have shown that,
Vimentin is expressed in the CTCs of patient with breast cancer, Vimentin expression up-regulation CTCs reacted with clinical treatment and disease into
Exhibition is related, prompts the molecular phenotype of the CTCs including EMT related to the Clinical Outcome of patient with breast cancer.
It has been reported that by CellSearch Profile Kit from metastatic breast cancer (metastatic breast
Cancer, MBC) CTCs that separates in patient's queue has unique miRNA express spectra, and this shows the miRNA expression pair of CTCs
The phenotype of CTCs has unique influence power, will have very big Research Prospects.However, about the miRNA and EMT phase in CTCs
The potential clinical correlation of molecule E-cadherin and Vimenti etc. are closed, current research is very few.
Summary of the invention
For the above-mentioned prior art, the purpose of the present invention is to provide a kind of detection breast cancer Peripheral Circulation tumour cells
In miRNA and EMT marker application.In addition, the invention further relates to used reagent and examinations in the application
Agent box, and the miRNA based on circulating tumor cell and EMT marker assessment metastatic breast cancer prognostic risk mould
Type.
The present invention is achieved by the following technical solutions: metastatic breast cancer prognosis risk assessment mRNA detection primer
Group, including the nucleotide sequence as shown in NO.1~6 SEQ ID.
Preferably, the metastatic breast cancer prognosis risk assessment mRNA detection primer group, further includes internal control primer, core
Nucleotide sequence is as shown in NO.7~10 SEQ ID.
Specific primer amplification sequence is as follows:
(1) primer of miR-106b is expanded:
Forward primer:
5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGATCTGC-3'
Reverse primer: 5'-ACACTCCAGCTGGGTAAAGTGCTGACAGTG-3'
(2) primer of E-cadherin mRNA is expanded:
Forward primer: 5'-TAGGTGTTCACATCATCGTCCGC-3'
Reverse primer: 5'-TCACGCTGTGTCATCCAACGG-3'
(3) primer of Vimentim mRNA is expanded:
Forward primer: 5'-CAGACAGGATGTTGACAATGCG-3'
Reverse primer: 5'-GATTTCCTCTTCGTGGAGTTTCTTC-3'
(4) internal reference U6 primer
Forward primer: 5'-GCTTCGGCAGCACATATACTAAAAT-3'
Reverse primer: 5'-CGCTTCACGAATTTGCGTGTCAT-3'；
(5) internal reference GAPDH primer
Forward primer: 5'-TGTCATCATATTTGGCAGGTTT-3'
Reverse primer: 5'-TTGGTATCGTGGAAGGACTCA-3'.
The present invention also provides a kind of metastatic breast cancer prognosis risk assessment mRNA detection kit, the kit packets
Include the primer as shown in NO.1~6 SEQ ID.
Preferably, the metastatic breast cancer prognosis risk assessment mRNA detection kit, further includes internal control primer, core
Nucleotide sequence is as shown in NO.7~10 SEQ ID
Preferably, the metastatic breast cancer prognosis risk assessment mRNA detection kit further include: for PCR reaction
SYBR Green mixed liquor, forward primer liquid, reverse primer liquid and nuclease free pure water.
Metastatic breast cancer prognosis wind is used for using detection kit as described above building the present invention also provides a kind of
The mathematical model nearly assessed, by detect miRNA, E-cadherin, Vimenti expression quantity, obtain relevant risk coefficient and
Cut-off value, the relevant risk coefficient are calculated by the following formula:
Wherein, wherein r=Risk Score；J=1,2,3 ... ... m；M is indicated: the quantity of covariant；I is indicated: a certain
A covariant；β is indicated: partial regression coefficient in model；X is indicated: covariant；
By the sum of risk factor finally obtained and the 3.214 multilevel iudge patient's metastatic breast cancer prognosis of cut-off value
Risk height, judgment criteria are as follows: low danger: Risk Sore < 3.214；It is high-risk: Sore >=3.214 Risk.
On the other hand, the present invention also provides the metastatic breast cancer prognosis risk assessment mRNA detection primer groups to turn
It is applied in shifting property breast cancer risk assessment.
The present invention provides the detection methods of metastatic breast cancer prognosis risk assessment mRNA a kind of, include the following steps:
(1) RNA is extracted from Peripheral Blood In Patients With Breast Cancer；
(2) reverse transcription obtains cDNA；
(3) fluorescent quantitative PCR；
(4) interpretation of result: judgement shifting property Prognosis in Breast Cancer risk is specifically according to following standard: low danger: Risk Sore <
3.214；It is high-risk: Sore >=3.214 Risk.
Beneficial effects of the present invention:
(1) present invention is with the marker E-cadherin of miR-106b, EMT in Peripheral Circulation tumour cell (CTC)
It is applied to metastatic breast cancer detection kit with the Vimentim new molecular model being combined into as combination molecule marker
Exploitation, and combine real time fluorescence quantifying PCR method, realize the prognosis evaluation of metastatic breast cancer, screening has recurrence high risk
Patient, instruct individualized clinical treatment, and improve the accuracy for the treatment of.
(2) metastatic breast cancer CTC Research of predicting markers detection kit of the invention has selected miR-106b, EMT to indicate
Object E-cadherin and Vimentim are combined, and detect that quick and convenient, detection sensitivity, specificity be high, at low cost, Ke Yiman
The detection of foot logarithm tumour patient big absolutely needs, and application range is extremely wide, and prediction accuracy is high according to clinical verification.
(3) metastatic breast cancer CTC Research of predicting markers detection kit of the invention is directed to Peripheral Circulation tumour cell
As the internal reference of quantitative reaction in sample, stable source of people RNA sequence U6 and GAPDH is introduced as internal reference and compares RNA, and excellent
Change the internal control primer devised for U6 and GAPDH, greatly improves miR-106b, E-cadherin and Vimentim in CTC
The accuracy of relative quantification when detection.
Detailed description of the invention
Fig. 1 is miR-106b and E-cadherin/Vimentim joint mark in kit joint-detection CTC of the invention
(wherein A figure indicates miR-106b and E- in modeling group for object and the ROC curve figure of individual molecule prediction DFS phase
Cadherin/Vimentim combines the ROC curve figure of marker and individual molecule prediction DFS phase；B figure indicates validation group
The ROC curve figure of miR-106b and E-cadherin/Vimentim joint marker and individual molecule prediction DFS phase).
Fig. 2 is miR-106b and E-cadherin/Vimentim joint mark in kit joint-detection CTC of the invention
(wherein A figure indicates miR-106b table in the CTC of disease progression patient to the comparison schematic diagram of object and individual molecule prediction chemotherapeutic efficacy
It is increased up to significant；B figure indicates that miR-106b and E-cadherin/Vimentim combines marker and single point in modeling group CTC
The comparison schematic diagram of son prediction chemotherapeutic efficacy；C figure indicates miR-106b and E-cadherin/Vimentim connection in validation group CTC
Close the comparison schematic diagram of marker and individual molecule prediction chemotherapeutic efficacy).
Specific embodiment
Embodiment 1
(1) circulating tumor cell (CTC) of patient with breast cancer is extracted
Using the separation of CellSearch system Peripheral Circulation tumour cell detector, enrichment, quantitive CT Cs, specific steps
It is as follows:
Human breast carcinoma peripheral blood in patients 10-50ml is taken, every 7.5ml peripheral blood is mixed with 6.5ml dilution buffer,
800g is centrifuged 10 minutes at room temperature, and the peripheral blood sample prepared and CTCs detection kit are in CellTracks AutoPrep
Upper operation after sample collection, is automatically transferred to CellTracks Analyzer system automatic camera, last artificial diagosis,
According to CTCs interpretation standard: in the epithelial cell of the Epcam+ of magnetic capture, while Immunofluorescence test confirms CD45-、CK+、
Dioc feminine gender channel no signal can determine whether to count the CTCs for meeting interpretation standard for CTCs.Pass through CellTracks AutoPrep
Operation profile kit can directly be enriched with CTCs in vitro.By the sample prepared together with Profile Kit in CellTrack
Sample is run on automatic system machine, the sample obtained after end of run is CTCs.
(2) total serum IgE is extracted from CTCs
A. the addition TRIzol lytic cell directly in the CTCs being enriched to, every 5~10 × 106Cell enters 1ml
TRIzol is inhaled repeatedly and is beaten.
B. homogenised sample is placed 5 minutes at (15-30 DEG C) of room temperature, is kept completely separate nucleic acid-protein compound.
C. every that 0.2ml chloroform is added using 1ml TRIzol, acutely oscillation 15 seconds, are placed at room temperature for 3 minutes.
D.2-8 DEG C 10000 × g is centrifuged 15 minutes.Sample is divided into three layers: bottom is yellow organic phase, and upper layer is colourless water
Mutually with a middle layer, for RNA mainly in water phase, water phase volume is about the 60% of TRIzol reagent used.
E. water phase is transferred in new pipe, such as DNA to be separated and protein can retain organic phase, and further operating is seen below,
With the RNA in isopropanol precipitating aqueous phase.It is every that 0.5ml isopropanol is added using 1ml TRIzol, it is placed at room temperature for 10 minutes.
F.2-8 DEG C 10000 × g is centrifuged 10 minutes, and RNA precipitate is not seen before centrifugation, is occurred after centrifugation in pipe side and tube bottom
Gelatinous precipitate removes supernatant.
G. with 75% ethanol washing RNA precipitate.It is every at least to add 75% ethyl alcohol of 1ml using 1ml TRIzol.2-8 DEG C does not surpass
It crosses 7500 × g to be centrifuged 5 minutes, abandons supernatant.
H. it is placed at room temperature for dry or vacuum and drains RNA precipitate, about dry in the air 5-10 minutes.
I. water or 0.5%SDS of the 25-200 μ l without RNase is added, is beaten several times with pipette tips suction, 55-60 DEG C is placed 10 minutes
Dissolve RNA, -70 DEG C of preservations obtain total RNA.
(3) reverse transcription prepares cDNA
A. electrophoresis checking R NA sample quality.
B. prepare Mix 1 (20uL system)
C. it is sub-packed in each pipe.
D. 2uL RNA sample (every pipe 50-1000ng) is added
E. 5 minutes are heated at 70 DEG C to be denaturalized the secondary structure for destroying RNA, end is placed on 2 minutes on ice.
F. it carries out preparing Mix 2 when initial denaturation
G. it dispenses into the pipe that each pipe initial denaturation terminates and cools down.If any bubble, slightly it is centrifuged.
H. room temperature is set 5 minutes only.
I. PCR instrument is set according to reverse transcriptase operating temperature, the General reactions time is 1 hour or so.Sample is put into, into
Row reaction.There should be 95 DEG C of 5 minutes inactivation steps at the end of reaction, reverse transcriptase is inactivated, prevent from influencing subsequent PCR anti-
It answers.
J. the first chain of cDNA synthesized can be stored in -20 DEG C.
(4) fluorescent quantitation q RT-PCR is detected
RT-PCR detection: (reverse transcription PCR of fluorescent quantitation technology)
A. PCR reaction solution (10ul system) is prepared by following composition.
B. reaction condition: common response condition
(A) reaction condition of 3Step PCR: the reaction condition of (B) 2Step PCR:
94℃ 30sec
55~65 DEG C of 30sec 30Cycles
72℃ 1min/kbp
(5) value-at-risk calculates
A. 2^- Δ Δ Ct method will be used in step (4), miR-106b, E-cadherin and Vimentim's of acquisition is opposite
Expression quantity.It is independent to repeat step (4) three times, take being averaged for the relative expression quantity of miR-106b, E-cadherin and Vimentim
Value substitutes into following formula:
Wherein, r=Risk Score；J=1,2,3 ... ... m；M is indicated: the quantity of covariant；I is indicated: some covariant
Amount；β is indicated: partial regression coefficient in model；X is indicated: covariant；
Parameter in equation
B. it is scored by the Risk score that above-mentioned formula obtains the present embodiment sample:
Risk score=2.253 (status of miR-106b)+1.544 (status of E-cadherin)+
1.439(status of Vimentim)；
C. high-risk patient screens judgment criteria
With 3.214 multilevel iudge patient's metastatic breast cancer prognostic risk height of cut-off value, judgment criteria are as follows: low
Danger: Risk Sore < 3.214；It is high-risk: Sore >=3.214 Risk.
The final Risk score of sample is 5.236, compared with cut-off value 3.214, Risk Sore in the present embodiment
> 3.214, therefore, the patient with breast cancer in the present embodiment are metastatic breast cancer high-risk patient.
Embodiment 2
It will be using miR-106b in kit joint-detection CTC of the invention and E-cadherin/Vimentim joint mark
The ROC curve figure of will object and individual molecule prediction DFS phase (progression free survival, PFS), is as a result shown in
Attached drawing 1.
A indicates that miR-106b and E-cadherin/Vimentim joint marker and individual molecule are pre- in modeling group in Fig. 1
Survey the ROC curve figure of DFS phase；B indicates that miR-106b and E-cadherin/Vimentim combines marker in validation group
With the ROC curve figure of individual molecule prediction DFS phase.MiR-106b and E-cadherin/Vimentim joint in modeling group
The AUC (95%, CI) of marker is 0.752 (0.658-0.847), is 0.726 (0.595-0.856) in validation group, and single
The AUC (95%, CI) of molecular prediction is below joint marker, it is seen that miR-106b and E-cadherin/ of the invention
It is high that Vimentim combines accuracy of the marker for Diagnosis of Breast cancer.
Embodiment 3
MiR-106b in kit joint-detection CTC of the invention and E-cadherin/Vimentim is combined into marker
ROC curve figure compared with individual molecule predicts chemotherapeutic efficacy, the results are shown in attached figure 2.
A indicates the significant raising of miR-106b expression in the CTC of disease progression patient in Fig. 2；B is indicated in modeling group CTC
MiR-106b and E-cadherin/Vimentim joint marker is compared with individual molecule predicts chemotherapeutic efficacy；C indicates verifying
MiR-106b and E-cadherin/Vimentim joint marker is compared with individual molecule predicts chemotherapeutic efficacy in group CTC.It builds
The AUC (95%, CI) of miR-106b and E-cadherin/Vimentim joint marker is 0.691 (0.598- in mould group
It 0.784), is 0.661 (0.519-0.804) in validation group, and the AUC (95%, CI) of individual molecule prediction is below joint mark
Will object, it is seen that miR-106b of the invention and E-cadherin/Vimentim joint marker is for predicting that mammary cancer chemotherapy is treated
The accuracy of effect is high.
Embodiment described above only expresses one of embodiment of the invention, and description is more specific and detailed
Carefully, but it cannot be understood as the limitations to patent of invention range.It should be pointed out that for the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the present invention
Protection scope.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
SEQUENCE LISTING
<110>Sun Yat-sen Memorial Hospital
<120>breast cancer circulating tumor cell miRNA and EMT markers in detecting kit and its application
<130> 2019.4.3
<160> 10
<170> PatentIn version 3.3
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Claims (8)
1. metastatic breast cancer prognosis risk assessment mRNA detection primer group, which is characterized in that including such as NO.1~6 SEQ ID
Shown in nucleotide sequence.
2. metastatic breast cancer prognosis risk assessment mRNA detection primer group as described in claim 1, which is characterized in that also wrap
Internal control primer is included, nucleotide sequence is as shown in NO.7~10 SEQ ID.
3. a kind of metastatic breast cancer prognosis risk assessment mRNA detection kit, which is characterized in that the kit includes such as
Primer shown in NO.1~6 SEQ ID.
4. metastatic breast cancer prognosis risk assessment mRNA detection kit as claimed in claim 3, which is characterized in that described
Kit further includes internal control primer, and nucleotide sequence is as shown in NO.7~10 SEQ ID.
5. metastatic breast cancer prognosis risk assessment mRNA detection kit as described in claim 3 or 4, which is characterized in that
The kit further include: SYBR Green mixed liquor, forward primer liquid, reverse primer liquid and free nucleic acid for PCR reaction
Enzyme pure water.
6. a kind of utilize if the described in any item detection kit buildings of claim 3~5 are for metastatic breast cancer prognosis
The mathematical model of risk assessment, which is characterized in that by detecting the expression quantity of miRNA, E-cadherin, Vimenti, obtain phase
It closes risk factor and cut-off value, the relevant risk coefficient is calculated by the following formula:
Wherein, wherein r=Risk Score；J=1,2,3 ... ... m；M is indicated: the quantity of covariant；I is indicated: some association
Variable；β is indicated: partial regression coefficient in model；X is indicated: covariant；
By the sum of risk factor finally obtained and 3.214 multilevel iudge patient's metastatic breast cancer prognostic risk of cut-off value
Just, judgment criteria are as follows: low danger: Risk Sore < 3.214；It is high-risk: Sore >=3.214 Risk.
7. a kind of metastatic breast cancer prognosis risk assessment mRNA detection primer group as claimed in claim 1 or 2 is in metastatic
Application in breast cancer risk assessment.
8. a kind of detection method of metastatic breast cancer prognosis risk assessment mRNA, which comprises the steps of:
(1) RNA is extracted from Peripheral Blood In Patients With Breast Cancer；
(2) reverse transcription obtains cDNA；
(3) fluorescent quantitative PCR；
(4) interpretation of result: judgement shifting property Prognosis in Breast Cancer risk is specifically according to following standard: low danger: Risk Sore < 3.214；
It is high-risk: Sore >=3.214 Risk.
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