CN108753964A - A kind of kit for being used for Diagnosis of Non-Small Cell Lung and shifting early warning - Google Patents
A kind of kit for being used for Diagnosis of Non-Small Cell Lung and shifting early warning Download PDFInfo
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- CN108753964A CN108753964A CN201810561095.2A CN201810561095A CN108753964A CN 108753964 A CN108753964 A CN 108753964A CN 201810561095 A CN201810561095 A CN 201810561095A CN 108753964 A CN108753964 A CN 108753964A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The present invention provides a kind of kits for early diagnosing NSCLC and predicting NSCLC transfers easy to use, quick, specificity is high.The marker of kit is that serum excretion body miR-320a, miR-622 or miR-320a combine CEA and Cyfra21 with miR-622；By the content for detecting marker in sample；Judgement sample whether there is non-small cell lung cancer risk.
Description
Technical field
The present invention relates to one kind being based on excretion body microRNAs：MiR-320a, miR-622, let-7f-5p's is non-small thin
Born of the same parents' lung cancer（NSCLC）The kit research and development of diagnosis and transfer early warning, belong to medicine and hygiene fields.
Background technology
Non-small cell lung cancer（Non-small cell lung cancer, NSCLC）Account for the 80% of all lung cancer, about 75%
Patient occurred DISTANT METASTASES IN, 5 years survival rates only 2% when medical.Therefore, there is an urgent need for a kind of more sensitive now
Diagnostic marker provides objective basis for early diagnosis, early treatment and the Index for diagnosis of NSCLC of DISTANT METASTASES IN occurs.
The excretion body in tumour cell source（Tomor-derived exosomes, TDEs）It has been proved to promote
Cancer progression, the cell-cell interaction mediated by excretion body may have important work in the generation of tumour and DISTANT METASTASES IN
With.The separation of excretion body and its in clinic using alternative invasive surgical to diagnose or the main cancer in NSCLC
Patient's is follow-up, wherein the availability of primary tumor tissue is difficult in Most patients.In addition, tolerant in excretion body
Such as miRNAs, from the degradation of enzyme under the protection of excretion body film, TDEs is easy to obtain in blood sample protein etc..Therefore
It can be arrived by stable detection in blood circulation, become the ideal biological marker of metastatic NSCLC diagnosis.
Include many kinds of substance in excretion body, wherein it is miRNA to study most commonly used（miRs / miRNAs）, it is one
The endogenic non-coding RNA molecule of class, is about 17-24 nt, and biological function is played by the regions 3'-UTR of target gene,
Since the region may participate in adjusting subcellular localization, nuclear translocation and stablize transcript, the controllable numerous target genes of miRNA molecule,
It has now been found that about>60% all human genes are regulated and controled by miRNA molecule.Previous studies have shown that miRNA is sent out in tumour
Oncogene or tumor suppressor may be used as to function in hair tonic exhibition and progression, this depends on the work of its target gene
With, and their unconventionality expressions in many human cancer types, including carcinoma of urinary bladder, gastric cancer and glioma etc..In short, this
It is a little to find to show that miRNA shows the potentiality of effective therapy target as treatment of cancer.
Existing clinical diagnosis technology such as X-ray, B ultrasound etc. can not be accomplished in clinical early detection, and the inspections such as CT, MRI and PET
It looks into, it can be found that smaller tumor tissues, but its specificity is low, it is still necessary to and the auxiliary of pathological biopsy can just clarify a diagnosis.Fibre branch
The diagnosis of non-small cell lung cancer has can be improved in the use of the endoscopes such as mirror, but is Interventional examination, to the damage of patient and
It is painful big, and height is required to operator's technology, also not all positions of intrapulmonary can get biopsy, not be suitable for large-scale
Crowd screens generaI investigation.Liquid biopsy has many advantages, such as that specificity is high, noninvasive, quick as novel detection means, is cancer inspection
Look into trend and the direction of development.
Invention content
Regarding to the issue above and present situation, the present invention provides a kind of easy to use, quick, specificity it is high for examining in early days
Disconnected NSCLC and the kit for predicting NSCLC transfers.
It is a kind of prediction non-small cell lung metastasis of cancer marker, the marker be serum excretion body miR-320a,
MiR-622 or miR-320a combine CEA and Cyfra21 with miR-622.
A kind of marker for early diagnosing non-small cell lung cancer and predicting metastases, the marker are selected from blood
Clear excretion body miR-320a, miR-622, let-7f-5p or miR-320a, miR-622, let-7f-5p joint CEA,
Cyfra21。
A kind of kit for early diagnosing non-small cell lung cancer and predicting metastases, the detection method is such as
Under：Detect the content of marker in sample；It is preliminary to judge if the content of marker is respectively greater than its critical value range in sample
There is provided the sample of the sample, there are larger non-small cell lung cancer risks.
The sample is serum, plasma sample.
It is a kind of early diagnosis non-small cell lung cancer and predict metastases in-vitro diagnosis and prediction kit, including：
It is coated on the anti-CEA first antibodies marked on the first chromatographic material, anti-Cyfra21 first antibodies；It is coated on the second chromatographic material
On label anti-CEA secondary antibodies, anti-Cyfra21 secondary antibodies；
Further include reverse transcription reaction system, quantitative fluorescent PCR reaction system and internal reference system；
The quantitative fluorescent PCR reaction system includes being directed to miR-320a, miR-622 or miR-320a and miR-622 respectively
Forward primer and reversed universal primer and reagent；The reagent of the quantitative fluorescent PCR reaction system includes：SYBR Green
Mixed liquor, forward primer liquid, reversed universal primer liquid and pure water；
The internal reference system includes internal reference miR-U6 and the forward and reverse primer for miR-U6；The reverse transcription reaction body
The reagent of system includes poly A polymerase, reverse transcriptase mixed liquor, reverse transcription buffer and nuclease free distilled water.
The antibody is marked using the method for horseradish peroxidase；The antibody is monoclonal antibody or more
Clonal antibody.
Advantageous effect of the present invention：
1, let-7f-5p expressions are remarkably decreased in NSCLC patients blood plasmas excretion body
Present invention firstly provides compared to health donors, under let-7f-5p expressions are notable in NSCLC patients blood plasma's excretion bodies
Drop, miR-622 and miR-320a contents therebetween without significant significant difference, ROC curve analysis shows that, AUC is
0.874, sensitivity 73.8%, specificity is 96.7%.
2, miR-622 expression is remarkably decreased in metastatic NSCLC patient, and miR-320a expressions significantly increase
Compared to metastatic NSCLC patient, miR-622 is expressed and is remarkably decreased in metastatic NSCLC patient, miR-320a expression
It is horizontal significantly to increase, there is the ability of good diagnosis transfer.ROC curve analysis shows that, miR-320a and miR-622 diagnosis
The AUC value of NSCLC transfers is respectively 0.802 and 0.851, and sensitivity is 62.5% and 80%, and specificity is 95% and 87.5%.
MiR-320a and miR-622 carries out Combining diagnosis, and AUC 0.855, sensitivity 85%, specificity is 80%.
3, Combining diagnosis effect is good
The ability of let-7f-5p, CEA and Cyfra21-1 triple combination's the diagnosis of NSCLC is better than individually diagnosis；miR-320a,
MiR-622, CEA and Cyfra21-1 Combining diagnosis metastatic NSCLC abilities are better than individually diagnosis.
The present invention combines serum excretion body let-7f-5p CEA and Cyfra21-1 verifications to the diagnostic of NSCLC, AUC
It is 0.981, sensitivity 94.7%, specificity is 93.3%.
The present invention combines serum excretion body miR-320a and miR-622 and CEA and Cyfra21-1 verification to NSCLC's
Diagnostic.NSCLC patient is divided into two groups, i.e. metastatic NSCLC patient and non-metastatic NSCLC patient, miR-320a,
The sensibility and specificity that miR-622, CEA, Cyfra21-1 combine CEA diagnosis of metastatic NSCLC is respectively 83.8% He
94.7%, significantly improve the sensitivity and specificity of diagnosis.
Description of the drawings
The expression figure of Fig. 1 NSCLC patient's excretion body total serum IgEs.
The expression figure of let-7f-5p and miR-320a in Fig. 2 NSCLC patients blood plasma's excretion bodies.
The expression figure of miR-622 in Fig. 3 NSCLC patients blood plasma's excretion bodies.
Expression figures of Fig. 4 let-7f-5p in early stage and middle and advanced stage NSCLC.
Expression figures of Fig. 5 miR-622 to metastatic NSCLC patient and non-metastatic NSCLC patient.
Expression figures of Fig. 6 miR-320 to metastatic NSCLC patient and non-metastatic NSCLC patient.
Expression figures of Fig. 7 let-7f-5p to metastatic NSCLC patient and non-metastatic NSCLC patient.
In Fig. 8 NSCLC patients blood plasma's excretion bodies, miR-320a, miR-622, Cyfra21-1 expression figure.
Expressions of Fig. 9 miR-320a and miR-622 to metastatic NSCLC patient and non-metastatic NSCLC patient
Figure.
Expression figures of Figure 10 CEA and Cyfra21-1 the joint let-7f-5p to NSCLC patients blood plasmas.
Figure 11 miR-320a, miR-622, CEA, Cyfra21-1 joint CEA diagnosis of metastatic NSCLC design sketch.
Specific implementation mode
Implement below for illustrating the present invention, but is not limited to the scope of the present invention.Unless otherwise specified, case study on implementation
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
Case study on implementation 1：microRNAs：Applications of the let-7f-5p in diagnosing early stage NSCLC
1, experimental design
The experimental design is that acquisition Shandong Tumor Hospital is diagnosed as cure the disease at the beginning of NSCLC people and healthy volunteer's serum sample,
Separation and concentration excretion body simultaneously obtains excretion body RNA.Let-7f-5p expression, its expression of statistical analysis are detected by the method for qPCR
Difference between healthy volunteer and NSCLC, early stage NSCLC, and analyze its diagnostic.
2, the involved patient of experiment and sample
Be included in 30 normal healthy controls persons that attached Shandong tumour hospital of Shandong University accepted for medical treatment in May, 2016 to 2017 for 2 months with
And 80 NSCLC patients, wherein metastatic NSCLC patient 40, non-metastatic NSCLC patient 40, and it is based on postoperative pathological
It learns and checks progress TNM classification.The clinical data that this research is included in includes mainly age, gender, AJCC neoplasm stagings（8th edition）,
The histological type of smoking history and NSCLC.NSCLC patient totally 80,24 ~ 84 years old age, normal healthy controls person totally 30, age 27
~ 81 years old.All patients receive fiberoptic bronchoscopy, percutaneous puncture or open surgical biopsy.In chemotherapy, radiotherapy and operative treatment
Preceding acquisition subject peripheral blood sample about 2ml is placed in mixing in the vacuum tube of the anti-coagulants containing EDTA, and is centrifuged at 3,000g, 4 DEG C
15 minutes.Then plasma sample is collected in centrifuge tube and be stored in -80 DEG C, it is spare.
3, in serum excretion body let-7f-5p levels measurement
The expression of excretion body let-7f-5p in the patient of NSCLC and the serum of healthy volunteer is analyzed using qPCR methods,
It is as follows：
3.1, the extraction of total serum IgE
Plasma sample is taken out from -80 DEG C of refrigerators, freeze thawing.0.8ml plasma samples are drawn in centrifuge tube, by 1:1 ratio
XBP is added in sample, overturns mixing immediately.It draws in mixing sample to centrifugal column, 500g centrifuges 1min, abandons liquid, will centrifuge
Column is put into collecting pipe again, is added in 3.5ml Buffer XWP to centrifugal column, and 500g centrifuges 5min, abandons liquid, and will centrifugation
Column is placed in new collecting pipe.700 μ l QIAzol, 5000g are added into centrifugal column and centrifuge 5min.Pyrolysis product is collected, is placed in
2ml is without in enzyme centrifuge tube.Be vortexed concussion, stands 5min at room temperature.3.5 μ l S/P Spike-in are added into centrifuge tube
Control.90 μ l chloroforms are added into centrifuge tube, acutely shake about 15s.It is incubated at room temperature 2 ~ 3min.12000g, 4 DEG C, centrifugation
15min, at this time liquid be divided into 3 layers.It draws in upper strata aqueous phase to new no enzyme pipe, then according to upper strata aqueous phase：100% anhydrous second
Alcohol=1:The absolute ethyl alcohol of certain volume is added in 2 ratio.It transferring samples in no enzyme centrifugal column, room temperature 8000g centrifuges 15s,
Abandon liquid.Above step is repeated, until all liq all filters.700 μ l Buffer RWT are added, room temperature 8000g centrifuges 15s,
Abandon liquid.700 μ l Buffer RPE are added, room temperature 8000g centrifuges 15s, abandons liquid.500 μ l Buffer RPE, room temperature is added
8000g centrifuges 2min, abandons liquid.Centrifugal column is transferred in new collecting pipe.The lid of centrifugal column is opened, centrifuges 5min at full speed,
Liquid is abandoned, centrifugal column is transferred in the new collecting pipes of 1.5ml.14 μ l are added without enzyme water, pillar is placed into 1min vertically, at full speed from
Heart 1min, eluted rna.Sample concentration and purity are detected with fluorescent quantitation microplate reader analyzer.
3.2, reverse transcription reaction and qPCR
By 5 × miScript HiSpec Buffer, 10 × miScript Nuclics Mix, miScript Reverse
Transcriptase Mix, DEPC treated water, 2 μ g mixings of total serum IgE, 42 DEG C of incubations 1h, 72 DEG C of incubation 10min.
According to sample size, according to the form below reaction system has configured mixed reaction solution, shakes mixing.
In in Biohazard Safety Equipment, the reaction solution that the above configuration is completed is separately added into the hole of corresponding PCR amplification plate
It is interior, pay attention to avoiding polluting, encloses hyaline membrane later.Notice that each sample standard deviation does 1 secondary orifices, experiment is repeated 3 times.Careful centrifugation,
And mixing.Operate the computer, be detected using 480 quantitative PCR apparatus of LightCycler, the program of according to the form below setting carries out instead
It answers.
4, mathematical statistics are analyzed
Statistical analysis is carried out using SPSS 22.0 and 6.0 softwares of GraphPad Prism.Two Sets of Measurement Data use Mann-
Whitney U are examined and t inspections are analyzed.Diagnosis combination is established using dualistic logistic regression, passes through Receiver Operating Characteristics
Curve（ROC curve）And area under the curve（AUC）Determine the independent of miRNAs, CEA and the Cyfra21-1 of differential expression or connection
Diagnostic is closed, and calculates true positives number of cases（TP）, true negative number of cases（TN）, false positive number of cases（FP）, false negative number of cases（FN）,
Sensibility, specificity, accuracy, positive predictive value（PPV）And negative predictive value（NPV）.There is statistics with p < 0.05 for difference
Meaning.
5, the summary of NSCLC cases feature and the relationship between let-7f-5p expressions
1 interpretation of result of table, the Clinical symptoms of 80 NSCLC patients, including age, gender, smoking state, histological type and
Neoplasm staging.Between metastatic and non-metastatic NSCLC, the expression of let-7f-5p, miR-320a and miR-622 with
Age, gender, smoking state and histological type are unrelated.
1 NSCLC case features of table are summarized and the relationship between let-7f-5p expressions
6, the relationship of excretion body total serum IgE clinical stages different from NSCLC
QPCR testing results show that the expression of NSCLC patient's excretion body total serum IgE is significantly higher than healthy volunteer.With non-turn
Shifting property NSCLC patient compares, and the excretion body total serum IgE of metastatic NSCLC patient then significantly increases.In addition, in normal healthy controls and non-
Also it observed significance difference differnce Fig. 1 between metastatic NSCLC.
7, serum excretion body let-7f-5p is in healthy volunteer and NSCLC patient levels' differences
It is quantified using the let-7f-5p levels in the excretion body of 110 subjects of method pair of qPCR, to determine let-
Whether 7f-5p can be used as the biomarker of the diagnosis of NSCLC.In 110 patients, including 80 NSCLC patients and 40 are strong
Health donor.
The expression of let-7f-5p is remarkably decreased in early and late NSCLC, and difference has apparent statistics
Meaning（p<0.0001 and p<0.0001）, see Fig. 4.
8, diagnostics of the ROC curve analysis serum excretion body let-7f-5p to NSCLC
The present invention so using ROC curve analyze, detection let-7f-5p to NSCLC diagnostics, ROC curve analysis shows that,
AUC is 0.874, sensitivity 73.8%, and specificity is 96.7%, sees Fig. 8.
9, serum excretion body let-7f-5p combines diagnostics of the CEA and Cyfra21-1 to NSCLC
The present invention combines diagnostic of serum excretion body let-7f-5p, CEA and the Cyfra21-1 verification to NSCLC in turn,
AUC is 0.981, sensitivity 94.7%, and specificity is 93.3%.The diagnostic threshold of CEA is 3.4 ng/mL, Cyfra21-1's
Diagnostic threshold is 3.3 ng/mL, sees Figure 10
Case study on implementation 2：microRNAs：Applications of the miR-320a and miR-6225p in NSCLC shifts early warning
1, experimental design
The experimental design is that acquisition Shandong Tumor Hospital is diagnosed as cure the disease at the beginning of NSCLC people and healthy volunteer's serum sample,
Separation and concentration excretion body simultaneously obtains excretion body RNA.MiR-320a and miR-622 expression, statistical are detected by the method for qPCR
Its difference of the expression between healthy volunteer and NSCLC, early stage NSCLC is analysed, and analyzes its diagnostic.
2, the involved patient of experiment and sample
80 NSCLC patients that attached Shandong tumour hospital of Shandong University accepted for medical treatment in May, 2016 to 2017 for 2 months are included in,
Middle metastatic NSCLC patient 40, non-metastatic NSCLC patient 40, and checked based on postoperative pathological and carry out TNM classification.
3, in serum excretion body miR-320a and miR-622 levels measurement
With embodiment 1
4, mathematical statistics are analyzed
With embodiment 1
5, the summary of NSCLC cases feature and the relationship between miR-320a and miR-622 expressions
With embodiment 1
6, serum excretion body miR-320a and miR-622 is in transfer and non-diverting NSCLC patient levels difference
Further analyze the expression of metastatic and non-metastatic NSCLC patient's excretion body miR-320a and miR-622.Knot
Fruit shows that the miR-320a and miR-622 expression in metastatic NSCLC patient are apparently higher than the outer of non-metastatic NSCLC patient
Secrete body.As a result there is significant difference, see Fig. 5 and Fig. 6.
7, ROC curve analysis serum excretion body miR-320a and miR-622 shifts NSCLC the efficiency of early warning
The present invention is analyzed using ROC curve in turn, and detection serum excretion body miR-320a and miR-622 shifts diagnosis to NSCLC
Efficiency, ROC curve analysis shows that, miR-320a and miR-622 the diagnosis of NSCLC transfer AUC value be respectively 0.802 He
0.851, sensitivity is 62.5% and 80%, and specificity is 95% and 87.5%.MiR-320a and miR-622 combine examining
Disconnected, AUC 0.855, sensitivity 85%, specificity is 80%.See Fig. 9.
8, serum excretion body miR-320a and miR-622 joint CEA and Cyfra21-1 are to the effect to NSCLC transfer early warning
Energy
The present invention combines serum excretion body miR-320a and miR-622 and CEA and Cyfra21-1 verification to NSCLC's in turn
Diagnostic.NSCLC patient is divided into two groups, i.e. metastatic NSCLC patient and non-metastatic NSCLC patient, miR-320a,
The sensibility and specificity that miR-622, CEA, Cyfra21-1 combine CEA diagnosis of metastatic NSCLC is respectively 83.8% He
94.7%, AUC 0.900.See Figure 11.
Claims (7)
1. a kind of marker of prediction non-small cell lung metastasis of cancer, which is characterized in that the marker is serum excretion body
MiR-320a, miR-622 or miR-320a combine CEA and Cyfra21 with miR-622.
2. a kind of early diagnosis non-small cell lung cancer and the marker for predicting metastases, which is characterized in that the marker
Selected from serum excretion body miR-320a, miR-622, let-7f-5p or serum excretion body miR-320a, miR-622, let-7f-
5p combines CEA, Cyfra21.
3. a kind of diagnostic method of kit using the marker described in claim 2, which is characterized in that the method is such as
Under：Detect the content of marker in sample；It is preliminary to judge if the content of marker is respectively greater than its critical value range in sample
There is provided the sample of the sample, there are larger non-small cell lung cancer risks.
4. kit according to claim 3, which is characterized in that the sample is serum, plasma sample.
5. a kind of kit using the marker described in claim 2, which is characterized in that including：It is coated on the first chromatography material
Anti- CEA first antibodies, the anti-Cyfra21 first antibodies marked on material；It is coated on the anti-CEA of the label on the second chromatographic material
Two antibody, anti-Cyfra21 secondary antibodies；
Further include reverse transcription reaction system, quantitative fluorescent PCR reaction system and internal reference system；
The quantitative fluorescent PCR reaction system includes being directed to miR-320a, miR-622 or miR-320a and miR-622 respectively
Forward primer and reversed universal primer and reagent；The reagent of the quantitative fluorescent PCR reaction system includes：SYBR Green
Mixed liquor, forward primer liquid, reversed universal primer liquid and pure water；
The internal reference system includes internal reference miR-U6 and the forward and reverse primer for miR-U6；The reverse transcription reaction body
The reagent of system includes poly A polymerase, reverse transcriptase mixed liquor, reverse transcription buffer and nuclease free distilled water.
6. kit according to claim 5, which is characterized in that the anti-CEA first antibodies of the label resist
The anti-CEA secondary antibodies of Cyfra21 first antibodies and label, anti-Cyfra21 secondary antibodies refer to using horseradish peroxidase
Method label antibody.
7. kit according to claim 5, which is characterized in that the antibody is monoclonal antibody or Anti-TNF-α
Body.
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Cited By (1)
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Application publication date: 20181106 |