CN110423817A - Detect application and the kit of the reagent of NUMB endocytosis adaptor protein expression - Google Patents

Detect application and the kit of the reagent of NUMB endocytosis adaptor protein expression Download PDF

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Publication number
CN110423817A
CN110423817A CN201910661289.4A CN201910661289A CN110423817A CN 110423817 A CN110423817 A CN 110423817A CN 201910661289 A CN201910661289 A CN 201910661289A CN 110423817 A CN110423817 A CN 110423817A
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numb
expression
breast cancer
reagent
detection
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夏伟
赵小芳
郝玲
胡明
李春明
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Jiangsu Vocational College of Medicine
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Jiangsu Vocational College of Medicine
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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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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    • C12Q2600/118—Prognosis of disease development
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    • C12Q2600/158—Expression markers
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    • G01—MEASURING; TESTING
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    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

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Abstract

The invention belongs to field of biotechnology, it is related to detecting application and the kit of the reagent of NUMB expression.Application of the reagent in preparation of the preparation for Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's Index for diagnosis more particularly, to detection NUMB expression and a kind of for Computer-aided Diagnosis of Breast Cancer and/or the kit of patient with breast cancer's Index for diagnosis.Clinical sample testing result shows that NUMB expression is significantly reduced compared with cancer beside organism in breast cancer；And NUMB low expression is unfavorable for patient with breast cancer's overall survival.Therefore, the reagent for detecting the changes in gene expression can be used for Prognosis in Breast Cancer or diagnosis, treatment.

Description

Detect application and the kit of the reagent of NUMB endocytosis adaptor protein expression
Technical field
The invention belongs to field of biotechnology, express water more particularly, to detection NUMB endocytosis adaptor protein (NUMB) Flat reagent is in preparation for the application and one kind in the preparation of Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's Index for diagnosis For Computer-aided Diagnosis of Breast Cancer and/or the kit of patient with breast cancer's Index for diagnosis.
Background technique
Breast cancer is the most common malignant tumour of women, and according to 2012 epidemiology statistics, the annual new diagnosis in the whole world is up to 1700000 patient with breast cancers, about 522000 people die of breast cancer, developed country become be only second to lung cancer it is important it is dead because Element.Breast cancer accounts for the 25% of all female tumors, and the 15% of all tumour associated deaths.In China, new breast cancer in 2013 278800 people of case is 42.02/100000 people by Chinese population standard disease incidence, and estimation death toll is 64600 people, dead Rate is 9.74/100000 people.In China as the change of socio-economic factor, breast cancer incidence and the death rate are all constantly increasing Add.With the understanding of the biological mechanism to breast cancer occurrence and development, neoplasm targeted therapy and endocrine therapy etc. are in breast cancer Important function is played in treatment.With the development of molecule parting and accurate medicine, new breast cancer key target is explored and found Gene is of great significance for breast cancer Individual Diagnosis and treatment.
Summary of the invention
The object of the present invention is to provide a kind of new prognostic marker for breast cancer NUMB endocytosis adaptor proteins (NUMB), thus The reagent for further providing for detection NUMB expression is sentenced in preparation for Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's prognosis Application in disconnected preparation and a kind of for Computer-aided Diagnosis of Breast Cancer and/or the kit of patient with breast cancer's Index for diagnosis.
NUMB is a kind of protein coding gene, is positioned at 14q24.2-q24.3, include 14 exons, Gene ID: 8650, coding protein is positioned at cell membrane, nucleus and cytoplasm.Currently, related NUMB gene is sent out in mammary gland carcinogenesis Effect in exhibition has not been reported.The present inventor by cancer and oncogene map (Cancer Genome Atlas, TCGA) high-throughput data mining discovery NUMB low expression is unfavorable for patient with breast cancer's prognosis, and is suffered from by collecting clinical breast cancer Person's sample and follow-up information further verify NUMB differential expression to the Prognostic of patient with breast cancer.
To achieve the goals above, the first aspect of the present invention provides detection NUMB endocytosis adaptor protein (NUMB) and expresses water Flat reagent is in preparation for the application in the preparation of Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's Index for diagnosis.
Further, the detection NUMB expression includes the gene expression dose and/or detection NUMB for detecting NUMB Protein expression level.
More specifically, the method for the detection NUMB expression includes: to detect breast cancer and cancer by RT-qPCR method The expression quantity of NUMB in the tissue of side；NUMB mrna expression amount in breast cancer and cancer beside organism is detected by molecular probe technology；It is logical Cross NUMB expressing quantity in immunohistochemistry or Western-Blot detection breast cancer and cancer beside organism.
More specifically, it is described detection NUMB expression reagent be targeting NUMB DNA sequences encoding oligonucleotide probe, PCR primer, or to target the antibody of NUMB.
In accordance with the present invention it is preferred that the reagent of the detection NUMB expression is with SEQ ID NO:1 and SEQ ID The real-time fluorescence quantitative PCR specific primer of nucleotide sequence shown in NO:2.
5 '-AGGTTGAGCCATGCAGTAGG-3 ', SEQ ID NO:1
5 '-GCTTGTTCAGTGGCTGTTGT-3 ', SEQ ID NO:2
The second aspect of the present invention provides a kind of examination for Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's Index for diagnosis Agent box, the kit include: the reagent for detecting NUMB expression.
Further, it is described detection NUMB expression reagent be targeting NUMB DNA sequences encoding oligonucleotide probe, PCR primer, or to target the antibody of NUMB.
Specifically, the reagent of the detection NUMB expression is with core shown in SEQ ID NO:1 and SEQ ID NO:2 The real-time fluorescence quantitative PCR specific primer of nucleotide sequence.
According to the present invention, the kit can also be containing other conventional reagents for real time fluorescent quantitative, preferably Ground, the kit further includes at least one of following components: Trizol, isopropanol, chloroform, dehydrated alcohol, without RNA enzyme Water, random primer, 5 × M-MLV buffer, dNTPs, RNase inhibitor, M-MLV reverse transcriptase, have SEQ ID NO:3 and The ACTB real-time fluorescence quantitative PCR specific primer of nucleotide sequence shown in SEQ ID NO:4.
5 '-GGCACCCAGCACAATGAAGA-3 ', SEQ ID NO:3
5 '-ACTCCTGCTTGCTGATCCAC-3 ', SEQ ID NO:4
Clinical sample testing result shows that NUMB expression significantly reduces (P < 0.001) compared with cancer beside organism in breast cancer；And NUMB low expression is unfavorable for patient with breast cancer's overall survival (P=0.018).Therefore, the reagent for detecting the changes in gene expression can For Prognosis in Breast Cancer or diagnosis, treatment.
Other features and advantages of the present invention will then part of the detailed description can be specified.
Detailed description of the invention
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and its Its purpose, feature and advantage will be apparent.
Fig. 1 shows the expression of NUMB in TCGA high throughput data analysis breast cancer.NUMB expression is aobvious in breast cancer tissue It writes and is lower than normal tissue (P < 0.001).
Fig. 2 shows the influences that TCGA high throughput data analysis NUMB low expression survives to patient with breast cancer.The low table of NUMB Up to being unfavorable for patient with breast cancer's overall survival (P=0.00435).
Fig. 3 shows the expression of NUMB in breast cancer tissue.NUMB expression is substantially less than cancer beside organism in breast cancer tissue (P=0.0037).
Fig. 4 shows the influence that NUMB low expression survives to patient with breast cancer in breast cancer tissue.NUMB low expression is unfavorable In patient with breast cancer's overall survival (P=0.018).
Specific embodiment
The preferred embodiment of the present invention is described in more detail below.Although the following describe preferred implementations of the invention Mode, however, it is to be appreciated that may be realized in various forms the present invention without that should be limited by the embodiments set forth herein.It is real The person that is not specified actual conditions in example is applied, is all carried out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same or instrument are not Production firm person is indicated, is the conventional products that can be obtained by commercially available purchase.
Embodiment 1
The present embodiment is used to illustrate the expression variation of NUMB in TCGA high throughput data analysis breast cancer.
1.TCGA high throughput data analysis process:
Login TCGA portal website UALCAN (http://ualcan.path.uab.edu/index.html) homepage, point It hits " Analysis ", inputs Gene Name " NUMB ", select TCGA dataset " Breast invasive carcinoma ", Retrieval is clicked " Expression ", and result is recorded.It is mapped using 12.0 software of GraphPad, statistical method is T inspection, P < 0.05 is statistically significant for difference.
2. result: the expression of NUMB significantly reduces (P < 0.001) compared with normal tissue in breast cancer tissue, as shown in Figure 1.
Embodiment 2
The present embodiment is for illustrating TCGA high throughput data analysis NUMB low expression and Prognosis in Breast Cancer relationship.
1.TCGA high throughput data analysis process:
It logs in TCGA portal website cBioPortal (http://www.cbioportal.org/), selects tumour data set " Breast Invasive Carcinoma (TCGA Provisional) ", group learn data and select " mRNA Expression z- Scores (RNA Seq V2 RSEM) ", gene input " NUMB:EXP≤- 2 ", retrieval, click in popup web page " Survival " records result.Kaplan-Meier method draws survivorship curve, and log-rank examines survivorship curve difference, P < 0.05 is statistically significant for difference.
2. result: NUMB low expression is unfavorable for patient with breast cancer's overall survival (P=0.00435) (Fig. 2).
Embodiment 3
The present embodiment is used to illustrate that the reagent of preparation detection NUMB expression quantity to be used to prepare the reagent of patient with breast cancer's prognosis Box (50 secondary response).
1.Trizol 50.0ml；
2. isopropanol 50.0ml；
3. chloroform 50.0ml；
4. dehydrated alcohol 50.0ml；
5. without RNA enzyme water 5.0ml
6. 1.0 μM of 50.0 μ l of random primer (Random primers)；
7. 5 × M-MLV buffer 2.0ml；
10.0mM triphosphoric acid base deoxynucleotide 8. (dNTPs) 100.0 μ l；
9. 50.0 μ l of 40U/ μ l RNase inhibitor；
10. 50.0 μ l of 200U/ μ l M-MLV reverse transcriptase；
11.ABI 2×PCR Mix 2.0ml；
12. 10.0 μM of 30.0 μ l of NUMB real-time fluorescence quantitative PCR specific primer, primer sequence are shown in Table 1；
13. 10.0 μM of 30.0 μ l of ACTB real-time fluorescence quantitative PCR specific primer, primer sequence are shown in Table 1；
1 fluorescence quantitative RT-PCR primer sequence of table
Embodiment 4
The present embodiment is used to illustrate the detection of clinical breast cancer tissue samples NUMB.
1. present study carries out under patient's informed consent.57 patient with breast cancer's clinical information are remembered from patient assessment Record.Breast cancer sample is divided into two parts: a part is freezed immediately in liquid nitrogen, is stored in -80 DEG C until carrying out RNA extraction, separately It is a part of then be used for histopathological evaluation.
2. Total RNAs extraction in tissue: this experiment carries out in ice bath.30~50mg is taken to organize (fresh or -70 DEG C and liquid nitrogen The tissue of middle preservation) it sets in 1.5ml centrifuge tube, 1ml Trizol is added and is sufficiently homogenized, is stored at room temperature 5min；Every pipe is added 200 μ l chloroforms acutely mix 30sec, stand 15min, and 4 DEG C of 12000rpm are centrifuged 15min；400 μ l of gentle aspiration supernatant liquid To in another new centrifuge tube, isometric isopropanol is added, is gently mixed by inversion, 4 DEG C of 12000rpm are centrifuged 10min；Supernatant is abandoned, 75% ethanol wash sediment of 1ml is added, 4 DEG C of 12000rpm are centrifuged 10min；Supernatant is discarded as far as possible, is dried at room temperature 10min, every pipe are added 10 μ l without RNA enzyme water, dissolve (65 DEG C of dissolution 10-15min).OD260 is measured, RNA concentration is calculated.
RNA (mg/ml)=40 × OD260× extension rate (n)/1000
3. reverse transcription: every 25 μ l reverse transcription system includes 100pmol random primer, 2 μ g total serum IgEs, 1 μ of M-MLV reverse transcriptase 0.625 1.25 μ l, 5 × M-MLV buffer of μ l, dNTPs (10mM) of l, RNase inhibitor, 5 μ l, no RNA enzyme water polishing to 25 μ l. Reaction condition are as follows: 37 DEG C of 1h, 95 DEG C of 5min.
4. quantitative PCR: every 20 μ l reaction system includes 2 × PCR Mix, 10 μ l, each 0.4 μ l, cDNA 1 of upstream and downstream primer μ l, ddH2O 8.2μl.Reaction condition are as follows: 94 DEG C of 2min, 94 DEG C of 15s, 60 DEG C of 40s, 40 circulations.
5.2-ΔΔCTMethod calculates NUMB relative expression quantity: this experiment detects in 57 breast cancer tissues and 18 cancer beside organisms The relative expression quantity of NUMB changes.ACTB is as reference gene, the target gene NUMB C that qPCR is measuredTValue with tissue-derived Reference gene ACTB CTValue subtracts each other to obtain Δ CT, then by Δ CTWith control group Δ CTSubtract each other to obtain Δ Δ CT(take sample by cancer ΔCTAverage value be Δ CTControl), every group of NUMB relative expression quantity is calculated using Power function in Excell table.Using soft Part GraphPad Prism 6.0 draws, NUMB differential expression by T check analysis breast cancer and cancer, and P < 0.05 is that difference has system Meter learns meaning.
6.NUMB low expression and patient with breast cancer's prognosis: follow-up of patients's time is 1-32 months, successfully receives follow-up patient Number is 57.NUMB relative expression quantity is low expression lower than 2 times of cancer beside organism's relative expression quantity mean, and totally 9, other are NUMB high expression, totally 48.Kaplan-Meier method draws survivorship curve, and log-rank examines survivorship curve difference, P < 0.05 It is statistically significant for difference.
7. result:
Clinical sample testing result shows that NUMB expression significantly reduces (P=0.0037) compared with cancer beside organism in breast cancer, such as Shown in Fig. 3；And NUMB low expression is unfavorable for patient with breast cancer's overall survival (P=0.018), as shown in Figure 4.
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill Many modifications and changes are obvious for the those of ordinary skill in art field.
Sequence table
<110>Jiangsu medical profession institute
<120>application and the kit of the reagent of NUMB endocytosis adaptor protein expression are detected
<130> BJI1900788JSYY
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aggttgagcc atgcagtagg 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gcttgttcag tggctgttgt 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ggcacccagc acaatgaaga 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
actcctgctt gctgatccac 20

Claims (9)

1. the reagent for detecting NUMB endocytosis adaptor protein (NUMB) expression is used for Computer-aided Diagnosis of Breast Cancer and/or cream in preparation Application in the preparation of adenocarcinoma patients' Index for diagnosis.
2. application according to claim 1, wherein the detection NUMB expression includes the gene expression for detecting NUMB Horizontal and/or detection NUMB protein expression level.
3. application according to claim 1, wherein the method for the detection NUMB expression includes: to pass through RT-qPCR Method detects the expression quantity of NUMB in breast cancer and cancer beside organism；It is detected in breast cancer and cancer beside organism by molecular probe technology NUMB mrna expression amount；NUMB protein expression in breast cancer and cancer beside organism is detected by immunohistochemistry or Western-Blot Amount.
4. application according to claim 1, wherein the reagent of the detection NUMB expression is targeting NUMB coding The oligonucleotide probe of DNA sequence dna, PCR primer, or to target the antibody of NUMB.
5. application according to claim 4, wherein the reagent of the detection NUMB expression is with SEQ ID NO: The real-time fluorescence quantitative PCR specific primer of nucleotide sequence shown in 1 and SEQ ID NO:2.
6. a kind of for Computer-aided Diagnosis of Breast Cancer and/or the kit of patient with breast cancer's Index for diagnosis, which includes: detection The reagent of NUMB expression.
7. kit according to claim 6, wherein the reagent of the detection NUMB expression is targeting NUMB coding The oligonucleotide probe of DNA sequence dna, PCR primer, or to target the antibody of NUMB.
8. kit according to claim 7, wherein the reagent of the detection NUMB expression is with SEQ ID The real-time fluorescence quantitative PCR specific primer of nucleotide sequence shown in NO:1 and SEQ ID NO:2.
9. kit according to claim 6, wherein the kit further includes at least one of following components: Trizol, isopropanol, chloroform, dehydrated alcohol, without RNA enzyme water, random primer, 5 × M-MLV buffer, dNTPs, RNA enzyme inhibit Agent, M-MLV reverse transcriptase, the ACTB real time fluorescent quantitative with nucleotide sequence shown in SEQ ID NO:3 and SEQ ID NO:4 PCR specific primer.
CN201910661289.4A 2019-07-22 2019-07-22 Detect application and the kit of the reagent of NUMB endocytosis adaptor protein expression Withdrawn CN110423817A (en)

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