CN110423813A - Detect application and the kit of the reagent of 1 expression of NudC domain protein - Google Patents
Detect application and the kit of the reagent of 1 expression of NudC domain protein Download PDFInfo
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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 NUDCD1 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 NUDCD1 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 NUDCD1 expression is significantly increased compared with cancer beside organism in breast cancer；And NUDCD1 high 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
Technical field
The invention belongs to field of biotechnology, express water more particularly, to detection NudC domain protein 1 (NUDCD1)
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 NudC domain protein 1 (NUDCD1), by
The reagent that this further provides for detection NUDCD1 expression is pre- for Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer in preparation
Application in the preparation judged afterwards and a kind of for Computer-aided Diagnosis of Breast Cancer and/or the reagent of patient with breast cancer's Index for diagnosis
Box.
NUDCD1 also known as CML66 or OVA66 is a kind of protein coding gene, is positioned at 8q23.1, comprising aobvious outside 12
Son, Gene ID:84955, coding protein are positioned at nucleus and cytoplasm.Currently, related NUDCD1 gene is in breast cancer
Effect in occurrence and development has not been reported.The present inventor passes through cancer and oncogene map (Cancer Genome
Atlas, TCGA) high throughput data mining discovery NUDCD1 high expression is unfavorable for patient with breast cancer's prognosis, and passes through collection clinic
Patient with breast cancer's sample and follow-up information further verify NUDCD1 differential expression to the Prognostic of patient with breast cancer.
To achieve the goals above, the first aspect of the present invention provides detection NudC domain protein 1 (NUDCD1) expression
Horizontal 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 NUDCD1 expression includes gene expression dose and/or the detection for detecting NUDCD1
The protein expression level of NUDCD1.
More specifically, it is described detection NUDCD1 expression method include: by RT-qPCR method detect breast cancer and
The expression quantity of NUDCD1 in cancer beside organism；NUDCD1mRNA expression in breast cancer and cancer beside organism is detected by molecular probe technology
Amount；NUDCD1 expressing quantity in breast cancer and cancer beside organism is detected by immunohistochemistry or Western-Blot.
More specifically, the reagent of the detection NUDCD1 expression is the oligonucleotide for targeting NUDCD1 DNA sequences encoding
Probe, PCR primer, or to target the antibody of NUDCD1.
In accordance with the present invention it is preferred that the reagent of the detection NUDCD1 expression is with SEQ ID NO:1 and SEQ
The real-time fluorescence quantitative PCR specific primer of nucleotide sequence shown in ID NO:2.
5 '-ATGCTTTATCTCCAGGGTTGG-3 ', SEQ ID NO:1
5 '-CCTAAGGCAGTGTCCAGCAT-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 NUDCD1 expression.
Further, the reagent of the detection NUDCD1 expression is the oligonucleotide for targeting NUDCD1 DNA sequences encoding
Probe, PCR primer, or to target the antibody of NUDCD1.
Specifically, the reagent of the detection NUDCD1 expression is with 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 NUDCD1 expression significantly increases (P < 0.001) compared with cancer beside organism in breast cancer；And
NUDCD1 high expression is unfavorable for patient with breast cancer's overall survival (P=0.0389).Therefore, the reagent of the changes in gene expression is detected
It can be used 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 NUDCD1 in TCGA high throughput data analysis breast cancer.NUDCD1 table in breast cancer tissue
Up to being significantly higher than normal tissue (P < 0.001).
Fig. 2 shows TCGA high throughput data analysis NUDCD1 high to express the influence survived to patient with breast cancer.NUDCD1
Height expression is unfavorable for patient with breast cancer's overall survival (P=0.0207).
Fig. 3 shows the expression of NUDCD1 in breast cancer tissue.NUDCD1 expression is significantly higher than by cancer in breast cancer tissue
It organizes (P < 0.001).
Fig. 4 shows NUDCD1 high in breast cancer tissue and expresses the influence survived to patient with breast cancer.NUDCD1 high expression
It is unfavorable for patient with breast cancer's overall survival (P=0.0389).
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 NUDCD1 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 " NUDCD1 ", select TCGA dataset " Breast invasive
Carcinoma ", retrieval are clicked " Expression ", and result is recorded.It is mapped using 12.0 software of GraphPad, statistical method
For T inspection, P < 0.05 is that difference is statistically significant.
2. result: the expression of NUDCD1 significantly increases (P < 0.001) compared with normal tissue in breast cancer tissue, as shown in Figure 1.
Embodiment 2
The present embodiment is for illustrating that TCGA high throughput data analyze NUDCD1 high 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 V2RSEM) ", gene input " NUDCD1:EXP >=2 ", it retrieves, is clicked 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: NUDCD1 high expression is unfavorable for patient with breast cancer's overall survival (P=0.0207) (Fig. 2).
Embodiment 3
The present embodiment is used to illustrate that the reagent of preparation detection NUDCD1 expression quantity to be used to prepare the examination of patient with breast cancer's prognosis
Agent 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 NUDCD1 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 NUDCD1.
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 NUDCD1 relative expression quantity: this experiment detects 57 breast cancer tissues and 18 cancer beside organisms
The relative expression quantity of middle NUDCD1 changes.ACTB is as reference gene, the target gene NUDCD1C that qPCR is measuredTIt is worth and same group
Knit the C of the reference gene ACTB in sourceTValue subtracts each other to obtain Δ CT, then by Δ CTWith control group Δ CTSubtract each other to obtain Δ Δ CT(take cancer
Other sample Δ CTAverage value be Δ CTControl), every group of NUDCD1 relative expression is calculated using Power function in Excell table
Amount.It is drawn using software GraphPad Prism 6.0, NUDCD1 differential expression by T check analysis breast cancer and cancer, P < 0.05
There is statistical significance for difference.
6.NUDCD1 high expression and patient with breast cancer's prognosis: follow-up of patients's time is 1-32 months, successfully receives follow-up trouble
Person's number is 57.It is that height is expressed that NUDCD1 relative expression quantity, which is higher than 2 times of cancer beside organism's relative expression quantity mean, totally 21,
He is NUDCD1 low expression, totally 36.Kaplan-Meier method draws survivorship curve, and log-rank examines survivorship curve difference, P
< 0.05 is that difference is statistically significant.
7. result:
Clinical sample testing result shows that NUDCD1 expression significantly increases (P < 0.001) compared with cancer beside organism in breast cancer, such as
Shown in Fig. 3；And NUDCD1 high expression is unfavorable for patient with breast cancer's overall survival (P=0.0389), 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 1 expression of NudC domain protein are detected
<130> BJI1900783JSYY
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atgctttatc tccagggttg g 21
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cctaaggcag tgtccagcat 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. detect the reagent of NudC domain protein 1 (NUDCD1) expression preparation for Computer-aided Diagnosis of Breast Cancer and/or
Application in the preparation of patient with breast cancer's Index for diagnosis.
2. application according to claim 1, wherein the detection NUDCD1 expression includes the gene for detecting NUDCD1
Expression and/or the protein expression level for detecting NUDCD1.
3. application according to claim 1, wherein the method for the detection NUDCD1 expression includes: to pass through RT-
QPCR method detects the expression quantity of NUDCD1 in breast cancer and cancer beside organism；It is detected by breast cancer and cancer by molecular probe technology
NUDCD1 mrna expression amount in tissue；It is detected in breast cancer and cancer beside organism by immunohistochemistry or Western-Blot
NUDCD1 expressing quantity.
4. application according to claim 1, wherein the reagent of the detection NUDCD1 expression is that targeting NUDCD1 is compiled
The oligonucleotide probe of code DNA sequence dna, PCR primer, or to target the antibody of NUDCD1.
5. application according to claim 4, wherein the reagent of the detection NUDCD1 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.
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 NUDCD1 expression.
7. kit according to claim 6, wherein the reagent of the detection NUDCD1 expression is targeting NUDCD1
The oligonucleotide probe of DNA sequences encoding, PCR primer, or to target the antibody of NUDCD1.
8. kit according to claim 7, wherein the reagent of the detection NUDCD1 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.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910635985.8A CN110423813A (en) | 2019-07-15 | 2019-07-15 | Detect application and the kit of the reagent of 1 expression of NudC domain protein |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050208500A1 (en) * | 2003-03-04 | 2005-09-22 | Erlander Mark G | Signatures of ER status in breast cancer |
| US20100152058A1 (en) * | 2004-09-30 | 2010-06-17 | Pier Paolo Di Fiore | Cancer markers |
-
2019
- 2019-07-15 CN CN201910635985.8A patent/CN110423813A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050208500A1 (en) * | 2003-03-04 | 2005-09-22 | Erlander Mark G | Signatures of ER status in breast cancer |
| US20100152058A1 (en) * | 2004-09-30 | 2010-06-17 | Pier Paolo Di Fiore | Cancer markers |
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Application publication date: 20191108 |