CN110373463A - Detect application and the kit of the reagent of cross-film P24 transport protein 9 expression - Google Patents
Detect application and the kit of the reagent of cross-film P24 transport protein 9 expression 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 cross-film P24 transport protein 9 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 TMED9 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 TMED9 expression is significantly increased compared with cancer beside organism in breast cancer；And TMED9 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 cross-film P24 transport protein 9 (TMED9)
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 (Breast cancer, BC) is most commonly seen gynecologic malignant tumor.In China, disease incidence reaches
25.89/10 ten thousand, account for about the 16.83% of all female malignant incidences.And China's breast cancer illness number and new diagnosis are suffered from
Person's number is just being increasing year by year in global ratio.With the promotion of operative treatment and chemicotherapy level, patient with breast cancer's existence
Time is obviously prolonged, but overall incidence and the death rate still occupy female tumor first place.In recent years, with high-throughput, multiple groups
The rapid growth of data, some novel markers are found related to mammary gland carcinogenesis and prognosis, have also prompted some new
The potential target spot with anti-breast cancer druggability, is breast cancer parting, and accurate prognosis and treatment breast cancer provide possibility.
Summary of the invention
The object of the present invention is to provide a kind of new prognostic marker for breast cancer cross-film P24 transport protein 9 (TMED9), by
The reagent that this further provides for detection TMED9 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.
TMED9 also known as GMP25 is a kind of protein coding gene, is positioned at 5q35.3, includes 5 exons, Gene
ID:54732, coding protein are primarily targeted for endoplasmic reticulum and golgiosome.Currently, related TMED9 gene is sent out in breast cancer
Raw developing effect has not been reported.The present inventor passes through cancer and oncogene map (Cancer Genome
Atlas, TCGA) high throughput data mining discovery TMED9 high expression is unfavorable for patient with breast cancer's prognosis, and passes through collection clinic cream
Adenocarcinoma patients' sample and follow-up information further verify TMED9 differential expression to the Prognostic of patient with breast cancer.
To achieve the goals above, the first aspect of the present invention provides detection cross-film P24 transport protein 9 (TMED9) 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 TMED9 expression includes gene expression dose and/or the detection for detecting TMED9
The protein expression level of TMED9.
More specifically, it is described detection TMED9 expression method include: by RT-qPCR method detect breast cancer and
The expression quantity of TMED9 in cancer beside organism；TMED9mRNA expression in breast cancer and cancer beside organism is detected by molecular probe technology
Amount；TMED9 expressing quantity in breast cancer and cancer beside organism is detected by immunohistochemistry or Western-Blot.
More specifically, the reagent of the detection TMED9 expression is the oligonucleotide spy for targeting TMED9 DNA sequences encoding
Needle, PCR primer, or to target the antibody of TMED9.
In accordance with the present invention it is preferred that the reagent of the detection TMED9 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 '-CTGGGTAGAGTGATGCGGAC-3 ', SEQ ID NO:1；
5 '-TCTCCGTCTCTCCGATGTGA-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 TMED9 expression.
Further, the reagent of the detection TMED9 expression is the oligonucleotide spy for targeting TMED9 DNA sequences encoding
Needle, PCR primer, or to target the antibody of TMED9.
Specifically, the reagent of the detection TMED9 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 TMED9 expression significantly increases (P < 0.001) compared with cancer beside organism in breast cancer；And
TMED9 high expression is unfavorable for patient with breast cancer's overall survival (P=0.0063).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 TMED9 in TCGA high throughput data analysis breast cancer.TMED9 is expressed in breast cancer tissue
It is significantly higher than normal tissue (P < 0.001).
Fig. 2 shows TCGA high throughput data analysis TMED9 high to express the influence survived to patient with breast cancer.TMED9 high
Expression is unfavorable for patient with breast cancer's overall survival (P=0.0035).
Fig. 3 shows the expression of TMED9 in breast cancer tissue.TMED9 expression is significantly higher than group by cancer in breast cancer tissue
Knit (P < 0.001).
Fig. 4 shows TMED9 high in breast cancer tissue and expresses the influence survived to patient with breast cancer.TMED9 high is expressed not
Conducive to patient with breast cancer's overall survival (P=0.0063).
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 TMED9 in TCGA high throughput data analysis breast cancer.
1.TCGA high throughput data analysis process:
Log in TCGA portal website UALCAN (h ttp://ualcan.path.uab.edu/index.html) homepage, point
It hits " Analysis ", inputs Gene Name " TMED9 ", 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 TMED9 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 TMED9 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 " TMED9: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: TMED9 high expression is unfavorable for patient with breast cancer's overall survival (P=0.0035) (Fig. 2).
Embodiment 3
The present embodiment is used to illustrate that the reagent of preparation detection TMED9 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 50.0 μ l of μM random primer (Random primers)；
7.5 × M-MLV buffer 2.0ml；
8.10.0mM 100.0 μ l of triphosphoric acid base deoxynucleotide (dNTPs)；
50.0 μ l of 9.40U/ μ l RNase inhibitor；
50.0 μ l of 10.200U/ μ l M-MLV reverse transcriptase；
11.ABI 2×PCR Mix 2.0ml；
12.10.0 μM 30.0 μ l of TMED9 real-time fluorescence quantitative PCR specific primer, primer sequence are shown in Table 1；
13.10.0 μM 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 TMED9.
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, is added
Enter 75% ethanol wash sediment of 1ml, 4 DEG C of 12000rpm are centrifuged 10min；Supernatant is discarded as far as possible, dries 10min at room temperature,
10 μ l are added without RNA enzyme water in every pipe, 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 TMED9 relative expression quantity: this experiment detects in 57 breast cancer tissues and 18 cancer beside organisms
The relative expression quantity of TMED9 changes.ACTB is as reference gene, the target gene TMED9C that qPCR is measuredTValue is come with tissue
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 sample by cancer
This Δ CTAverage value be Δ CTControl), every group of TMED9 relative expression quantity is calculated using Power function in Excell table.Benefit
It is drawn with software GraphPad Prism 6.0, TMED9 differential expression by T check analysis breast cancer and cancer, P < 0.05 is difference
With statistical significance.
6.TMED9 high 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.It is high expression that TMED9 relative expression quantity, which is higher than 2 times of cancer beside organism's relative expression quantity mean, and totally 10, other are
TMED9 low expression, totally 47.Kaplan-Meier method draws survivorship curve, and log-rank examines survivorship curve difference, P <
0.05 is statistically significant for difference.
7. result:
Clinical sample testing result shows that TMED9 expression significantly increases (P < 0.001) compared with cancer beside organism in breast cancer, such as
Shown in Fig. 3；And TMED9 high expression is unfavorable for patient with breast cancer's overall survival (P=0.0063), 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 cross-film P24 transport protein 9 expression are detected
<130> BJI1900769JSYY
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ctgggtagag tgatgcggac 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tctccgtctc tccgatgtga 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 cross-film P24 transport protein 9 (TMED9) 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 TMED9 expression includes the gene table for detecting TMED9
Up to horizontal and/or detection TMED9 protein expression level.
3. application according to claim 1, wherein the method for the detection TMED9 expression includes: to pass through RT-
QPCR method detects the expression quantity of TMED9 in breast cancer and cancer beside organism；It is detected by breast cancer and cancer by molecular probe technology
TMED9 mrna expression amount in tissue；TMED9 in breast cancer and cancer beside organism is detected by immunohistochemistry or Western-Blot
Expressing quantity.
4. application according to claim 1, wherein the reagent of the detection TMED9 expression is targeting TMED9 coding
The oligonucleotide probe of DNA sequence dna, PCR primer, or to target the antibody of TMED9.
5. application according to claim 4, wherein the reagent of the detection TMED9 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 TMED9 expression.
7. kit according to claim 6, wherein the reagent of the detection TMED9 expression is that targeting TMED9 is compiled
The oligonucleotide probe of code DNA sequence dna, PCR primer, or to target the antibody of TMED9.
8. kit according to claim 7, wherein the reagent of the detection TMED9 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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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2163649A1 (en) * | 2008-09-11 | 2010-03-17 | Fédération Nationale des Centres de Lutte Contre le Cancer | Molecular classifier for evaluating the risk of metastasic relapse in breast cancer |
| WO2014071279A2 (en) * | 2012-11-05 | 2014-05-08 | Genomic Health, Inc. | Gene fusions and alternatively spliced junctions associated with breast cancer |
| CN105339797A (en) * | 2013-04-18 | 2016-02-17 | 建喾立嗣股份公司 | Genetic marker for early breast cancer prognosis prediction and diagnosis, and use thereof |
-
2019
- 2019-07-08 CN CN201910611438.6A patent/CN110373463A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2163649A1 (en) * | 2008-09-11 | 2010-03-17 | Fédération Nationale des Centres de Lutte Contre le Cancer | Molecular classifier for evaluating the risk of metastasic relapse in breast cancer |
| WO2014071279A2 (en) * | 2012-11-05 | 2014-05-08 | Genomic Health, Inc. | Gene fusions and alternatively spliced junctions associated with breast cancer |
| CN105339797A (en) * | 2013-04-18 | 2016-02-17 | 建喾立嗣股份公司 | Genetic marker for early breast cancer prognosis prediction and diagnosis, and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| SONAKSHI MISHRA ET AL.: "The protein secretion modulator TMED9 drives CNIH4/TGFα/GLI signaling opposing TMED3-WNT-TCF to promote colon cancer metastases", 《ONCOGENE》 * |
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Application publication date: 20191025 |