CN110373464A - Detect application and the kit of the reagent of Derlin1 protein expression level - Google Patents
Detect application and the kit of the reagent of Derlin1 protein expression level 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 Derlin1 protein expression level.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 DERL1 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 DERL1 expression is significantly increased compared with cancer beside organism in breast cancer；And DERL1 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, more particularly, to detection Derlin1 albumen (DERL1) expression
Application and one kind of the reagent in the preparation that preparation is used for Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's Index for diagnosis are used for
The kit of Computer-aided Diagnosis of Breast Cancer and/or 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 Derlin1 albumen (DERL1), thus into one
The reagent that step provides detection DERL1 expression is used for Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's Index for diagnosis in preparation
Preparation in application and a kind of for Computer-aided Diagnosis of Breast Cancer and/or the kit of patient with breast cancer's Index for diagnosis.
DERL1 also known as DER1 is a kind of protein coding gene, is positioned at 8q24.13, includes 9 exons, Gene
ID:79139, coding protein are primarily targeted for endoplasmic reticulum and cell membrane.Currently, related DERL1 gene is in mammary gland carcinogenesis
Developing effect has not been reported.The present inventor passes through cancer and oncogene map (Cancer Genome
Atlas, TCGA) high throughput data mining discovery DERL1 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 DERL1 differential expression to the Prognostic of patient with breast cancer.
To achieve the goals above, the first aspect of the present invention provides detection Derlin1 albumen (DERL1) expression
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 DERL1 expression includes gene expression dose and/or the detection for detecting DERL1
The protein expression level of DERL1.
More specifically, it is described detection DERL1 expression method include: by RT-qPCR method detect breast cancer and
The expression quantity of DERL1 in cancer beside organism；DERL1mRNA expression in breast cancer and cancer beside organism is detected by molecular probe technology
Amount；DERL1 expressing quantity in breast cancer and cancer beside organism is detected by immunohistochemistry or Western-Blot.
More specifically, the reagent of the detection DERL1 expression is the oligonucleotide spy for targeting DERL1 DNA sequences encoding
Needle, PCR primer, or to target the antibody of DERL1.
In accordance with the present invention it is preferred that the reagent of the detection DERL1 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 '-GATTTGCATCGTGATTACTGGCT-3 ', SEQ ID NO:1；
5 '-GCCTTAAATCGTGTTCCAAACCA-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 DERL1 expression.
Further, the reagent of the detection DERL1 expression is the oligonucleotide spy for targeting DERL1 DNA sequences encoding
Needle, PCR primer, or to target the antibody of DERL1.
Specifically, the reagent of the detection DERL1 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 DERL1 expression significantly increases (P < 0.001) compared with cancer beside organism in breast cancer；And
DERL1 high expression is unfavorable for patient with breast cancer's overall survival (P=0.0301).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 DERL1 in TCGA high throughput data analysis breast cancer.DERL1 is expressed in breast cancer tissue
It is significantly higher than normal tissue (P < 0.001).
Fig. 2 shows TCGA high throughput data analysis DERL1 high to express the influence survived to patient with breast cancer.DERL1 high
Expression is unfavorable for patient with breast cancer's overall survival (P < 0.001).
Fig. 3 shows the expression of DERL1 in breast cancer tissue.DERL1 expression is significantly higher than group by cancer in breast cancer tissue
Knit (P < 0.001).
Fig. 4 shows DERL1 high in breast cancer tissue and expresses the influence survived to patient with breast cancer.DERL1 high is expressed not
Conducive to patient with breast cancer's overall survival (P=0.0301).
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 DERL1 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 " DERL1 ", 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 DERL1 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 DERL1 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 V2 RSEM) ", gene input " DERL1: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: DERL1 high expression is unfavorable for patient with breast cancer's overall survival (P < 0.001) (Fig. 2).
Embodiment 3
The present embodiment is used to illustrate that the reagent of preparation detection DERL1 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 DERL1 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 DERL1.
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 DERL1 relative expression quantity: this experiment detects in 57 breast cancer tissues and 18 cancer beside organisms
The relative expression quantity of DERL1 changes.ACTB is as reference gene, the target gene DERL1C 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 DERL1 relative expression quantity is calculated using Power function in Excell table.Benefit
It is drawn with software GraphPad Prism 6.0, DERL1 differential expression by T check analysis breast cancer and cancer, P < 0.05 is difference
With statistical significance.
6.DERL1 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 DERL1 relative expression quantity, which is higher than 2 times of cancer beside organism's relative expression quantity mean, and totally 18, other are
DERL1 low expression, totally 39.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 DERL1 expression significantly increases (P < 0.001) compared with cancer beside organism in breast cancer, such as
Shown in Fig. 3；And DERL1 high expression is unfavorable for patient with breast cancer's overall survival (P=0.0301), 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 Derlin1 protein expression level are detected
<130> BJI1900770JSYY
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gatttgcatc gtgattactg gct 23
<210> 2
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gccttaaatc gtgttccaaa cca 23
<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 Derlin1 albumen (DERL1) expression is used for Computer-aided Diagnosis of Breast Cancer and/or breast cancer in preparation
Application in the preparation of patient's Index for diagnosis.
2. application according to claim 1, wherein the detection DERL1 expression includes the gene table for detecting DERL1
Up to horizontal and/or detection DERL1 protein expression level.
3. application according to claim 1, wherein the method for the detection DERL1 expression includes: to pass through RT-
QPCR method detects the expression quantity of DERL1 in breast cancer and cancer beside organism；It is detected by breast cancer and cancer by molecular probe technology
DERL1 mrna expression amount in tissue；DERL1 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 DERL1 expression is targeting DERL1 coding
The oligonucleotide probe of DNA sequence dna, PCR primer, or to target the antibody of DERL1.
5. application according to claim 4, wherein the reagent of the detection DERL1 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 DERL1 expression.
7. kit according to claim 6, wherein the reagent of the detection DERL1 expression is that targeting DERL1 is compiled
The oligonucleotide probe of code DNA sequence dna, PCR primer, or to target the antibody of DERL1.
8. kit according to claim 7, wherein the reagent of the detection DERL1 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 (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140322243A1 (en) * | 2013-04-26 | 2014-10-30 | The Translational Genomics Research Institute | Methods of detecting breast cancer brain metastasis with genomic and epigenomic biomarkers |
| CN105209636A (en) * | 2013-03-15 | 2015-12-30 | 麦塔马克基因股份有限公司 | Compositions and methods for cancer prognosis |
-
2019
- 2019-07-08 CN CN201910611439.0A patent/CN110373464A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105209636A (en) * | 2013-03-15 | 2015-12-30 | 麦塔马克基因股份有限公司 | Compositions and methods for cancer prognosis |
| US20140322243A1 (en) * | 2013-04-26 | 2014-10-30 | The Translational Genomics Research Institute | Methods of detecting breast cancer brain metastasis with genomic and epigenomic biomarkers |
Non-Patent Citations (2)
| Title |
|---|
| JIAO WANG;HUI HUA;YULIANG RAN: "Derlin-1 is overexpressed in human breast carcinoma and protects cancer cells from endoplasmic reticulum stress-induced apoptosis", 《BREAST CANCER RESEARCH》 * |
| KLOPFLEISCH R等: "Increased Derlin-1 expression in metastases of canine mammary adenocarcinomas.", 《JOURNAL OF COMPARATIVE PATHOLOGY》 * |
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