CN110527727A - Detect application and the kit of the reagent of zinc finger protein 28 expression - Google Patents
Detect application and the kit of the reagent of zinc finger protein 28 expression Download PDFInfo
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- CN110527727A CN110527727A CN201910601484.8A CN201910601484A CN110527727A CN 110527727 A CN110527727 A CN 110527727A CN 201910601484 A CN201910601484 A CN 201910601484A CN 110527727 A CN110527727 A CN 110527727A
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- 230000014509 gene expression Effects 0.000 title claims abstract description 59
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- 101710160496 Zinc finger protein 28 Proteins 0.000 title claims description 73
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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 ZNF28 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 ZNF28 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 ZNF28 expression is significantly increased compared with cancer beside organism in breast cancer；And ZNF28 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 the examination of detection zinc finger protein 28 (ZNF28) expression
Application and one kind of the agent 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 for cream
The kit of gland cancer auxiliary diagnosis and/or patient with breast cancer's Index for diagnosis.
Background technique
Breast cancer is most commonly seen gynecologic malignant tumor.Global cancer epidemiology statistical data shows, the world in 2012
In range breast cancer new cases sum 1677000, be only second to lung cancer the second high-incidence tumour and women disease incidence and
The highest malignant tumour of the death rate.China's breast cancer incidence is up to 25.89/10 ten thousand, accounts for about all female malignant morbidities
The 16.83% of rate.Moreover, China's breast cancer illness number and new diagnosis patient's number are just being increasing year by year in global ratio, and
Show morbidity rejuvenation trend, the physical and mental health for seriously affecting women, even threat to life.Therefore, pathogenesis of breast carcinoma mechanism
Research with prognosis is of far-reaching significance to patient with breast cancer.Breast cancer is in origin of cell, Histological Study, disease classification, clinical table
Existing, therapeutic response and metastatic potential etc. all show great complexity and heterogeneity, limit Prognosis in Breast Cancer mark
The popularity of will object application.Therefore, there is an urgent need to develop more targeted prognostic marker for breast cancer, to meet clinical need
It asks.
With quickling increase for the high-throughput data of multiple groups, some molecular biology markers are found and mammary gland carcinogenesis
Related with prognosis, this makes it possible more acurrate, effectively diagnosis and treatment breast cancer.
Summary of the invention
The object of the present invention is to provide a kind of new prognostic marker for breast cancer zinc finger protein 28s (ZNF28), thus into one
The reagent that step provides detection ZNF28 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.
ZNF28 also known as KOX24 is a kind of protein coding gene, is positioned at 19q13.41, includes 7 exons, Gene
ID:7576, coding protein are primarily targeted for nucleus.Currently, the work in relation to ZNF28 gene in breast cancer occurrence and development
With having not been reported.The present inventor passes through cancer and oncogene map (Cancer Genome Atlas, TCGA) high pass
Amount data mining discovery ZNF28 high expression is unfavorable for patient with breast cancer's prognosis, and by collect clinical breast cancer clinical samples and
Follow-up information further verifies ZNF28 differential expression to the Prognostic of patient with breast cancer.
To achieve the goals above, the first aspect of the present invention provides detection zinc finger protein 28 (ZNF28) 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 ZNF28 expression includes gene expression dose and/or the detection for detecting ZNF28
The protein expression level of ZNF28.
More specifically, it is described detection ZNF28 expression method include: by RT-qPCR method detect breast cancer and
The expression quantity of ZNF28 in cancer beside organism；ZNF28mRNA expression in breast cancer and cancer beside organism is detected by molecular probe technology
Amount；ZNF28 expressing quantity in breast cancer and cancer beside organism is detected by immunohistochemistry or Western-Blot.
More specifically, the reagent of the detection ZNF28 expression is the oligonucleotide spy for targeting ZNF28 DNA sequences encoding
Needle, PCR primer, or to target the antibody of ZNF28.
In accordance with the present invention it is preferred that the reagent of the detection ZNF28 expression is with SEQ IDNO:1 and SEQ ID
The real-time fluorescence quantitative PCR specific primer of nucleotide sequence shown in NO:2.
5 '-TGACATTCAGGGACGTGGC-3 ', SEQ ID NO:1；
5 '-TTGCCTTGCCCTGTTGAGAA-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 ZNF28 expression.
Further, the reagent of the detection ZNF28 expression is the oligonucleotide spy for targeting ZNF28 DNA sequences encoding
Needle, PCR primer, or to target the antibody of ZNF28.
Specifically, the reagent of the detection ZNF28 expression is with core shown in SEQ ID NO:1 and SEQID 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 ZNF28 expression significantly increases (P < 0.001) compared with cancer beside organism in breast cancer；And
ZNF28 high expression is unfavorable for patient with breast cancer's overall survival (P=0.0347).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 ZNF28 in TCGA high throughput data analysis breast cancer.ZNF28 is expressed in breast cancer tissue
It is significantly higher than normal tissue (P < 0.001).
Fig. 2 shows TCGA high throughput data analysis ZNF28 high to express the influence survived to patient with breast cancer.ZNF28 high
Expression is unfavorable for patient with breast cancer's overall survival (P=0.00743).
Fig. 3 shows the expression of ZNF28 in breast cancer tissue.ZNF28 expression is significantly higher than group by cancer in breast cancer tissue
Knit (P < 0.001).
Fig. 4 shows ZNF28 high in breast cancer tissue and expresses the influence survived to patient with breast cancer.ZNF28 high is expressed not
Conducive to patient with breast cancer's overall survival (P=0.0347).
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 ZNF28 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 " ZNF28 ", select TCGAdataset " Breastinvasive carcinoma ", inspection
Rope 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 ZNF28 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 ZNF28 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 " mRNAExpression z-
Scores (RNA Seq V2RSEM) ", gene input " ZNF28: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: ZNF28 high expression is unfavorable for patient with breast cancer's overall survival (P=0.00743) (Fig. 2).
Embodiment 3
The present embodiment is used to illustrate that the reagent of preparation detection ZNF28 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 ZNF28 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 ZNF28.
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 ZNF28 relative expression quantity: this experiment detects in 57 breast cancer tissues and 18 cancer beside organisms
The relative expression quantity of ZNF28 changes.ACTB is as reference gene, the target gene ZNF28C 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 ZNF28 relative expression quantity is calculated using Power function in Excell table.Benefit
It is drawn with software GraphPad Prism 6.0, ZNF28 differential expression by T check analysis breast cancer and cancer, P < 0.05 is difference
With statistical significance.
6.ZNF28 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 ZNF28 relative expression quantity, which is higher than 2 times of cancer beside organism's relative expression quantity mean, and totally 9, other are
ZNF28 low expression, totally 48.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 ZNF28 expression significantly increases (P < 0.001) compared with cancer beside organism in breast cancer, such as
Shown in Fig. 3；And ZNF28 high expression is unfavorable for patient with breast cancer's overall survival (P=0.0347), 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 zinc finger protein 28 expression are detected
<130> BJI1900765JSYY
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tgacattcag ggacgtggc 19
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttgccttgcc ctgttgagaa 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 zinc finger protein 28 (ZNF28) 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 ZNF28 expression includes the gene table for detecting ZNF28
Up to horizontal and/or detection ZNF28 protein expression level.
3. application according to claim 1, wherein the method for the detection ZNF28 expression includes: to pass through RT-
QPCR method detects the expression quantity of ZNF28 in breast cancer and cancer beside organism；It is detected by breast cancer and cancer by molecular probe technology
ZNF28 mrna expression amount in tissue；ZNF28 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 ZNF28 expression is targeting ZNF28 coding
The oligonucleotide probe of DNA sequence dna, PCR primer, or to target the antibody of ZNF28.
5. application according to claim 4, wherein the reagent of the detection ZNF28 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 ZNF28 expression.
7. kit according to claim 6, wherein the reagent of the detection ZNF28 expression is that targeting ZNF28 is compiled
The oligonucleotide probe of code DNA sequence dna, PCR primer, or to target the antibody of ZNF28.
8. kit according to claim 7, wherein the reagent of the detection ZNF28 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910601484.8A CN110527727B (en) | 2019-07-05 | 2019-07-05 | Application of reagent for detecting zinc finger protein 28 expression level and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910601484.8A CN110527727B (en) | 2019-07-05 | 2019-07-05 | Application of reagent for detecting zinc finger protein 28 expression level and kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110527727A true CN110527727A (en) | 2019-12-03 |
| CN110527727B CN110527727B (en) | 2021-10-29 |
Family
ID=68659610
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910601484.8A Expired - Fee Related CN110527727B (en) | 2019-07-05 | 2019-07-05 | Application of reagent for detecting zinc finger protein 28 expression level and kit |
Country Status (1)
| Country | Link |
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| CN (1) | CN110527727B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1307024A (en) * | 2000-01-26 | 2001-08-08 | 上海博道基因技术有限公司 | Polypeptide-haman HIT structural sequence motif-contg. zinc finger protein 28 and polynucleotide for coding said polypeptide |
| CN1320642A (en) * | 2000-04-27 | 2001-11-07 | 上海博德基因开发有限公司 | Polypeptide-human zinc finger protein 28 and polynucleotide for coding it |
| US20100285995A1 (en) * | 2008-01-02 | 2010-11-11 | Jose Russo | Identification and Characterization of Pregnancy-Associated Genetic Signatures and Use Thereof for Diagnosis and Treatment of Breast Cancer |
| CN105518153A (en) * | 2013-06-20 | 2016-04-20 | 因姆内克斯普雷斯私人有限公司 | Biomarker identification |
| CN109797219A (en) * | 2019-01-08 | 2019-05-24 | 江苏医药职业学院 | Detect application and the kit of the reagent of ABRACL expression |
-
2019
- 2019-07-05 CN CN201910601484.8A patent/CN110527727B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1307024A (en) * | 2000-01-26 | 2001-08-08 | 上海博道基因技术有限公司 | Polypeptide-haman HIT structural sequence motif-contg. zinc finger protein 28 and polynucleotide for coding said polypeptide |
| CN1320642A (en) * | 2000-04-27 | 2001-11-07 | 上海博德基因开发有限公司 | Polypeptide-human zinc finger protein 28 and polynucleotide for coding it |
| US20100285995A1 (en) * | 2008-01-02 | 2010-11-11 | Jose Russo | Identification and Characterization of Pregnancy-Associated Genetic Signatures and Use Thereof for Diagnosis and Treatment of Breast Cancer |
| CN105518153A (en) * | 2013-06-20 | 2016-04-20 | 因姆内克斯普雷斯私人有限公司 | Biomarker identification |
| CN109797219A (en) * | 2019-01-08 | 2019-05-24 | 江苏医药职业学院 | Detect application and the kit of the reagent of ABRACL expression |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110527727B (en) | 2021-10-29 |
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Effective date of registration: 20221116 Address after: Room 5029, Floor 5, Building C, China Merchants Jiangwan International Center, Qiaokou District, Wuhan, Hubei 430030 Patentee after: Wuhan Aobo Technology Center Address before: 224008 no.283, Jiefang South Road, Yancheng City, Jiangsu Province Patentee before: JIANGSU VOCATIONAL College OF MEDICINE |
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