CN107515305A - Uterine neck carcinoma marker EBP50 and its application - Google Patents
Uterine neck carcinoma marker EBP50 and its application Download PDFInfo
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- CN107515305A CN107515305A CN201610420509.0A CN201610420509A CN107515305A CN 107515305 A CN107515305 A CN 107515305A CN 201610420509 A CN201610420509 A CN 201610420509A CN 107515305 A CN107515305 A CN 107515305A
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- ebp50
- egfr
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- 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
- G01N33/57411—Specifically defined cancers of cervix
Abstract
The present invention relates to molecular biology and medical oncology field, is related to a kind of applications of new special uterine neck carcinoma marker EBP50 in cervical carcinoma prognosis and molecular targeted therapy.The present invention has found EBP50 notable low expressions in cervical cancer tissues using the method analysis of bioinformatics joint biological experiment, and with suppression tumor cell proliferation and the ability in cycle.EGFR paths are significant for treatment of human cervical cancer, and EBP50 can be with EGFR interactions.It has been found that EBP50 can suppress the activation of EGFR/ERK paths, the effect of tumor suppressor gene is played in cervical carcinoma.Finally by prognostic analysis, new approaches can be provided, also provide new target spot for the treatment of cervical carcinoma as a kind of new special uterine neck carcinoma marker, the prognosis of research and prediction Patients with Cervical Cancer for cervical cancer pathogenesis mechanism by specifying EBP50.
Description
Technical field
The invention belongs to oncomolecularbiology field, and in particular to a kind of tumor markers EBP50 is in prediction cervical carcinoma prognosis
In application, and applications of the EBP50 as uterine neck carcinoma marker in molecular targeted agents are prepared.
Background technology
Cervical carcinoma is the common malignant tumour of women.The new hair cervical carcinoma 528 000 in the whole world in 2012, dead 266 000, its
In 85% cervical carcinoma betide developing country.China is the second country occurred frequently of cervical carcinoma, annual new cases about 13.15 ten thousand,
The 28.7% of global new cases is accounted for, death toll accounts for 1/3rd of global death, and is in rejuvenation trend.It can be seen that palace
Neck cancer health of the serious threat to China women.The early cervical examination that some areas are implemented at home at present contributes to
Cervical lesionses are found in time and carry out treatment, block vicious transformation of the cervical lesionses to cervical carcinoma.But for remote poverty-stricken area,
Early screening work can not be completely covered, and patient once makes a definite diagnosis the cervical carcinoma middle and advanced stage stage that has often been in, and treat
Effect is poor.
For other malignant tumours, cervical carcinoma by early screening, as TCT (Thinprepcytologic test, newly
Bai Shi liquid based cytologies technology) cytology detection, HPV detections, vaginoscopy and the methods of cervical tissue pathological examination
Cervical lesionses can be found.Prevention and the poor area of medical condition in China, have been found that cervical cell cancer during patient assessment more
Become, there are about 2/3rds in patient just medical after cervical carcinoma symptom occurs three months has developed into cancer of late stage.Cervical carcinoma
Surgical resection therapy usually due to tumour transfer and can not thorough tumor resection tissue so that clinical treatment is relatively difficult,
It is postoperative also usually to recur (5 years survival rates of human invasive cervix neoplasms are only 20%-50%, Postoperative recurrent rate about 35%)；Radiotherapy
Range of exposures must be expanded because of metastases again, the damage to normal body is correspondingly increased, influence prognosis.So have
The prognosis of effect ground prediction cervical carcinoma and searching novel tumor molecular targeted agents are particularly important as essential therapeutic arsenals.
Development of Novel uterine neck carcinoma marker is as the focus that prognostic indicator and novel therapeutic target spot are always cervical carcinoma research.
The surgical resection therapy of cervical carcinoma usually due to tumour transfer and can not thorough tumor resection tissue so that clinical treatment
It is relatively difficult, it is postoperative also usually to recur (5 years survival rates of human invasive cervix neoplasms are only 20%-50%, Postoperative recurrent rate about 35%).
Therefore, it is the important scientific problems that cervical carcinoma research is badly in need of solving to predict palace prognosis and carry out corresponding auxiliary treatment.Tumour
The discovery of mark and rationally application are the premises of tumour prediction prognosis and auxiliary treatment.Therefore for finding out more preferable cervical carcinoma
Prognosis molecule mark, also the molecular targeted therapy for cervical carcinoma provide important theoretical foundation.
So far, there is not yet effectively predicting the molecular marker for being available for clinical practice of cervical carcinoma prognosis.Using EBP50 as
Mark is used for the reagent and method of cervical carcinoma detection, all will verify EBP50 and the phase of cervical carcinoma for further investigation and on a large scale
Guan Xing, prognosis situation is predicted cervical carcinoma and provides supplemental treatment regimens for individual patient there is important clinical meaning.
The content of the invention
It is an object of the invention to provide EBP50 new application.The invention provides EBP50 as cervical carcinoma prognosis molecule mark
Purposes, and the application using EBP50 as target sieving auxiliary treatment of cervical carcinoma medicine, ginseng is provided for cervical carcinoma prognosis and treatment
Examine foundation.
The invention provides purposes of the EBP50 as uterine neck carcinoma marker, mainly for cervical carcinoma.
The expression quantity of com-parison and analysis cervical cancer tissues sample and EBP50 in normal control sample.
The part of test results of the present invention analyzes (Gene set enrichment analysis, GSEA) research with genetic enrichment
The biological function that EBP50 is played in Patients with Cervical Cancer, the permutation test of 1000 times is performed, calculate FDR values, FDR<0.05 recognizes
It is statistically significant for tool.
Using Pull down and Co-IP checking EBP50 mutant DD and EGFR interaction abilities.
Using the situation of western blot detection EGFR and ERK phosphorylations, it is found that EBP50 is obvious in Cervical Cancer HeLa Cells
Suppress EGFR and the ERK in downstream phosphorylation level.Experimental result is consistent with bioinformatics result, shows that EBP50 can be adjusted
Save EGFR/ERK signal paths.Illustrate from another angle, EBP50 can be as the new target of screening uterine neck cancer drug in the present invention
Point.
K-M survivorship curve analysis results are shown：Patient's prognosis mala of EBP50 low expressions, prompts it can also serve as new
Cervical carcinoma prognosis molecule mark.
Brief description of the drawings
Fig. 1 is that EBP50 protein expressions are lowered and promote cell propagation and cyclic graph in cervical cancer tissues
Fig. 2 is EBP50 expression and the negatively correlated figure of EGFR-ERK pathway activities in cervical carcinoma
Fig. 3 is that EBP50 mutant DD is weakened and EGFR binding ability figures
Fig. 4 is that EBP50 mutant DD weakens to EGFR-ERK path rejection ability figures
Fig. 5 is that EBP50 expression can predict Patients with Cervical Cancer prognosis figure
Specific implementation method
Embodiment one：Analyze the expression change of EBP50 albumen in cervical cancer tissues and normal structure
(1) material and method
1. material
Download the IHC results of cervical cancer tissues and normal structure in The human protein atlas databases
2. method
With the IOD values of the software statistics ImmunohistochemistryResults Results of Image Pro Plus 6, then by SPSS to tumor group and normal
Group carries out statistical analysis.
(2) result
The sample of 11 cervical carcinomas and 3 normal structures, pass through the expression feelings of Immunohistochemical study sample EBP50 albumen
Condition, as shown in Figure 1A, visible EBP50 albumen positive expression in normal structure, and EBP50 protein stainings in cervical cancer tissues
Close to feminine gender (Figure 1A).
Embodiment two：Analyze the expression change of EBP50 albumen in cervical cancer tissues and normal structure
(1) material and method
1. material
Download the sequencing result of the RNA Seq v2 of cervical carcinoma in TCGA databases, and Molecular Signatures
Two gene set (GO in Database:0042127 and GO:0045787)
2. method
The sequencing result of the RNA Seq v2 of cervical carcinoma in TCGA databases is divided into EBP50 height expression groups by EBP50 expression quantity
With low expression group, then with GSEA software analysis cell proliferation genes collection (GO:And cell cycle gene collection (GO 0042127):
0045787) enrichment condition of gene expression in.
(2) result
The gene significant enrichment that cell proliferation genes collection and cell cycle gene are concentrated EBP50 low expressions group (figure in cervical carcinoma
1B-C), show that EBP50 can significantly inhibit propagation and the cycle of cervical cancer cell.
Embodiment three：Analyze influence of the EBP50 expression to EGFR/ERK signal paths in cervical carcinoma
(1) material and method
1. material
Download the result of the RNA Seq v2 of cervical carcinoma sequencing result and RPPA protein chip in TCGA databases
Cell line used in experiment is that the stabilization of this laboratory structure strikes low EBP50 Cervical Cancer HeLa Cells system and HeLa cell lines
PAGE gel
Pvdf membrane
Skimmed milk power
Primary antibody EBP50 antibody (CST, 1：1000), primary antibody pERK antibody (abcam, 1：1000), primary antibody ERK antibody (abcam, 1：
1000) and GAPDH antibody (abcam, 1：1000)
TBST
Secondary antibody (1：10000)
EGF
2. method
The sequencing result of the RNA Seq v2 of cervical carcinoma in TCGA databases is divided into EBP50 height expression groups by EBP50 expression quantity
With low expression group, then with GSEA software analysis cell proliferation genes collection (GO:And cell cycle gene collection (GO 0042127):
0045787) enrichment condition of gene expression in.
Inoculating cell：On the packet of each 6 orifice plate rims labelling experiment and date, the cell diluted of addition respective handling per hole
Suspension 2ml, i.e. cell number are 150,000/hole.6 orifice plates are put into incubator (at 37 DEG C, under conditions of 5%CO2) culture relaying
Continuous culture.Second day serum starvation is stimulated 5 minutes after 24 hours with EGF, collects cell.
Cell pyrolysis liquid is taken to carry out 10%SDS-PAGE electrophoresis, albumen is transferred on pvdf membrane (100V, 80min), with 5% degreasing
Milk powder room temperature closes 1h；Primary antibody EBP50 antibody, pERK antibody, ERK antibody and GAPDH antibody distinguish 4 DEG C of overnight incubations, TBST
Wash film 3 times；With secondary antibody (1：10000) 1h is incubated at room temperature, TBST washes film 3 times, exposes.
(2) result
The gene set significant enrichment that EGFR Pathway Activations are lowered EBP50 height expression group (Fig. 2A) in cervical carcinoma, shows that EBP50 can
To significantly inhibit the EGFR signal paths in cervical carcinoma.Pearson correlations and regression analysis show, EBP50 expression
It is significantly negatively correlated (Fig. 2 B) with c-RAF phosphorylated protein expression, prompt EBP50 to inhibit EGFR downstream signals point really
The activity of son.GSEA analysis results show, ERK pathway gene collection significant enrichments EBP50 low expressions group (Fig. 2 C) in cervical carcinoma,
Further illustrate that EBP50 can suppress the activation of downstream ERK paths of EGFR mediations.Western Blotting results show, surely
Surely the HeLa cells for having struck EBP50 low ERK phosphorylation level when EGF is stimulated 5 minutes significantly raises, and shows that EBP50 can be with
Suppress the ERK signal paths (Fig. 2 D) of EGFR mediations.
Example IV：Verify EBP50 mutant and EGFR interaction ability
(1) material and method
1. material
PBK-CMV-HA-EBP50 and pBK-CMV-HA-EBP50 mutant (S279D/S301D, DD) plasmid
Lip2000
Gst fusion protein
Coomassie brilliant blue
Ponceau S dyestuffs
HA beads
Cell line used in experiment is HeLa and COS7 cell lines
Primary antibody HA antibody (CST, 1：1000), an anti-egfr antibodies (CST, 1：1000) and GST antibody (abcam, 1：1000)
TBST
Secondary antibody (1：10000)
2. method
Each culture dish is collected into 1.5ml centrifuge tubes with 1ml cell pyrolysis liquids after HeLa or COS-7 cell transient transfections, 4 DEG C
Rotation is incubated 40-60min；4 DEG C, 13000rpm centrifugation 15min, take supernatant into new centrifuge tube, leave and take a small amount of as immune
Trace compares；30 μ l HA beads are added in remaining sample, 4 DEG C of rotations are incubated 3h；Added after lavation buffer solution rinsing appropriate
Albumen sample-loading buffer, 95 DEG C of heating 5min, centrifugation draw supernatant, obtain immunoblotting assay sample；Sample is immunized
Engram analysis.
(2) result
GST-Pull down and Co-IP experiment all show that EBP50 mutant DD and EGFR interaction weaken (Fig. 3).
Embodiment five：Western Blotting detect influence of the EBP50 mutant to EGFR/ERK signal paths
(1) material and method
1. material
Lip2000
Cell line used in experiment is HeLa cell lines
PAGE gel
Pvdf membrane
Skimmed milk power
Primary antibody pEGFR antibody (CST, 1：1000), an anti-egfr antibodies (CST, 1：1000), primary antibody pERK antibody (abcam, 1：
1000), primary antibody ERK antibody (abcam, 1：1000), primary antibody HA antibody (CST, 1：1000) and GAPDH antibody (abcam, 1：
1000)
TBST
Secondary antibody (1：10000)
EGF
2. method
Cell pyrolysis liquid is taken to carry out 10%SDS-PAGE electrophoresis, albumen is transferred on pvdf membrane (100V, 80min), with 5% degreasing
Milk powder room temperature closes 1h；Primary antibody pEGFR antibody (CST, 1：1000), an anti-egfr antibodies (CST, 1：1000), primary antibody pERK resists
Body (abcam, 1：1000), primary antibody ERK antibody (abcam, 1：1000), primary antibody HA antibody (CST, 1：And GAPDH antibody 1000)
(abcam, 1：1000) 4 DEG C of overnight incubations, TBST wash film 3 times respectively；With secondary antibody (1：10000) 1h is incubated at room temperature, TBST washes film 3
It is secondary, exposure.
(2) result
With the EBP50 mutant DD of EGFR interaction reduced capabilities EGFR and ERK phosphorylation level can be promoted to raise (figure
4) EBP50, is shown by the interaction with EGFR to adjust EGFR and downstream signaling molecule activity.
Embodiment six：Influences of the K-M tracing analysis EBP5 to cervical cancer patient prognosis
(1) material and method
1. material
Download the sequencing result and clinical patient information of the RNA Seq v2 of cervical carcinoma in TCGA databases
2. method
It is not morphed for EGFR gene group using Prism5 analyses EBP50 expression and do not carried out chemicotherapy
Patient accumulate survival probability
Influence.
(2) result
Analysis result finds that patient's prognosis of EBP50 low expression groups is worse (Fig. 5), shows that EBP50 does not occur in EGFR gene group
Really can be as the prognosis molecule mark of cervical carcinoma in the patient of variation.
Claims (6)
- Purposes of the 1.EBP50 in tumor markers is prepared, wherein described tumour is cervical carcinoma.
- 2. application as claimed in claim 1, it is characterised in that EBP50 is called NHERF1 and SLC9A3R1.
- 3. large biological molecule EBP50 albumen as claimed in claim 1 or mRNA are as palace as the feature of tumor markers The index of neck cancer prognosis.
- 4. weaken the EBP50 mutant DD with EGFR interactions（S279D/S301D）Structure and identification.
- 5. EBP50 mutant DD as claimed in claim 4（S279D/S301D）For EGFR downstream signaling pathways and cell work( The research system of energy.
- 6.EBP50 is preparing the application of auxiliary treatment of cervical carcinoma cancer targeting molecules agents.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114395625A (en) * | 2021-12-29 | 2022-04-26 | 广东省人民医院 | Application of COPA in preparation of cervical cancer diagnosis biomarker and/or cervical cancer drug development |
Citations (2)
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| CN102575290A (en) * | 2009-07-13 | 2012-07-11 | 萨里大学 | Biomarker ER2 for gynaecological cancer |
| CN103472121A (en) * | 2013-09-09 | 2013-12-25 | 济南大学 | Sandwich type electrochemical immunosensor for simultaneously detecting markers of cervical cancer |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102575290A (en) * | 2009-07-13 | 2012-07-11 | 萨里大学 | Biomarker ER2 for gynaecological cancer |
| CN103472121A (en) * | 2013-09-09 | 2013-12-25 | 济南大学 | Sandwich type electrochemical immunosensor for simultaneously detecting markers of cervical cancer |
Non-Patent Citations (4)
| Title |
|---|
| CHAOYUAN SUN,ET AL: "EBP50 phosphorylation by Cdc2-Cyclin B kinase affects actin cytoskeleton reorganization and regulates functions of human breast cancer cell line MDA-MB-231", 《MOL. CELLS》 * |
| JUNFANG ZHENG,ET AL: "Abstract 1935: EBP50 inhibits breast cancer cell proliferation by blocking EGFR phosphorylation", 《PROCEEDINGS OF THE 102ND ANNUAL MEETING OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH》 * |
| JUNQI HE,ET AL: "Phosphorylation and Cell Cycle-dependent Regulation of Na+/H+Exchanger Regulatory Factor-1 by Cdc2 Kinase", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114395625A (en) * | 2021-12-29 | 2022-04-26 | 广东省人民医院 | Application of COPA in preparation of cervical cancer diagnosis biomarker and/or cervical cancer drug development |
| CN114395625B (en) * | 2021-12-29 | 2023-08-04 | 广东省人民医院 | Application of COPA in preparation of cervical cancer diagnosis biomarker and/or development of cervical cancer drug |
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