CN110004228A - One kind diagnosis marker relevant to breast cancer molecular parting and application thereof - Google Patents
One kind diagnosis marker relevant to breast cancer molecular parting and application thereof Download PDFInfo
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- CN110004228A CN110004228A CN201910264400.6A CN201910264400A CN110004228A CN 110004228 A CN110004228 A CN 110004228A CN 201910264400 A CN201910264400 A CN 201910264400A CN 110004228 A CN110004228 A CN 110004228A
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Abstract
The invention discloses a kind of diagnosis marker relevant to breast cancer molecular parting, which is USP30, and diagnosis efficiency is high, can be realized the parting detection of breast cancer.The diagnosis marker can prepare for Diagnosis of Breast cancer or judge the reagent or kit of Prognosis in Breast Cancer, can prepare reagent or kit for judging breast cancer classification.And USP30 can prepare the reagent or kit for inhibiting breast cancer cell to shift, and lay a good foundation for the exploitation of breast cancer medicines.
Description
Technical field
The invention belongs to biotechnology and oncology technical field, be related to it is a kind of it is relevant to breast cancer molecular parting go it is general
Elementization enzyme and its application.
Background technique
Breast cancer is the most common female cancer, accounts for 28.7% and lethality of all cancers being diagnosed to be in women
Second high malignant tumour.With the progress of breast cancer biological diagnosis and treatment aspect, 5 years survival rates of breast cancer have increased
To more than 85%.However, most of patient with breast cancer dies of postoperative cerebral transfer progress, especially Basaloid in 5 years
The brain metastes of type (Basal-like, Basal) breast cancer.
Research points out that brain metastes eventually occur for the patient with breast cancer of about 50% progressive stage, and are more common in Basal type and people
Epidermal growth factor acceptor 2 overexpression type (HER2overexpression, Her2).The breast cancer diagnosis mark of breast cancer at this stage
Will object mainly has the circRNA such as circTADA2A-E6, circTADA2A-E5/E6, circNOL10, cfDNA methylation sites
Methylation level and the plasma proteinase C1 inhibitor of N-Acetyl-D-glucosamine glycosyl etc..These markers can not identify trouble
The breast cancer classification of person, not high in people's Level of Expression of Retinoic Acid, unstable in vitro, the standard of detection level is difficult to formulate.Therefore,
There is an urgent need to a kind of identification biomarkers to screen high-risk patient and predict breast cancer result and traditional clinical pathology
Feature.
Deubiquitinating enzymes (DUBs) are a kind of large numbers of protease families.It mainly passes through hydrolysis Ubiquitin carboxy
The slave chain of ubiquitin molecule specificity is connected to the protein or precursor protein water of ubiquitin by ester bond, peptide bond or the isopeptide bond of end
Solution is got off.Deubiquitinating enzymes pass through the degradation of the adjustable protein of deubiquitination；Coordinate the cellular localization of protein；Activation and
Inactivate protein；And regulatory protein matter-protein interaction is broken up, apoptosis, autophagy to influence cell Proliferation.Remove ubiquitin
The occurrence and development for changing enzyme and cancer are closely related.The study found that deubiquitinating enzymes express imbalance in kinds cancer tissue, pass through
Regulation promotees cancer or presses down the activity of cancer signal path, plays the function of tumor suppressor or proto-oncogene.
Research finds that a variety of deubiquitinating enzymes are abnormal in breast carcinoma, and related with breast cancer occurrence and development.Knot
Fruit shows that the expression of Breast Cancer-Specific deubiquitinating enzymes and breast cancer molecular subtypes, the diagnosis of breast cancer and prognostic evaluation have
Substantial connection.The study found that the deubiquitinating enzymes USP30 in the present invention can overcome above-mentioned existing breast cancer diagnosis marker to lack
Point, USP30 are substantially less than other types of breast cancer, and USP30 albumen in the expression of base type (three is negative) breast cancer
Also relatively stable in vitro, detection method is simple, and immunohistochemistry can be realized, and the standard of detection level is also easy to formulate.
Summary of the invention
First mesh of the invention is: for the deficiencies of the prior art, providing a kind of relevant to tumor cells parting examine
Disconnected marker, diagnosis marker USP30.The diagnosis marker diagnosis efficiency is high, suffers from addition to can accurately identify tumour/non-tumour
Person, additionally it is possible to realize the parting detection of breast cancer；Sensibility is high, and the standard of detection level is also easy to formulate, and detection method is simple,
Simple immunohistochemistry can be realized.
Second object of the present invention is: providing the purposes of diagnosis marker relevant to tumor cells parting.
Third object of the present invention is: providing Diagnosis of Breast cancer, judges Prognosis in Breast Cancer or judge breast cancer classification
Reagent or kit.
Fourth object of the present invention is: providing the reagent or kit for inhibiting breast cancer cell transfer.
The technical scheme adopted by the invention is that a kind of diagnosis marker relevant to tumor cells parting, the diagnosis mark
Will object is USP30.
A kind of diagnosis marker relevant to tumor cells parting for diagnosing tumour or judges tumor prognosis in preparation
Purposes in reagent or kit.
Further, tumour is breast cancer.
Further, breast cancer is triple negative breast cancer.
A kind of diagnosis marker relevant to tumor cells parting is preparing reagent or reagent for judging tumour classification
Purposes in box.
Further, tumour classification is judged for the classification that judges breast cancer.
Further, judge the classification of breast cancer for difference triple negative breast cancer and other type breast cancer, wherein other
Type breast cancer is Luminal A type, Luminal Type B and HER2 positive.
A kind of Diagnosis of Breast cancer judges Prognosis in Breast Cancer or judges the reagent or kit of breast cancer classification, reagent or examination
It include USP30 in agent box.
A kind of reagent of diagnosis marker relevant to tumor cells parting in preparation for inhibiting breast cancer cell to shift
Or the purposes in kit.
A kind of reagent or kit inhibiting breast cancer cell transfer includes USP30 in reagent or kit.
The beneficial effects of the present invention are: the present invention provides a kind of novel breast cancer diagnosis molecular marker, diagnosis
It is high-efficient, in addition to can accurately identify tumour/non-tumor patient, additionally it is possible to realize the parting detection of breast cancer；Sensibility is high, detection
Horizontal standard is also easy to formulate, and detection method is simple, and simple immunohistochemistry can be realized.
Detailed description of the invention
Fig. 1 is USP30 expression analysis figure in breast cancer tissue in the embodiment of the present invention 1, wherein figure a is analysis cancer base
Because of a group map TCGA breast cancer expression database, including 1102 tumor tissues and 113 normal tissues, in tumor tissues
The expression of USP30 is substantially less than normal tissue, and figure b is analysis cancer gene group map TCGA breast cancer expression database,
Including 803 lumen type breast cancer, 27 human epidermal growth factor acceptor Her2 positive breast cancers and 76 three negative creams
Gland cancer, expression of the USP30 in triple negative breast cancer are lower than other all types breast cancer, and figure c is analysis gene expression data
GSE76275 data set in the GEO of library, including 198 triple negative breast cancers and 67 other type of mammary cancers, USP30 exists
Expression in triple negative breast cancer is lower than other type of mammary cancers；
Fig. 2 is USP30 immunohistochemical experiment figure in breast cancer tissue and cancer beside organism in the embodiment of the present invention 2；
Fig. 3 is that breast cancer cell migrates lab diagram in the embodiment of the present invention 3, wherein figure a1 is MDA- after being overexpressed USP30
MB-231 cell migration reduced capability, figure a2 are after striking low USP30, and MDA-MB-231 cell migration ability enhancing, figure b is each group
The statistical value of migrating cell number, migrating cell number substantially reduces after being overexpressed USP30, P < 0.0001；It is migrated carefully after striking low USP30
Born of the same parents' digital display work increases, P < 0.0001；
Fig. 4 is patient with breast cancer's survival Analysis figure, wherein figure a is no recurrence breast cancer patient five year survival rate, USP30
Highly expressed survival is higher than USP30 low expression patient, and figure b is triple negative breast cancer without patients with recurrent five year survival rate,
The highly expressed survival of USP30 is higher than USP30 low expression patient.
Specific embodiment
Technical scheme is described further with attached drawing combined with specific embodiments below, but it is understood that this hair
Bright protection scope is not limited by specific embodiment.
USP30 expression analysis in 1 breast cancer tissue of embodiment
Present inventor downloads cream from the cancer gene group map official website (The Cancer Genome Atlas, TCGA)
The RNA-Seq data of gland cancer are analyzed, and the expression quantity of USP30 in 1102 tumor tissues and 113 normal breasts is analyzed,
And 803 lumen type breast cancer, 27 human epidermal growth factor acceptor Her2 positive breast cancers and 76 triple negative breast cancers
With the expression quantity of USP30 in 113 normal breasts, as a result as shown in figure 1 shown in a, b, table of the USP30 in breast cancer tissue is shown
Up to substantially less than normal galactophore tissue, especially triple negative breast cancer；
Present inventor is further through in gene expression data base (Gene Expression Omnibus, GEO)
GSE76275 data set is to 198 triple negative breast cancers and 67 other type of mammary cancers (including Luminal A type, Luminal
Type B and HER2 are positive) in tissue the expression of USP30 analyzed, as a result as shown in figure 1 shown in c, display triple negative breast cancer group
The expression for knitting middle USP30 is substantially less than other type of mammary cancerous tissues.
In conclusion USP30 expression quantity in breast cancer tissue, especially triple negative breast cancer tissue significantly reduces.
2 breast cancer tissue's immunohistochemical experiment of embodiment
By 30 breast cancer tissues with clinical tissue slice by 30 breast cancer cancers through the aquation, phosphate buffer of dewaxing
After the series of steps such as PBS cleaning, antigen retrieval, serum closing, then 1:250 5%BSA (fetal calf serum) volume ratio is used respectively
4 DEG C of refrigerator overnights of diluted USP30 antibody are incubated for, and after 37 DEG C of constant-temperature incubation 30min of secondary antibody of biotin labeling, develop the color, is multiple
It contaminates, clean, being dehydrated transparent rear mounting.
Experimental result is as shown in Fig. 2, the expression of USP30 is significantly lower than cancer beside organism in display breast cancer tissue.
The migration experiment of 3 breast cancer cell of embodiment
USP30 will be overexpressed respectively and strike the MDA-MB-231 cell inoculation of low USP30 in cell, for 24 hours poststaining, meter
The cell of number migration, steps are as follows:
500,000 MDA-MB-231 cell kind plate is used Lipofectamine 3000 by the day before transfection respectively afterwards for 24 hours
USP30-GFP and SiRNA-USP30 are transferred to prepare overexpression USP30 and strike low USP30 to cell with RNAimax transfection reagent
MDA-MB-231 cell.After transfecting 36h, 2 cell μ m diameter 6.5mm of aperture is put into 24 orifice plates, upper chamber is added 500ul's
The culture medium that 750ul contains 10% fetal calf serum is added in serum free medium, lower room.5 × 10^ is then taken respectively4It is overexpressed USP30
With strike the MDA-MB-231 cell inoculation of low USP30 in cell in (healthy and free from worry, article No. 3422).After inoculation for 24 hours, with 0.01%
Violet staining takes five visuals field to take pictures at random, and the cell of five visuals field migration is counted with ImageJ, counts the flat of five visuals field
Mean value.Wherein GFP is control plasmid, and USP30-GFP is the overexpression plasmid that USP30 coded sequence has been cloned on GFP carrier,
CONsiRNA is negative control RNA interfering, and USP30siRNA is USP30 RNA interfering.
The influence migrated by migrating experimental analysis USP30 to breast cancer cell, experimental result is as shown in figure 3, USP30 shadow
Breast cancer cell migration is rung, height expression USP30 inhibits cell migration, and low expression promotes cell migration.
4 patient with breast cancer's survival Analysis of embodiment
With Kaplan-meier analysis without recurrence breast cancer patient's five year survival rate, by 1764 patient with breast cancers, according to
The median of the mRNA expression of the USP30 of patient with breast cancer divides into USP30 low expression patient with breast cancer (n=885),
USP30 high expresses patient with breast cancer (n=879).Simultaneously with Kaplan-meier analysis without recurrence triple negative breast cancer patient five
Year survival rate, by 360 triple negative breast cancer patients, according to the median of the mRNA expression of the USP30 of patient with breast cancer
It divides into USP30 low expression patient (n=180), USP30 high expresses patient (n=180).
Experimental result as shown in figure 4, in no recurrence breast cancer patient and without in recurrence triple negative breast cancer patient, express by height
Its five year survival rate of the patient of USP30 is higher than the patient of low expression USP30.Illustrate that USP30 can inhibit the generation and development of tumour,
It is potential tumor suppressor gene.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Claims (10)
1. a kind of diagnosis marker relevant to tumor cells parting, which is characterized in that the diagnosis marker is USP30.
2. diagnosis marker relevant to tumor cells parting according to claim 1 for diagnosing tumour or is sentenced in preparation
The reagent of disconnected tumor prognosis or the purposes in kit.
3. purposes according to claim 2, which is characterized in that the tumour is breast cancer.
4. purposes according to claim 3, which is characterized in that the breast cancer is triple negative breast cancer.
5. diagnosis marker relevant to tumor cells parting according to claim 1 is in preparation for judging tumour classification
Reagent or kit in purposes.
6. purposes according to claim 5, which is characterized in that described to judge tumour classification for the classification that judges breast cancer.
7. purposes according to claim 6, which is characterized in that the classification for judging breast cancer is three negative breast of difference
Cancer and other type breast cancer, other described type breast cancer are Luminal A type, Luminal Type B and HER2 positive.
8. a kind of Diagnosis of Breast cancer judges Prognosis in Breast Cancer or judges the reagent or kit of breast cancer classification, which is characterized in that
It include USP30 in the reagent or kit.
9. diagnosis marker relevant to tumor cells parting according to claim 1 is in preparation for inhibiting breast cancer thin
The purposes in reagent or kit that dysuria with lower abdominal colic is moved.
10. a kind of reagent or kit for inhibiting breast cancer cell transfer, which is characterized in that include in the reagent or kit
USP30。
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Cited By (4)
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| CN111948395A (en) * | 2020-08-19 | 2020-11-17 | 复旦大学附属金山医院 | Quadruple marker for diagnosing immune regulation subtype of triple negative breast cancer and application thereof |
| CN113943803A (en) * | 2021-10-13 | 2022-01-18 | 深圳市人民医院 | Application of HTR6 in diagnosis and prognosis of breast cancer |
| CN114277148A (en) * | 2021-12-30 | 2022-04-05 | 深圳康华君泰生物科技有限公司 | Biomarker for breast cancer typing and application thereof |
| CN114672555A (en) * | 2020-12-24 | 2022-06-28 | 清华大学深圳国际研究生院 | Cancer prognosis marker and application thereof |
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