CN110004228B - Diagnostic marker related to breast cancer molecular typing and application thereof - Google Patents
Diagnostic marker related to breast cancer molecular typing and application thereof Download PDFInfo
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Abstract
The invention discloses a diagnostic marker related to breast cancer molecular typing, which is USP30, has high diagnostic efficiency and can realize the typing detection of breast cancer. The diagnostic marker can be used for preparing a reagent or a kit for diagnosing breast cancer or judging prognosis of breast cancer, and can be used for preparing a reagent or a kit for judging breast cancer category. And USP30 can be used for preparing a reagent or a kit for inhibiting breast cancer cell metastasis, which lays a foundation for the development of breast cancer medicaments.
Description
Technical Field
The invention belongs to the technical fields of biotechnology and oncology, and relates to deubiquitinase related to breast cancer molecular typing and application thereof.
Background
Breast cancer is the most common female cancer, accounting for 28.7% of all cancers diagnosed in females, and is also the second highest mortality malignancy. With advances in biological diagnosis and treatment of breast cancer, the 5-year survival rate of breast cancer has increased to over 85%. However, most breast cancer patients die within 5 years from post-operative brain metastasis progression, particularly brain metastasis of Basal-like (Basal) breast cancer.
Studies indicate that about 50% of advanced breast cancer patients eventually develop brain metastases and are most commonly found in Basal and human epidermal growth factor receptor 2 over-expression (HER 2). The breast cancer diagnosis markers of the breast cancer at the present stage mainly comprise circRNA such as circTADA2A-E6, circTADA2A-E5/E6, circNOL10 and the like, methylation level of cfDNA methylation sites, N-acetylglucosaminyl plasma proteinase C1 inhibitor and the like. These markers are not capable of identifying the breast cancer class of the patient, are not high in expression level in the human body, are unstable in vitro, and the standard of the detection level is difficult to formulate. Thus, there is an urgent need for identification of biomarkers to screen high risk patients and predict breast cancer outcome as well as traditional clinical pathology.
Deubiquitinase (DUBs) is a large family of proteases. It hydrolyzes ubiquitin molecule-specific from ubiquitin-linked proteins or precursors by mainly hydrolyzing the carboxyl-terminal ester, peptide or isopeptide bond of ubiquitin. Deubiquitinase can regulate protein degradation by deubiquitination; coordinating cellular localization of proteins; activating and inactivating proteins; and regulates protein-protein interactions, thereby affecting cell proliferation, differentiation, apoptosis, autophagy. Deubiquitinase is closely related to the development and progression of cancer. It was found that deubiquitinase is dysregulated in expression in various cancer tissues and functions as a tumor suppressor or protooncogene by modulating the activity of a pro-or cancer-suppressing signaling pathway.
Several deubiquitinating enzymes have been found to be abnormally expressed in breast cancer and are involved in the development of breast carcinogenesis. The results show that the expression of the breast cancer specific deubiquitinase has close relation with the diagnosis and prognosis evaluation of breast cancer molecular subtype and breast cancer. The research shows that the deubiquitinase USP30 can overcome the defects of the existing breast cancer diagnosis markers, the expression level of the USP30 in basal (triple negative) breast cancer is obviously lower than that of other types of breast cancer, the USP30 protein is relatively stable in vitro, the detection method is simple, the immunohistochemistry can be realized, and the standard of the detection level is easy to formulate.
Disclosure of Invention
The first object of the present invention is: in order to overcome the defects in the prior art, a diagnostic marker related to tumor molecular typing is provided, wherein the diagnostic marker is USP30. The diagnosis marker has high diagnosis efficiency, can accurately identify tumor/non-tumor patients, and can realize the parting detection of breast cancer; the sensitivity is high, the standard of the detection level is easy to formulate, the detection method is simple, and simple immunohistochemistry can be realized.
The second object of the invention is: the use of diagnostic markers associated with the typing of tumor molecules is provided.
A third object of the invention is: reagents or kits for diagnosing breast cancer, determining prognosis of breast cancer, or determining the class of breast cancer are provided.
A fourth object of the invention is: provided are reagents or kits for inhibiting metastasis of breast cancer cells.
The technical scheme adopted by the invention is that a diagnosis marker related to tumor molecular typing is USP30.
Use of a diagnostic marker associated with the molecular typing of a tumour in the manufacture of a reagent or kit for diagnosing a tumour or for determining the prognosis of a tumour.
Further, the tumor is breast cancer.
Further, the breast cancer is triple negative breast cancer.
Use of a diagnostic marker associated with the molecular typing of a tumour in the manufacture of a reagent or kit for determining the class of a tumour.
Further, the determination of the tumor type is a determination of the type of breast cancer.
Further, the classification of breast cancer is judged as distinguishing triple negative breast cancer from other types of breast cancer, wherein the other types of breast cancer are luminel type a, luminel type B and HER2 positive.
A reagent or kit for diagnosing breast cancer, determining prognosis of breast cancer, or determining class of breast cancer, comprising USP30.
Use of a diagnostic marker associated with tumor molecular typing in the manufacture of a reagent or kit for inhibiting metastasis of breast cancer cells.
A reagent or kit for inhibiting metastasis of breast cancer cells, comprising USP30.
The beneficial effects of the invention are as follows: the invention provides a novel breast cancer diagnosis molecular marker which has high diagnosis efficiency, can accurately identify tumor/non-tumor patients, and can realize the parting detection of breast cancer; the sensitivity is high, the standard of the detection level is easy to formulate, the detection method is simple, and simple immunohistochemistry can be realized.
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FIG. 1 is a graph showing the analysis of the expression level of USP30 in breast cancer tissue in example 1 of the present invention, wherein FIG. a is a database of the analysis of the genome map of cancer TCGA breast cancer, which comprises 1102 tumor tissues and 113 normal tissues, the expression level of USP30 in tumor tissues is significantly lower than that in normal tissues, FIG. b is a database of the analysis of the genome map of cancer TCGA breast cancer, which comprises 803 luminal breast cancers, 27 human EGF receptor Her2 positive breast cancers and 76 triple negative breast cancers, the expression level of USP30 in triple negative breast cancers is lower than that in all other types of breast cancers, and FIG. c is a database of GSE76275 in the analysis of the gene expression database GEO, which comprises 198 triple negative breast cancers and 67 other types of breast cancers, and the expression level of USP30 in triple negative breast cancers is lower than that in other types of breast cancers;
FIG. 2 is a diagram showing the USP30 immunohistochemistry of breast cancer tissue and paracancestral tissue in example 2 of the present invention;
FIG. 3 is a graph showing the migration experiment of breast cancer cells in example 3 of the present invention, wherein FIG. a1 shows that the migration ability of MDA-MB-231 cells is reduced after the USP30 is overexpressed, FIG. a2 shows that the migration ability of MDA-MB-231 cells is enhanced after the USP30 is knocked down, and FIG. b shows the statistics of the number of the migration cells in each group, the number of the migration cells is significantly reduced after the USP30 is overexpressed, and P <0.0001; the migration cell number is significantly increased after knocking down USP30, and P is less than 0.0001;
FIG. 4 is a graph showing the survival rate analysis of breast cancer patients, wherein FIG. a shows the five-year survival rate of patients with no recurrence of breast cancer, the survival rate of patients with high USP30 expression is higher than that of patients with low USP30 expression, and FIG. b shows the five-year survival rate of patients with no recurrence of triple negative breast cancer, and the survival rate of patients with high USP30 expression is higher than that of patients with low USP30 expression.
Detailed Description
The technical scheme of the present invention will be further described with reference to specific embodiments and drawings, but it should be understood that the scope of the present invention is not limited by the specific embodiments.
EXAMPLE 1 analysis of USP30 expression level in breast cancer tissue
The inventor downloads RNA-Seq data of breast cancer from a cancer genome map (The Cancer Genome Atlas, TCGA) functional network for analysis, and analyzes the expression level of USP30 in 1102 tumor tissues and 113 normal breast, 803 luminal breast cancer, 27 human epidermal growth factor receptor Her2 positive breast cancer, 76 triple negative breast cancer and the expression level of USP30 in 113 normal breast, and the results show that the expression level of USP30 in the breast cancer tissues is obviously lower than that of the normal breast tissues, especially triple negative breast cancer, as shown in the results in fig. 1 a and b;
the inventors of the present application, in turn, analyzed the expression of USP30 in 198 triple negative breast cancers and 67 other types of breast cancers (including luminel type a, luminel type B and HER2 positive) tissues via GSE76275 dataset in the gene expression database (Gene Expression Omnibus, GEO), and as a result, as shown in fig. 1 c, showed that the expression level of USP30 in triple negative breast cancer tissues was significantly lower than that in other types of breast cancer tissues.
In conclusion, the expression level of USP30 in breast cancer tissue, particularly triple negative breast cancer tissue, was significantly reduced.
Example 2 mammary cancer tissue immunohistochemical experiment
30 breast cancer tissues and 30 clinical tissue sections beside the breast cancer are subjected to a series of steps of dewaxing, hydration, phosphate buffer PBS cleaning, antigen retrieval, serum sealing and the like, then are respectively incubated with USP30 antibody diluted by 1:250 percent BSA (fetal bovine serum) in a volume ratio at 4 ℃ overnight in a refrigerator, and are subjected to biotin-labeled secondary antibody constant temperature incubation at 37 ℃ for 30min, and then are subjected to color development, counterstaining, cleaning, dehydration and transparency and sealing.
The results of the experiment are shown in FIG. 2, which shows that the expression level of USP30 in breast cancer tissue is significantly lower than that in paracancestral tissue.
Example 3 Breast cancer cell migration experiment
MDA-MB-231 cells overexpressing USP30 and knocked down USP30, respectively, were seeded into the chamber, stained after 24h, and the migrated cells were counted as follows:
50 ten thousand MDA-MB-231 cells were plated one day before transfection, and 24 hours later, USP30-GFP and SiRNA-USP30 were transferred to the cells using Lipofectamine 3000 and RNAimax transfection reagents, respectively, to prepare MDA-MB-231 cells that overexpressed USP30 and knocked down USP30. After 36h of transfection, a chamber with a diameter of 2 μm and a diameter of 6.5mm was placed in a 24-well plate, 500ul of serum-free medium was added to the upper chamber, and 750ul of medium containing 10% fetal bovine serum was added to the lower chamber. Then respectively taking 5 x 10 ≡ 4 Overexpression of USP30 and knock-down of USP30MDA-MB-231 cells were seeded in the chamber (Corning, cat. No. 3422). 24h after inoculation, five fields were randomly photographed with 0.01% crystal violet staining, five field migrated cells were counted with ImageJ, and the average of the five fields was counted. Wherein GFP is a control plasmid, USP30-GFP is an over-expression plasmid in which USP30 coding sequence is cloned on GFP vector, CONsiRNA is negative control interfering RNA, and USP30siRNA is USP30 interfering RNA.
The effect of USP30 on breast cancer cell migration was analyzed by migration experiments, and the experimental results are shown in FIG. 3, in which USP30 affects breast cancer cell migration, high expression of USP30 inhibits cell migration, and low expression promotes cell migration.
Example 4 survival analysis of breast cancer patients
Five-year survival of recurrence-free breast cancer patients was analyzed by Kaplan-meier, and 1764 breast cancer patients were classified as USP30 low-expressing breast cancer patients (n=885) and USP30 high-expressing breast cancer patients (n=879) according to the median of the mRNA expression levels of USP30 in the breast cancer patients. Meanwhile, five-year survival rate of recurrence-free triple-negative breast cancer patients is analyzed by Kaplan-meier, 360 triple-negative breast cancer patients are divided into USP30 low-expression patients (n=180) and USP30 high-expression patients (n=180) according to the median value of the mRNA expression level of USP30 of the breast cancer patients.
The experimental results are shown in fig. 4, and the five-year survival rate of the patients with high expression of USP30 is higher than that of the patients with low expression of USP30 in the patients with no recurrence of breast cancer and the patients with no recurrence of triple negative breast cancer. It is demonstrated that USP30 can inhibit the occurrence and development of tumors and is a potential oncogene.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
Claims (1)
1. Use of a plasmid overexpressing USP30 in the preparation of an agent for inhibiting metastasis of breast cancer cells.
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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 |
| 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 |
| 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 |
| CN114277148B (en) * | 2021-12-30 | 2024-03-08 | 深圳康华君泰生物科技有限公司 | Biomarker for breast cancer typing and application thereof |
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| CN1989411A (en) * | 2004-06-21 | 2007-06-27 | 普罗吉安拉公司 | Diagnostic and screening methods and kits associated with proteolytic activity |
| WO2014031859A2 (en) * | 2012-08-24 | 2014-02-27 | University Of Utah Research Foundation | Compositions and methods relating to blood-based biomarkers of breast cancer |
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| CN105327350A (en) * | 2014-07-23 | 2016-02-17 | 中国科学院上海巴斯德研究所 | Application of ubiquitin pathway related factor in regulating function of helper T cells |
| CN108144049A (en) * | 2016-12-02 | 2018-06-12 | 中国科学院上海巴斯德研究所 | Application of the ubiquitination pathway correlation factor in regulating regulatory T cell function |
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| CN1989411A (en) * | 2004-06-21 | 2007-06-27 | 普罗吉安拉公司 | Diagnostic and screening methods and kits associated with proteolytic activity |
| WO2014031859A2 (en) * | 2012-08-24 | 2014-02-27 | University Of Utah Research Foundation | Compositions and methods relating to blood-based biomarkers of breast cancer |
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