CN114034866A - Breast cancer diagnosis marker and application thereof - Google Patents
Breast cancer diagnosis marker and application thereof Download PDFInfo
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- CN114034866A CN114034866A CN202111431631.5A CN202111431631A CN114034866A CN 114034866 A CN114034866 A CN 114034866A CN 202111431631 A CN202111431631 A CN 202111431631A CN 114034866 A CN114034866 A CN 114034866A
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
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- G01N2333/4748—Details p53
Abstract
The invention discloses a breast cancer diagnosis marker and application thereof. The invention detects the expression of ADRM1 protein in breast cancer tissues by an immunohistochemical method, and the result shows that ADRM1 is obviously up-regulated in the cancer tissues of breast cancer patients and is obviously related to the tumor size. Meanwhile, the UALCAN database analysis shows that the expression level of ADRM1 in TP53 mutant type and non-mutant type breast cancer is obviously higher than that in normal breast cancer, the expression of ADRM1 is closely related to the expression of TP53 in the breast cancer, and the expression of ADRM1 and TP53 proteins in the tissues of a breast cancer patient can be detected by combining the ADRM1 protein and the TP53 protein, so that the accuracy is high.
Description
Technical Field
The invention relates to the field of tumor diagnosis, in particular to a breast cancer diagnosis marker and application thereof.
Background
The breast cancer is one of common malignant tumors of women, and global tumor epidemiological statistics show that the number of new breast cancer cases is about 170 ten thousand in 2012, and the number of death cases is about 52.2 ten thousand in 2012. In China, breast cancer has become the first of the high-incidence tumors in women, and the incidence and mortality of breast cancer accounts for the second of all tumors in western countries such as the United states. With the continuous progress of the modern medical treatment level, the cure rate and survival rate of the breast cancer are remarkably improved, but the metastasis and recurrence of the breast cancer are still serious risk factors threatening breast cancer patients, and the occurrence of the metastasis and recurrence not only increases the treatment difficulty, but also reduces the survival time of the patients. The literature reports that the 5-year survival rate of the first breast cancer patient can reach 60% -70%, and the 5-year survival rate of the patient after local recurrence of breast cancer is only 42% -49%. Therefore, the intensive research on the mechanism of the breast cancer recurrence and metastasis, the search for new key factors influencing the prognosis of the breast cancer, and the development of effective measures for preventing and treating the breast cancer recurrence and metastasis on the basis of the new key factors are important scientific problems in the current breast cancer research, and have very important clinical significance for further improving the overall treatment level of the breast cancer.
Current approaches to treating solid tumors include direct attacks such as chemotherapy, radiation therapy, and surgery. However, these approaches can cause some damage to normal tissue. Unlike standard chemotherapy, targeted treatment strategies aim to avoid damage to normal tissues by better targeting tumors. Targeted drugs induce tumor cell necrosis and apoptosis by recognizing oncogenic sites (cell surface antigens, membrane protein molecules or gene fragments) of tumor cells. The food and drug administration has approved a number of targeted therapies to treat certain types of cancer. Endocrine Therapy (ET) has revolutionized the treatment of breast cancer, especially in luminal tumor patients, with ET accounting for a large portion of the gains in adjuvant systemic therapy. Estrogen Receptor (ER) and HER2 are major targets for therapy and the focus of drug development. Although currently available ET drugs, such as tamoxifen and trastuzumab, are generally effective and well tolerated, not all patients equally benefit. Therefore, there is a need to identify novel genes as breast cancer biomarkers and therapeutic targets for breast cancer patients to improve early diagnosis and therapeutic outcomes.
ADRM1 was identified as adhesion-modulating molecule-1 in metastatic tumor cells, encoding an intact plasma membrane protein involved in development, cell adhesion, deubiquitination and proteolysis. As one of the two major ubiquitin receptors on the 26S proteasome, ADRM1 can selectively mediate the degradation of key proteins and participate in a variety of cellular processes. Studies have shown that the potential role of human ADRM1 in tumorigenesis, whose overexpression is significantly associated with early cancer recurrence and low survival, is also identified as an amplification target in cancer. Drugs targeting ADRM1 may be a good strategy for cancer treatment. TP53 tumor suppressor protein acts as a cellular stress sensor and is activated under stress conditions such as DNA damage and oncogene activation (oncogenic stress). This may inhibit cell division or survival through transactivation of downstream target genes. In addition, TP53 is also involved in a variety of other cellular processes, including DNA repair, differentiation, and stem cell renewal. Germline mutations in TP53 may result in a higher risk of breast cancer. Mutant TP53 has several attractive features as a target for breast cancer therapy. First, TP53 mutated in approximately 30% of all cases and approximately 80% of Triple Negative (TN) tumors (i.e., ER, PR, and HER2 negative tumors), suggesting TP53 is the most common mutant gene in breast cancer. Second, the gene is mutated in almost 90% of breast cancer patients with brain metastases. Finally, the TP53 mutation is clonal and should be present in all malignant cells of breast cancer.
Disclosure of Invention
The invention aims to provide a breast cancer diagnosis marker and application thereof, wherein both ADRM1 and TP53 protein diagnosis markers are positively correlated with the incidence rate of breast cancer and are highly expressed in breast cancer tissues, and the ADRM1 and TP53 proteins in tumor patient tissues are jointly detected through the ADRM1 protein and the TP53 protein, so that the accuracy is high.
The technical scheme adopted by the invention for solving the technical problems is as follows:
ADRM1 and TP53 protein biomarkers for breast cancer diagnosis, and the ADRM1 and TP53 protein biomarkers can be used for detecting the expression of ADRM1 and TP53 proteins in tissues of tumor patients.
The ADRM1 and TP53 proteins can be used for preparing a product for combined diagnosis of breast cancer, and the ADRM1 and TP53 protein biomarkers for breast cancer diagnosis can be used for auxiliary diagnosis of breast cancer.
Preferably, the breast cancer diagnostic product comprises an immunohistochemical method for detecting the expression levels of ADRM1 and TP53 proteins in a biological sample.
Preferably, the immunohistochemical detection reagent comprises xylene, ethanol, phosphate buffer, DBA color reagent, hematoxylin and gum.
Preferably, the method for detecting the expression level of a protein comprises: (1) detecting the expression level of ADRM1 and TP53 proteins in a biological sample; (2) elevated expression levels of ADRM1 and TP53 proteins were associated with breast cancer.
The invention has the beneficial effects that:
the invention discloses an ADRM1 protein biomarker for breast cancer diagnosis and application thereof for the first time, wherein the ADRM1 protein biomarker is highly expressed in breast cancer tissues and positively correlated with the occurrence of breast cancer, and is a potential biomarker for breast cancer diagnosis. The development of the ADRM1 protein biomarker is expected to become a new means for auxiliary diagnosis and targeted therapy of breast cancer.
The invention discovers that the expression level of ADRM1 in TP53 mutant type and non-mutant type breast cancers is obviously higher than that in normal breast cancers for the first time, and shows that the expression of ADRM1 is closely related to the expression of TP53 in the breast cancers. The expression of ADRM1 and TP53 proteins in the tissues of tumor patients is jointly detected by ADRM1 and TP53 proteins, and the detection result has high accuracy.
Drawings
Figure 1ADRM1 expression at different cohorts was assessed based on clinical parameters of breast cancer patients using the UALCAN database, (a) specimen type, (B) age, (C) race, (D) lymph node status, (E) histological grade, (F) breast cancer classification, (G) TP53 mutation status, # p <0.05, # p <0.01, # p < 0.001.
Figure 2 the expression of TP53 was assessed in different cohorts based on clinical parameters of breast cancer patients using the UALCAN database, (a) sample type, (B) age, (C) race, (D) lymph node status, (E) histological grade, (F) breast cancer classification, # p <0.05, # p <0.01, # p < 0.001.
FIG. 3 is a diagram of a gene-gene interaction network for constructing ADRM1 and TP53 using cytoscape.
Figure 4 is a graph of survival analysis of ADRM1 in breast cancer patients.
FIG. 5 is a graph of survival analysis of hsa-let-7b in breast cancer patients.
FIG. 6 is a graph of a survival assay for hsa-miR-125b in breast cancer patients.
FIG. 7 is a base sequence diagram of the matching of hsa-let-7b and hsa-miR-125b with ADRM1 and TP 53.
FIG. 8 is a graph showing expression of hsa-let-7b-5p and hsa-miR-125b-5p in breast cancer tissue and normal tissue.
Fig. 9 is an enrichment analysis diagram of target genes GO and KEGG of mirnas.
Detailed Description
The present invention will be further described with reference to the structures or terms used herein. The description is given for the sake of example only, to illustrate how the invention may be implemented, and does not constitute any limitation on the invention.
Example 1 determination of expression level of ADRM1 protein in Breast cancer tissue
(1) Sample collection
148 samples of the breast cancer patients with pathological diagnosis in the hospital after surgical excision of tissue are collected, and the patient enrollment criteria are as follows: the surgical resection pathology was confirmed to be breast cancer; before operation, radiotherapy, chemotherapy, hormone and biological target treatment are not carried out.
(2) Immunohistochemical method of detection
Continuously slicing a breast cancer nest tissue paraffin specimen by 4 mu m, carrying out conventional dewaxing treatment, washing by PBS (phosphate buffer solution), operating according to the instruction of an immunohistochemical kit, and diluting a mouse anti-human ADRM1 by l: 500, mouse anti-human TP53 dilution l: 100, Phosphate Buffered Saline (PBS) was used as a negative control instead of primary antibody. DAB color development at room temperature for 5-l0min, washing with tap water, hematoxylin counterstaining, gradient ethanol dehydration, xylene transparency, gum sealing, and optical microscope observation.
(3) Analysis of immunohistochemical results by semi-quantitative method
The staining intensity of the tumor cells was scored in combination with the proportion of positive cells. Staining intensity scores were based on the staining status exhibited by most cells, and scoring criteria: the non-staining is counted as 0 point, the light yellow is counted as 1 point, the tan is counted as 2 points, and the tan is counted as 3 points. The positive cell proportion score is obtained by randomly selecting 5 high power lens fields (400 x) under an optical microscope, calculating the percentage of tumor cells presenting positive staining to all tumor cells in the fields, taking the mean value of each field as the percentage of positive cells, and scoring standard: the staining of the tumor-free cells is counted for 0min, 1-30% of the tumor cells are stained for 1 min, 30-60% of the tumor cells are stained for 2 min, and the staining of the > 60% of the tumor cells is counted for 3 min. The final score is the product of the staining intensity score and the positive cell proportion score. The scores were scored to avoid tumor necrotic regions and tumor margins, the scores included only tumor cells, and tumor stromal cells were not counted. Final scores of 0-3 were defined as low expression and scores of 4-9 as high expression.
(4) Immunohistochemical outcome determination
Two high-age clinical pathologists use the same pathological microscope to independently perform the judgment, and for the section with inconsistent results, the other pathologist is invited to judge by the two original doctors in a collective way.
(5) Correlation analysis of ADRM1 expression with clinical features in breast cancer tissues
Describing measurement data by x +/-s, and performing t test by using two independent samples; counting the data utilization rate (or percentage) to carry out statistical description, and adopting two independent sample chi-square tests; the data sorting and analysis process is carried out in SPSS 22.0 software, and the data with P less than 0.05 (two sides) has statistical significance, and the results are shown in Table 1.
TABLE 1 relationship of ADRM1 and TP53 expression to breast cancer clinicopathological parameters
The results show that, among 148 breast cancer patients, the ADRM1 is highly expressed in 83 cases and is lowly expressed in 65 cases, the expression of the ADRM1 protein has a significant correlation with the clinical stage of the breast cancer patient, and the higher the expression level of the ADRM1 protein is, the higher the expression level of the ADRM1 protein is, the higher the malignancy degree of the breast cancer is.
Example 2 differential expression of ADRM1 and TP53 genes in Breast cancer tissue and Normal tissue
The expression level of ADRM1 in breast cancer was analyzed using the UALCAN database (http:// UALCAN. path. uab. edu/index. html). The screening conditions were as follows: gene symbol ═ ADRM 1; TCGA dataset is break innovative cardio. 1097 breast cancer samples and 114 normal tissue samples were tested for the condition. The relationship of ADRM1 to age, ethnicity, lymph node metastasis stage, histological grade, and breast cancer classification, as well as expression in both mutant and non-mutant TP53 breast cancer samples, were further analyzed using the UALCAN database, and the results are shown in FIG. 1.
The expression level of ADRM1 was elevated in breast cancer patients compared to the normal control group (p <0.001, fig. 1A), and the expression level of ADRM1 was significantly correlated with age, race, lymph node metastasis stage, histological grade, and breast cancer classification (fig. 1B-F). Furthermore, the expression level of ADRM1 in both TP53 mutant and non-mutant breast cancers was significantly higher than in normal breast cancers (fig. 1G), indicating that expression of ADRM1 was closely related to expression of TP53 in breast cancers.
The expression level of TP53 in breast cancer was analyzed using the UALCAN database (http:// UALCAN. path. uab. edu/index. html). The screening conditions were as follows: gene symbol ═ ADRM 1; TCGA dataset is break innovative cardio. 1097 breast cancer samples and 114 normal tissue samples satisfying the conditions. The relationship of TP53 to age, race, lymph node metastasis stage, histological grade, and breast cancer classification was further analyzed using the UALCAN database, and the results are shown in FIG. 2. The expression level of TP53 was elevated in breast cancer patients compared to the normal control group (p <0.001, fig. 2A), and the expression level of TP53 was significantly correlated with age, race, lymph node metastasis stage, histological grade, and breast cancer classification (fig. 2B-F).
Example 3 screening method for miRNA of ADRM1 and TP53
(1) Screening for MiRNAs acting in concert on ADRM1 and TP53
The interaction of ADRM1 and TP53 with miRNA is predicted by online databases mirtarBase (http:// www.mirbase.org /), mirtarBase (http:// mirtarbasise. cuhk. edu. cn/php/index. php), TargetScan (http:// www.targetscan.org/mamm _31/), and TransmiR (http:// www.cuilab.cn/Transmir). The Cytoscape software (www.cytoscape.org /) was used to construct and visualize the regulatory network of mirnas on ADRM1 and TP 53. As a result, 15 miRNAs including hsa-let-7b-5p, hsa-miR-125b-5p, hsa-miR-24-3p, hsa-miR-1233-5p, hsa-miR-5693, hsa-miR-149-3p, hsa-miR-6766-5p, hsa-miR-4728-5p, hsa-miR-6756-5p, hsa-miR-19b-3, hsa-miR-6778-5p, hsa-miR-194-3p, hsa-miR-6785-5p, hsa-miR-19a-3p and hsa-miR-6883-5 co-target ADRM1 and TP53 (FIG. 3).
(2) Further screening for miRNAs acting in concert on ADRM1 and TP53
Kaplan-Meier (www.kmplot.com) was used to assess prognostic significance of expression levels of ADRM1, TP53 and interacting miRNAs in breast cancer. Data were divided into high expression and low expression groups according to gene expression level, and Overall Survival (OS) was analyzed for different HER, ER and lymph node status. Hazard ratios with 95% confidence intervals and log rank P values were calculated with significance set at P < 0.05. Based on the characteristic that the expression trend of miRNA is opposite to that of target gene, miRNA influencing breast cancer progression through regulating ADRM1 and TP53 expression are further screened.
Kaplan-Meier survival curves show that high expression of ADRM1 miRNA is associated with poor OS (HR ═ 1.41, 95% CI:1.26-1.57, P < 0.001). Group analysis of HER2, ER, and lymph node status showed that high expression of ADRM1 significantly reduced the overall survival of patients who were ER positive (HR ═ 1.52, 95% CI:1.09-2.11, P ═ 0.012), HER2 negative (HR ═ 1.37, 95% CI:1.1-1.71, P ═ 0.0047), lymph node negative (HR ═ 1.44, 95% CI:1.02-2.03, P ═ 0.038) (fig. 4).
The invention also performs survival analysis of miRNA. Analysis shows that the expression of hsa-let-7b and hsa-miR-125b is positively correlated with the overall survival rate. That is, the higher the expression of hsa-let-7b and hsa-miR-125b, the better the overall survival rate (hsa-let-7b: HR ═ 0.66, 95% CI:0.54-0.8, P ═ 4.1 e-05; hsa-miR-125b: HR ═ 0.72, 95% CI:0.58-0.88, P ═ 0.0015). Subsequent cohort analysis also showed that low expression of hsa-let-7b significantly reduced overall survival in ER-positive (HR ═ 0.72, 95% CI:0.57-0.92, P ═ 0.0089), HER 2-positive (HR ═ 0.65, 95% CI:0.49-0.86, P ═ 0.0026), lymph node-positive (HR ═ 0.6395% CI:0.47-0.83, P ═ 0.0012) patients (fig. 5). As shown in fig. 6, low expression of hsa-miR-125b significantly reduced the overall survival of patients with ER-positive (HR ═ 0.62, 95% CI:0.47-0.820, P ═ 0.00071), ER-negative (HR ═ 0.63, 95% CI:0.48-0.83, P ═ 0.00078), HER 2-positive (HR ═ 0.71, 95% CI:0.53-0.94, P ═ 0.018), lymph node-positive (HR ═ 0.78, 95% CI:0.6-1.01, P ═ 0.061), lymph node-negative (HR ═ 0.55, 95% CI:0.39-0.79, P ═ 0.00078).
(3) Searching base sequences matched with ADRM1 and TP53 on the miRNA obtained in (2), and verifying whether the miRNA screened in (2) is a common regulatory gene of ADRM1 and TP53
The miRNA sequence screened finally is searched on the NCBI (https:// www.ncbi.nlm.nih.gov /) homepage, and the region matching with ADRM1 and TP53 is searched by adopting a sequence alignment method. By aligning the sequences, two miRNAs, hsa-let-7b-5p and hsa-miR-125b-5p, were found to have matching binding sites to TP53 and ADRM1 (FIG. 7).
(4) Analyzing the expression level of miRNA in breast cancer and normal tissues, and further verifying whether the screened miRNA is a common regulatory gene of ADRM1 and TP53
The expression levels of the screened miRNAs in breast cancer and normal tissues were analyzed using an online database ENCORI (http:// starbase. syssu. edu. cn/panMirDeffExp. php.). The screening conditions were as follows: miRNA is Hsa-let-7b-5p, and Cancer is Breast innovative cardio; miRNA is Hsa-miR-125b-5p, and Cancer is Breast innovative carcinosoma. 1085 breast cancer samples satisfying the conditions, and 104 normal tissue samples. The results showed a significant reduction in hsa-let-7b-5p and hsa-miR-125b-5p in breast cancer tissue compared to normal tissue (FIG. 8). These data indicate that hsa-let-7b-5p and hsa-miR-125b-5p are potential regulatory genes for ADRM1 and TP53 associated with breast cancer progression.
(5) Enrichment analysis of target genes of screened miRNAs
GO (GO: http:// genetic on tology. org /) functional enrichment analysis and KEGG (KEGG: http:// http:// www.kegg.jp/KEGG /) signal channel analysis are carried out by using DAVID (https:// DAVID. ncifcrf. gov /) comprehensive analysis tool, the biological functions mainly exerted by the target genes of miRNA are excavated, and P <0.05 has statistical significance. In GO analysis, the most important enrichment pathways are regulation of stress activated MAPK cascade, positive regulation of stress activated MAPK cascade, signal transduction of TP53 type mediators, positive regulation of MAP kinase activity, miRNA metabolic process, generation of small RNAs in RNA silencing genes and generation of miRNAs participating in miRNA gene silencing. In the KEGG analysis, target genes were significantly enriched in cancer pathways, micrornas in cancer, Wnt signaling pathway, adhesion protein binding proteins, MAPK signaling pathway, and TGF- β signaling pathway (fig. 9). These results suggest that these target genes may be involved in the development of cancer.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (4)
- Application of ADRM1 and TP53 proteins in preparing a product for combined diagnosis of breast cancer.
- 2. The use of claim 1, wherein: the combined diagnosis breast cancer product comprises an immunohistochemical method for detecting the expression level of ADRM1 and TP53 proteins in a biological sample.
- 3. Use according to claim 2, characterized in that: the immunohistochemical detection reagent comprises xylene, ethanol, phosphate buffer solution, DBA color reagent, hematoxylin and gum.
- 4. Use according to claim 3, characterized in that: the method for detecting the expression level of the protein comprises the following steps: (1) detecting the expression level of ADRM1 and TP53 proteins in a biological sample; (2) elevated expression levels of ADRM1 and TP53 proteins were associated with breast cancer.
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| US20080182246A1 (en) * | 2006-09-05 | 2008-07-31 | Yixin Wang | Methods of predicting distant metastasis of lymph node-negative primary breast cancer using biological pathway gene expression analysis |
| CN103981273A (en) * | 2014-05-30 | 2014-08-13 | 复旦大学附属肿瘤医院 | Mutant gene group for mammary cancer risk assessment and detection kit thereof |
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