CN114660291A - Bladder cancer prognosis marker, prognosis evaluation system and application of prognosis evaluation system - Google Patents

Bladder cancer prognosis marker, prognosis evaluation system and application of prognosis evaluation system Download PDF

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CN114660291A
CN114660291A CN202210444452.3A CN202210444452A CN114660291A CN 114660291 A CN114660291 A CN 114660291A CN 202210444452 A CN202210444452 A CN 202210444452A CN 114660291 A CN114660291 A CN 114660291A
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bladder cancer
otud6a
prognosis
cdc6
expression
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崔剑锋
史本康
邹永新
陈守臻
朱耀丰
柳晓晨
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Qilu Hospital of Shandong University
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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
    • 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
    • G01N33/57484—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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse

Abstract

The invention provides a bladder cancer prognosis marker, a prognosis evaluation system and application thereof, belonging to the technical field of tumor prognosis evaluation. According to the invention, the expression quantity of OTUD6A and CDC6 in bladder cancer is obviously increased, and meanwhile, the research shows that the expression conditions of OTUD6A and CDC6 are closely related to the overall survival rate of a bladder cancer patient, so that the purposes of judging the malignancy degree of the bladder cancer and the prognosis of the patient can be realized by detecting the expression of OTUD6A and CDC6, the survival rate of the bladder cancer patient is improved, a new thought is provided for clinical accurate treatment decision of the bladder cancer, and the clinical application value is good.

Description

Bladder cancer prognosis marker, prognosis evaluation system and application of prognosis evaluation system
Technical Field
The invention belongs to the technical field of tumor prognosis evaluation, and particularly relates to a bladder cancer prognosis marker, a prognosis evaluation system and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Urothelial cancer is one of the common clinical diseases in urinary surgery, and comprises bladder cancer, renal pelvis cancer, ureteral cancer and the like, wherein the bladder cancer accounts for more than 90% of the whole urothelial cancer, and the disease is usually better to be developed in male and female, and the ratio of the disease development in male and female is about 4: 1. The bladder cancer has high recurrence and death risk, and the 5-year survival rate of the muscle invasive bladder cancer is less than 50 percent. According to the latest global cancer statistics in 2022, the incidence of bladder cancer in men was 4 th and the mortality was 9 th. However, in addition to the currently known tumor grading, staging, lymph node metastasis and ki-67 staining, which can be used to determine the malignancy of a tumor and the prognosis of a patient, there are no clear indicators for assessing the prognosis of a patient.
Immunohistochemistry (IHC) detection is a research that utilizes the principle of antigen-antibody specific binding, and uses a chemical reaction to color a color-developing agent (fluorescein, enzyme, metal ion, isotope) of a labeled antibody to determine the antigens (polypeptide and protein) in tissues or cells, and to localize, qualitatively and relatively quantitatively the antigens. Since immunohistochemistry has the characteristics of strong specificity, high sensitivity, accurate positioning and the like, and can organically combine morphological research and functional research together, it has been widely applied to many fields of biological and medical research. In pathological studies, the role and significance of immunohistochemistry is clearly more important. However, the inventors found that there are only few reports on methods for assessing the malignancy and prognosis of bladder cancer by immunohistochemical detection.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a bladder cancer prognostic marker, a prognostic evaluation system and application thereof. According to the invention, the expression quantity of OTUD6A and CDC6 in bladder cancer is remarkably increased, and the research shows that the expression conditions of OTUD6A and CDC6 are closely related to the overall survival rate of bladder cancer patients, so that the aim of judging the malignancy degree of the bladder cancer and the prognosis of the patients can be realized by detecting the expression of OTUD6A and CDC6, and the invention is completed based on the research results.
The technical scheme of the invention is as follows:
in a first aspect of the present invention, there is provided a prognostic marker for bladder cancer, which includes OTUD6A and CDC 6.
In a second aspect of the present invention, there is provided a use of a substance for detecting the above prognostic marker in the preparation of a product for detecting bladder cancer.
The bladder cancer related detection product comprises a bladder cancer immunohistochemical detection kit which can be used for judging the malignancy degree of bladder cancer and the prognosis condition of a patient.
In a third aspect of the invention, there is provided a product comprising a substance for detecting the above-mentioned prognostic marker, for use in the prognostic assessment of bladder cancer.
In a fourth aspect of the invention, the application of the prognostic markers and products in the preparation of a bladder cancer prognostic evaluation system is provided.
In a fifth aspect of the present invention, there is provided a bladder cancer prognosis evaluation system, comprising: an analysis unit and an evaluation unit;
wherein the analysis unit is used for obtaining the results of the subjects OTUD6A and CDC 6;
the evaluation unit gives corresponding evaluation scores to obtain total scores according to the obtained results of the OTUD6A and the CDC6, and outputs the corresponding prognosis states of the subjects;
the beneficial technical effects of one or more technical schemes are as follows:
according to the technical scheme, immunohistochemical staining is carried out on CDC6 and OTUD6A proteins through a patient tissue specimen, the expression levels of the CDC6 and the OTUD6A are detected, prognosis is judged according to the expression levels of the CDC6 and the OTUD A, and the malignancy degree of a tumor is evaluated; meanwhile, the prognosis condition of the patient can be evaluated, so that the survival rate of the patient with bladder cancer is improved, a new thought is provided for clinical accurate treatment decision of bladder cancer, and the method has good clinical application value.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 shows the expression levels of OTUD6A and CDC6 proteins (upper) and the relative expression levels of OTUD6A protein (lower left) and CDC6 protein (lower right) in tumor tissues and tissues adjacent to the cancer in 20 samples of bladder cancer and tissues adjacent to the cancer in the present example.
FIG. 2 is a correlation analysis of the expression of OTUD6A and CDC6 proteins in bladder tumor tissue in the present example.
FIG. 3 shows the mRNA expression levels of OTUD6A (left) and CDC6 (right) in tumor and tissue specimens near cancer in examples of the present invention.
FIG. 4 is CDC6 and OTUD6A expression (left) and correlation analysis of protein expression levels (right) in normal urothelial and bladder cancer cell lines in examples of the present invention.
FIG. 5 is a photograph of immunohistochemical staining of OTUD6A and CDC6 in a bladder cancer tissue chip according to an embodiment of the present invention.
FIG. 6 is a survival curve for patients with high and low expression of OTUD6A in an example of the invention.
FIG. 7 is a survival curve for patients with high and low expression of CDC6 in an example of the invention.
FIG. 8 is a survival curve for determining patient prognosis for OTUD6A in combination with CDC6 expression in an example of the invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In one embodiment of the present invention, there is provided a prognostic marker for bladder cancer, which includes OTUD6A and/or CDC 6.
Specifically, the research of the invention finds that the expression amounts of OTUD6A and CDC6 in bladder cancer are obviously higher than those of normal bladder epithelium, and the expression amounts of OTUD6A and CDC6 proteins have obvious positive correlation; and the results of the overall survival rate analysis of the OTUD6A and CDC6 by using the patient prognosis information in the bladder cancer tissue chip show that the overall survival rate of the OTUD6A low-expression bladder cancer patient is obviously higher than that of the high-expression group, and the overall survival rate of the CDC6 low-expression bladder cancer patient is also obviously higher than that of the high-expression group. The combined application of the OTUD6A and the CDC6 protein predicts the survival condition of the bladder cancer patient, and the survival analysis result shows that the overall survival rate of the patient with high OTUD6A and CDC6 is lowest, the overall survival rate of the patient with low OTUD6A and CDC6 is highest, and the overall survival rate of the patient with low OTUD6A and high CDC6 is lower than that of the patient with high OTUD6A and low CDC 6.
In another embodiment of the present invention, there is provided a use of the substance for detecting the prognostic marker described above in the preparation of a product for detecting bladder cancer.
The bladder cancer related detection product comprises a bladder cancer immunohistochemical detection kit which can be used for judging the malignancy degree of bladder cancer and the prognosis condition of a patient.
In yet another embodiment of the present invention, there is provided a product comprising a substance for detecting the above-mentioned prognostic marker for use in the prognostic assessment of bladder cancer.
The product comprises but is not limited to a preparation or a kit for detecting the expression of OTUD6A and/or CDC6 in a sample to be detected.
The test sample can be a human sample, and more particularly, the test sample comprises bladder (cancer) tissue of a subject.
In yet another embodiment of the present invention, the product may be an immunohistochemical detection kit. Thus, the kit may contain a primary antibody, a secondary antibody, an endogenous hydrogen peroxide scavenger, a color-developing reagent (e.g., DAB solution) that reacts with horseradish peroxidase and develops color, a reagent (e.g., hematoxylin) that specifically stains cell nuclei in the tissue, and the like.
The primary antibody is specifically an OTUD6A and CDC6 specific antibody prepared against human OTUD6A and CDC6 antigens, such as rabbit anti-human OTUD6A polyclonal antibody and rabbit anti-human CDC6 specific antibody, and is not specifically limited herein.
Correspondingly, the secondary antibody only needs to be matched with the species, class or subclass of the primary antibody, for example, when the primary antibody is rabbit anti-human OTUD6A or CDC6 polyclonal antibody, the secondary antibody can be goat anti-rabbit IgG.
In another embodiment of the present invention, the application of the prognostic markers and products described above in the preparation of a system for prognosis of bladder cancer is provided.
The bladder cancer prognosis evaluation system can be used for evaluating the malignancy and survival rate of bladder cancer of a subject.
In yet another embodiment of the present invention, there is provided a bladder cancer prognosis evaluation system, comprising: an analysis unit and an evaluation unit;
wherein the analysis unit is used for obtaining the results of the subjects OTUD6A and/or CDC 6;
the evaluation unit gives a total score to the corresponding evaluation score according to the obtained results of OTUD6A and/or CDC6, and outputs the corresponding prognosis state of the subject;
more specifically, the results of the subjects OTUD6A and/or CDC6 were obtained staining intensity after immunohistochemical staining and area of positive cells in the whole tissue.
The specific evaluation flow of the evaluation unit comprises the following steps: dividing the staining intensity into negative 0, weak 1, medium 2 and strong positive 3; dividing the area of the whole tissue occupied by the positive cells into 0 point less than 5%, 1 point 5% -25%, 2 points 26% -50%, 3 points 51% -75% and 4 points more than 75%; multiplying the two fractions; low expression was judged in 0-7 points and high expression was judged in 8-12 points.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
The method for evaluating the malignancy of the bladder cancer and the prognosis of a patient by using the immunohistochemical detection kit comprises the following steps:
(1) the bladder cancer tissue taken out of the patient body is embedded and sliced to obtain a paraffin section.
(2) The paraffin sections were placed in an oven at 65 ℃ for 30 minutes to 1 hour until the residual liquid paraffin on the tissue completely fell off.
(3) Dewaxing was carried out in the order of the following table
Figure BDA0003615954520000061
Figure BDA0003615954520000071
(4) Diluting 50 Xantigen repairing solution with three distilled water to 1X, placing in microwave oven, heating with high fire for 5 min, placing slide in boiling antigen repairing solution, heating with low fire for 17 min, and naturally cooling at room temperature.
(5) Slides were washed three times with PBS for 3 minutes each.
(6) The tissue was encircled with a pen, endogenous hydrogen peroxide scavenger was added dropwise in the dark, left to stand at room temperature for 20 minutes, and then the slide was washed three times with PBS for 3 minutes each.
(7) 5% BSA solution was added dropwise to the tissue and allowed to stand at room temperature for 1 hour for blocking.
(8) CDC6 and OTUD6A primary antibody (rabbit anti-human OTUD6A polyclonal antibody (Proteitech, Inc., cat. 24486-1-AP) and recombinant rabbit anti-human CDC6 antibody (Abcam, cat. ab109315) were diluted with PBS.
) The blocking solution was poured off and primary antibody was added dropwise to cover the tissue completely and incubated overnight at 4 ℃.
(9) The next day, the slide was removed and allowed to re-warm at room temperature for 1 hour.
(10) After dropping off the primary antibody, the slides were washed three times with PBS, 3 minutes each.
(11) Adding a secondary antibody which is goat anti-rabbit IgG, Jackson ImmunoResearch, Cat 711-035-152, and standing and incubating for 1 hour at room temperature.
(12) Slides were washed three times with PBS for 3 minutes each.
(13) mu.L of 20 × DAB solution was mixed with 190. mu.L DAB substrate solution (scaled up or down in equal proportion depending on the size and number of slide tissues).
(14) 1 XDAB solution was added dropwise to the tissue and observed under a microscope, and after staining was moderate, the tissue was placed in PBS.
(15) Hematoxylin was added dropwise to the tissue for 30 seconds and rinsed in running water for 5 minutes.
(16) The dehydration treatment was carried out in the order as shown in the following table
Figure BDA0003615954520000081
(17) Neutral gum was added drop wise to the slide for mounting.
(18) The next day observation was performed under an upright microscope and images were recorded.
(19) Staining scores were according to the following criteria: the staining intensity can be divided into 0 point of negative, 1 point of weak, 2 points of medium and 3 points of strong positive; the area of the positive cells in the whole tissue can be divided into 0 point of less than 5%, 1 point of 5% -25%, 2 points of 26% -50%, and 3 points of 51% -75%. Greater than 75% of 4 points. The two fractions are multiplied. Low expression was judged in 0-7 points and high expression was judged in 8-12 points.
20 Western blot detection of the expression of OTUD6A and CDC6 in bladder cancer and tissues adjacent to the cancer. The results are shown in figure 1, and compared with the para-carcinoma tissues, the expression of OTUD6A and CDC6 proteins is up-regulated in bladder cancer tissues. As shown in fig. 2, the expression of OTUD6A and CDC6 proteins showed a significant positive correlation. RT-qPCR resultsIt was shown that the mRNA expression levels of OTUD6A and CDC6 were higher in bladder cancer tissues than in paraneoplastic tissues (FIG. 3). The results of detecting CDC6 and OTUD6A proteins in different bladder cancer cell lines and urothelial cell lines are shown in FIG. 4, and the OTUD6A and CDC6 protein expression are in obvious positive correlation (r20.7302, P0.0302). Chips containing 68 cases of bladder cancer and 40 cases of normal bladder epithelial tissue were subjected to OTUD6A and CDC6 immunohistochemical staining, and immunohistochemical scoring was performed according to staining intensity and area. As a result, the expression level of the OTUD6A and CDC6 in cancer is obviously higher than that of normal bladder epithelium, and the expression levels of the OTUD6A and CDC6 proteins have obvious positive correlation (r2＝0.2905,P<0.0001, fig. 5). The above results indicate that OTUD6A and CDC6 protein expression have a positive correlation in bladder cancer.
The overall survival rate analysis (Kaplan-Meier plot) is carried out on OTUD6A and CDC6 by using patient prognosis information in the bladder cancer tissue chip, and the results show that the overall survival rate of OTUD6A low-expression bladder cancer patients is remarkably higher than that of high-expression patients (figure 6), and the overall survival rate of CDC6 low-expression bladder cancer patients is also remarkably higher than that of high-expression patients (figure 7). The combined application of the OTUD6A and the CDC6 protein predicts the survival condition of the bladder cancer patient, the survival analysis result shows that the overall survival rate of the patient with high OTUD6A and CDC6 is the lowest, the overall survival rate of the patient with low OTUD6A and CDC6 is the highest, the overall survival rate of the patient with low OTUD6A and CDC6 is lower than that of the patient with high OTUD6A and low CDC6 (P0.0307, figure 8), and the combination of OTUD6A and the expression of CDC6 has the value of guiding the prognosis of the bladder cancer patient.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (10)

1. A bladder cancer prognostic marker comprising OTUD6A and/or CDC 6.
2. Use of a substance for detecting the prognostic marker or markers according to claim 1, in the preparation of a product for detecting bladder cancer-related diseases.
3. The use of claim 2, wherein the bladder cancer-related test product comprises a bladder cancer immunohistochemical test kit for determining bladder cancer malignancy and patient prognosis.
4. A product comprising a substance for detecting the prognostic marker of claim 1, for use in the prognostic assessment of bladder cancer.
5. The product of claim 4, wherein the product comprises a preparation or kit for detecting the expression of OTUD6A and CDC6 in a test sample;
preferably, the sample to be tested is a human sample, and further, the sample to be tested comprises bladder (cancer) tissues of a subject.
6. The product of claim 4, wherein the product is an immunohistochemical detection kit.
7. Use of the prognostic marker according to claim 1 and the products according to claims 4 to 6 for the preparation of a system for the prognostic evaluation of bladder cancer.
8. A bladder cancer prognosis evaluation system, comprising: an analysis unit and an evaluation unit;
wherein the analysis unit is adapted to obtain the outcome of the prognostic marker of claim 1 from the subject;
the evaluation unit gives a total score to the corresponding evaluation score according to the result of obtaining the prognostic marker, and outputs the prognostic state corresponding to the subject.
9. The system of claim 8, wherein the prognostic marker is obtained as a result of immunohistochemical staining followed by intensity of staining and area of positive cells in the whole tissue.
10. The system of claim 8, wherein the evaluation unit is configured to perform an evaluation process comprising: dividing the staining intensity into negative 0, weak 1, medium 2 and strong positive 3; dividing the area of the whole tissue occupied by the positive cells into 0 point less than 5%, 1 point 5% -25%, 2 points 26% -50%, 3 points 51% -75% and 4 points more than 75%; multiplying the two fractions; low expression was judged in 0-7 points and high expression was judged in 8-12 points.
CN202210444452.3A 2022-04-26 2022-04-26 Bladder cancer prognosis marker, prognosis evaluation system and application of prognosis evaluation system Pending CN114660291A (en)

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