CN111679072B - Application of KDM6B protein in breast cancer prognosis assessment kits and diagnostic kits - Google Patents

Abstract

The invention provides application of KDM6B protein in a breast cancer prognosis evaluation kit and a breast cancer diagnosis kit, and belongs to the technical field of biology. The application of the KDM6B protein in a breast cancer prognosis evaluation kit comprises the steps of taking the KDM6B protein as a molecular marker, utilizing a KDM6B monoclonal antibody or a KDM6B polyclonal antibody, combining an experimental reagent, and detecting the relative expression quantity of the KDM6B protein in a breast cancer tissue. The invention has the advantages that the KDM6B protein is used as a molecular marker for detecting the expression level of the KDM6B protein and can be used for guiding the prognosis judgment of breast cancer.

Description

Application of KDM6B protein in breast cancer prognosis assessment kits and diagnostic kits

technical field

The invention belongs to the field of biotechnology, and particularly relates to the application of KDM6B protein in breast cancer prognosis evaluation kits and diagnostic kits.

Background technique

Breast cancer is the most common malignancy in women, second only to lung cancer in incidence worldwide, and more than 1.2 million women are estimated to be diagnosed with breast cancer each year. The incidence of breast cancer in my country has risen to the first among female malignant tumors, and the mortality rate ranks fifth, becoming one of the greatest threats to women's health. The early symptoms of breast cancer are not obvious, and the diagnosis is already in the middle and late stages. Although after surgery, chemotherapy, and radiotherapy, the curative effect of many breast cancer patients is not satisfactory. Early diagnosis, early treatment and prognostic evaluation of breast cancer are the keys to improving the survival rate and prolonging the life of patients.

Breast cancer is a highly heterogeneous malignant tumor, mainly divided into four subtypes: Luminal A, Luminal B, Her-2 positive and triple negative breast cancer (TNBC). Among them, triple negative breast cancer refers to a subtype of breast cancer with negative expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth receptor 2 (HER-2). It is highly susceptible to early metastasis and chemotherapy resistance, which seriously affects the survival of breast cancer patients. The 5-year survival rate of patients is less than 15%. Due to the lack of endocrine and anti-HER-2 targeted therapy receptors, the treatment methods for triple-negative breast cancer are limited, and the prognosis of patients is relatively poor. At present, there is no effective indicator to evaluate its prognosis.

Therefore, there is an urgent need in the field to provide new markers for the diagnosis and treatment of breast cancer, and to search for genes and/or proteins for the prognosis of breast cancer (especially triple-negative breast cancer). Relapse is important.

The classification of breast cancer is complex, especially triple-negative breast cancer lacks effective clinical treatment targets and has a very poor prognosis. Therefore, it is difficult to find effective indicators to reasonably evaluate the therapeutic effect and prognosis of different types of breast cancer. At present, little is known about the role of KDM6B in breast cancer, the expression of KDM6B in different types of breast cancer tissues is not clear, and the application of KDM6B expression in breast cancer cells in the prognosis of patients has not been reported.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide new applications of KDM6B protein, especially the application of KDM6B protein in breast cancer prognosis assessment kits and diagnostic kits.

The first object of the present invention can be achieved through the following technical solutions: the application of KDM6B protein in breast cancer prognosis assessment kit, KDM6B protein is used as molecular marker, KDM6B monoclonal antibody or KDM6B polyclonal antibody is used, combined with experimental reagents, The relative expression of KDM6B protein in breast cancer tissues was detected.

Preferably, the experimental reagent is an immunohistochemical experimental reagent.

Preferably, the KDM6B polyclonal antibody is prepared by immunizing rabbits with KDM6B protein.

Preferably, the immunohistochemical reagents include phosphate buffered saline solution, sodium citrate solution, ethanol solution, 3% methanol-H ₂ O ₂ solution, blocking solution, DAB chromogenic solution, biotin-labeled goat anti-rabbit solution IgG.

Preferably, the described kit is used to detect the expression of KDM6B protein in breast cancer tissue in vitro, and its detection method comprises the following steps:

S01: Use the phosphate buffered saline solution, sodium citrate solution, ethanol solution, 3% methanol-H ₂ O ₂ solution, blocking solution, DAB chromogenic solution, and biotin-labeled goat anti-rabbit IgG in the kit to separate the mammary glands. Immunohistochemical staining of cancer tissue sections;

S02: use an image analysis system to collect the dyed image;

S03: Score the staining results by immunohistochemical scoring method respectively;

S04: According to the score, KDM6B protein molecules in breast cancer tissue are divided into high expression and low expression.

The second object of the present invention can be achieved through the following technical scheme: the application of KDM6B protein in a breast cancer diagnostic kit, characterized in that the breast cancer diagnostic kit uses KDM6B protein as a diagnostic marker.

Principle of the present invention: The present invention mainly describes the expression characteristics of KDM6B in different types of breast cancer tissues, the relationship between KDM6B expression and clinicopathological factors, and focuses on the correlation between KDM6B and patient prognosis. The main content of the present invention is to provide a new application of KDM6B protein, use immunohistochemical method to detect the expression of KDM6B protein in breast cancer tissue, analyze the correlation between the expression of KDM6B and the clinicopathological indicators of breast cancer, based on the relative expression of KDM6B protein The correlation between the expression level and breast cancer, the detection of the expression level of the protein as a molecular marker can be used to guide the prognosis judgment of breast cancer, and the present invention is completed on this basis.

Compared with the prior art, the present invention has the following advantages:

(1) The present invention describes a protein KDM6B that is highly expressed in breast cancer cells. The significant increase in the expression of KDM6B indicates the existence of breast cancer and provides a new marker for the diagnosis of breast cancer.

(2) The present invention proves that there is a correlation between KDM6B protein and clinicopathological factors of breast cancer through retrospective analysis, and KDM6B protein is closely related to the occurrence and development of breast cancer.

(3) The present invention shows through survival curve analysis that different expression levels of KDM6B in triple-negative breast cancer and non-triple-negative breast cancer have different effects on postoperative survival of patients, and the two show opposite trends. In breast cancer, those with low KDM6B expression have a better prognosis, but in non-triple-negative breast cancer, those with high KDM6B expression have a relatively better prognosis.

(4) The present invention is further verified by OSbrca database, which proves that KDM6B has different prognostic effects on triple-negative breast cancer and non-triple-negative breast cancer patients.

Description of drawings

Fig. 1 is that the positive expression site of KDM6B protein in breast cancer of the present invention is tan (200X);

Fig. 2 is the expression (400X) of KDM6B of the present invention in the cytoplasm of breast cancer cells;

Fig. 3 is the negative expression (200X) of KDM6B in breast cancer of the present invention;

Fig. 4 is the negative expression (200X) of KDM6B in benign breast tumor of the present invention;

Figure 5 is a graph showing the expression of KDM6B of the present invention and the survival time of breast cancer patients (P>0.05);

Figure 6 is a graph showing the expression of KDM6B of the present invention and the survival time of triple-negative breast cancer patients (P>0.05);

Fig. 7 is the curve of KDM6B expression of the present invention and survival time of non-triple-negative breast cancer patients (P>0.05);

Figure 8 shows that in triple-negative breast cancer, patients with "high 25%" KDM6B expression have shorter overall survival, TCGA (OS, HR (95%CI)=8.6175 (1.0024-74.0813), P=0.0497);

Figure 9 shows that in non-triple-negative breast cancer, patients with "25% higher" KDM6B expression have longer overall survival when ER is positive, GSE7390 (OS, HR (95%CI)=0.3164 (0.1205-0.8308), P=0.0195);

Figure 10 shows that in non-triple-negative breast cancer with positive ER and PR expression, patients with "high 25%" KDM6B expression have longer disease-free survival, GSE21653 (DFS, HR (95%CI)=0.3496 (0.1341-0.9113 ), P=0.0315).

Detailed ways

The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

Collection of clinical case information

Between 2011 and 2014, all 160 patients had surgical specimens from the Second Affiliated Hospital of Wenzhou Medical University with histologically confirmed breast cancer and corresponding molecular subtypes. The clinical information of the patients was recorded: age, gender, detailed past hospitalization information, tumor size, tumor type and grade, number of lymph node metastases and follow-up information (patient survival, date and cause of death, etc.). Our study was approved by an institutional ethics committee, and all patients obtained written informed consent and authorized surgical removal of tissue for scientific purposes.

Clinical tissue samples

The tumor tissue of patients with primary breast cancer is taken from the solid or hard part of the tumor, and the necrotic part should be avoided as much as possible. All patients did not receive any anti-tumor therapy such as radiotherapy or chemotherapy before surgery. Histological grading was determined by two independent pathologists, and the clinical stage of the tumor was determined according to a clinical classification system. The 160 case samples in this study consisted of 80 triple-negative breast cancers, 70 non-triple-negative breast cancers, and 10 benign tissues.

Reagent preparation

(1) Phosphate buffered saline solution (0.01mol/L PBS, pH 7.4): Dissolve 2 L of PBS solid powder slowly in 1800 mL of double distilled water, then adjust the pH of the solution to 7.4 with HCl, and add water to the volume to 2L, store at room temperature for use.

(2) Sodium citrate solution: Dissolve sodium citrate buffer (0.01mol/L, pH 6.0) in 800mL of double distilled water, adjust the pH value to 6.0, add water to make up to 1L, and store at room temperature for use.

(3) Configure different concentrations of ethanol:

95% ethanol: The preparation ratio is the volume of absolute ethanol: the volume of distilled water = 19:1, that is, 475 mL of absolute ethanol is added to 25 mL of distilled water, and after mixing, it is stored at room temperature for use.

90% ethanol: The preparation ratio is the volume of absolute ethanol: the volume of distilled water = 9:1, that is, 450 mL of absolute ethanol is added to 50 mL of distilled water, and after mixing, it is stored at room temperature for use.

85% ethanol: The preparation ratio is the volume of absolute ethanol: the volume of distilled water = 17:3, that is, 425 mL of absolute ethanol is added to 75 mL of distilled water, and after mixing, it is stored at room temperature for use.

75% ethanol: The preparation ratio is the volume of absolute ethanol: the volume of distilled water = 3:1, that is, 375 mL of absolute ethanol is added to 125 mL of distilled water, mixed and stored at room temperature for use.

(4) 3% methanol-H ₂ O ₂ solution (abbreviated as 3% H ₂ O ₂ ): prepare a solution of the required volume in the ratio of 30% H ₂ O ₂ : methanol: water=1:1:8.

(5) DAB chromogenic solution: add 2 drops of B solution (DAB concentrated chromogenic solution) and 1 drop of C solution (enzyme-labeled goat anti-mouse/rabbit IgG polymer) to 1 mL of solution A (DAB substrate buffer). ), ready to use.

Tumor tissue paraffin section

The selected breast cancer paraffin block samples were placed on the ice table, pre-cooled for 45 minutes, and the paraffin block samples were sectioned using a manual paraffin microtome. Placed in warm water at about 40°C, heated evenly in warm water and spread out flat, and fixed the tissue on a detachment-proof glass slide for staining.

Immunohistochemical SP method to detect the expression of KDM6B protein

(1) Bake the detachment-resistant glass slide containing the tissue section in a 60°C incubator for 90 minutes.

(2) Dewaxing and hydration of paraffin sections: soak in xylene for 10 min twice each, then soak in 75%, 85%, 90%, and 95% alcohol for 5 min each, after hydration, use phosphate buffered saline solution on a shaker Rinse 3 times, 3min each time.

(3) Incubate the intact tissue on the section with 3% methanol-H ₂ O ₂ solution for 25 min in the dark at room temperature to inactivate endogenous peroxidase in the tissue, and then rinse with phosphate buffered saline on a shaker 3 times, 3min each time.

(4) Antigen heat recovery: Immerse the tissue section in the prepared antigen recovery solution (0.01M citrate buffer), place it in a pressure cooker and heat it to boiling, continue heating for 10 minutes after boiling, take it out and place it on ice to cool to At room temperature (about 20 min), the tissue sections were taken out and washed with phosphate buffered saline three times on a shaker for 5 min each time.

(5) Add about 100 μL of 10% goat serum to each tissue section to seal, so that the goat serum can completely cover the tissue, incubate at room temperature for 25 minutes, shake off the excess serum, and wipe off the residual serum around the tissue with absorbent paper.

(6) Rabbit anti-human KDM6B primary antibody was diluted proportionally with PBS, the titer was 1:500, and about 100 μL of the diluted primary antibody was added dropwise to each slice, so that the primary antibody could completely cover the tissue. Rabbit anti-human KDM6B primary antibody is rabbit KDM6B polyclonal antibody (which can recognize KDM6B protein in mammals such as human and mouse).

(7) Place the tissue sections in a humidified box, keep the slides flat, to avoid the primary antibody slipping and not covering the tissue completely, and place in a 4°C refrigerator for 16-24 hours.

(8) Take out the glass slide and wash it with phosphate buffered saline three times on the shaker, 5 min each time. Then, 50 μL of secondary antibody (biotin-labeled goat anti-rabbit IgG) was added dropwise to the sections, placed in a constant temperature water bath at 37°C for 30 min, and washed with phosphate buffered saline three times on a shaker for 5 min each time.

(9) Add 100 μL of DAB solution to each section dropwise, and develop color at room temperature for about 2-3 minutes. Control the color development intensity under microscope observation. When the desired staining intensity is reached, rinse off the DAB solution with double distilled water to stop the color development. , and tap water to rinse the residual DAB solution on the slices.

(10) Hematoxylin counterstaining for 1 min, rinsed with tap water for 15 min.

(11) Dehydrate tissue sections with 95%, 90%, 85%, and 75% alcohol concentration gradients respectively, soak in xylene for 2 times for 5 min each time, and finally seal with neutral gum and cover glass.

Immunohistochemical staining results

The staining results were scored according to the immunohistochemical scoring method (IRS). KDM6B was expressed in the cytoplasm and nucleus of breast cancer, and the positive expression was brown-yellow granules. The scoring standard of immunohistochemical staining should consider both the staining intensity of immunoreaction (staining intensity, SI) and the percentage of positive cells (positive percent, PP).

The SI score was 4, with 0: no positive cells; 1: weakly positive cells at the strongest positive staining; 2: positively stained cells; 3: strongly positive cells.

PP is divided into 5 grades, and the scoring criteria are as follows: 0 points: no positive staining area; 1 point: the area of the positive staining area accounts for ≤25% of the total tissue area; 2 points: the area of the positive staining area accounts for 26% of the total tissue area ～50%; 3 points: the percentage of the positive staining area in the total tissue area is between 51% and 75%; 4 points: the positive staining area is >75%.

The immunohistochemical score was equal to SI multiplied by PP, and the results were divided into high expression group (score>4 points) and low expression group (0≤score≤4 points) according to the score.

Image acquisition and analysis

Immunohistochemical staining images were acquired by Leica-Q550CW image analysis system.

Statistical analysis

According to the count data, the χ ² test was selected, the Kaplan-Meier (KM) method was used to detect the survival curve, and the Log-rank test was used to compare the differences in survival rates between groups. The prognostic factors were evaluated by univariate and multivariate analysis (Cox proportional hazards regression model), and the data were analyzed by SPSS19.0 software, P<0.05 was statistically significant.

Breast Cancer Database Analysis

The gene expression profiles and clinical data in the OSbrca breast cancer gene database are stored and managed by the US Microsoft SQL Server database. The breast cancer gene expression profile database is mainly composed of The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (Gene Expression Omnibus, GEO), data were included in the analysis according to the following four criteria: (1) The cohort must have at least 50 breast cancer cases. (2) The cohort must contain individual clinical follow-up information. (3) Probe annotation should be done, or probes can be converted to gene symbols by ID conversion. (4) If the cohort has multiple platforms, only platforms with more than 50 individual samples are selected. Survival analysis was performed on the biomarker KDM6B reported in OSbrca, and the data were divided into "25% higher vs. 25% lower" expression, and the data were divided into triple-negative breast cancer and non-cancerous breast cancer according to the expression of ER, PR, and Her-2. For the triple negative breast cancer groups, Kaplan-Meier survival curves with log-rank P values were generated, respectively, and HR and 95% confidence intervals (95% CI) were calculated using univariate Cox regression analysis.

Expression of KDM6B in breast cancer tissues

Immunohistochemical results showed that KDM6B protein was expressed in both the cytoplasm and nucleus of breast cancer (as shown in Figure 1-4). The high expression of KDM6B in 150 cases of breast cancer tissues was 79 cases and 10 cases of benign breast tissues. The number of cases with high expression of KDM6B was 1 case, and the chi-square (χ ² ) analysis showed that the difference between the two expressions was statistically significant (P<0.05), suggesting that the expression of KDM6B in breast cancer tissue was higher than that in benign tumors. As shown in Table 1.

Table 1 Differences of KDM6B expression between benign breast tumors and breast cancer

Further analysis of the positive expression results of KDM6B showed that 23 of 80 cases of triple-negative breast cancer were highly expressed, and 56 of 70 cases of non-triple-negative breast cancer were highly expressed. Chi-square analysis of the expression differences of KDM6B among the three triple-negative breast cancers showed that the differences among the three were statistically significant (P<0.05), suggesting that compared with triple-negative breast cancers, KDM6B in non-triple-negative breast cancer The expression was higher in tissues, and the results are shown in Table 2.

Table 2 Differences of KDM6B expression among benign breast tumors, non-triple-negative and triple-negative breast cancers

The relationship between KDM6B expression and breast cancer-related clinicopathological factors

According to the statistical analysis of the chi-square test, in the tissue samples of 150 breast cancer patients, the expression of KDM6B protein was correlated with the expression of ER, PR, Her-2 and Ki-67 in the tissues of breast cancer patients, and the difference was statistically significant. (P<0.05) (as shown in Table 3); but there was no significant correlation with the patient's age, tumor diameter, and lymph node metastasis (P>0.05).

Table 3 The relationship between KDM6B expression and related clinicopathological factors in breast cancer patients

The relationship between the expression of KDM6B and the prognosis of breast cancer patients

In order to discuss the relationship between the expression of KDM6B and the prognosis of breast cancer patients, according to the immunohistochemical staining results of KDM6B expression in breast cancer, the Kaplan-Meier survival analysis results showed that among the related clinicopathological factors in 150 breast cancer patients, the patients Age, lymph node metastasis, PR expression were correlated with prognosis of breast cancer patients (P<0.05) (as shown in Table 5), tumor diameter, ER, Her-2, Ki-67 and KDM6B expression were correlated with There was no statistical correlation between the prognosis of breast cancer patients (P>0.05). Cox regression model analysis was constructed to evaluate the correlation between KDM6B expression and age, tumor diameter, lymph node metastasis, ER, PR, Her-2, and Ki-67. Independent factors for postoperative patient prognosis (as shown in Table 6).

Table 5 Relationship between clinicopathological factors and prognosis in 150 breast cancer patients

Table 6 Multivariate analysis results of prognosis of 150 breast cancer patients

The relationship between the expression of KDM6B and the overall survival of breast cancer

According to the immunohistochemical staining results of KDM6B expression in breast cancer, combined with the follow-up of breast cancer patients, to understand the expression of KDM6B in breast cancer and the prognosis of breast cancer patients, and draw the overall survival curve. The curve analysis showed that among all breast cancer patients, patients with different KEM6B expression levels had different postoperative survival time, but the difference in survival time between the high expression group and the low expression group was not statistically significant (P>0.05) (Fig. 5). The expression of KDM6B in triple-negative and non-triple-negative breast cancer and the prognosis of breast cancer patients were further analyzed, and the overall survival curve was drawn. The curve analysis showed that there were differences in postoperative survival among patients with different KDM6B expression levels. The prognosis of patients with high KDM6B expression in the late follow-up period was relatively good, but in triple-negative and non-triple-negative breast cancer, there was no significant difference in the survival time between the high-expression group and the low-expression group (P>0.05) (Figures 6 and 7). Show). Longer survival time means better prognosis.

Comparison of breast cancer databases to analyze the relationship between KDM6B and prognosis of breast cancer patients

The prognostic value of KDM6B gene in breast cancer was analyzed using existing high-throughput data in the OSbrca database. The results showed that in triple-negative breast cancer with negative expression of ER, PR, and Her-2, patients with "high 25%" expression of KDM6B had worse prognosis than "low 25%" expression, and overall survival (Overall Survival, OS) Shorter, the difference was statistically significant: TCGA (OS, HR (95% CI) = 8.6175 (1.0024-74.0813), P = 0.0497) (as shown in Figure 8), in ER-positive non-triple-negative breast cancer , KDM6B "high 25%" expression in patients with longer overall survival than "low 25%" expression, the difference is statistically significant: GSE7390 (OS, HR (95%CI) = 0.3164 (0.1205-0.8308), P = 0.0195) (As shown in Figure 9); in non-triple-negative breast cancer with positive ER and PR expression, the prognosis of patients with KDM6B expression "high 25%" is better than that of "low 25%" expression, and the disease-free survival (Disease- free survival, DFS) was longer, and the difference was statistically significant: GSE21653 (DFS, HR (95%CI)=0.3496 (0.1341-0.9113), P=0.0315) (as shown in Figure 10). Validation analysis of OSbrca database showed that KDM6B has different predictive value in different types of breast cancer. High KDM6B expression in triple-negative breast cancer indicates poor prognosis, while high KDM6B expression in non-triple-negative breast cancer predicts longer survival. The difference was statistically significant, indicating that the function of KDM6B may be related to the expression of ER, PR and Her-2.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which the present invention pertains can make various modifications or additions to the described specific embodiments or substitute in similar manners, but will not deviate from the spirit of the present invention or go beyond the definition of the appended claims range.

Claims (5)

1. The application of KDM6B protein in preparing a triple-negative and non-triple-negative breast cancer prognosis evaluation kit is characterized in that KDM6B protein is used as a molecular marker, a KDM6B monoclonal antibody or a KDM6B polyclonal antibody is used in combination with an experimental reagent, and the relative expression quantity of the KDM6B protein in breast cancer tissues is detected;

   performing survival analysis on a biomarker KDM6B reported in OSbrca, dividing data into expressions with 25% higher VS and 25% lower, dividing the data into two groups of triple negative breast cancer and non-triple negative breast cancer according to the expression conditions of ER, PR and Her-2, respectively generating a Kaplan-Meier survival curve with a logarithmic rank P value, and calculating HR and 95% confidence interval (95% CI) by using single-factor Cox regression analysis; high KDM6B expression in triple negative breast cancers suggests poor prognosis, whereas high KDM6B expression in non-triple negative breast cancers predicts longer survival.
2. 2. The use of claim 1, wherein the test agent is an immunohistochemical test agent.
3. 3. The use of claim 1, wherein said KDM6B polyclonal antibody is prepared from KDM6B protein by rabbit immunization.
4. 4. The use of claim 2, wherein the immunohistochemical assay reagent comprises phosphate buffered saline, sodium citrate solution, ethanol solution, 3% methanol-H₂O₂Solution, confining liquid, DAB developing liquid and biotin-labeled goat anti-rabbit IgG.
5. 5. The use according to claim 1, wherein the kit is used for detecting the expression level of KDM6B protein in breast cancer tissues in vitro, and the detection method comprises the following steps:

   s01: phosphate buffer salt solution, sodium citrate solution, ethanol solution and 3% methanol-H in the kit are utilized₂O₂Carrying out immunohistochemical staining on the breast cancer tissue section by using solution, confining liquid, DAB developing liquid and biotin labeled goat anti-rabbit IgG;

   s02: collecting the dyed image by using an image analysis system;

   s03: scoring the staining results respectively by an immunohistochemical integral scoring method;

   s04: and (3) dividing KDM6B protein molecules in the breast cancer tissues into high expression quantity and low expression quantity according to the scores.

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