Prostate cancer marker NEK5 reagent and application thereof

Abstract

The invention relates to the technical field of prostate cancer detection, in particular to a prostate cancer marker NEK5 reagent and application thereof, and discloses application of molecules for detecting NEK5 protein in preparation of a kit or a detection reagent for predicting the malignancy degree or prognosis degree of prostate cancer. The invention also discloses a kit for predicting the malignancy degree or prognosis degree of the prostate cancer, which comprises an endogenous peroxidase blocking agent, animal non-immune serum, a primary antibody, a biotin-labeled secondary antibody, streptomyces avitin protein-peroxidase, a DAB color developing agent and a phosphate buffer salt solution, the relation between the expression level of the NEK5 and the Gleason score of the prostate cancer patient is elaborated for the first time, the kit applies the expression of the NEK5 protein to the prostate cancer patient for the first time, and a reliable method can be provided for predicting the malignancy degree of the prostate cancer patient according to the expression of the NEK5 protein, so that the malignancy degree of the prostate cancer patient is predicted.

Description

Technical Field

[0001] The present invention relates to the technical field of prostate cancer detection, in particular to a prostate cancer marker NEK5 reagent and application thereof. Background Art

[0003] Although the medical community currently has a variety of effective treatments for prostate cancer, including hormone therapy, surgery, and radiotherapy, many prostate cancer patients still die within 1 to 2 years after treatment due to disease progression (such as biochemical recurrence or postoperative tumor metastasis).

[0004] The degree of malignancy of prostate cancer can be assessed by histological grading. The most commonly used is the Gleason score (GS) scoring system, which divides the malignancy of prostate cancer into 2-10 points based on the sum of the scores of the main structural area and the secondary structural area in the prostate cancer tissue. The best differentiation is 1+1=2 points, and the worst is 5+5=10 points.

[0005] Currently, the biochemical recurrence of prostate cancer patients is assessed by serum prostate-specific antigen (PSA). However, the usage and diagnostic strategy of serum prostate-specific antigen (PSA) were developed in European and American populations. Although studies on Asian populations have verified its diagnostic efficacy, some differences have also been found in clinical applications. The main difference is that the normal reference value of PSA in healthy Asian populations is significantly lower than that in European and American populations.

[0006] In view of this grim situation, it is particularly important to find new molecular markers to promptly detect high-risk prostate cancer patients, predict the malignancy of prostate patients through markers, conduct timely follow-up and early intervention in order to improve patients' quality of life and prognosis. Summary of the invention

[0007] In order to predict the malignancy of prostate patients through new prostate cancer markers, conduct timely follow-up and early intervention, the present application provides a prostate cancer marker NEK5 reagent and its application.

[0008] To achieve the above object, the technical solution adopted by the present invention is: to provide a use of a molecule for detecting NEK5 protein in preparing a kit or a detection reagent for predicting the malignancy or prognosis of prostate cancer.

[0009] As a preferred embodiment of the use of the present invention, the kit or detection reagent is used for the detection of surgically removed tissue samples or puncture biopsy tissue samples.

[0010] As a preferred embodiment of the use of the present invention, the molecule for detecting NEK5 protein refers to a molecule that can specifically detect whether NEK5 protein is expressed.

[0011] As a preferred embodiment of the use of the present invention, the molecule capable of specifically detecting whether the NEK5 protein is expressed is a nucleic acid or a protein.

[0012] As a preferred embodiment of the use of the present invention, the protein is an antibody.

[0013] As a preferred embodiment of the use of the present invention, the antibody is a rabbit anti-NEK5 polyclonal antibody.

[0014] The present invention also provides a kit for predicting the malignancy or prognosis of prostate cancer, wherein the kit comprises a molecule for detecting NEK5 protein.

[0015] As a preferred embodiment of the kit of the present invention, the kit comprises: an endogenous peroxidase blocker, animal non-immune serum, a primary antibody, a biotin-labeled secondary antibody, streptavidin-peroxidase, a DAB color developer and a phosphate buffered saline solution, and the primary antibody is a rabbit anti-NEK5 polyclonal antibody.

[0016] As a preferred embodiment of the kit of the present invention, the endogenous peroxidase blocker is a hydrogen peroxide solution with a mass concentration of 3%.

[0017] As a preferred embodiment of the kit of the present invention, the mass concentration of the animal non-immune serum is 5%.

[0018] As a preferred embodiment of the kit of the present invention, the phosphate buffered saline solution is 1×PBS buffer with a concentration of 0.01M.

[0019] As a preferred embodiment of the kit of the present invention, the dilution multiple of the primary antibody is 1:100.

[0020] As a preferred embodiment of the kit of the present invention, the dilution factor of the secondary antibody is 1:300-500.

[0021] The beneficial effects of the present invention are:

[0022] The present invention first describes that the expression level of NEK5 is related to the Gleason score (GS) of prostate cancer patients, and can provide a reliable method for predicting the malignancy of prostate cancer patients.

[0023] The kit of the present invention applies NEK5 protein expression to prostate cancer patients for the first time, predicts the malignancy of prostate cancer patients according to NEK5 protein expression, conducts follow-up early, improves the survival rate of prostate cancer patients, and reduces the mortality rate of prostate cancer patients.

[0024] The kit of the present invention is simple and rapid to operate, has high detection sensitivity and specificity, can realize qualitative and semi-quantitative detection of selected clinical samples, does not require other special instruments, and all are commonly used instruments. The clinical samples can be preserved for a long time, and can be completed in general hospital laboratories or pathology rooms. The kit is expected to become a new technology for postoperative disease diagnosis of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a comparison chart of the differences between the protein IR expression score of NEK5 protein in human prostate cancer tissue and the Gleason score (GS) score grouping. DETAILED DESCRIPTION

[0026] In the present invention, NEK5 protein is also called NIMA Related Kinase 5 (NEK5). As a member of the serine / threonine protein kinase family, it can affect the occurrence, development and proliferation of tumor cells by binding to and phosphorylating the activity of target proteins. Studies have shown that NEK5 is closely related to the malignant progression and poor prognosis of tumors in various tumors, and can be used as one of the predictive factors for tumor progression and prognosis.

[0027] In the present invention, the malignancy of the prostate cancer can be evaluated by histological grading. The most commonly used is the Gleason grading system, which is also called the Gleason score (GS) grading system. The Gleason grading was first proposed by pathologist Professor Gleason in 1974. According to the degree of differentiation of the glandular cells of prostate cancer, it can be divided into 1-5 grades, and the cancer acinus at this level with the largest area is defined as the most common growth type, followed by the second most common growth type. The sum of the two scores is the Gleason score. The malignancy of prostate cancer is divided into 2 to 10 points based on the sum of the scores of the main structural area and the secondary structural area in the prostate cancer tissue. The best differentiation is 1+1=2 points, and the worst is 5+5=10 points. Gleason grading is a widely used method for histological grading of prostate cancer. Because the Gleason grading is well correlated with biological behavior and prognosis, it has gradually been recognized and used more and more widely, becoming an important reference indicator for formulating prostate cancer treatment plans. Since the 1990s, the prostate cancer treatment guidelines recommended by the American Comprehensive Cancer Network have listed Gleason grade, prostate-specific antigen (PSA) level and tumor stage as the most important indicators for determining treatment options.

[0028] In order to accurately help clinicians diagnose and treat prostate cancer, the present invention provides a new prediction technology for prostate cancer progression, specifically a detection reagent / kit for prostate cancer progression. The main advantages of the present invention are: for the first time, it is explained that the expression level of NEK5 is related to the Gleason score (GS) of prostate cancer patients, which can provide a reliable method for predicting the malignancy of prostate cancer patients, and the operation is simple and fast, without the need for other special instruments, and can be applied to general hospital laboratories or pathology rooms, and is expected to become a new technology for the diagnosis of prostate cancer.

[0029] In some embodiments, the present invention provides a detection kit for prostate cancer progression, which can be used for the diagnosis of NEK5 in the Gleason score (GS) of prostate cancer. The kit includes: 50ul of endogenous peroxidase blocker; 50ul of animal non-immune serum; 1ul of rabbit anti-NEK5 polyclonal antibody as the primary antibody; 50ul of biotin-labeled secondary antibody; 50ul of streptavidin-peroxidase; 100ul of DAB colorimetric reagent; and 1000ul of phosphate buffered saline (PBS buffer).

[0030] The above kit can be used to detect the expression level of NEK5 in tumor tissue and diagnose the possibility of Gleason score (GS) of prostate cancer patients, thereby distinguishing different disease progression and prognosis diagnosis of clinical prostate cancer patients.

[0031] In some embodiments, the present invention provides a detection kit capable of detecting the expression status of NEK5 in clinical samples of prostate cancer and predicting the progression of prostate cancer, wherein the reagent composition is as follows:

[0032] Endogenous peroxidase blocker (3% hydrogen peroxide solution, purchased from Beibo Biotechnology Co., Ltd.) 50ul; animal non-immune serum (5%, purchased from Beibo Biotechnology Co., Ltd.) 50ul; primary antibody is rabbit anti-NEK5 polyclonal antibody 1ul, dilution ratio is 1:100; biotin-labeled secondary antibody 50ul, dilution ratio is 1:300; streptavidin-peroxidase 50ul; DAB colorimetric reagent 100ul; phosphate buffered saline (1XPBS buffer, 0.01M) 1000ul.

[0033] The kit is simple and rapid to operate, with high detection sensitivity and specificity. It can realize qualitative and semi-quantitative detection of selected clinical samples without the need for other special instruments. Clinical samples can be preserved for a long time and can be completed in general hospital laboratories or pathology rooms. The kit is expected to become a new technology for the diagnosis of prostate cancer after surgery.

[0034] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the reagent concentrations in the present invention are all mass concentrations. Unless otherwise specified, the reagents or materials in the present invention can be obtained from the market or other public channels. Unless otherwise specified, the experimental methods in the present invention are all conventional methods.

[0035] Embodiment 1:

[0036] An embodiment of the kit for detecting the progression of prostate cancer of the present invention comprises the following components:

[0037] Endogenous peroxidase blocker (3% hydrogen peroxide solution, purchased from Beibo Biotechnology Co., Ltd.) 50ul; animal non-immune serum (5%, purchased from Beibo Co., Ltd.) 50ul; primary antibody is rabbit anti-NEK5 polyclonal antibody (purchased from China ABclonal Co., Ltd.) 1ul; biotin-labeled secondary antibody 50ul; streptavidin-peroxidase 50ul; DAB colorimetric reagent 100ul; phosphate buffered saline (1XPBS buffer, 0.01M) 1000ul.

[0038] Embodiment 2:

[0039] This example uses clinical chip samples of prostate cancer patients to perform polyclonal antibody staining tests on NEK5, and combines clinical sample data to further demonstrate the beneficial effect of the kit of Example 1 on predicting prostate cancer patients and Gleason scores (GS), proving that the kit of the present invention can effectively predict the disease progression of prostate cancer patients.

[0040] The method for using the kit of Example 1 comprises the following steps:

[0041] 1) Taking out the clinical tissue of prostate cancer from liquid nitrogen and storing it in an embedding box;

[0042] 2) Rinse with tap water for 5 hours continuously;

[0043] 3) Place the tissue in a Leica automatic dehydrator for dehydration;

[0044] 4) Embed tissue in paraffin;

[0045] 5) Serially slice the paraffin-embedded tissue blocks to a thickness of approximately 4 μm;

[0046] 6) Place the slices in a constant temperature oven at 60°C for about 2 hours, specifically 2 hours ± 20 minutes;

[0047] 7) Place the tissue sections in a Leica automatic dewaxing machine for dewaxing (including xylene and gradient alcohol dewaxing, specifically, xylene dewaxing first, then xylene dewaxing);

[0048] 8) Rinse with distilled water, place the slices in citric acid antigen repair solution (make sure there is enough liquid to prevent it from drying out during microwave heating) for antigen repair, microwave for 3 minutes, take out and cool (specifically 20 seconds), heat again for 1 minute, take out and cool at room temperature (specifically 20 seconds), heat again for 1 minute, and cool to room temperature;

[0049] 9) Use a water-blocking pen to circle the sliced ​​tissue and soak it in PBS;

[0050] 10) Add 50ul of peroxidase blocking solution to each slice to inactivate endogenous peroxidase in the tissue and incubate at room temperature for 15 minutes;

[0051] 11) Rinse or soak in PBS for 3 × 3 minutes (repeat 3 times, 3 minutes each time);

[0052] 12) Remove the PBS solution, add 50ul of non-immune animal serum to each tissue section to block non-specific binding, and incubate at room temperature for 10-15 minutes;

[0053] 13) Drain the serum and add 200ul of the first antibody diluted in PBS to each section. It should be noted that the first antibody is a mixture of 1ul of rabbit anti-NEK5 polyclonal antibody and 100ul of PBS buffer;

[0054] 14) Place in an incubation box at 4°C overnight, specifically for 7 to 8 hours;

[0055] 15) The next day, rewarm the slices overnight at room temperature for about 30 minutes, specifically 30 minutes ± 5 minutes;

[0056] 16) Rinse or soak in PBS for 3 × 10 minutes;

[0057] 17) Remove the PBS solution, add 50ul of biotin-labeled secondary antibody (dilution factor 1:300) to each slice, and incubate at room temperature for 30 minutes. It is worth mentioning that the secondary antibody is the anti-rabbit antibody of the primary antibody;

[0058] 18) Rinse or soak in PBS for 3 × 10 minutes (repeat 3 times, 10 minutes each time);

[0059] 19) Remove the PBS solution, add 50ul of streptavidin-peroxidase solution to each slice, and incubate at room temperature for 15 minutes;

[0060] 20) Rinse or soak in PBS for 3 × 3 minutes;

[0061] 21) Shake off the PBS solution, add 100ul of freshly prepared diaminobenzidine (DAB) colorimetric solution to each slice, observe under a microscope, and immediately immerse the slice in distilled water to stop staining when yellow staining is observed;

[0062] 22) Place the sections on a Leica automatic pathology machine for hematoxylin counterstaining and sealing (ethanol, xylene);

[0063] 23) Let it dry and observe under a Leica microscope;

[0064] 24) Immunohistochemistry test results: Under a Leica fluorescence microscope, five high-power fields were randomly selected from each section to observe cells. Positive cells were those with brown or brown granules in the cytoplasm and / or nucleus. The experimental results were determined based on the staining intensity of positive cells and the proportion of positive cells in the total cells.

[0065] Score by staining intensity: colorless for 0 points, light yellow for 1 point, brown for 2 points, and brown for 3 points; then score by positive cell rate: no positive staining in tumor cells for 0 points, positive cell rate ≤ 5% for 0 points, 6% to 25% for 1 point, 26% to 50% for 2 points, 51-75% for 3 points, > 75% for 4 points. A quantitative method is used for NEK5 scoring, that is, the sum of the two scores is used as the final staining score, ranging from 0 to 7 points.

[0066] The results showed that in the NEK5 immunoscore of tissue microarrays prepared from specimens of 95 patients who underwent radical prostatectomy (see Table 1), NEK5 protein was mainly stained in the cytoplasm of prostate cancer cells, and high expression of NEK5 was associated with high GS scores of patients (P < 0.05, such as Figure 1 ).

[0067] Table 1 Correlation between NEK5 expression and clinical indicators of prostate cancer patients

[0068]

[0069] Figure 1 In the figure, the vertical axis IRS (immunohistochemical scores) represents the sum of the staining intensity score and the staining range score in immunohistochemistry. The bar graph shows that NEK5 is highly expressed in prostate cancer tissues with high malignancy and lowly expressed in prostate cancer tissues with low malignancy. It can be obtained that the protein expression of NEK5 is positively correlated with the GS score, which means that NEK5 can predict the malignancy of prostate patients.

[0070] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed in the present application, and these modifications or replacements should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. Use of a molecule for detecting NEK5 protein in the preparation of a kit or a detection reagent for predicting the malignancy or prognosis of prostate cancer.

2. The use according to claim 1, characterized in that: The kit or detection reagent is used for detecting surgically removed tissue samples or puncture biopsy tissue samples.

3. The use according to claim 1, characterized in that: The molecule for detecting NEK5 protein refers to a molecule that can specifically detect whether NEK5 protein is expressed.

4. The use according to claim 3, characterized in that: The molecule capable of specifically detecting whether NEK5 protein is expressed is a nucleic acid or a protein.

5. The use according to claim 4, characterized in that: The protein is an antibody.

6. The use according to claim 5, characterized in that: The antibody is a rabbit anti-NEK5 polyclonal antibody.

7. A kit for predicting the malignancy or prognosis of prostate cancer, characterized in that: The kit includes molecules for detecting NEK5 protein.

8. A kit for predicting the malignancy or prognosis of prostate cancer according to claim 7, characterized in that: The invention comprises an endogenous peroxidase blocker, animal non-immune serum, a primary antibody, a biotin-labeled secondary antibody, streptavidin-peroxidase, a DAB color developer and a phosphate buffered saline solution, wherein the primary antibody is a rabbit anti-NEK5 polyclonal antibody.

9. A kit for predicting the malignancy or prognosis of prostate cancer according to claim 8, characterized in that: The endogenous peroxidase blocker is a hydrogen peroxide solution with a mass concentration of 3%, the mass concentration of the animal non-immune serum is 5%, and the phosphate buffered saline solution is a 1×PBS buffer solution.

10. A kit for predicting the malignancy or prognosis of prostate cancer according to claim 8 or 9, characterized in that: The dilution multiple of the primary antibody is 1:100, and the dilution multiple of the secondary antibody is 1:300-500.

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