PHF6 index-based bladder cancer neuroendocrine subtype diagnosis and prognosis reagent and kit thereof

Abstract

The invention relates to an immunohistochemical detection reagent and kit for bladder cancer neuroendocrine subtype diagnosis and prognosis based on a PHF6 index. The reagent comprises a PHF6 primary antibody, a PHF6 secondary antibody, an antigen repair solution, blocking serum, a DAB developing solution, a hematoxylin staining solution, a hydrochloric acid-alcohol differentiation solution, an ammonia water blue-returning solution and a neutral gum mounting medium. The obvious correlation between the expression level of the PHF6 and the neuroendocrine subtype of the bladder cancer is determined by detecting the expression condition of the PHF6 in the bladder cancer tissue and combining a series of cell experiments and clinical pathological analysis. The invention provides a novel and effective diagnosis means which can help a clinician to accurately judge the neuroendocrine subtype of bladder cancer, so that a more accurate individualized treatment scheme is formulated for a patient, and more reliable prognosis evaluation is provided.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to new uses of PHF6, specifically reagents and kits for the diagnosis and prognosis of neuroendocrine subtypes of bladder cancer based on the PHF6 index. Background Technology

[0002] PHF6, short for Plant Homeodomain Finger Protein 6, is a protein molecule with a unique structure. Encoded by a specific gene, this protein contains unique structural motifs such as plant homeodomains. These domains play a crucial role in the interaction between the protein and DNA, RNA, or other proteins, thus exerting important functions in various biological processes, including intracellular signal transduction and gene expression regulation.

[0003] Bladder cancer, a common malignant tumor of the urinary system, has an increasing incidence rate worldwide. Molecular subtyping studies of bladder cancer have revealed multiple subtypes, including a neuroendocrine subtype. The neuroendocrine subtype of bladder cancer possesses unique biological characteristics, including high expression of neuroendocrine-related markers, p53 mutations, and RB1 deletions. Furthermore, it exhibits significant differences from other subtypes in disease progression, treatment response, and prognosis.

[0004] However, current diagnostic and prognostic methods for neuroendocrine subtypes of bladder cancer (NEBC) still face significant technical limitations, failing to meet clinical needs. Existing technologies primarily rely on multimodal methods such as histopathological examination, gene testing, imaging examinations, and biomarker detection for bladder cancer diagnosis and prognosis. However, these methods present numerous challenges in diagnosing NEBC. For example, pathological diagnosis depends on the pathologist's experience, exhibiting subjectivity and variability; imaging diagnosis has a high false-positive rate, easily leading to overdiagnosis and overtreatment. Regarding prognostic assessment, effective prognostic biomarkers are lacking; existing prognostic assessments mainly rely on pathological grading and staging, but these methods have low specificity for NEBC, making it difficult to accurately predict patient prognosis. Furthermore, NEBC responds poorly to traditional chemotherapy and immunotherapy, lacks effective targeted therapies, and suffers from insufficient individualization of treatment choices, resulting in limited treatment outcomes. Therefore, there is an urgent need for more novel and effective biomarkers and detection methods to overcome the current technological bottlenecks.

[0005] There are currently no publicly available research reports on the association between PHF6 and the neuroendocrine subtype of bladder cancer (NEBC) and its mechanism of action. Summary of the Invention

[0006] The purpose of this invention is to address the challenges faced in the diagnosis and prognosis of neuroendocrine subtypes of bladder cancer (NEBC) in the existing technology, particularly the lack of in-depth understanding and technical means regarding the association between PHF6 and NEBC. This invention provides a PHF6-based biomarker and reagents and kits for the diagnosis and prognosis of NEBC. It aims to solve the problem of insufficient research on the role and mechanism of action of PHF6 in NEBC, providing clinicians with a new diagnostic tool and solution for accurately identifying and assisting in the diagnosis of NEBC neuroendocrine subtypes, and for assessing patient prognosis, thereby providing important guidance for treatment strategies.

[0007] This invention provides a reagent and kit for the diagnosis and prognosis of neuroendocrine subtypes of bladder cancer based on the PHF6 index, filling a gap in this research. By integrating clinical case data and in-depth basic biological experimental data, this invention explores the relationship between PHF6 and the neuroendocrine transformation subtype (NEBC) of bladder cancer. The kit of this invention allows for the quantitative or qualitative determination of PHF6 expression levels in samples, thereby providing a basis for clinical diagnosis and prognostic assessment.

[0008] The technical solution of the present invention:

[0009] This invention first provides an immunohistochemical detection reagent for the diagnosis and prognosis of neuroendocrine subtypes of bladder cancer based on the PHF6 index. The reagent includes a PHF6 primary antibody, a secondary antibody, an antigen retrieval solution, blocking serum, DAB chromogenic solution, hematoxylin staining solution, hydrochloric acid-alcohol differentiation solution, ammonia-blue solution, and a neutral resin mounting medium. The amount of reagent used is determined based on the tissue to be covered.

[0010] ① Antigen retrieval solution: 0.01 mol / L citrate buffer (pH=6.0). Weigh 2.94 g of trisodium citrate, add about 900 ml of deionized water, adjust the pH to 6.0 with citric acid, and then bring the volume to 1000 ml.

[0011] ②Blocking serum: Normal goat serum. Dilute the normal goat serum with PBS at a ratio of 1:5, and prepare fresh before use.

[0012] ③ DAB chromogenic reagent: DAB chromogenic reagent kit (solution A, solution B, solution C). Mix solutions A and B in the proportions specified in the instructions. Add 50 μL of solution A to 1 ml of solution B, mix well, and use immediately.

[0013] ④ Hematoxylin staining solution: Weigh 2g of hematoxylin, dissolve it in 100ml of anhydrous ethanol, add 17.6g of potassium aluminum sulfate, heat to dissolve, then add 0.2g of sodium iodate, stir well, cool and filter, and add distilled water to 500ml.

[0014] ⑤ Hydrochloric acid-ethanol differentiation solution: Add 1 ml of concentrated hydrochloric acid to 99 ml of 70% ethanol and mix well.

[0015] ⑥ Ammonia blueing solution: Add 5ml of concentrated ammonia to 1000ml of distilled water and mix well.

[0016] ⑦ Neutral resin mounting tablets: Commercially available regular neutral resin mounting tablets.

[0017] ⑧ PHF6 primary antibody: Rabbit anti-human PHF6 monoclonal antibody, diluted 1:100 with antibody dilution buffer before use. The antibody dilution buffer is PBS buffer containing 1% BSA. Weigh 1g of BSA and dissolve it in 100ml of PBS (0.01mol / L, pH=7.4).

[0018] ⑨ Secondary antibody: Biotin-labeled goat anti-rabbit IgG, diluted with antibody diluent at a ratio of 1:150 before application.

[0019] The reagent can be used to detect the content of PHF6 in bladder cancer tissue from patients. This invention uses immunohistochemistry (IHC) to detect PHF6.

[0020] This invention also provides a kit for the diagnosis and prognosis of neuroendocrine subtypes of bladder cancer based on the PHF6 index, the kit comprising the reagents described above. The kit serves as an auxiliary tool for sample processing and reagent dispensing, as well as internal standards for stability and quality control.

[0021] In some specific embodiments of the present invention, the primary antibody in the reagent is IH. C The existing PHF6 antibody used in the experiment.

[0022] According to some specific embodiments of the present invention, when the PHF6 level in bladder cancer tissue is higher than the average level in normal individuals, that is, higher than the diagnostic threshold, the bladder cancer patient undergoing the test is diagnosed as a neuroendocrine subtype patient of bladder cancer; when the PHF6 level in bladder cancer tissue is higher than the average level in normal individuals, that is, higher than the prognostic threshold, the prognosis of the patient diagnosed as a neuroendocrine subtype patient of bladder cancer is poor.

[0023] Advantages and beneficial effects of the present invention:

[0024] Using the kit of this invention, a strong correlation between PHF6 and the neuroendocrine subtype of bladder cancer can be obtained based on previous clinical pathological data and basic biological experimental data. Based on this correlation between PHF6 protein expression and the neuroendocrine subtype of bladder cancer, using this protein as a molecular marker to detect its expression level allows for quantitative or qualitative determination of PHF6 expression levels in samples, thus providing a basis for clinical diagnosis and prognostic assessment. This kit can be used to rapidly guide the diagnosis of patients with the neuroendocrine subtype of bladder cancer. Attached Figure Description

[0025] Figure 1 To detect the expression levels of PHF6 in Normal (normal bladder cancer) and NEBC (neuroendocrine bladder cancer) and the expression levels of NEBC (neuroendocrine bladder cancer) markers NSE and Syn by immunohistochemistry;

[0026] Figure 2 A represents the transfection efficiency of PHF6 after overexpression in bladder cancer neuroendocrine cell lines. Figure 2 B represents the transfection efficiency of PHF6 after knockdown in bladder cancer neuroendocrine cell lines;

[0027] Figure 3 A represents the effect of PHF6 overexpression on the proliferation of bladder cancer neuroendocrine cells. Figure 3 B represents the effect of PHF6 knockdown on the proliferative capacity of bladder cancer neuroendocrine cells.

[0028] Figure 4 A represents the effect of PHF6 overexpression on the invasive ability of bladder cancer neuroendocrine cells. Figure 4 B represents the effect of PHF6 knockdown on the invasive ability of bladder cancer neuroendocrine cells. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention are only used to explain the present invention and do not mean to limit the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] Example 1: Study on the expression of PHF6 in the neuroendocrine subtype of bladder cancer tissue.

[0033] This embodiment uses immunohistochemistry to detect the expression of PHF6 in bladder cancer tissue, as detailed below:

[0034] 1. Dewaxing and hydration: Before dewaxing, fresh bladder cancer neuroendocrine subtype tissue sections were baked in a 65℃ constant temperature oven for 1-2 hours (the baking time was increased appropriately depending on the thickness of the wax seal). Then, the paraffin sections were dewaxed in xylene 3 times, 10 minutes each time. Then, they were hydrated in 100%, 95%, 90%, 80%, and 70% alcohol for 5 minutes each.

[0035] 2. Antigen retrieval: Place the slide in the antigen retrieval solution, microwave on high until boiling, then reduce to medium heat and maintain boiling for 10-15 minutes, then let it cool naturally to room temperature.

[0036] 3. Blocking: Rinse the sections three times with PBS for 5 minutes each time, then add blocking serum to cover the tissue and incubate at room temperature for 30 minutes.

[0037] 4. Add primary antibody: Pour off the blocking serum, do not rinse, add diluted PHF6 primary antibody to cover the tissue, and incubate overnight at 4°C.

[0038] 5. Add secondary antibody: The next day, take out the slides, bring them to room temperature, wash them 3 times with PBS for 5 minutes each time, add diluted secondary antibody to cover the tissue, and incubate at room temperature for 30 minutes.

[0039] 6. DAB color development: Rinse three times with PBS for 5 minutes each time, add freshly prepared DAB color development solution, observe the color development under a microscope, and stop the color development when appropriate.

[0040] 7. Hematoxylin counterstaining: After rinsing the sections with tap water, immerse them in hematoxylin staining solution for 3-5 minutes, then rinse with tap water.

[0041] 8. Differentiation and Blueing: Place the slices in hydrochloric acid-alcohol differentiation solution for a few seconds, rinse with tap water, then place them in ammonia blueing solution for a few seconds, and rinse with tap water.

[0042] 9. Dehydration, clearing and mounting: Dehydrate the sections by immersing them in 70%, 80%, 90%, 95% and 100% alcohol for 5 minutes each, then clear them in xylene three times for 5 minutes each time, and finally mount them with a neutral resin mounting medium.

[0043] 10. Judgment of Staining Results: Positive staining is defined as brownish-red staining within the cell nucleus. Semi-quantitative scoring is performed based on two aspects: the percentage of positive tumor cells and the staining intensity. Staining intensity scores are: 0 points for no staining, 1 point for light brownish-yellow, 2 points for moderate staining, and 3 points for dark brownish-yellow. Positive tumor cell percentage scores are: 1 point for 0% ≤ positive cell percentage ≤ 25%; 2 points for 25% < positive cell percentage ≤ 50%; 3 points for 50% < positive cell percentage ≤ 75%; and 4 points for 75% < positive cell percentage ≤ 100%. The final score is calculated based on the percentage of positive tumor cells and staining intensity: H-Score = Percentage of positive tumor cells × Staining intensity score.

[0044] Test results as follows Figure 1 As shown, PHF6 is highly expressed in the neuroendocrine subtype of bladder cancer compared to normal tissue.

[0045] Example 2: Transfection efficiency of PHF6 after knockdown and overexpression in bladder cancer neuroendocrine-related cell lines

[0046] 1. Experiment Agent

[0047] Lipofectamine 2000 transfection reagent (Invitrogen, USA); PHF6 knockdown plasmid (Anhui General Biotechnology Co., Ltd., China); PHF6 overexpression plasmid (Anhui General Biotechnology Co., Ltd., China).

[0048] 2. Cell culture: The UMUC3 cell line, a neuroendocrine-related cell line for bladder cancer, was cultured in MEM medium containing 10% FBS at 37°C and in a 5% CO2 incubator.

[0049] 3. Transfection: Transfect UMUC3 cells, a neuroendocrine-related cell line for bladder cancer, at a concentration of 1×10⁻⁶ cells / year. 4 The cells were seeded into 24-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for 24 hours. The medium was then replaced with MEM medium containing 10% FBS and free of antibiotics. The cells were transfected according to the instructions of Liposome Transfection Reagent 2000 (purchased from Invitrogen). The plasmids of the experimental group and the control group were transfected into the corresponding cells.

[0050] 4. Western blot detection of PHF6 receptor protein expression level: 48 h after cell transfection, cells were collected using cell lysis buffer (150 mM sodium chloride + 1% NP-40 (detergent) + 0.1% SDS (detergent) + 2 μg / ml Aprotinin (protease inhibitor) (added before use) + 2 μg / ml Leupeptin (protease inhibitor) (added before use) or 1 mM PMSF (protease inhibitor) + 1.5 mM EDTA (protease inhibitor) + 1 mM NaVanadate (phospholipase inhibitor)). Protein concentration was determined using a BCA protein detection kit. 50 μg of protein from each experimental group was transferred to an SDS-PAGE membrane, blocked with 5% skim milk powder, and then incubated overnight at 4°C with PHF6 receptor antibody and internal control antibody. The membrane was washed three times with TBST solution and then incubated at room temperature for 1 h with horseradish peroxidase (HRP)-labeled goat anti-mouse (Zhongshan Jinqiao Co., Ltd., 1:5000). The sample was incubated with ECL immunoblotting chemiluminescence reagent for 5 minutes, then exposed, developed, and fixed. The housekeeping protein GAPDH was used as an internal control protein.

[0051] 5. Results

[0052] like Figure 2 As shown, overexpression plasmids can promote the expression of PHF6, while knockdown plasmids can inhibit the expression of PHF6.

[0053] Example 3: Effect of PHF6 on the proliferative capacity of neuroendocrine subtypes of bladder cancer

[0054] 1. Steps:

[0055] (1) First, the UMUC3 cell line, a neuroendocrine-related cell line for bladder cancer, was cultured in a medium containing 10% fetal bovine serum to a density of 70-80%. Then, plasmids from the experimental and control groups were transfected into the corresponding cells using transfection reagents. 24 hours after transfection, the cell count was determined using a cell counter or manual counting method, and the seeding concentration was adjusted to an appropriate range, controlling the cell count to be within the range of 400-1000 cells / well, and seeded into 6-well plates; culture continued for 14 days, or until the cell count in most individual clones exceeded 50. During the culture process, the medium was changed every 3 days, and the cell status was observed; after the colony formation was completed, the cells were fixed with 1 mL of 4% paraformaldehyde for 15-30 minutes. Finally, the cells were washed with PBS; 1 mL of crystal violet staining solution was added to each well, and the staining time was controlled within 10-20 minutes; the cells were washed multiple times with PBS, and photographs were taken (the entire 6-well plate and each well were photographed individually).

[0056] 2. Results

[0057] The results are as follows Figure 3 As shown, PHF6 levels affect the proliferative capacity of the neuroendocrine subtype of bladder cancer, and there is a strong correlation between the two.

[0058] Example 4: Effect of PHF6 on the invasive ability of neuroendocrine subtypes of bladder cancer

[0059] 1. Procedure: First, the UMUC3 cell line, a neuroendocrine-related cell line for bladder cancer, was cultured to a density of 70-80% in medium containing 10% fetal bovine serum. Then, plasmids from the experimental and control groups were transfected into the corresponding cells using transfection reagents. Forty-eight hours after transfection, Matrigel matrix was prepared, mixed with culture medium at a specific ratio, coated onto the polycarbonate membrane of a Transwell chamber, and incubated at 37°C to form a matrix layer. Next, the transfected cell suspension was adjusted to an appropriate concentration and seeded into Transwell chambers, which were then placed in 24-well plates containing culture medium and incubated at 37°C and 5% CO2 for 24-48 hours. After incubation, the chambers were washed with PBS, and the invading cells were fixed with fixative and then stained. Cells penetrating the matrix layer were observed and photographed under a microscope. Finally, the number of cells penetrating the matrix layer was quantitatively analyzed using image analysis software to assess the effect of PHF6 on the invasive ability of UMUC3 cells.

[0060] 2. Results

[0061] The results are as follows Figure 4 As shown, PHF6 levels affect the invasive ability of neuroendocrine subtypes of bladder cancer, and there is a strong correlation between the two.

[0062] Although the invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the invention fall within the scope of protection claimed by the invention.

Claims

1. An immunohistochemical detection reagent for the diagnosis and prognosis of neuroendocrine subtypes of bladder cancer based on the PHF6 index, characterized in that, The reagents include PHF6 primary antibody, secondary antibody, antigen retrieval solution, blocking serum, DAB chromogenic solution, hematoxylin staining solution, hydrochloric acid alcohol differentiation solution, ammonia water blueing solution, and neutral resin mounting medium.

2. The reagent according to claim 1, characterized in that, This reagent is used to detect the expression of PHF6 in bladder cancer tissue.

3. The reagent according to claim 1, characterized in that, This reagent is used to detect PHF6 using immunohistochemistry.

4. The reagent according to claim 1, characterized in that, The primary antibody for PHF6 is rabbit anti-human PHF6 monoclonal antibody; the secondary antibody is biotin-labeled goat anti-rabbit IgG; the antigen retrieval solution is 0.01 mol / L citrate buffer, pH=6.0; the blocking serum is normal goat serum; the DAB chromogenic solution is a DAB chromogenic kit, including solution A, solution B, and solution C; the hematoxylin staining solution is prepared by dissolving 2 g of hematoxylin in 100 ml of anhydrous ethanol, adding 17.6 g of potassium aluminum sulfate, heating to dissolve, then adding 0.2 g of sodium iodate, stirring evenly, cooling, filtering, and adding distilled water to 500 ml; the hydrochloric acid-ethanol differentiation solution is prepared by adding 1 ml of concentrated hydrochloric acid to 99 ml of 70% ethanol; the ammonia-based blueing solution is prepared by adding 5 ml of concentrated ammonia to 1000 ml of distilled water; the neutral resin mounting medium is a commercially available conventional neutral resin mounting medium.

5. An immunohistochemical detection kit for the diagnosis and prognosis of neuroendocrine subtypes of bladder cancer based on the PHF6 index, characterized in that, The kit contains the reagent as described in any one of claims 1 to 4.

6. The reagent kit according to claim 5, characterized in that, It also includes tissue slicers for sample processing, anti-detachment slides and coverslips, as well as positive control tissue slices and negative control tissue slices to ensure the stability and quality control of the test.

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