Use of a detection reagent for crot protein in a product for prognosing the risk of nasopharyngeal carcinoma

Abstract

The present application relates to the technical field of medical diagnosis, and particularly relates to the use of a detection reagent of CROT protein in a product for prognosis of nasopharyngeal carcinoma risk, wherein the amino acid sequence of the CROT protein is shown as SEQ ID NO. 1. The subject for prognosis of nasopharyngeal carcinoma risk is a human, and the sample for detection is a tissue of the subject. The high expression of the CROT protein is related to the occurrence and malignant growth behavior of nasopharyngeal carcinoma, and compared with a healthy control, the high expression of the CROT protein indicates that the subject has nasopharyngeal carcinoma or has the risk of nasopharyngeal carcinoma; the expression of the CROT protein in a tissue of a nasopharyngeal carcinoma patient is increased, and it is determined as a poor prognosis; the expression of the CROT protein in a tissue of a nasopharyngeal carcinoma patient is decreased, and it is determined as a good prognosis.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostic technology, specifically to the use of a CROT protein detection reagent in products for predicting nasopharyngeal carcinoma risk. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is a highly malignant epithelial tumor with significant geographical clustering and racial susceptibility, posing a serious public health problem globally. According to the latest epidemiological data, there were approximately 120,434 new cases of NPC and 73,485 deaths worldwide in 2022. Although studies have identified the main pathogenic factors of NPC, including Epstein-Barr virus (EBV) infection, genetic susceptibility, and environmental exposure, distant metastasis and tumor recurrence remain the leading causes of death and have long constrained improvements in clinical treatment outcomes. Therefore, developing a highly efficient, specific, and clinically applicable molecular biomarker for NPC to enable early diagnosis, prognostic assessment, and targeted therapy is of great significance for improving patient survival rates.

[0003] Carnitine octanoyltransferase (CROT) is a key metabolic enzyme located in peroxisomes and mitochondria, belonging to the carnitine acyltransferase family. It plays a crucial role in the β-oxidation pathway of medium- and long-chain fatty acids in cells, responsible for transferring medium- and long-chain acyl groups such as octanoyl from coenzyme A to carnitine, thereby promoting the entry of fatty acids into different organelles for metabolic breakdown. This process plays a central regulatory role in maintaining intracellular lipid homeostasis and energy balance. Recent studies have shown that abnormal CROT expression is closely related to the development and progression of various malignant tumors, including melanoma and ovarian cancer.

[0004] However, the role of CROT in nasopharyngeal carcinoma is still unclear, and it is necessary to investigate the role of CROT protein in the development and progression of nasopharyngeal carcinoma and its clinical significance. Summary of the Invention

[0005] To address the above issues, this invention provides the use of a CROT protein detection reagent in products for predicting nasopharyngeal carcinoma risk.

[0006] This invention is achieved through the following technical solution:

[0007] The use of a CROT protein detection reagent in a product for prognostic nasopharyngeal carcinoma risk, wherein the amino acid sequence of the CROT protein is shown in SEQ ID NO.1.

[0008] SEQ ID NO.1:

[0009] Preferably, the subject with the prognostic risk of nasopharyngeal carcinoma is a human being.

[0010] Preferably, the sample being tested is the subject's tissue.

[0011] Preferably, the tissue is cancerous tissue.

[0012] Preferably, the detection reagent is a reagent for quantitatively detecting the expression level of the CROT protein.

[0013] Preferably, the detection reagent is a CROT protein antibody.

[0014] Preferably, the method for quantitatively detecting the expression level of the CROT protein is immunohistochemistry.

[0015] Preferably, the specific steps of the immunohistochemical experimental method are as follows:

[0016] (1) Prepare tissue microarrays from the tissue to be tested, and then bake, dewax, and retrieve antigens from the tissue microarrays.

[0017] (2) Rinse with PBS buffer and incubate with primary antibody.

[0018] (3) Rinse with PBS buffer and incubate with secondary antibody.

[0019] (4) After washing with PBS buffer, add DAB colorimetric solution, develop color for 10 minutes, and then rinse with tap water.

[0020] (5) Counterstain, mount, and examine under a microscope.

[0021] Preferably, the primary antibody is a rabbit-derived antibody.

[0022] Preferably, the secondary antibody is a biotin-labeled goat anti-rabbit antibody.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention uses immunohistochemistry to detect the expression of CROT protein in nasopharyngeal carcinoma tissues. The results show that the expression of CROT protein in nasopharyngeal carcinoma tissues is significantly higher than that in adjacent normal tissues. p <0.05; Univariate analysis of survival using Kaplan-Meier survival analysis and log-rank statistical test showed that high expression of CROT protein in cancer tissue was significantly positively correlated with poor prognosis in nasopharyngeal carcinoma. p =0.0253; Multivariate Cox regression analysis further confirmed that CROT protein expression is a prognostic factor independent of other clinical variables. This indicates that high expression of CROT protein in this invention is associated with the occurrence and malignant growth behavior of nasopharyngeal carcinoma, and can be used as an indicator of poor prognosis of nasopharyngeal carcinoma, suggesting that CROT protein is a potential therapeutic target for nasopharyngeal carcinoma. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a graph showing the comparison of CROT expression in nasopharyngeal carcinoma and adjacent normal tissues according to the present invention. * indicates statistical significance. p <0.05.

[0027] Figure 2 This is a graph showing the correlation between CROT expression levels and the prognosis of nasopharyngeal carcinoma patients. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0030] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0031] Example 1

[0032] 1. Materials and Methods

[0033] 1.1 Source of tissue samples

[0034] The nasopharyngeal carcinoma patient samples were obtained from the tissue sample bank of Shanghai Chimchao Biotechnology Co., Ltd. The surgical period was from January 2008 to September 2013, and the final follow-up date was March 2019. All cases were pathologically confirmed as nasopharyngeal carcinoma and had not received any preoperative treatment. There were 81 males and 45 females. The age range was 18-76 years, with a mean age of 51 years. Of these 140 samples, 14 contained both cancerous tissue and matching adjacent tissue within 1.5 cm of the cancer, while the remaining 112 samples contained only cancerous tissue.

[0035] 1.2 Tissue chip fabrication

[0036] The tissue microarray was fabricated by Shanghai Xinchao Biotechnology Co., Ltd. All donor tissue blocks underwent routine pathological sectioning and HE staining, followed by secondary diagnosis by pathologists, who marked typical pathological sites on the HE sections. Using a tissue microarray fabrication instrument (Beecher Instruments, Inc.), 1.5mm diameter holes were punched in the recipient paraffin blocks (blank blocks). Then, according to the markings on the HE slides, target tissue cores were obtained from the corresponding positions on the donor paraffin blocks and placed into the array wells of the recipient paraffin blocks. This process was repeated to ultimately fabricate a 140-point array block (HNasN140Su01) matching nasopharyngeal carcinoma tissue and adjacent normal tissue. Serial sections (4-5µm thickness) were prepared using a microtome (Leica, Germany) and mounted onto imported glass slides treated to prevent detachment to fabricate the tissue microarray.

[0037] 1.3 Immunohistochemical experiments

[0038] Immunohistochemical experiments were performed using the EnVision two-step method.

[0039] Reagents and equipment:

[0040] Constant temperature drying oven, model number: PH-070A, product standard number: 140612457, manufacturer: Shanghai Yiheng Scientific Instruments Co., Ltd.

[0041] Fully Automated Immunohistochemical Staining System Specifications: Autostainer Link 48, Product Standard Number: YZB / USA2016-2012, Manufacturer: Dako North America, Inc.

[0042] Centrifuge, model number: 1-14, manufacturer: Saetorius.

[0043] Fully automated immunohistochemical pretreatment system, model: PT Link, product standard number: YZB / USA0528-2012, manufacturer: Dako North America, Inc.

[0044] ST5010 Fully Automatic Dyeing Instrument (Dewaxing), Model: LEICAST5010, Manufacturer: LEICA.

[0045] Immunohistochemistry kit, product name: EnVision™ FLEX+, Mouse, High pH, ​​(Link), brand: Dako, catalog number: K8002.

[0046] Hematoxylin, product number: SLBT4555, brand: Sigma Aldrich.

[0047] Antibody diluent, catalog number: S2022, brand: Dako. Aperio scanner, model: Aperio XT, manufacturer: LEICA.

[0048] Experimental procedure:

[0049] (1) Place the tissue chip in an oven, set the temperature to 63 degrees, and bake the wax for one hour.

[0050] (2) Tissue dewaxing: After the slides are baked, they are taken out of the oven and placed in a fully automatic staining machine for dewaxing; the dewaxing process is as follows:

[0051] Two cylinders for xylene, 15 minutes per cylinder, according to the instrument's set time.

[0052] Two cylinders of anhydrous ethanol, 7 minutes per cylinder, according to the instrument's set time.

[0053] 90% alcohol, 1 tank, 5 minutes, according to the instrument's set time.

[0054] 80% alcohol, 1 tank, 5 minutes, according to the instrument's set time.

[0055] 70% alcohol, 1 tank, 5 minutes, according to the instrument's set time.

[0056] (3) Antigen retrieval

[0057] Repair was performed using the DAKO fully automated immunohistochemical pretreatment system.

[0058] (4) Primary antibody incubation

[0059] Add primary antibody (rabbit-derived)

[0060] After repair, remove the slide and rinse it three times with PBST buffer, one minute each time. Take the primary antibody out of the refrigerator and centrifuge it at 7200 rpm for at least 30 seconds. Take out the primary antibody and dilute it with antibody diluent according to the dilution ratio. Add the primary antibody and incubate at room temperature for 30 minutes or overnight at 4 degrees Celsius.

[0061] (0) Secondary antibody and colorimetric assay

[0062] Add secondary antibiotics (goat)

[0063] Rinse the slides three times with PBST buffer, 1 minute each time. If the slides were incubated overnight at 4°C, allow them to warm to room temperature for at least 30 minutes before rinsing with PBST. Add biotin-labeled goat anti-rabbit protein (from Fuzhou Maixin UltraSensitive SP kit) for 10 minutes; after 10 minutes, rinse three times with PBST, 1 minute each time. Add streptomycin-peroxidase (from Fuzhou Maixin UltraSensitive SP kit) for 10 minutes; after 10 minutes, rinse three times with PBST, 1 minute each time.

[0064] (6) DAB color development

[0065] Take the DAB kit out of the refrigerator and prepare it by adding 1 drop of DAB diluent and 1 drop of DAB chromogen. Add the diluted DAB to the slide and observe the color intensity. The maximum color development time is 10 minutes. After that, rinse with tap water for 5 minutes.

[0066] (7) Hematoxylin counterstaining

[0067] Add SIGMA (Hascher's hematoxylin) to the slide for 1 minute. After that time, immerse the slide in 0.25% hydrochloric acid alcohol (400ml 70% alcohol + 1ml concentrated hydrochloric acid) for at least 2 seconds. Rinse with tap water for more than 2 minutes, air dry at room temperature, and then seal the slide.

[0068] Three high-power fields were randomly observed under an optical microscope. The number of positive cells in at least 3 × 100 cells was recorded, and the staining positivity rate was calculated as the percentage of positive cells out of the total number of cells. The total score was calculated by multiplying the "staining intensity score" and the "staining positivity rate score" to form a group: <12 points were classified as the low antibody expression group, and ≥12 points were classified as the high antibody expression group.

[0069] 1.4 Statistical Analysis

[0070] The expression of CROT protein in nasopharyngeal carcinoma and adjacent normal tissues was analyzed using the chi-square test. The correlation between CROT expression and clinical indicators in nasopharyngeal carcinoma patients was analyzed using the chi-square test. Correlation analysis of CROT protein with nasopharyngeal carcinoma prognosis: univariate survival analysis was performed using Kaplan-Meier survival analysis and log-rank statistical tests. Variables with statistical significance in the univariate analysis were included in a multivariate Cox proportional hazards regression model to assess the independent impact of each variable on overall survival (OS). p <0.05 is statistically significant.

[0071] 2 Experimental Results

[0072] 2.1 Expression analysis of CROT protein in nasopharyngeal carcinoma and adjacent normal tissues

[0073] Immunohistochemical results showed that CROT protein was expressed in the cytoplasm of nasopharyngeal carcinoma and adjacent normal tissues. Chi-square test analysis showed that CROT expression was significantly higher in nasopharyngeal carcinoma than in adjacent normal tissues. p <0.05), the analysis results are shown in […]. Figure 1 .

[0074] 2.2 Correlation between CROT expression and clinical indicators in nasopharyngeal carcinoma patients

[0075] Chi-square test analysis showed that CROT expression had no statistically significant relationship with clinical indicators such as age, sex, pathological grade, N stage, and TNM stage. P >0.05), see Table 1.

[0076] Table 1. Correlation between CROT expression and clinical indicators in nasopharyngeal carcinoma patients

[0077]

[0078] Note: " / " indicates that this item is not present.

[0079] Table 1 (continued)

[0080]

[0081] Note: " / " indicates that this item is not present.

[0082] 2.3 Correlation analysis of CROT protein expression and prognosis of nasopharyngeal carcinoma

[0083] The follow-up data for nasopharyngeal carcinoma patients are as follows: 29 patients died from nasopharyngeal carcinoma, with a median follow-up time of 59 months (range 43–86 months); 97 patients remained alive, with a median follow-up time of 92 months (range 68–125 months). Univariate analysis of survival using Kaplan-Meier survival analysis and log-rank statistical test showed that nasopharyngeal carcinoma patients with low CROT expression in their cancer tissue had longer overall survival. p =0.0253), and their 5-year survival rate was significantly higher than that of nasopharyngeal carcinoma patients with high CROT expression, such as Figure 2 As shown in Table 2, the univariate analysis results of survival time were consistent with the actual follow-up results, indicating that CROT can be used as an indicator of poor prognosis in nasopharyngeal carcinoma.

[0084] 2.4 Cox Multivariate Regression Analysis

[0085] Multivariate Cox regression analysis further confirmed that CROT protein expression is a prognostic factor independent of other clinical variables and can be used in conjunction with clinical indicators for patient prognostic assessment, as shown in Table 2.

[0086] Table 2 Univariate and Multivariate Analysis

[0087]

[0088] Note: " / " indicates that this item is not present.

[0089] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0090] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. The use of a CROT protein detection reagent in the preparation of products for predicting nasopharyngeal carcinoma risk, characterized in that, The amino acid sequence of the CROT protein is shown in SEQ ID NO.1; The detection reagent is a reagent for quantitatively detecting the expression level of the CROT protein; the detection reagent is a CROT protein antibody.

2. The use according to claim 1, characterized in that, The subjects whose prognostic risk of nasopharyngeal carcinoma were human.

3. The use according to claim 2, characterized in that, The sample being tested is the subject's tissue.

4. The use according to claim 3, characterized in that, The tissue in question is cancerous.

5. The use according to claim 1, characterized in that, The method for quantitatively detecting the expression level of the CROT protein is immunohistochemistry.

6. The use according to claim 5, characterized in that, The specific steps of the immunohistochemical experimental method are as follows: (1) Prepare tissue microarrays from the tissue to be tested, and then bake, dewax, and retrieve antigens from the tissue microarrays. (2) Rinse with PBS buffer and incubate with primary antibody; (3) Rinse with PBS buffer and incubate with secondary antibody; (4) After washing with PBS buffer, add DAB colorimetric solution, develop the color for 10 minutes, and then rinse with tap water. (5) Counterstain, mount, and examine under a microscope.

7. The use as described in claim 6, characterized in that, The primary antibody is a rabbit-derived antibody.

8. The use as described in claim 6, characterized in that, The secondary antibody is a biotin-labeled goat anti-rabbit antibody.

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