Use of a VAPA protein detection reagent in the preparation of a product for diagnosing and prognosing colon cancer risk

Abstract

The present invention relates to the field of medical diagnostic technology, and more specifically to the use of a detection reagent for VAPA protein in the preparation of a product for diagnosing and prognosing the risk of colon cancer. The amino acid sequence of the VAPA protein is shown in SEQ ID NO.1, the subject of diagnosis and prognosis is a human, and the sample for detection is the tissue of the subject. The low expression of the VAPA protein described in the present invention is associated with the occurrence and malignant growth behavior of colon cancer. Compared with healthy controls, low expression of the VAPA protein indicates that the subject has colon cancer or is at risk of developing colon cancer; the expression of VAPA protein in tissue samples of colon cancer patients is reduced, which is judged as a poor prognosis; the expression of VAPA protein in tissue samples of colon cancer patients is increased, which is judged as a good prognosis. The results suggest that VAPA protein is a potential target for the treatment of colon cancer.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis technology, and more specifically to use of a detection reagent for VAPA protein in preparing products for diagnosing and prognosing colon cancer risk. Background Art

[0002] Colorectal cancer, CRC for short, is one of the most malignant tumors, and its morbidity and mortality rates have shown a clear increasing trend. Most CRCs grow slowly and are caused by precursor lesions such as adenomatous polyps or sessile serrated lesions. This slow growth, of about 10 years, makes it possible to screen for early cancers and precursor lesions. Therefore, screening and early standardized diagnosis and treatment are the key to improving the prognosis of CRC patients and reducing the burden of CRC disease. At present, the diagnosis of colon cancer mainly relies on methods such as colonoscopy and abdominal CT, but most patients are already in the late stage of the tumor when they are discovered, and have lost the best time for treatment. Therefore, finding specific key molecules and therapeutic targets is of great significance for the early diagnosis, prognosis, new drug development and clinical treatment of colon cancer.

[0003] VAPA (Vesicle Associated Membrane Protein Associated Protein A), also known as VAMP-associated protein A, is a vesicle transport-associated protein located between the endoplasmic reticulum and the Golgi apparatus. It belongs to the SNARE protein family and is involved in vesicle transport processes. It is closely related to the occurrence and development of various neurodegenerative diseases and tumors. For example, a study showed that based on RNA-seq data analysis of 118 normal subjects and SCLC patients, it was found that the expression level of circRNA-circVAPA from exons 2-4 of the VAPA gene was higher in SCLC than in the control group. At the molecular mechanism level, circVAPA activates the phosphoinositide 3-kinase PI3K / protein kinase BAKT signaling pathway by regulating the miR-377-3p and miR-494-3p / insulin-like growth factor 1 receptor IGF1R axis, thereby accelerating the progression of small cell lung cancer. Furthermore, circVAPA loss significantly enhanced the inhibitory effect of the IGF1R kinase inhibitor BMS-536924 in cells and xenograft mouse models. At the same time, compared with normal tissues, circ-VAPA expression was significantly upregulated in tumor tissues of gastric cancer and colorectal cancer patients. In vitro experiments showed that circ-VAPA plays the role of a pro-oncogene in gastric cancer and colorectal cancer cells, and can serve as a new potential diagnostic biomarker and therapeutic target for gastric cancer and colorectal cancer. In addition, there are literature reports that compared with healthy people and liver cancer patients without bone metastasis, the VAPA level in the serum of liver cancer patients with bone metastasis is significantly increased; and compared with normal liver tissue and liver cancer tissue without bone metastasis, the VAPA expression level in liver cancer tissue with bone metastasis is significantly increased. However, the role of VAPA in colorectal cancer is still unclear, and it is necessary to develop the role of VAPA protein in the occurrence of colorectal cancer and its clinical significance. Summary of the Invention

[0004] To solve the above problems, the present invention provides the use of a detection reagent for VAPA protein in the preparation of products for diagnosing and prognosing colon cancer risk, so as to seek effective prognostic monitoring indicators and lay the foundation for improving the prognosis of colon cancer patients.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Use of a detection reagent for VAPA protein in preparing a product for diagnosing and prognosing colon cancer risk, wherein the amino acid sequence of the VAPA protein is shown in SEQ ID NO.1.

[0007] SEQ ID NO.1:

[0008] masasgamak heqilvldpp tdlkfkgpft dvvttnlklr npsdrkvcfk vkttaprrycvrpnsgiidp gstvtvsvml qpfdydpnek skhkfmvqti fappntsdme avwkeakpde lmdsklrcvfempnendkln dmepskavpl naskqdgpmp kphsvslndt etrklmeeck rlqgemmkls eenrhlrdeglrlrkvahsd kpgststasfrdnvtsplps llvviaaifi gfflgkfil.

[0009] Preferably, the NCBI accession number of the VAPA protein is AAH02992.

[0010] Furthermore, in the above use, the subject for diagnosis and prognosis of colon cancer risk is human.

[0011] In the above use, as a preferred embodiment, the sample to be detected is a tissue of a subject.

[0012] Preferably, the tissue is cancer tissue.

[0013] In the above use, the detection reagent is a detection reagent for quantitatively detecting the expression amount of the VAPA protein.

[0014] Preferably, the detection reagent is a VAPA protein antibody.

[0015] Preferably, the VAPA protein antibody is purchased from Proteintech, product number 15275-1-AP.

[0016] Preferably, the method for quantitatively detecting the expression level of the VAPA protein is immunohistochemistry.

[0017] Preferably, the immunohistochemistry experimental method comprises the following specific steps:

[0018] (1) The tissue to be tested is made into a tissue chip, and the tissue chip is waxed, dewaxed, and antigen repaired.

[0019] (2) Rinse with PBS buffer and incubate with primary antibody.

[0020] (3) Rinse with PBS buffer and incubate with secondary antibody.

[0021] (4) After washing with PBS buffer, add DAB color developing solution and rinse with tap water after color development for 10 minutes.

[0022] (5) Restain, seal, and examine under a microscope.

[0023] Preferably, the primary antibody is a rabbit antibody.

[0024] Preferably, the secondary antibody is biotin-labeled goat anti-rabbit.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention detected the expression of VAPA protein in colorectal cancer tissues using immunohistochemistry. The results showed that the expression of VAPA protein in colon cancer tissues was significantly lower than that in adjacent tissues (p < 0.001). The correlation between VAPA protein expression and clinical indicators of colon cancer patients was tested, and the results showed that VAPA protein expression was closely correlated with N stage, TNM stage, and PDL1 group stroma (p < 0.05). Univariate analysis of survival using Kaplan-Meier survival analysis and log-rank statistical test showed that low VAPA protein expression in cancer tissues was significantly positively correlated with poor prognosis of colon cancer (p = 0.0041). This indicates that low VAPA protein expression in the present invention is associated with the occurrence and malignant growth behavior of colon cancer, can be used as an indicator for early diagnosis of colon cancer and poor prognosis, and suggests that VAPA protein is a potential therapeutic target for colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 Comparison of VAPA expression in colon cancer and adjacent adjacent tissues. ***Statistically significant (p<0.001);

[0029] Figure 2 This is the ROC analysis result of predicting colon cancer occurrence by VAPA protein based on the SVM prediction model in the present invention;

[0030] Figure 3 This is the correlation between the VAPA expression level of the present invention and the prognosis of colon cancer patients. DETAILED DESCRIPTION

[0031] To facilitate understanding of the present invention, the present invention will be described more fully below, along with preferred embodiments thereof. 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 more thorough and comprehensive understanding of the disclosure of the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0033] The invention of the present invention is as follows:

[0034] Currently, the diagnosis of colon cancer relies primarily on methods such as colonoscopy and abdominal CT scans. However, most patients are already in the late stages of the disease by the time it is discovered, missing the optimal window for treatment. Therefore, identifying specific key molecules and therapeutic targets is crucial for the early diagnosis, prognosis, new drug development, and clinical treatment of colon cancer.

[0035] VAPA protein belongs to the SNARE protein family and is involved in vesicle trafficking. It is closely related to the occurrence and development of various neurodegenerative diseases and tumors. However, the role of VAPA in colorectal cancer is still unclear. The role of VAPA protein in colorectal cancer and its clinical significance need to be explored.

[0036] Based on this, the present invention provides a VAPA protein detection reagent for the preparation of a product for the diagnosis and prognosis of colon cancer, the amino acid sequence of the VAPA protein is shown in SEQ ID NO.1. The expression of the VAPA protein in colorectal cancer tissue was detected by immunohistochemistry, and the results showed that the expression of VAPA protein in colon cancer tissue was significantly lower than that in adjacent tissues, p < 0.001; the correlation between VAPA protein expression and clinical indicators of colon cancer patients was detected, and the results showed that VAPA protein expression was closely related to N stage, TNM stage, and PDL1 group stroma, p < 0.05; the Kaplan-Meier survival analysis method and log-rank statistical test were used for survival univariate analysis, which showed that low expression of VAPA protein in cancer tissue was significantly positively correlated with poor prognosis of colon cancer, p = 0.0041. This shows that low expression of VAPA protein in the present invention is related to the occurrence and malignant growth behavior of colon cancer, can be used as an indicator for early diagnosis of colon cancer and poor prognosis, and suggests that VAPA protein is a potential target for colon cancer treatment.

[0037] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0038] Example 1

[0039] 1. Materials and Methods

[0040] 1.1 Source of tissue samples

[0041] 101 samples of colorectal cancer patients were collected from the tissue sample bank of Shanghai Xinchao Biotechnology Co., Ltd. The surgery time was from July 2006 to May 2007, and the final follow-up time was July 2015. All cases were pathologically confirmed to have colon cancer and did not receive any preoperative treatment. There were 56 males, 44 females, and 1 unknown case. The age ranged from 24 to 90 years old, with a median age of 57 years old. Among these 101 samples, 79 contained cancer tissue and matching adjacent cancer tissue 1.5 cm away from the cancer, and the remaining 22 contained only cancer tissue. The clinical pathological data of the 101 colorectal cancer patients are shown in Table 1 。

[0042] Table 1 Clinical and pathological data of 1101 patients with colorectal cancer

[0043]

[0044]

[0045] Note: “ / ” means no item.

[0046] 1.2 Tissue Microarray Fabrication

[0047] The production of tissue microarrays was completed by Shanghai Xinchao Biotechnology Co., Ltd. All donor tissue wax blocks were subjected to routine pathological sectioning and HE staining, and a pathologist made a secondary diagnosis and marked the typical pathological sites on the HE sections. Using a tissue microarray production instrument, the English name is Beecher Instruments. Inc., a hole with a diameter of 1.5 mm was punched on the recipient wax block, that is, the blank wax block. Then, according to the marking range on the HE film, the target tissue core was obtained at the corresponding position of the donor tissue wax block and placed in the array hole of the recipient wax block. The above steps were repeated to finally produce a 180-point array block HCol-Ad e180Sur-06 that matched colon cancer tissue and adjacent tissue. A microtome was purchased from Leica in Germany, and continuous sections were made with a thickness of 4um to 5um. The sections were mounted on imported slides that had been treated to prevent detachment to make tissue microarrays.

[0048] 1.3 Immunohistochemistry experimental method

[0049] Immunohistochemistry experiments were performed using the EnVision two-step method.

[0050] Reagents and equipment:

[0051] Constant temperature drying oven, specification model: PH-070A, product standard number: 140612457, manufacturer: Shanghai Yiheng Scientific Instrument Co., Ltd. Automatic immunohistochemistry staining system, specification model: Autostainer Link48, product standard number: YZB / USA2016-2012, manufacturer: Dako North America, Inc. Centrifuge, specification model: 1-14, manufacturer: Saetorius. Automatic immunohistochemistry pretreatment system, specification model: PT Link, product standard number: YZB / USA0528-2012, manufacturer: Dako North America, Inc. ST5010 automatic staining instrument (dewaxing), specification model: LEICAST5010, manufacturer: LEICA. Immunohistochemistry kit, product name: EnVision TM FLEX+ Mouse, HighpH (Link), Brand: Dako, Catalog No.: K8002. Hematoxylin, Catalog No.: SLBT4555, Brand: Sigma Aldrich, Antibody Diluent, Catalog No.: S2022, Brand: Dako. Aperio Scanner, Model: Aperio XT, Manufacturer: LEICA.

[0052] Experimental operation:

[0053] (1) Place the tissue chip in an oven, adjust the temperature to 63 degrees, and bake with wax for one hour.

[0054] (2) Tissue dewaxing: After the slides are baked, they are taken out of the oven and placed in an automatic staining machine for dewaxing. The dewaxing process is as follows:

[0055] Two cylinders of xylene, 15 minutes each cylinder, according to the time set by the instrument.

[0056] Two cylinders of anhydrous ethanol, 7 minutes per cylinder, according to the time set by the instrument.

[0057] 90% alcohol (1 cylinder) for 5 minutes, according to the instrument setting time.

[0058] 80% alcohol (1 cylinder) for 5 minutes, according to the instrument setting time.

[0059] 70% alcohol (1 cylinder) for 5 minutes, according to the instrument setting time.

[0060] (3) Antigen retrieval

[0061] The cells were repaired using the DAKO fully automated immunohistochemistry pretreatment system.

[0062] (4) Primary antibody incubation

[0063] Add primary antibody (rabbit source)

[0064] After repair, remove the slide and rinse it with PBST buffer three times, 1 minute each time; remove the primary antibody from the refrigerator and centrifuge it at 7200 rpm for at least 30 seconds; remove the primary antibody and dilute it with antibody diluent according to the dilution; add the primary antibody, incubate at room temperature for 30 minutes or in a refrigerator at 4 degrees overnight.

[0065] (5) Secondary antibody and color development

[0066] Add secondary antibody (goat)

[0067] Rinse the slides three times with PBST buffer for 1 minute each. If the slides were stored overnight at 4°C, warm them to room temperature for at least 30 minutes before rinsing with PBST. Add biotinylated goat anti-rabbit (from the Fuzhou Maixin UltraSensitive SP kit) for 10 minutes. After the time is up, rinse the slides three times with PBST for 1 minute each. Add streptavidin-peroxidase (from the Fuzhou Maixin UltraSensitive SP kit) for 10 minutes. After the time is up, rinse the slides three times with PBST for 1 minute each.

[0068] (6) DAB color development

[0069] Take out the DAB test kit from the refrigerator and prepare it by adding 1 ml of DAB diluent and 1 drop of DAB chromogen. Add the diluted DAB to the slide and observe the color intensity. The color development time is up to 10 minutes. After that, rinse with tap water for 5 minutes.

[0070] (7) Hematoxylin counterstaining

[0071] Add SIGMA Hematoxylin to the slide for 1 minute. After the time is up, 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, dry at room temperature, and then seal the slide.

[0072] Randomly observe three high-power fields under an optical microscope, record the number of positive cells in at least 3 × 100 cells, and calculate the staining positivity rate (the percentage of positive cells to the total number of cells). Group the cells using the product of the "staining intensity score" and the "staining positivity rate score" as the total score: a score of <12 is considered the low antibody expression group, and a score of ≥12 is considered the high antibody expression group.

[0073] 1.4 Statistical analysis

[0074] The expression of VAPA protein in colon cancer and adjacent tissues was analyzed using the chi-square test. The correlation between VAPA expression and clinical indicators in colon cancer patients was analyzed using the chi-square test. The correlation between VAPA protein and colon cancer prognosis was analyzed using the Kaplan-Meier survival analysis and the log-rank test for univariate analysis of survival. A p < 0.05 was considered statistically significant.

[0075] 2. Experimental Results

[0076] 2.1 Analysis of VAPA protein expression in colon cancer and adjacent tissues

[0077] Immunohistochemistry results showed that VAPA protein was expressed in the cytoplasm of colon cancer and adjacent tissues. Chi-square test analysis showed that VAPA expression in colon cancer was significantly lower than that in adjacent tissues, p < 0.001. Figure 1 .

[0078] 2.2 Correlation between VAPA expression and clinical indicators in patients with colon cancer

[0079] We first used the MetaboAnalyst 5.0 online tool and the SVM prediction model to evaluate the AUC value and accuracy of VAPA protein for predicting colon cancer. The results showed that its AUC value was 0.878 and its accuracy was as high as 82.4%. Figure 2 shown.

[0080] Furthermore, chi-square test analysis showed that VAPA expression was closely associated with N stage, TNM stage, and PDL1 group stroma (P < 0.05). Furthermore, there was no significant statistical correlation between VAPA expression and age, sex, pathological grade, tumor size, T stage, M stage, TNM stage, MLH1, MSH2, MSH6, MSH2, PMS2, or PDL-1 (P > 0.05, see Table 2).

[0081] Table 2 Correlation between VAPA expression and clinical indicators in patients with colon cancer

[0082]

[0083]

[0084] Note: “ / ” means no item.

[0085] 2.3 Correlation analysis between VAPA protein expression and prognosis of colon cancer

[0086] The follow-up of colon cancer patients is as follows: the surgery time was from July 2006 to May 2007, and the final follow-up time was July 2015. During the follow-up period, 60 patients died of colon cancer, with a median follow-up time of 28 months (1 to 102 months); 41 patients are still alive, with a median follow-up time of 102 months (98 to 108 months). The univariate analysis of survival using the Kaplan-Meier survival analysis method and the log-rank statistical test showed that colon cancer patients with high VAPA expression in cancer tissue had a longer overall survival, p = 0.0041, and their 5-year survival rate was significantly higher than that of colon cancer patients with low VAPA expression. The results are as follows Figure 3 The results of univariate analysis of survival were consistent with the actual follow-up results, indicating that VAPA can be used as an indicator for judging poor prognosis of colon cancer.

[0087] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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.

[0088] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Use of a VAPA protein detection reagent in the preparation of a product for prognosing colon cancer risk, characterized in that: The amino acid sequence of the VAPA protein is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The subject for prognosticating colon cancer risk is a human.

3. The use according to claim 2, characterized in that The sample to be tested is a tissue of a subject.

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

5. The use according to claim 1, characterized in that The detection reagent is a detection reagent for quantitatively detecting the expression amount of the VAPA protein.

6. The use according to claim 5, characterized in that The detection reagent is VAPA protein antibody.

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

8. The use according to claim 7, characterized in that The specific steps of the immunohistochemistry experimental method are as follows: (1) The tissue to be tested is made into a tissue chip, and the tissue chip is waxed, dewaxed, and antigen repaired; (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 and rinse with tap water after color development for 10 minutes; (5) Restain, seal, and examine under a microscope.

9. The use according to claim 8, characterized in that The primary antibody is a rabbit antibody.

10. The use according to claim 8, characterized in that The secondary antibody is biotin-labeled goat anti-rabbit.

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