Application of VAPA protein detection reagent in preparation of product for diagnosis and prognosis of colon cancer risk

Abstract

The invention relates to the technical field of medical diagnosis, in particular to application of a VAPA protein detection reagent in preparation of products for diagnosis and prognosis of colon cancer risks. The amino acid sequence of the VAPA protein is shown as SEQ ID NO.1, the diagnosis and prognosis subject is a human, and the detected sample is the tissue of the subject. The low expression of the VAPA protein is related to the occurrence and malignant growth behavior of the colon cancer, and compared with a healthy control, the low expression of the VAPA protein indicates that a subject suffers from the colon cancer or has the risk of suffering from the colon cancer; if the VAPA protein expression in the tissue sample of the colon cancer patient is reduced, judging that the colon cancer patient has poor prognosis; if the VAPA protein expression in the tissue sample of the colon cancer patient is increased, good prognosis is determined. The result shows that the VAPA protein is a potential colon cancer treatment target.

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 a product 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 have 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, 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 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 protein (Vesicle Associated Membrane Protein Associated Protein A), also known as VAMP-associated protein A, is a vesicle transport-related protein located between the endoplasmic reticulum and the Golgi apparatus. It belongs to the SNARE protein family and is involved in the membrane vesicle transport process. It is closely related to the occurrence and development of various neurodegenerative diseases and tumors. For example, a study showed that based on the analysis of RNA-seq and other data from 118 normal people and SCLC patients, it was found that the expression level of circRNA-circVAPA from exons 2-4 of the VAPA gene in SCLC was higher than that 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, accelerating the progression of small cell lung cancer. Furthermore, circVAPA deficiency 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 played the role of a pro-oncogene in gastric cancer and colorectal cancer cells, and can be used 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 was 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 was 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 a product for diagnosing and prognosing colon cancer risk, so as to seek effective prognostic monitoring indicators and lay a 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-mentioned 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, catalog 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 detects the expression of VAPA protein in colorectal cancer tissue by immunohistochemistry, and the results show that the expression of VAPA protein in colon cancer tissue is significantly lower than that in adjacent tissue, p<0.001; the correlation between VAPA protein expression and clinical indicators of colon cancer patients is detected, and the results show that VAPA protein expression is closely related to N stage, TNM stage, and PDL1 group stroma, p<0.05; the univariate analysis of survival period using Kaplan-Meier survival analysis and log-rank statistical test shows that low expression of VAPA protein in cancer tissue is significantly positively correlated with poor prognosis of colon cancer, p=0.0041. It shows that the low expression of VAPA protein in the present invention is related to the occurrence and malignant growth behavior of colon cancer, and can be used as an indicator for early diagnosis of colon cancer and poor prognosis, and it is suggested that VAPA protein is a potential target for colon cancer treatment. 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

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

[0029] Figure 2 The ROC analysis result of predicting the occurrence of colon cancer 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] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[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 the specification of the present invention herein 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] In the existing technology, the diagnosis of colon cancer mainly relies on colonoscopy and abdominal CT, but most patients are already in the late stage of tumor when they are discovered, and have lost the best treatment opportunity. 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.

[0035] VAPA protein belongs to the SNARE protein family, participates in the membrane vesicle transport process, and 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, and it is necessary to explore the role of VAPA protein in colorectal cancer and its clinical significance.

[0036] Based on this, the present invention provides a use of a VAPA protein detection reagent in the preparation of a product for diagnosis and prognosis of colon cancer, and 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 univariate analysis of survival period using Kaplan-Meier survival analysis and log-rank statistical test showed that low expression of VAPA protein in cancer tissue was significantly positively correlated with poor prognosis of colon cancer, p=0.0041. It shows that the low expression of VAPA protein in the present invention is related to the occurrence and malignant growth behavior of colon cancer, and can be used as an indicator for early diagnosis of colon cancer and poor prognosis, and it 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 in conjunction with specific embodiments.

[0038] Example 1

[0039] 1. Materials and Methods

[0040] 1.1 Source of tissue samples

[0041] 101 samples of colon 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 be 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 patients with colorectal cancer are shown in Table 1 。

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

[0043]

[0044]

[0045] Note: “ / ” means no item.

[0046] 1.2 Tissue microarray fabrication

[0047] The tissue chip was made by Shanghai Xinchao Biotechnology Co., Ltd. All donor tissue wax blocks were routinely sectioned and stained with HE. A pathologist made a secondary diagnosis and marked the typical pathological sites on the HE sections. Using a tissue chip maker, the English name is Beecher Instruments. Inc., holes were punched on the recipient wax block, i.e., the blank wax block, with a hole diameter of 1.5 mm. 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 make a 180-point array block HCol-Ad e180Sur-06 that matched colon cancer tissue and adjacent tissue. A slicer was used, purchased from Leica, 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 chips.

[0048] 1.3 Immunohistochemistry experimental method

[0049] EnVision two-step method was used for immunohistochemistry experiments.

[0050] Reagents and Equipment:

[0051] Constant temperature drying oven, specification model: PH-070A, product standard number: 140612457, manufacturer: Shanghai Yiheng Scientific Instrument Co., Ltd. Fully automatic immunohistochemical staining system instrument specification model: Autostainer Link48, product standard number: YZB / USA2016-2012, manufacturer: Dako North America, Inc. Centrifuge, specification model: 1-14, manufacturer: saetorius. Fully automatic immunohistochemical pretreatment system instrument, specification model: PT Link, product standard number: YZB / USA0528-2012, manufacturer: Dako North America, Inc. ST5010 fully automatic staining instrument (dewaxing), specification model: LEICAST5010, manufacturer: LEICA. Immunohistochemical kit, product name: EnVision TM FLEX+, Mouse, HighpH, (Link), Brand: Dako, Product No.: K8002. Hematoxylin, Product No.: SLBT4555, Brand: Sigma Aldrich, Antibody Diluent, Product 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 for one hour.

[0054] (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:

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

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

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

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

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

[0060] (3) Antigen retrieval

[0061] The DAKO fully automated immunohistochemistry pretreatment system was used for instrument repair.

[0062] (4) Primary antibody incubation

[0063] Add primary antibody (rabbit source)

[0064] After repair, take out the slide and rinse it with PBST buffer three times, 1 minute each time; take out the primary antibody from the refrigerator and centrifuge it at 7200 rpm for no less than 30 seconds; take out 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 store in a refrigerator at 4 degrees overnight.

[0065] (5) Secondary antibody and color development

[0066] Add secondary antibody (goat)

[0067] Rinse the slides with PBST buffer 3 times, 1 minute each time; if the slides are left overnight at 4 degrees, before the experiment, the slides need to be warmed up at room temperature for more than 30 minutes and then rinsed with PBST; add biotin-labeled goat anti-rabbit, from Fuzhou Maixin UltraSensitive SP kit, for 10 minutes; after the time is up, rinse with PBST 3 times, 1 minute each time. Add streptavidin-peroxidase, from Fuzhou Maixin UltraSensitive SP kit, for 10 minutes; after the time is up, rinse with PBST 3 times, 1 minute each time.

[0068] (6) DAB color development

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

[0070] (7) Hematoxylin counterstaining

[0071] Add SIGMA Hastelloy's hematoxylin to the slide for 1 minute. After the time is up, immerse it in 0.25% hydrochloric acid alcohol (400ml70% alcohol + 1ml concentrated hydrochloric acid) for no less than 2 seconds, rinse with tap water for more than 2 minutes, dry it at room temperature and then seal the slide.

[0072] Randomly observe 3 high-power fields under an optical microscope, record the number of positive cells in no less than 3×100 cells, and calculate the staining positive rate of the number of positive cells to the total number of cells. The product of the "staining intensity score" and the "staining positive rate score" was used as the total score for grouping, with <12 points as the antibody low expression group and ≥12 points as the antibody high expression group.

[0073] 1.4 Statistical analysis

[0074] The expression of VAPA protein in colon cancer and adjacent tissues was analyzed by chi-square test. The correlation between VAPA expression and clinical indicators of colon cancer patients was analyzed by chi-square test. Correlation analysis of VAPA protein with prognosis of colon cancer: Kaplan-Meier survival analysis and log-rank statistical test were used for univariate analysis of survival. p<0.05 was statistically significant.

[0075] 2. Experimental results

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

[0077] The results of immunohistochemistry experiments 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. The analysis results are shown in 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, the results of chi-square test analysis showed that VAPA expression was closely related to N stage, TNM stage and PDL1group stroma, p < 0.05. In addition, there was no significant statistical relationship between VAPA expression and age, gender, pathological grade, tumor size, T stage, M stage, TNM stage, MLH1, MSH2, MSH6, MSH2, PMS2, and 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 poor prognosis of colon cancer.

[0087] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a detection reagent for VAPA protein in the preparation of a product for diagnosing and prognosing the risk of colon cancer, 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 diagnosis and prognosis of 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 amount 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) preparing a tissue chip from the tissue to be tested, waxing, dewaxing, and antigen repairing the tissue chip; (2) Wash with PBS buffer and incubate with primary antibody; (3) Wash 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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