A method based on the phosphorylation of colon cancer-related factor SIRT2 and its application

Abstract

The present invention discloses a method based on the phosphorylation of SIRT2, a colon cancer-related factor, and its application, and relates to the field of biomedicine technology. The present invention confirms the relationship between the phosphorylation of SIRT2 Y191 and colorectal cancer through in vitro and in vivo experiments, and reveals related applications targeting SIRT2 Y191 phosphorylation. The applications specifically include: using phosphorylation antibodies specific to the SIRT2 Y191 site to detect SIRT2 Y191 phosphorylation in colon cancer cell exosomes as a biomarker for early diagnosis of colon cancer, and using SIRT2 Y191 phosphorylation blockers in the preparation of anti-colon cancer drugs. SIRT2 Y191 phosphorylation is expected to become a new biological marker for colon cancer screening, personalized treatment and prognosis monitoring, providing a new direction for the treatment of colon cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for phosphorylating a colon cancer-related factor SIRT2 and an application thereof. Background Art

[0002] Colorectal cancer (CRC) is a common digestive tract malignancy, with a morbidity and mortality rate that ranks among the highest among all cancers. Early symptoms of CRC are often subtle, and most patients are diagnosed in the advanced stages, missing the optimal window for treatment. Early detection and intervention, as a secondary prevention strategy for cancer, are among the most important measures to reduce mortality. Most CRCs develop from benign lesions, following a progression from normal epithelium to adenoma to adenocarcinoma. Progression from neoplasia to malignancy typically takes 5 to 10 years, providing a crucial window for early diagnosis and treatment of CRC. Therefore, in-depth research into the mechanisms of CRC occurrence and progression is crucial to identify valuable biomarkers that can inform CRC diagnosis and prevention.

[0003] Tumor exosomes are a crucial component of the tumor microenvironment. They are bilayer vesicles approximately 50-150 nm in diameter, released by mammalian cells into the extracellular space and body fluids. They contain a variety of proteins, lipids, and nucleic acids. Tumor-associated exosomes may participate in immunosuppression, antigen presentation, angiogenesis, cell migration, cell differentiation, cell death, and tumor invasion. Their exploration into early diagnosis, prognosis, and therapeutic potential has become a hot topic. A growing number of studies have demonstrated that various proteins in exosomes play a crucial role in early tumor diagnosis. For example, GPC1 expression is significantly elevated in circulating exosomes from patients with pancreatic ductal carcinoma and CRC. Tumor exosome-derived GPC1 could be used as an early detection tool for pancreatic cancer. Exosomes can be easily collected from body fluids, and their analysis is relatively straightforward. Therefore, protein analysis in exosomes holds the potential to serve as a novel biomarker for early colorectal cancer screening.

[0004] The histone deacetylase SIRT2, primarily present in the cytoplasm, deacetylates several important molecules, including p53, FOXO1, FOXO3a, α-tubulin, histone H4, and SMC1A. It has a wide range of physiological functions, particularly in regulating the cell cycle and tumorigenesis. Early studies have linked aberrant SIRT2 expression to various malignancies, suggesting its dual role as a tumor suppressor or promoter, a topic of debate. Our recent findings also demonstrate that SIRT2 inhibits mitotic catastrophe in colon cancer cells by deacetylation and phosphorylation of the cell cycle checkpoint and genomic stability maintenance factor SMC1A, thereby promoting the early development of colon cancer. These findings also reveal the anti-colon cancer therapeutic mechanism of SIRT2 inhibitors, further suggesting that SIRT2 may serve as a biomarker for monitoring therapeutic efficacy and predicting prognosis in colorectal cancer patients, as well as a target for molecular therapy. However, to date, most research has focused on identifying downstream target molecules of SIRT2. Whether post-translational modifications of the SIRT2 protein itself can be secreted into the extracellular space and exocytic pathways, as well as the biological role of SIRT2 in the colon cancer microenvironment and its diagnostic value in colon cancer, have not been studied. Currently, only a few studies have found that cyclin-dependent kinases (CDKs) can phosphorylate SIRT2, regulating cell mitosis and affecting cell migration and proliferation. Therefore, the development of drugs targeting the SIRT2 phosphorylation site Y191 for the treatment of colon cancers characterized by abnormal LCK activation is groundbreaking. Summary of the Invention

[0005] In response to the problems existing in the prior art, the present invention aims to provide a method for phosphorylating the colon cancer-related factor SIRT2 and its application. It also relates to the release of SIRT2 Y191 phosphorylated in tumor exosomes and its application in predicting and detecting early colon cancer.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0007] The present invention discloses an application of a reagent for detecting SIRT2 phosphorylation in colon cancer cells in the preparation of a product for diagnosing and screening colon cancer, characterized in that the phosphorylation is SIRT2 Y191 site-specific phosphorylation.

[0008] Furthermore, the SIRT2 Y191 phosphorylated protein is used as a biomarker to prepare products for diagnosing and screening the occurrence of colon cancer.

[0009] Furthermore, the product detects the expression level of SIRT2 Y191 phosphorylated protein in the sample.

[0010] Furthermore, the sample is tissue, cell or exosome.

[0011] The present invention also discloses a product for colon cancer prognosis assessment based on SIRT2 Y191 phosphorylation, which is characterized in that the prognosis of colon cancer patients is assessed by detecting the expression level of SIRT2 Y191 phosphorylated protein in the sample, wherein high expression of SIRT2 Y191 phosphorylation is associated with poor prognosis.

[0012] Furthermore, the sample is tissue, cell or extracellular vesicles.

[0013] The present invention also discloses an anti-colon cancer drug based on SIRT2 Y191 phosphorylation, characterized in that the drug contains a SIRT2 Y191 phosphorylation blocker, which can inhibit the phosphorylation of SIRT2 Y191, thereby inhibiting the proliferation of colon cancer cells.

[0014] The present invention also discloses an application of a SIRT2 phosphorylation blocker in the preparation of an anti-colon cancer drug, characterized in that the SIRT2 Y191 phosphorylation blocker is used to inhibit clinical colon cancer with abnormal LCK activation.

[0015] The present invention also discloses a method for inhibiting colon cancer cell proliferation based on SIRT2 Y191 phosphorylation, which is characterized in that the proliferation ability of colon cancer cells is reduced by inhibiting the phosphorylation of SIRT2 Y191. The method includes using an LCK kinase inhibitor or a SIRT2 Y191 phosphorylation blocker.

[0016] The present invention also discloses the use of a SIRT2 Y191 phosphorylation blocker in the preparation of a drug for inhibiting the proliferation and invasion ability of colon cancer cells.

[0017] Compared with the prior art, the present invention has the following beneficial effects.

[0018] The present study discovered that LCK activation in colon cancer cells phosphorylates SIRT2 at tyrosine 191 (Y191). Phosphorylated SIRT2 can be secreted into the extracellular space in the form of exosomes. SIRT2 Y191 phosphorylation promotes colon cancer cell proliferation, and colon cancer-derived exosomes stably express phosphorylated SIRT2 Y191. Therefore, this phosphorylation site (SIRT2 Y191) could be a potential target for drug development to treat malignancies characterized by abnormal LCK activation. Furthermore, the present study confirmed that SIRT2 Y191 phosphorylation correlates with colorectal cancer progression and poor prognosis. SIRT2 Y191 phosphorylation has the potential to become a novel biomarker for colorectal cancer screening, personalized treatment, and prognosis monitoring, providing a new direction for colorectal cancer treatment. Therefore, reagents for detecting SIRT2 Y191 phosphorylation levels could be used for early diagnosis or prognosis of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Identification of SIRT2 Y191 phosphorylation in exosomes derived from human colon cancer cells. Figure A shows the size of exosomes detected by Nano-tracker; Figure B shows the characteristic morphology of SIRT2 Y191 phosphorylated protein in exosomes using transmission electron microscopy; and Figure C shows the peaks of SIRT2 in MS / MS.

[0020] Figure 2 Western blotting detection results of SIRT2 Y191 phosphorylation expression in exosomes derived from human colon cancer cells.

[0021] Figure 3 Western blotting results of SIRT2Y191 phosphorylation expression in exosomes derived from normal human colon epithelial cells (NCM60) and colon cancer cells (SW620).

[0022] Figure 4 Figure 2 shows the proliferation of colon cancer cells expressing wild-type WT SIRT2 and phosphorylated Y191F SIRT2.

[0023] Figure 5 Figure 2. SIRT2 Y191 phosphorylation protein expression in a human colon cancer tissue microarray. A shows immunohistochemical staining of SIRT2 Y191 phosphorylation protein in colon cancer and adjacent adjacent tissues. Scale bar: 25 μm. B shows the statistical results of Figure A.

[0024] Figure 6 The relationship between SIRT2 Y191 phosphorylation protein expression level and clinical prognosis in human colon cancer tissue microarray.

[0025] Figure 7Figure 1 shows the inhibitory effect of LCK kinase inhibitors. A shows the effect of LCK kinase inhibitor PP2 on SIRT2 phosphorylation levels; B shows the effect of LCK kinase inhibitor PP2 on the proliferation of human colon cancer cells. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments, as all technologies implemented based on the present invention fall within the scope of the present invention.

[0027] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0028] Example 1 Detection of colon cancer driver factors inducing SIRT2 Y191 phosphorylation and exosome exudation.

[0029] 1. Figure 1 As shown in Figures AC, exosomes were extracted from the human colon cancer cell line SW620 using ultrahigh-speed centrifugation. Nanoparticle tracking analysis (NTA) and mass spectrometry were performed (Shanghai Zhongke New Life Science Co., Ltd.). Colloidal gold immunoelectron microscopy was then performed on mouse colon cancer cells MC38 and human colon cancer cells SW620. All of these experiments identified the presence of SIRT2 Y191 phosphorylated protein.

[0030] The specific rabbit polyclonal anti-SIRT2 Y191 phosphorylation antibody was custom-made by Abmart Biotechnology.

[0031] Ultra-high-speed centrifugation method to extract exosomes:

[0032] (1) Collect the cell culture medium, centrifuge at 2000 rpm for 5 minutes at room temperature, and discard the precipitate to remove cell debris;

[0033] (2) Centrifuge at 10,000 g for 30 minutes at 4°C, discard the pellet, and remove apoptotic bodies.

[0034] (3) Centrifuge at 100,000 g for 70 minutes at 4°C, discard the supernatant, and resuspend the pellet in chilled PBS;

[0035] (4) Centrifuge the resuspended pellet at 100,000 g for 70 minutes at 4°C and discard the supernatant. The pellet is the exosomes. Resuspend in PBS and freeze at -80°C or add the corresponding volume of 6× loading buffer and denature at high temperature for use in Western Blot experiments.

[0036] Exosome colloidal gold immunoelectron microscopy staining steps:

[0037] (1) Prepare materials: 30 μL exosome liquid, 0.1 g / L poly-lysine, 40 g / L paraformaldehyde, PBS, 50 mM glycine (PBS preparation), blocking solution: 10 g / L cold water fish gelatin, rabbit anti-SIRT2 Y191 primary antibody, colloidal gold-conjugated goat anti-rabbit secondary antibody, washing solution: 1 g / L cold water fish gelatin, 1% glutaraldehyde, sealing film, clean tweezers, and a small ring for scooping;

[0038] (2) After thawing the exosomes, add an equal volume of 40 g / L paraformaldehyde to fix the exosomes;

[0039] (3) Place two drops of 200 μL poly-lysine on the sealing film and place four copper meshes on the poly-lysine droplets for activation for 2 minutes. Place 10 μL exosomes on the sealing film, absorb the activated copper mesh and place it on the exosome droplets for adsorption for 10 minutes. Place the copper mesh after adsorption of the sample on 200 μL PBS and wash the fixative twice, 2 minutes each time.

[0040] (4) Transfer the copper mesh to a 200 μL droplet of 50 mM glycine and incubate three times for 2 minutes each to block the aldehyde groups;

[0041] (5) Transfer the copper mesh to 200 μL of blocking solution and block for 10 minutes;

[0042] (6) Dilute SIRT2 Y191 primary antibody in blocking solution at 1:50, transfer the copper grid to 10 μL of primary antibody and incubate for 30 minutes; the negative control is incubated with blocking solution only, and two copper grids are incubated with negative control and primary antibody respectively;

[0043] (7) Use cleaning solution to clean the copper mesh 6 times, 1 minute each time;

[0044] (8) Dilute colloidal gold secondary antibody with PBS at a ratio of 1:50, transfer the copper grid to 10 μL of secondary antibody and incubate for 30 minutes;

[0045] (9) Wash the secondary antibody 6 times with PBS, 1 minute each time;

[0046] (10) Transfer the copper mesh to 1% glutaraldehyde for 5 minutes to fix the marker and exosomes to prevent loss of the marker;

[0047] (11) Start the 4°C operation: Wash the copper mesh with water 6 times, 1 minute each time, to avoid the reaction between phosphate and uranium stain;

[0048] (12) Transfer the copper mesh to a 40 g / L uranyl acetate droplet for 10 minutes;

[0049] (13) Transfer the copper mesh to a 10 g / L methylcellulose droplet for 5 minutes and draw the wire with a small ring;

[0050] (14) Electron microscope inspection.

[0051] 2. To further explore the effect of colon cancer driver (hypoxia) on the exocytosis of SIRT2 Y191 phosphorylation, we stimulated SW620 cells with hypoxia (1% O2, 5% CO2) for 36 hours, extracted SW620 exosome samples by ultra-high speed centrifugation, and performed protein immunoblotting. The results are shown in Figure 2. Figure 2 As shown in the results, it was found that under hypoxic conditions, the intracellular SIRT2 Y191 phosphorylation protein level was significantly increased, and the SIRT2 Y191 phosphorylation in exosomes also showed an increasing trend.

[0052] Western blotting experiments:

[0053] (1) After the sample concentration was determined by the BCA quantitative method, the corresponding volume of 6× SDS-PAGE Loading Buffer was added at a ratio of 1:6, and the sample was incubated in a 100°C dry bath for 10 minutes, and then frozen in a -80°C refrigerator for subsequent experiments;

[0054] (2) Prepare stacking gel and separation gel of appropriate concentrations and add the denatured protein sample to the lane;

[0055] (3) Electrophoresis and protein transfer: Place the SDS-PAGE in the electrophoresis tank, fill the tank with running buffer, vortex the protein sample to mix, and add an appropriate volume to the pores of the stacking gel. Perform electrophoresis at a constant voltage of 80V, and adjust the constant voltage to 120V after the protein sample enters the separation gel. After the electrophoresis is completed, perform protein transfer. Place the PVDF membrane in methanol to activate it, remove the gel plate, and transfer the protein from the negative electrode to the positive electrode at a constant current of 200mA in the order of splint-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-splint. Select the transfer time and current size according to the molecular weight of the target protein;

[0056] (4) Blocking and antibody incubation: After the transfer is completed, remove the PVDF membrane and block it with 5% BSA or skim milk powder prepared in TBST at room temperature for 1 hour. Cut the PVDF membrane according to the molecular weight of the protein. Prepare the primary antibody with antibody diluent or TBST and incubate at 4°C overnight. The primary antibodies include: SIRT2 Y191 phosphorylation antibody (custom-made by Abmart), SIRT2 antibody (Abcam, USA, catalog number ab211033), Alix antibody (Proteintech, China, catalog number 12422-1-AP), CD9 antibody (Proteintech, China, catalog number 20597-1-AP), TSG101 antibody (Proteintech, China, catalog number 28283-1-AP), Calnexin antibody (Proteintech, China, catalog number 10427-2-AP);

[0057] (5) Wash three times with TBST, 10 minutes each time. Select the appropriate secondary antibody according to the requirements of the primary antibody and incubate at room temperature for 1 hour. Wash three times with TBST, 10 minutes each time;

[0058] (6) Exposure imaging: Use commercial chemiluminescent liquid for exposure imaging and save the original image.

[0059] Example 2 SIRT2 Y191 phosphorylation is highly expressed in exosomes derived from colon cancer cells.

[0060] We collected culture media from normal colon epithelial cells HCoEpiC and human colon cancer cells SW620, extracted exosomes by ultra-high-speed centrifugation, and performed immunoblotting. The results were as follows: Figure 3 As shown, SIRT2 Y191 phosphorylation is highly expressed in exosomes derived from human colon cancer cells.

[0061] Example 3 SIRT2 Y191 phosphorylation promotes colon cancer cell proliferation.

[0062] Human colon cancer cells HCT116 stably expressing SIRT2 WT (wild type) or SIRT2 Y191F (phosphorylated inactive type) were passaged in vitro to prepare cell suspensions and counted. Colon cancer cell suspensions were inoculated in 96-well plates, with approximately 100 μL of 3,000 cells per well. The same sample was repeated three times. The culture plates were placed in an incubator for culture (37°C, 5% CO2). One plate was taken out on each of the 1-3 days and 10 μL of CCK-8 solution was added to each well. The culture plates were placed in an incubator for incubation for 1-4 hours, and the absorbance (OD) at 450 nm was measured using an enzyme reader. The results are shown below. Figure 4 As shown, compared with wild-type SIRT2 WT, SIRT2 Y191F inactivated form significantly inhibited the proliferation of colon cancer cells.

[0063] Construction of point mutation plasmid:

[0064] (1) Primer design

[0065] Table 1 Primers related to constructing point mutation plasmids

[0066]

[0067] Primers ordered from Sangon were centrifuged at 13,000 rpm for 1 minute, dissolved in deionized water according to the instructions, and diluted tenfold to a working concentration of 10 μM;

[0068] (2) PCR amplification of target plasmid

[0069]

[0070] The reaction procedure is as follows:

[0071]

[0072] (2) Take a small amount of amplified product for agarose gel electrophoresis detection. If the target plasmid is amplified correctly, proceed to the next step of the experiment;

[0073] (3) Digest the amplified product to remove the methylated template plasmid. Take 50 μL of the amplified product, add 1 μL of DpnI digestion enzyme, and digest at 37°C for 1 hour.

[0074] (4) Recombination reaction to form a circularized plasmid;

[0075] Table 3 Digestion reaction system

[0076]

[0077] The above reaction system was reacted at 37°C for 30 minutes and then immediately cooled on ice;

[0078] (5) Transformation: 10 μL of the above product was transformed into 50 μL of DH5α;

[0079] (6) Pick a single clone, extract the plasmid, and send it for sequencing.

[0080] Example 4 Expression of SIRT2 Y191 phosphorylation in colon cancer patients.

[0081] We further used immunohistochemistry to incubate human colon cancer tissue chips with specific SIRT2 Y191 phosphorylation antibodies. Figure 5 A shows that SIRT2 Y191 phosphorylated protein is mainly distributed in the cell membrane and cytoplasm, and is expressed in brown. The expression intensity in human colon cancer tissue is higher than that in adjacent non-tumor tissue ( Figure 5 A). After quantification of samples in tissue microarrays, statistical analysis showed that the expression level of SIRT2 Y191 phosphorylated protein in 28 colon cancer tissues was significantly higher than that in 27 non-tumor tissues (P < 0.05, Figure 5 B).

[0082] 1. Clinical specimens: The human colon cancer specimen chip (HCol-Ade060CS-01) used in this invention was purchased from Shanghai Xinchao Biotechnology Co., Ltd.

[0083] 2. Immunohistochemistry

[0084] (1) Baking: Place the tissue slices in a 65°C oven for 1-2 hours to prevent the tissue from falling off;

[0085] (2) Dewaxing: xylene I and xylene II, dewaxing twice, each time for 10 minutes, to ensure that the paraffin can be completely dissolved;

[0086] (3) Gradient hydration: 100%, 95%, 85%, 75% alcohol, 5 minutes per tank. Take out and place in single distilled water, and gently shake on a shaker for 5 minutes;

[0087] (4) Fixation: shake with 4% paraformaldehyde at room temperature for 10 minutes, then wash with PBS for 5 minutes each time, three times;

[0088] (5) Repair: Repair according to the antibody repair method, usually with citric acid; citric acid configuration: 150mL ddH2O + 1.5mL 100× citric acid;

[0089] Before adding the slices, place the plastic box containing the citric acid in a pressure cooker and boil it (preheat). Then add the washed slices. Boil under high pressure for 2 minutes, then remove and allow to cool to room temperature.

[0090] (6) Wash with PBS three times, 5 minutes each time;

[0091] (7) Add solution A from the immunohistochemistry kit and incubate at room temperature for 10 minutes to inactivate endogenous peroxidase and eliminate infection caused by endogenous peroxidase;

[0092] (8) Wash with PBS three times, 5 minutes each time;

[0093] (9) Add the normal goat non-immune serum B solution provided by the kit and incubate at room temperature for 1 hour;

[0094] (10) Remove serum and add primary antibody (SIRT2 Y191, 1:2000) at 4°C overnight; primary antibody preparation: use 0.25% TritonX100;

[0095] (11) Wash with PBS four times, 5 minutes each time;

[0096] (12) Add biotin-labeled secondary antibody C dropwise and incubate at room temperature for 30 minutes;

[0097] (13) Wash with PBS four times, 5 minutes each time;

[0098] (14) Add streptavidin peroxidase D dropwise and incubate at room temperature for 20 minutes;

[0099] (15) Wash with PBS four times, 5 minutes each time;

[0100] (16) Add 1-2 drops of DAB colorimetric solution (keep away from light and prepare immediately before use) to each slide, observe the color development under a microscope and time the time; DAB preparation: 850 μL ddH2O + 50 μL A + 50 μL B + 50 μL C; Note: Do not develop the color for too long, and ensure that the color development of each slide is consistent to prevent the background from being too dark;

[0101] (17) Stain the nucleus with hematoxylin for 2-3 minutes, then rinse with tap water for 10 minutes to return to blue.

[0102] (18) Gradient dehydration: alcohol 75%, 85%, 95%, 100% 1, 100% 2, xylene I, xylene II, 5 minutes / tank;

[0103] (19) Seal the slides with neutral resin, taking care to avoid bubbles, air dry, and store in a slide box.

[0104] Example 5 Correlation between SIRT2 Y191 phosphorylation level and prognosis of colon cancer patients.

[0105] To determine the relationship between SIRT2 Y191 phosphorylation and the prognosis of colon cancer patients, we further divided 76 colon cancer tissues into SIRT2 Y191 phosphorylation high expression group and SIRT2 Y191 low expression group according to the median expression level of SIRT2 Y191 phosphorylation protein. The Kaplan-Meier method was used to analyze the survival prognosis of colon cancer patients. The analysis results showed that the overall survival rate of colon cancer patients in the SIRT2 Y191 phosphorylation high expression group was significantly shorter than that in the SIRT2 Y191 phosphorylation low expression group (P < 0.05, Figure 6 ).

[0106] The human colon cancer survival rate specimen chip (HColA180Su17) used in the present invention was purchased from Shanghai Xinchao Biotechnology Co., Ltd.

[0107] Example 6 LCK kinase inhibitors inhibit SIRT2 phosphorylation and colon cancer cell proliferation.

[0108] Human colon cancer SW620 cells were co-transfected with Flag-SIRT2 and Myc-LCK plasmids, and the cells were treated with LCK kinase inhibitor PP2 (20 μM / mL) for 8 hours. After the cells were collected, immunoprecipitation and Western blotting were used to detect the effect of inhibiting LCK activity on SIRT2 phosphorylation. Figure 7 A shows that PP2 significantly inhibited the phosphorylation level of SIRT2.

[0109] We prepared the human colon cancer cell SW620 into a cell suspension by passage in vitro and counted them. The colon cancer cell suspension was inoculated in a 96-well plate, with about 3000 cells in 100μL per well, and the same sample was repeated 3 times. The culture plate was placed in an incubator and cultured for 24 hours (37°C, 5% CO2). PP2 (20μM / mL) inhibitor was added to treat the cells. One plate was taken out on 1-3 days and 10μL CCK-8 solution was added to each well. The culture plate was placed in an incubator and incubated for 1-4 hours. The absorbance (OD) at 450nm was measured by an enzyme-labeled instrument. The results are as follows: Figure 7As shown in Figure B, compared with the control group, the PP2 inhibitor treatment group significantly inhibited the proliferation of colon cancer cells.

[0110] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting SIRT2 phosphorylation in colon cancer cells in the preparation of a product for diagnosing and screening colon cancer, characterized in that: The phosphorylation is SIRT2 Y191 site-specific phosphorylation.

2. The use according to claim 1, characterized in that The SIRT2 Y191 phosphorylated protein is used as a biomarker to prepare products for diagnosing and screening the occurrence of colon cancer.

3. The use according to claim 1, characterized in that The product detects the expression level of SIRT2 Y191 phosphorylated protein in the sample.

4. The use according to claim 3, characterized in that The sample is tissue, cell or exosome.

5. A product for colon cancer prognosis assessment based on SIRT2 Y191 phosphorylation, characterized in that: The prognosis of colon cancer patients was evaluated by detecting the expression level of SIRT2 Y191 phosphorylated protein in the samples, among which high expression of SIRT2 Y191 phosphorylation was associated with poor prognosis.

6. The product according to claim 5, characterized in that The sample is tissue, cell or exosome.

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