Phosphorylation based on colon cancer related factor SIRT2 and application thereof

Abstract

The invention discloses phosphorylation based on a colon cancer related factor SIRT2 and application of phosphorylation, and relates to the technical field of biological medicines. The relation between SIRT2 Y191 phosphorylation and colorectal cancer is confirmed through in-vivo and in-vitro experiments, related application with SIRT2 Y191 phosphorylation as a target spot is disclosed, and the application specifically includes the steps that the SIRT2 Y191 site specific phosphorylation antibody is used for detecting SIRT2 Y191 phosphorylation in colorectal cancer cell exosomes and serves as a biomarker for early diagnosis of colorectal cancer, and the application of the SIRT2 Y191 site specific phosphorylation antibody to the colorectal cancer cell exosomes is developed. The invention also discloses an application of the phosphorylation blocker of SIRT2 Y191 in preparation of anti-colon cancer drugs. SIRT2 Y191 phosphorylation is expected to become a novel biological marker for colon cancer screening, individualized treatment and prognosis monitoring, and a new direction is provided for colon cancer treatment.

Description

Technical Field

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

[0002] Colorectal cancer (CRC) is a common malignant tumor of the digestive tract, and its incidence and mortality rate are among the highest among all types of cancer. The early symptoms of CRC are not obvious, and most patients are already in the middle and late stages when they are discovered, thus losing the best time for treatment. Early detection and intervention as a secondary prevention strategy for tumors is one of the most important measures to reduce mortality. Most CRCs develop from benign lesions and follow the development sequence of normal epithelium-adenoma-adenocarcinoma. It often takes 5 to 10 years for neoplasia to progress to malignant tumors, which provides an important screening time window for early diagnosis and treatment of CRC. Therefore, in-depth research on the occurrence and development mechanism of CRC is needed to find valuable biomarkers from it, so as to provide a reference for the diagnosis and prevention of CRC.

[0003] Tumor exosomes are an important part of the tumor microenvironment. They are double-layer vesicles with a diameter of about 50-150nm. They are released by mammalian cells into the extracellular and body fluids, and contain a variety of proteins, lipids, nucleic acids and other components. Tumor-associated exosomes can participate in immunosuppression, antigen presentation, angiogenesis, cell migration, cell differentiation, cell death, tumor invasion and other aspects. In addition, the exploration of early diagnosis, prognosis and treatment of tumors has become a research hotspot. More and more studies have shown that various proteins in exosomes play an important role in the early diagnosis of tumors. For example, in the circulating exosomes of patients with pancreatic ductal carcinoma and CRC, the expression level of GPC1 is significantly increased, and tumor exosome-derived GPC1 can be used as an early detection tool for pancreatic cancer. Exosomes can be easily collected from body fluids, and their analysis is relatively simple, so protein analysis in exosomes has the potential to become a new biomarker for early screening of colon cancer.

[0004] Histone deacetylase SIRT2 is mainly present in the cytoplasm and can deacetylate multiple important molecules such as p53, FOXO1, FOXO3a, α-tubulin, histone H4, SMC1A, etc. It has a wide range of physiological functions, especially in regulating the cell cycle and tumor formation. Early studies found that abnormal expression of SIRT2 is associated with a variety of malignant tumors, playing a dual role as a tumor suppressor or promoter, and has become a focus of debate. Our recent research results also show that SIRT2 inhibits mitotic catastrophe in colon cancer cells by deacetylation of cell cycle checkpoints and genome stability maintenance factor SMC1A and regulating its phosphorylation, thereby promoting the early occurrence of colon cancer. At the same time, it reveals the anti-colon cancer treatment mechanism of SIRT2 inhibitors, further indicating that SIRT2 may become a biomarker for efficacy monitoring and prognosis prediction in patients with colorectal cancer and a target for molecular therapy. However, so far, most of the research has focused on finding the downstream target molecules of SIRT2, while whether the post-translational modification of SIRT2 protein itself can be secreted into the extracellular 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 small number of studies have found that cyclin-dependent kinases (CDKs) can phosphorylate SIRT2, regulate the process of cell mitosis, and affect cell migration and proliferation. Therefore, it is of pioneering significance to study the development of drugs targeting the SIRT2 phosphorylation site Y191 for the treatment of colon cancers with abnormal LCK activation. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method based on the phosphorylation of SIRT2, a colon cancer-related factor, and its application, and also to release SIRT2 Y191 phosphorylation in the form of tumor exosomes and its application in predicting and discovering 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 preparing a product for diagnosing and screening colon cancer, wherein 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 comprises a SIRT2 Y191 phosphorylation blocker, and the blocker 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 the proliferation of colon cancer cells based on SIRT2 Y191 phosphorylation, characterized in that the proliferation ability of colon cancer cells is reduced by inhibiting the phosphorylation of SIRT2 Y191, and the method comprises using an LCK kinase inhibitor or a SIRT2 Y191 phosphorylation blocker.

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

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0018] The present invention has found that after LCK activation in colon cancer cells, SIRT2 protein tyrosine 191 (Y191) is phosphorylated, and phosphorylated SIRT2 can be secreted outside the cell in the form of exosomes. SIRT2 Y191 phosphorylation promotes the proliferation of colon cancer cells, and colon cancer-derived exosomes can stably express SIRT2 Y191 phosphorylation. Therefore, drugs can be developed targeting this phosphorylation site (SIRT2 Y191) for the treatment of malignant tumors such as abnormal LCK activation in clinical practice. In addition, the present invention also confirms that the progression and poor prognosis of colorectal cancer are related to SIRT2Y191 phosphorylation, and SIRT2 Y191 phosphorylation is expected to become a new biological marker for colon cancer screening, individualized treatment and prognosis monitoring, providing a new direction for the treatment of colon cancer. Therefore, reagents for detecting the phosphorylation level of SIRT2 Y191 can 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. A is a Nano-tracker detection image of the exosome particle size; B is an electron transmission microscopy image of SIRT2 Y191 phosphorylated protein with characteristic morphology of exosomes; C is a secondary mass spectrometry peak image of SIRT2.

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

[0021] Figure 3 Western blotting detection 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 The expression of SIRT2 Y191 phosphorylated protein in human colon cancer tissue microarray. A is the immunohistochemical staining result of SIRT2 Y191 phosphorylated protein in colon cancer and adjacent tissues, the scale is 25μm. B is the statistical result 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 7The detection of the inhibitory effect of LCK kinase inhibitors. A is the detection of SIRT2 phosphorylation level by LCK kinase inhibitor PP2; B is the detection of human colon cancer cell proliferation by LCK kinase inhibitor PP2. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to 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 AC, the exosome samples of human colon cancer cell line SW620 were extracted by ultra-high speed centrifugation. Nanoparticle Tracking Analysis (NTA) and mass spectrometry detection (Shanghai Zhongke New Life Co., Ltd.) were performed, and then colloidal gold immunoelectron microscopy staining analysis was performed on mouse colon cancer cells MC38 and human colon cancer cells SW620. The above experiments all 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: (1) Collect the cell culture medium, centrifuge at 2000 rpm for 5 minutes at room temperature, and discard the precipitate to remove cell debris; (2) Centrifuge at 10,000 g for 30 min at 4°C, discard the precipitate, and remove apoptotic bodies. (3) Centrifuge at 100,000 g for 70 min at 4°C, discard the supernatant, and resuspend the pellet in chilled PBS; (4) Centrifuge the resuspended precipitate at 100,000 g for 70 minutes at 4°C and discard the supernatant. The precipitate is the exosomes. Resuspend it in PBS and freeze it at -80°C or add the corresponding volume of 6× loading buffer and denature it at high temperature for Western Blot experiments.

[0032] Exosome colloidal gold immunoelectron microscopy staining steps: (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, cleaning solution: 1 g / L cold water fish gelatin, 1% glutaraldehyde, sealing film, clean tweezers, and a small ring for scooping slices; (2) After thawing the exosomes, add an equal volume of 40 g / L paraformaldehyde to fix the exosomes; (3) Drop 2 drops of 200 μL poly-lysine on the sealing film, and place 4 copper meshes on the poly-lysine droplets for activation for 2 minutes. Drop 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 to wash the fixative twice, 2 minutes each time; (4) Transfer the copper mesh to a 200 μL 50 mM glycine droplet and incubate three times to block the aldehyde group, each time for 2 minutes; (5) Transfer the copper mesh to 200 μL of blocking solution and block for 10 minutes; (6) Dilute SIRT2 Y191 primary antibody with blocking solution at 1:50, transfer the copper grid to 10 μL primary antibody and incubate for 30 minutes; the negative control is incubated with blocking solution only, and the negative control and primary antibody are incubated with 2 copper grids each; (7) Use cleaning solution to clean the copper mesh 6 times, 1 minute each time; (8) Dilute colloidal gold secondary antibody with PBS at 1:50, transfer the copper grid to 10 μL of secondary antibody and incubate for 30 minutes; (9) Wash the secondary antibody 6 times with PBS, 1 minute each time; (10) Transfer the copper grid to 1% glutaraldehyde for fixation for 5 minutes to cross-link the marker and exosomes to avoid loss of the marker; (11) Start operation at 4°C: Wash the copper mesh with water six times, 1 minute each time, to avoid reaction between phosphate and uranium stain; (12) Transfer the copper mesh to a 40 g / L uranyl acetate droplet for 10 minutes; (13) Transfer the copper mesh to a 10 g / L methylcellulose droplet for 5 minutes and draw the wire with a small ring; (14) Inspection using electron microscope.

[0033] 2. To further explore the effect of colon cancer driver (hypoxia) on SIRT2 Y191 phosphorylation exocytosis, 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 2As 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.

[0034] Western Blotting: (1) After the concentration of the sample was detected by the BCA quantitative method, the corresponding volume of 6× SDS-PAGE Loading Buffer was added at a ratio of 1:6, incubated in a 100°C dry bath for 10 minutes, and then frozen in a -80°C refrigerator for subsequent experiments; (2) Prepare stacking gel and separation gel of appropriate concentrations and add the denatured protein sample to the lane; (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 amount of volume to the concentrated gel channel. Perform electrophoresis at a constant voltage of 80V, and adjust to a constant voltage of 120V after the protein sample enters the separation gel. After the electrophoresis, perform protein transfer. Activate the PVDF membrane in methanol, remove the gel plate, and transfer 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-sponge. Select the transfer time and current size according to the molecular weight of the target protein; (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 overnight at 4°C. 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); (5) Wash three times with TBST, 10 minutes each time. Select appropriate secondary antibody according to the requirements of primary antibody, and incubate at room temperature for 1 hour. Wash three times with TBST, 10 minutes each time; (6) Exposure imaging: Use commercial chemiluminescent liquid for exposure imaging and save the original image.

[0035] Example 2 SIRT2 Y191 phosphorylation is highly expressed in exosomes derived from colon cancer cells.

[0036] We collected culture media from normal colon epithelial HCoEpiC and human colon cancer cell SW620, extracted exosomes by ultra-high speed centrifugation, and performed immunoblotting. The results are as follows: Figure 3 As shown, SIRT2 Y191 phosphorylation is highly expressed in exosomes derived from human colon cancer cells.

[0037] Example 3 SIRT2 Y191 phosphorylation promotes the proliferation of colon cancer cells.

[0038] 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 about 100 μL of 3,000 cells per well, and the same sample was repeated 3 times. The culture plates were placed in an incubator for culture (37°C, 5% CO2). Take out one plate each day for 1-3 days, add 10 μL of CCK-8 solution to each well, place the culture plates in an incubator for incubation for 1-4 hours, and use an enzyme reader to measure the absorbance (OD) at 450 nm. The results are as follows: Figure 4 As shown, compared with wild-type SIRT2 WT, SIRT2 Y191F inactivated form significantly inhibited the proliferation of colon cancer cells.

[0039] Construction of point mutation plasmid: (1) Primer design Table 1 Primers related to constructing point mutation plasmids Primers ordered from Bioengineering were centrifuged at 13,000 rpm for 1 min, dissolved in deionized water according to the instructions, and diluted tenfold to a working concentration of 10 μM; (2) PCR amplification of target plasmid The reaction procedure is as follows: (2) Take a small amount of the amplified product for agarose gel electrophoresis detection. If the target plasmid is amplified correctly, proceed to the next step of the experiment; (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; (4) Recombination reaction to form a circularized plasmid; Table 3 Digestion reaction system The above reaction system was reacted at 37°C for 30 minutes and then immediately cooled on ice; (5) Transformation: transform 10 μL of the above product into 50 μL DH5α; (6) Pick a single clone, extract the plasmid, and send it for sequencing.

[0040] Example 4 Expression of SIRT2 Y191 phosphorylation in colon cancer patients.

[0041] We further used immunohistochemistry to incubate human colon cancer tissue microarrays with specific SIRT2 Y191 phosphorylation antibodies. Figure 5 A shows that SIRT2 Y191 phosphorylated protein is mainly distributed in the cell membrane and cytoplasm, expressed in brown, and the expression intensity in human colon cancer tissue is higher than that in adjacent non-tumor tissue ( Figure 5 A). After quantifying the samples in the tissue chip, 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).

[0042] 1. Clinical specimens: The human colon cancer specimen chip (HCol-Ade060CS-01) used in the present invention was purchased from Shanghai Xinchao Biotechnology Co., Ltd.

[0043] 2. Immunohistochemistry (1) Baking: Place the tissue slices in a 65°C oven for 1-2 hours to prevent the tissue from falling off; (2) Dewaxing: xylene I and xylene II, dewaxing twice, each time for 10 minutes, to ensure that the paraffin can be completely dissolved; (3) Gradient hydration: 100%, 95%, 85%, 75% alcohol, 5 minutes in each tank. Take it out and put it in single distilled water, and shake it gently on a shaker for 5 minutes; (4) Fixation: shake 4% paraformaldehyde at room temperature for 10 minutes, then wash with PBS, 5 minutes each time, three times; (5) Repair: Repair according to the antibody repair method, usually with citric acid; citric acid configuration: 150mLddH2O+1.5mL 100× citric acid; Before putting the slices in, put the plastic box containing citric acid into the pressure cooker to boil (preheat). Then add the washed slices. Boil at high pressure for 2 minutes, take out and cool naturally to room temperature; (6) Wash three times with PBS, 5 minutes each time; (7) Add solution A in the immunohistochemistry kit and incubate at room temperature for 10 minutes to inactivate endogenous peroxidase and eliminate infection by endogenous peroxidase; (8) Wash with PBS three times, 5 minutes each time; (9) Add the normal goat non-immune serum B solution provided by the kit and incubate at room temperature for 1 hour; (10) Remove serum and add primary antibody (SIRT2 Y191, 1:2000) at 4°C overnight; preparation of primary antibody: use 0.25% TritonX100; (11) Wash with PBS four times, 5 minutes each time; (12) Add biotin-labeled secondary antibody C and incubate at room temperature for 30 minutes; (13) Wash with PBS four times, 5 minutes each time; (14) Add streptavidin peroxidase D and incubate at room temperature for 20 minutes; (15) Wash with PBS four times, 5 minutes each time; (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 reaction; DAB preparation: 850 μL ddH2O + 50 μL A + 50 μL B + 50 μL C; Note: Do not allow the color development to take too long and ensure that the color development of each slide is consistent to prevent the background from being too dark; (17) Stain the nucleus with hematoxylin for 2-3 minutes, and rinse with tap water to blue for 10 minutes; (18) Gradient dehydration: alcohol 75%, 85%, 95%, 100% 1, 100% 2, xylene I, xylene II, 5 min / tank; (19) Seal the slides with neutral resin, taking care not to create air bubbles. Air dry the slides and store them in a slide box.

[0044] Example 5 Correlation between SIRT2 Y191 phosphorylation level and prognosis of colon cancer patients.

[0045] To determine the relationship between SIRT2 Y191 phosphorylation and the prognosis of patients with colon cancer, we further divided 76 cases of 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 patients with colon cancer. The analysis results showed that the overall survival rate of patients with colon cancer 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 ).

[0046] The human colon cancer survival rate specimen chip (HColA180Su17) used in the present invention was purchased from Shanghai Xinchao Biotechnology Co., Ltd.

[0047] Example 6 LCK kinase inhibitors inhibit SIRT2 phosphorylation and colon cancer cell proliferation.

[0048] Flag-SIRT2 and Myc-LCK plasmids were co-transfected into human colon cancer SW620 cells, and the cells were treated with LCK kinase inhibitor PP2 (20 μM / mL) for 8 hours. After the cells were collected, immunoprecipitation experiments 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.

[0049] We prepared the cell suspension by in vitro passage of human colon cancer cells SW620 and counted them. The colon cancer cell suspension was inoculated in a 96-well plate, with about 100 μL of 3,000 cells per well, and the same sample was repeated 3 times. The culture plate was placed in an incubator for culture (37°C, 5% CO2) for 24 hours, and PP2 (20 μM / mL) inhibitor was added to treat the cells. One plate was taken out on 1-3 days, and 10 μL of CCK-8 solution was added to each well. The culture plate was placed in an incubator for incubation for 1-4 hours, and the absorbance value (OD) at 450 nm was measured by an enzyme reader. The results are shown as follows: Figure 7 As shown in B, the PP2 inhibitor treatment group significantly inhibited the proliferation of colon cancer cells compared with the control group.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope 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 a product 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 cell 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 use according to claim 5, characterized in that: The sample is tissue, cell or cell exosome.

7. An anti-colon cancer drug based on SIRT2 Y191 phosphorylation, characterized in that: The drug comprises a SIRT2Y191 phosphorylation inhibitor, which can inhibit the phosphorylation of SIRT2 Y191, thereby inhibiting the proliferation of colon cancer cells.

8. Use of a SIRT2 phosphorylation blocker in the preparation of an anti-colon cancer drug, characterized in that: The SIRT2Y191 phosphorylation inhibitor is used to inhibit clinical colon cancer with abnormal LCK activation.

9. A method for inhibiting colon cancer cell proliferation based on SIRT2 Y191 phosphorylation, characterized in that: The proliferation ability of colon cancer cells is reduced by inhibiting the phosphorylation of SIRT2 Y191. The method comprises using an LCK kinase inhibitor or a SIRT2 Y191 phosphorylation blocker.

10. Application of SIRT2 Y191 phosphorylation blocker in the preparation of drugs for inhibiting the proliferation and invasion ability of colon cancer cells.

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