Application of SIRT6 K170 site acetylation modification as marker in colorectal cancer

Abstract

The invention belongs to the technical field of tumor markers, and particularly relates to application of SIRT6K170 site acetylation modification as a marker in colorectal cancer. The SIRT6K170 site acetylation modification is taken as a marker, the prognosis of the colon cancer can be evaluated by detecting the SIRT6K170 site acetylation modification, and the SIRT6K170 site is taken as a target spot to design a medicine, so that the proliferation and / or invasion of colon cancer cells can be inhibited, and the colon cancer can be treated. Results of the embodiment show that acetylation modification of the SIRT6K170 site can improve the stability of SIRT6 protein and significantly inhibit the proliferation and invasion ability of colon cancer cells, and the inhibition effect is further improved after the K170 site of SIRT6 is mutated into R.

Description

Technical Field

[0001] This invention belongs to the field of tumor marker technology, specifically relating to the application of SIRT6 K170 site acetylation modification as a marker in colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors, with a global incidence rate as high as 10.2%, making it the third most common cancer worldwide, and its incidence rate is showing an increasing trend globally. Although research into colorectal cancer has deepened, drugs have been continuously updated, and our understanding of its pathogenesis has improved, with emerging methods such as surgery and immunotherapy being proposed, leading to some success in the treatment of colorectal cancer, the five-year survival rate for early-stage patients has now increased to 90%. However, current diagnostic criteria, methods, and techniques for early-stage colorectal cancer are still immature and have considerable limitations. Furthermore, the early onset of colorectal cancer is often insidious, with obvious symptoms only appearing in later stages. Therefore, by the time patients receive treatment, the vast majority are already in the late stages of the disease, often with distant metastases, resulting in a still relatively poor overall survival rate.

[0003] The Silent Regulatory Protein (SIRT) family is a class of proteases involved in protein deacetylation and ADP-ribosyltransferase. Highly conserved evolutionarily, they are widely distributed from yeast to mammals. The Sirtuin family plays a crucial role in a range of physiological processes, including metabolic regulation and oxidative stress. Sirtuin 6 (SIRT6), a member of the mammalian Sirtuin family, possesses deacetylase, deacylase, and mono-ADP-ribosyltransferase activities. Primarily located in the cell nucleus, SIRT6 is a 355-amino acid NAD+-dependent deacetylase. This protein has a structurally conserved NAD+-binding domain called the Rossmann fold domain, and a less homologous zinc-binding domain within its catalytic core. Numerous studies have reported that SIRT6 participates in regulating various intracellular functions, playing a vital role in the regulation of DNA damage repair, aging, and cellular metabolism. Currently, there are no studies on SIRT6 and the occurrence and development of colorectal diseases. Clarifying the relationship between SIRT6 and the occurrence and development of colorectal diseases is of great significance for developing targeted drugs for the treatment of colorectal diseases and for finding specific biomarkers for the prognostic assessment of colorectal patients. Summary of the Invention

[0004] The purpose of this invention is to provide the application of SIRT6 K170 acetylation modification as a biomarker in colorectal cancer. Using SIRT6 K170 acetylation modification as a biomarker, the prognosis of colorectal cancer can be assessed by detecting SIRT6 K170 acetylation modification; and SIRT6 K170 site can be used as a target to inhibit the proliferation and / or invasion of colorectal cancer cells, thereby treating colorectal cancer; drugs targeting SIRT6 K170 site provide a basis for tumor treatment.

[0005] This invention provides the application of SIRT6 K170 site acetylation modification as a marker in one or more of the following:

[0006] (1) Screening and / or preparing products for the treatment of colon cancer;

[0007] (2) Screening and / or preparing products for colorectal cancer prognostic assessment;

[0008] (3) Screening and / or preparing products that improve the prognosis of colorectal cancer;

[0009] (4) Screening and / or preparing products that improve the stability of SIRT6 protein.

[0010] This invention also provides the use of reagents and / or methods for detecting acetylation modification at the SIRT6 K170 site in one or more of the following:

[0011] (1) Screening and / or preparing products for the treatment of colon cancer;

[0012] (2) Screening and / or preparing products for colorectal cancer prognostic assessment;

[0013] (3) Screening and / or preparing products that improve the prognosis of colorectal cancer;

[0014] (4) Screening and / or preparing products that improve the stability of SIRT6 protein.

[0015] Preferably, the method includes immunoprecipitation or mass spectrometry; the reagent includes SIRT6 K170 acetylated antibody.

[0016] Preferably, the SIRT6 K170 acetylated antibody is obtained by immunizing animals with the SIRT6 K170 polypeptide; the amino acid sequence of the SIRT6 K170 polypeptide is TVAKARGLRACRGELRDT, wherein K is acetylated.

[0017] Preferably, the animal includes a rabbit;

[0018] The number of immunizations is 3 to 5, and the dose for each immunization is 400 to 600 μg per animal.

[0019] This invention also provides the application of the SIRT6 K170 site as a target in the preparation of products for treating colon cancer.

[0020] The present invention also provides a protein for treating colon cancer, wherein the protein has a lysine mutation at position 170 to arginine compared to the wild-type SIRT6 protein.

[0021] This invention also provides the use of wild-type SIRT6 protein or the protein described in the above-described technical solutions in the preparation of products for treating colon cancer.

[0022] Preferably, the product for treating colon cancer includes a product that inhibits the proliferation and / or invasion of colon cancer cells; the colon cancer cells include human colon cancer HCT116 cells.

[0023] The present invention also provides a medicament for treating colon cancer, wherein the active ingredient of the medicament comprises one or more of the following: wild-type SIRT6 protein, the protein described in the above-described technical solution, a product overexpressing wild-type SIRT6 protein, and a product overexpressing the protein described in the above-described technical solution.

[0024] Beneficial effects:

[0025] This invention uses acetylation modification at the SIRT6 K170 site as a biomarker. By detecting this modification, the prognosis of colorectal cancer can be assessed. Furthermore, drugs targeting the SIRT6 K170 site can be designed to inhibit the proliferation and / or invasion of colorectal cancer cells, thus treating colorectal cancer. The results of these examples demonstrate that acetylation modification at the SIRT6 K170 site increases the stability of the SIRT6 protein and significantly inhibits the proliferation and invasion of colorectal cancer cells. The inhibitory effect is further enhanced after the SIRT6 K170 site is mutated to R.

[0026] Furthermore, this invention utilizes the peptide shown in SEQ ID NO.1 to immunize animals, and the resulting antibody can effectively recognize the acetylated SIRT6 K170 site. Proteins that are not acetylated at the K170 site will not be recognized. It has good specificity and good immunogenicity, with an antiserum titer of 1:243000. It can be used as a reagent to detect acetylation modification at the SIRT6 K170 site, enabling prognostic assessment of colorectal cancer, screening and / or preparation of products for treating colorectal cancer, and providing a basis for the treatment of colorectal cancer. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0028] Figure 1Figure 1 shows the detection results of SIRT6 acetylation modification after HDAC inhibitor treatment;

[0029] Figure 2 Figure showing the changes in SIRT6 protein after HDAC inhibitor treatment;

[0030] Figure 3 The figure shows the changes in SIRT6 protein stability after HDAC RNAi.

[0031] Figure 4 Figure 1 shows the SIRT6 ubiquitination detection results after HDAC inhibitor treatment;

[0032] Figure 5 This is a graph showing the mass spectrometry detection results;

[0033] Figure 6 Figure 1 shows the detection results of acetylation modification at different sites of SIRT6;

[0034] Figure 7 The image shows the experimental results for CCK8.

[0035] Figure 8 The image shows the results of the clone formation experiment;

[0036] Figure 9 This is a diagram showing the results of dot hybridization.

[0037] Figures 1-9 In the diagram, NC represents the untreated group, Ac-K represents lysine acetylation modification, and input represents the whole-cell NP40 lysate protein without antibody and bead treatment. Detailed Implementation

[0038] This invention provides the use of SIRT6 K170 site acetylation modification as a marker in one or more of the following: (1) screening and / or preparing products for the treatment of colorectal cancer; (2) screening and / or preparing products for the prognostic assessment of colorectal cancer; (3) screening and / or preparing products for improving the prognosis of colorectal cancer; (4) screening and / or preparing products for improving the stability of SIRT6 protein.

[0039] The present invention also provides the use of reagents and / or methods for detecting SIRT6 K170 site acetylation modification in one or more of the following: (1) screening and / or preparing products for treating colorectal cancer; (2) screening and / or preparing products for colorectal cancer prognostic assessment; (3) screening and / or preparing products for improving colorectal cancer prognosis; (4) screening and / or preparing products for improving SIRT6 protein stability.

[0040] In this invention, the method preferably includes immunoprecipitation or mass spectrometry; the reagent preferably includes an SIRT6K170 acetylated antibody. The product of this invention preferably includes a drug.

[0041] In this invention, the SIRT6 K170 acetylated antibody is preferably obtained by immunizing animals with the SIRT6 K170 peptide; the amino acid sequence of the SIRT6 K170 peptide is preferably TVAKARGLRACRGELRDT (SEQ ID NO.1), wherein K is acetylated. The animals used in this invention preferably include rabbits; the number of immunizations is preferably 3 to 5, and the dose for each immunization is preferably 400 to 600 μg / animal. The SIRT6 K170 peptide provided by this invention has good immunospecificity, and antibodies with similar specificity and immunogenicity can be obtained by immunizing different batches of animals with the SIRT6 K170 peptide. The results of the examples show that the obtained SIRT6 K170 acetylation antibody can effectively recognize the acetylated SIRT6 K170 site, while proteins that are not acetylated at the K170 site will not be recognized. It has good specificity and good immunogenicity, with an antiserum titer of 1:243000. It can be used as a reagent to detect acetylation modification at the SIRT6 K170 site, enabling prognostic assessment of colorectal cancer, screening and / or preparation of products for the treatment of colorectal cancer.

[0042] This invention also provides the application of the SIRT6 K170 site as a target in the preparation of products for treating colorectal cancer. This invention targets the SIRT6 K170 site, increasing the acetylation level of the SIRT6 K170 site, or mutating the SIRT6 K170 site to arginine, which can inhibit the proliferation and / or invasion of colorectal cancer cells, thus treating colorectal cancer. Acetylation modification of the SIRT6 K170 site can inhibit its ubiquitination modification, thereby increasing the stability of the SIRT6 protein and inhibiting the proliferation of colorectal cancer cells. While mutating the SIRT6 K170 site to arginine reduces acetylation modification at the SIRT6 K170 site, ubiquitination modification at this site is also completely blocked. Therefore, mutating the SIRT6 K170 site to arginine can inhibit the proliferation and invasion of colorectal cancer cells.

[0043] The present invention also provides a protein for treating colon cancer, wherein the protein has a lysine mutation at position 170 to arginine compared to the wild-type SIRT6 protein.

[0044] This invention also provides the use of wild-type SIRT6 protein or the protein described in the above-described technical solutions in the preparation of products for treating colon cancer. In this invention, the wild-type SIRT6 protein has the NCBI accession number Q8N6T7, and its preferred amino acid sequence is as shown in SEQ ID NO.2, specifically MSVNYAAGLSPYADKGKCGLPEIFDPPEELERKVWELARLVWQSSSVVFHTGAGISTASGIPDFRGPHGVWTMEERGLAPKFDTTFESARPTQTHMALVQLERVGLLRFLVSQNVDGLHVRSGFPRDKLAELHGNMFVEECAKCKTQYVRDTVVGTMGLKATGRLCTVAKARGLRACRGELRDTILDWEDSLPDRDLALADEASRNADLSITLGTSLQIRPSGNLPLATKRRGGRLVIVNLQPTKHDRHADLRIHGYVDEVMTRLMKHLGLEIPAWDGPRVLERALPPLPRPPTPKLEPKEESPTRINGSIPAGPKQEPCAQHNGSEPASPKRERPTSPAPHRPPKRVKKAKAVPS.

[0045] Wild-type SIRT6 protein inhibits the proliferation and invasion of colorectal cancer cells by competitively inhibiting ubiquitination through acetylation, thus treating colon cancer. Mutating lysine at position 170 of wild-type SIRT6 protein to arginine prevents ubiquitination at this site, thereby inhibiting the proliferation and invasion of colorectal cancer cells and treating colon cancer.

[0046] In this invention, the product for treating colon cancer preferably includes a product that inhibits the proliferation and / or invasion of colon cancer cells; the colon cancer cells preferably include human colon cancer HCT116 cells.

[0047] This invention also provides a medicament for treating colon cancer, the active ingredient of which includes one or more of the following: wild-type SIRT6 protein, the protein described in the above-described technical solution, a product overexpressing wild-type SIRT6 protein, and a product overexpressing the protein described in the above-described technical solution. Overexpression of wild-type SIRT6 in HCT116 cells inhibits cell growth and proliferation, and this inhibitory effect is more pronounced after a mutation to R at the K170 site.

[0048] To further illustrate the present invention, the application of SIRT6K170 site acetylation modification as a marker in colorectal cancer is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] Identification of SIRT6 acetylation modification sites

[0051] 1. Construct the following plasmid:

[0052] (1) Insert the SIRT6 encoding gene (accession number in NCBI: GeneID: 51548) into the multiple cloning site (MCS) region of the pcDNA3.1-flag plasmid to obtain the SIRT6 wild-type plasmid with the flag tag, namely Flag-SIRT6WT.

[0053] (2) The lysine (K) at position 145 of SIRT6 is mutated to arginine (R), and the corresponding coding gene is inserted into the multiple cloning site (MCS) region of the pcDNA3.1-flag plasmid to obtain the SIRT6 protein K145 KR mutant plasmid with the flag tag, namely Flag-SIRT6K145R.

[0054] (3) Mutate lysine (K) at position 170 of SIRT6 to arginine (R), and insert the corresponding coding gene into the multiple cloning site (MCS) region of pcDNA3.1-flag plasmid to obtain the SIRT6 protein K170 KR mutant plasmid with flag tag, namely Flag-SIRT6K170R.

[0055] (4) Mutate lysine (K) at position 145 of SIRT6 to arginine (R) and lysine (K) at position 170 to arginine (R), and insert the corresponding coding gene into the multiple cloning site (MCS) region of pcDNA3.1-flag plasmid to obtain the SIRT6 protein K170 KR mutant plasmid with flag tag, namely Flag-SIRT6K145+170R.

[0056] 2. SIRT6 itself undergoes acetylation modification. Therefore, human colon cancer (HCT116) cells were treated with the 5 μM HDAC2 inhibitor CAY10683 for 24 h. Cells were collected, and proteins were extracted using NP-40 lysis buffer. Immunoprecipitation (IP) was performed using a SIRT6-specific antibody to detect SIRT6 acetylation modification (Ace). The results showed that SIRT6 acetylation modification increased in HCT116 cells after treatment with CAY10683. Figure 1 It was also found that SIRT6 protein accumulated after CAY10683 treatment. Figure 2 ), and found that it works by affecting the stability of the SIRT6 protein. Figure 3Mechanistic studies have found that HDAC2 inhibitor treatment reduces the ubiquitination level of SIRT6 protein, leading to SIRT6 accumulation. Figure 4 ).

[0057] 3. To further determine the SIRT6 acetylation modification site, human colon cancer (HCT116) cells were treated with 5 μM HDAC2 inhibitor CAY10683 and then transfected with Flag-SIRT6WT obtained in step 1. After 24 h of transfection, cells were collected, and proteins were extracted using NP-40 lysis buffer. Mass spectrometry analysis was performed according to existing techniques (Chen, H., Li, Y., Li, H. et al. NBS1 lactylation is required for efficient DNA repair and chemotherapy resistance. Nature 631, 663–669 (2024). https: / / doi.org / 10.1038 / s41586-024-07620-9). The results showed that the K145 and K170 sites of SIRT6 are likely the acetylation sites of SIRT6, and these sites are highly evolutionarily conserved. Figure 5 ).

[0058] 4. To verify whether the two sites obtained in step 3 mass spectrometry are acetylation sites of SIRT6, human colon cancer (HCT116) cells were treated with 5 μM DAC2 inhibitor CAY10683 and then transfected with the SIRT6 plasmid from step 1. After 24 h of transfection, cells were collected, and proteins were extracted using NP-40 lysis buffer. Immunoprecipitation (IP) was performed using a specific antibody against flag. The results showed that the addition of CAY10683 increased the acetylation levels on SIRT6 in both wild-type and K145 mutant SIRT6 cells; however, after the K170 mutation, even with the addition of CAY10683, the acetylation level at this site did not increase further, indicating that the K170 site of SIRT6 is the acetylation modification site of this protein. Figure 6 Therefore, at the K170 site of SIRT6, acetylation and ubiquitination of SIRT6 compete with each other. The acetylation site at K170 of SIRT6 occupies the position, affecting the ubiquitination modification of SIRT6, thereby increasing the stability of SIRT6 protein.

[0059] Example 2

[0060] The effect of SIRT6 acetylation modification on colorectal cancer tumors

[0061] (1) Add 8 μg of the transfection plasmid to a 1.5 mL EP tube, then add 8 μL of liposomes. After incubating at room temperature for 5 min, add 500 μL of Opti-MEM serum-free medium, mix by pipetting, and incubate at room temperature for 15 min. Then add the mixture to a 10 cm dish containing HCT116 colon cancer cells. Change the medium once after 8 h. After transfection for 24 h, use trypsin to collect the cells into centrifuge tubes and mix thoroughly by pipetting. Count the cells under a microscope and seed them into 96-well plates at a density of 200 cells per well. Each treatment group is seeded into 3 replicate wells for CCK8 assay analysis. The transfection plasmid is an empty vector with a flag tag, flag-vector (denoted as Control), Flag-SIRT6WT (denoted as SIRT6OE) constructed in step 3 of Example 2, or Flag-SIRT6K170R (denoted as SIRT6 K170R) constructed in step 3 of Example 2.

[0062] After cells adhered to the 96-well plates, 10 μL of CCK-8 reagent was added to the culture medium, and the plates were incubated for 2 hours. The absorbance of the cells at 450 nm was measured. Thereafter, the absorbance at 450 nm was measured every 24 hours using the same method, and the results were obtained after three consecutive measurements. The results showed that overexpression of wild-type SIRT6 in HCT116 cells inhibited cell growth and proliferation, and this inhibitory effect was more pronounced after the K170 site was mutated to R. Figure 7 ).

[0063] (2) To further verify the conclusion of step (1), a colony formation experiment was performed. Specifically, 8 μg of plasmid for transfection was added to a 1.5 mL EP tube, followed by 8 μL of liposomes. After incubation at room temperature for 5 min, 500 μL of liposomes was added. Opti, mix thoroughly by pipetting, and after standing at room temperature for 15 min, add the mixture to a 10 cm dish containing colon cancer HCT116 cells. Change the culture medium after 8 h. After 24 h of transfection, use trypsin to collect the cells into centrifuge tubes and mix thoroughly by pipetting. Count the cells under a microscope and seed them into 6-well plates at a density of 300 cells per well. Add 2 ml of complete culture medium to each well of the 6-well plate, shake in a figure-eight motion to evenly disperse the cells in the 6-well plate, and incubate in a cell culture incubator. The transfection plasmids used are the empty vector flag-vector (denoted as NC) with a flag tag, Flag-SIRT6WT (denoted as SIRT6WT) constructed in step 3 of Example 2, or Flag-SIRT6K170R (denoted as SIRT6 KR) constructed in step 3 of Example 2.

[0064] After culturing for 10–14 days, the cells were stained with 0.1% crystal violet solution and incubated at room temperature for 30 minutes. The crystal violet solution was discarded, and residual staining was washed with PBS until the background was clear. Clonal counts were performed by photographing the cells. The results showed that overexpression of SIRT6 in HCT116 cells inhibited cell growth and proliferation, and mutation of SIRT6 at the K170 site to R further inhibited cell growth. Figure 8 ).

[0065] Example 3

[0066] Preparation of SIRT6 acetylated antibody

[0067] 1. Preparation of immune peptides

[0068] The protein sequences before and after the K170 site of SIRT6 protein were synthesized artificially to serve as a coating peptide; the amino acid sequence of the coating peptide is: TVAKARGLRACRGELRDT (SEQ ID NO.1), wherein K is acetylated.

[0069] 2. Preparation of post-immunization serum

[0070] The artificially synthesized coated peptide from step 1 was reacted with activated mcKLH (Pierce) at room temperature for 2 hours to obtain an antigen solution.

[0071] (1) First immunization: The antigen solution was emulsified with an equal volume of Freund's complete adjuvant at an immunization dose of 600 μg / rabbit, and then injected subcutaneously at multiple points (6 points) into different batches of experimental rabbits.

[0072] (2) Secondary immunization: Three weeks after the first immunization, the antigen solution was emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points (4 points) into the experimental rabbits after the first immunization, according to the immunization dose of 400 μg / rabbit.

[0073] (3) Three immunizations: Two weeks after the second immunization, the antigen solution was emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points (4 points) into the experimental rabbits after the second immunization, according to the immunization dose of 400 μg / rabbit.

[0074] (4) Four immunizations: One week after the three immunizations, the antigen solution was emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points (4 points) into the experimental rabbits after the three immunizations, according to the immunization dose of 400 μg / rabbit.

[0075] (5) Five immunizations: One week after the four immunizations, the antigen solution was emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points (4 points) into the experimental rabbits after the four immunizations, according to the immunization dose of 400 μg / rabbit.

[0076] (6) One week after five immunizations, serum was collected, which is the post-immunization serum.

[0077] 3. Indirect ELISA detection of antibody titer

[0078] (1) Add 2 μg / mL of the immunopeptide obtained in step 1 to the microplate, incubate overnight at 4°C, wash 3 times with PBST, block with 5% skim milk, block at 37°C for 2 hours, and wash again with PBST.

[0079] (2) Add the pre-immunization animal serum (negative) and post-immunization serum (positive) from step 2 to the ELISA plate according to the designed dilution concentrations (Table 1), incubate at 37°C for 1 h, wash three times with PBST, add HRP-labeled goat anti-rabbit IgG antibody diluted to an appropriate concentration to the ELISA plate, incubate at 37°C for 40 min, wash with PBST, add freshly prepared TMB substrate chromogenic working solution, develop for 10 min, stop the chromogenic process with 2M sulfuric acid, and detect the OD value at 450 nm. If the OD value is... 阳 / OD 阴 >2.1, and OD 阳 A value > 0.1 indicates a valid result. The test results are shown in Table 1.

[0080] Table 1. Results of serum titer (OD value) detected by indirect ELISA.

[0081]

[0082]

[0083] The blank control consists of reaction wells without any sample added.

[0084] As shown in Table 1, the antiserum titer of experimental animal JXR5501 was 1:24300, and the antiserum titer of experimental animal JXR5502 was also 1:24300. The synthetic polypeptide acetylated at the SIRT6 K170 site had a good immunogenicity.

[0085] 4. On the second day after the fifth immunization, whole blood was collected from the experimental rabbits for antigen affinity purification. Specifically, the antigen affinity column was equilibrated with Binding Buffer until the baseline was stable. The rabbit serum was loaded onto the column for acetylated antibody affinity and non-acetylated antibody absorption. The acetylated antibodies were collected. The purified antibodies were dialyzed against 0.01M PBS (pH 7.2) and stored in 0.01M PBS (pH 7.2). The dialyzed antibodies were concentrated, and the specific antibody concentration was determined using a protein quantification instrument. A preservation solution (PBS solution at pH 7.4 + 0.09% NaN3) was prepared, and the antibodies were stored in the preservation solution.

[0086] Example 4

[0087] Sensitivity of SIRT6 acetylated antibodies

[0088] The artificially synthesized coated peptide from step 1 of Example 3 was reacted with activated mcKLH (Pierce) at room temperature for 2 hours to obtain an artificial antigen solution.

[0089] Wild-type SIRT6 protein was reacted with activated mcKLH at room temperature for 2 hours to obtain a control antigen solution.

[0090] The artificial antigen solution and control antigen solution were diluted with 0.01M PBS (pH 7.4), and 2 μL of the diluent was taken. The diluted antigen solution was dried in a 37°C incubator for 30 min. After blocking with blocking buffer (TBST solution containing 5% skim milk) in a 37°C incubator for 10 min, the solution was washed 1 to 2 times with washing buffer and then blotted dry with filter paper. The K170 site acetylated antibody obtained from rabbit JXR5501 in Example 3 was diluted 1:500 with diluent (0.01M PBS, pH 7.4) and added to an incubator. The solution was incubated in a humidified chamber at 37°C for 30 min. The solution was washed again with washing buffer three times with shaking for 3 min each time, and then the remaining washing buffer was blotted dry. Goat anti-rabbit IgG serum diluted 1:10000 was added and incubated in a humidified chamber at room temperature for 10 min. After washing and blotting dry, the substrate colorimetric reaction was performed with chromogenic buffer for 10 min. The reaction was then stopped by washing with water and the results were observed. The presence of distinct colored spots indicates a positive result. This suggests that the acetylation reaction at the K170 site can be well recognized and detected by the antibody, while proteins that do not undergo acetylation at the K170 site are not recognized, indicating that the antibody has good specificity. Figure 9 ).

[0091] As can be seen from the above, acetylation modification of the SIRT6 K170 site can increase the stability of the SIRT6 protein and significantly inhibit the proliferation and invasion of colon cancer cells. The inhibitory effect is further enhanced after the SIRT6 K170 site is mutated to R.

[0092] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of SIRT6 K170 acetylation modification as a marker in one or more of the following: (1) Screening and / or preparing products for the treatment of colon cancer; (2) Screening and / or preparing products for colorectal cancer prognostic assessment; (3) Screening and / or preparing products that improve the prognosis of colorectal cancer; (4) Screening and / or preparing products that improve the stability of SIRT6 protein.

2. The use of reagents and / or methods for detecting acetylation modification at the SIRT6 K170 site in one or more of the following: (1) Screening and / or preparing products for the treatment of colon cancer; (2) Screening and / or preparing products for colorectal cancer prognostic assessment; (3) Screening and / or preparing products that improve the prognosis of colorectal cancer; (4) Screening and / or preparing products that improve the stability of SIRT6 protein.

3. The application according to claim 2, characterized in that, The method includes immunoprecipitation or mass spectrometry; the reagent includes SIRT6 K170 acetylated antibody.

4. The application according to claim 3, characterized in that, The SIRT6 K170 acetylated antibody was obtained by immunizing animals with the SIRT6 K170 peptide; the amino acid sequence of the SIRT6 K170 peptide is TVAKARGLRACRGELRDT, where K is acetylated.

5. The application according to claim 4, characterized in that, The animals include rabbits; The number of immunizations is 3 to 5, and the dose for each immunization is 400 to 600 μg per animal.

6. Application of SIRT6 K170 site as a target in the preparation of products for treating colorectal cancer.

7. A protein for treating colon cancer, characterized in that, Compared to the wild-type SIRT6 protein, this protein has a lysine mutation at position 170, replacing arginine.

8. Use of wild-type SIRT6 protein or the protein of claim 7 in the preparation of a product for treating colon cancer.

9. The application according to claim 6 or 8, characterized in that, The products for treating colon cancer include those that inhibit the proliferation and / or invasion of colon cancer cells; the colon cancer cells include human colon cancer HCT116 cells.

10. A drug for treating colon cancer, characterized in that, The active ingredient of the drug includes one or more of the following: wild-type SIRT6 protein, the protein of claim 7, a product overexpressing wild-type SIRT6 protein, and a product overexpressing the protein of claim 7.

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