Application of FH acetylation in diagnosis, staging, treatment and prognosis prediction of colorectal cancer

Abstract

The application discloses application of FH acetylation in diagnosis, staging, treatment and prognosis prediction of colorectal cancer (CRC). The application screens acetylated non-histone proteins and acetylation sites related to CRC in a systematic manner, and for the first time finds that FH K80 acetylation modification level regulates the malignant phenotype of CRC, the FH K80 acetylation modification level presents significant difference in CRC patients, and is related to prognosis, and a high acetylation state (FH K80Q) can inhibit the proliferation, migration and invasion of CRC cells. The application provides a new direction for diagnosis, prognosis prediction and effective treatment of CRC, and has a wide application prospect in clinic.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically involving the application of FH acetylation in the diagnosis, staging, treatment and prognosis prediction of colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is a malignant tumor with leading incidence and mortality rates worldwide, and its pathogenesis is complex and not yet fully understood. However, for patients with advanced and metastatic CRC, clinical treatment options are limited and ineffective. In-depth exploration of the molecular mechanisms of CRC development and progression, and the search for new therapeutic targets and strategies, have become a research hotspot and an urgent problem to be solved in the fields of basic medicine and clinical oncology.

[0003] Recent studies have shown that post-translational modifications of proteins, especially non-histone acetylation, have attracted much attention due to their crucial roles in regulating protein function, stability, and interactions. As a reversible post-translational modification, acetylation can significantly alter the biological properties of proteins, thereby affecting cellular physiological and pathological processes. Previous studies have confirmed that abnormal acetylation modifications of various non-histone proteins are closely related to tumorigenesis and development. However, current research on key non-histone acetylation modifications in CRC still has many gaps, particularly lacking systematic studies to reveal the roles and regulatory mechanisms of specific acetylation sites in CRC progression. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides the application of FH acetylation in the diagnosis, staging, treatment, and prognosis prediction of colorectal cancer.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides the use of a reagent for detecting FH K80 acetylation levels in the preparation of products for diagnosing colorectal cancer / diagnosing colorectal cancer staging / predicting colorectal cancer prognosis.

[0007] Furthermore, the reagent includes a specific antibody against FH K80.

[0008] Furthermore, the reagents also include reagents used to detect FH K80 acetylation levels by ELISA, Western Blot, immunoprecipitation, mass spectrometry, and immunocytochemistry.

[0009] Furthermore, the reagent also includes detectable markers.

[0010] Furthermore, the detectable markers include radioactive isotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent components, magnetic particles, and bioluminescent components.

[0011] A second aspect of the present invention provides a product for diagnosing colorectal cancer / diagnosing colorectal cancer staging / predicting colorectal cancer prognosis, said product comprising a reagent for detecting FH K80 acetylation levels.

[0012] Furthermore, the products include chips, test strips, reagent kits, or nucleic acid membrane strips.

[0013] Furthermore, the kit also includes instructions.

[0014] Furthermore, the kit also includes a buffer solution.

[0015] The third aspect of the present invention provides any one of the following substances:

[0016] (1) A polypeptide for treating colorectal cancer, wherein the 80th amino acid of the polypeptide is mutated from lysine to glutamine compared to wild-type FH protein;

[0017] (2) The nucleic acid encoding the polypeptide described in (1);

[0018] (3) A carrier comprising the nucleic acid described in (2);

[0019] (4) A host cell comprising the nucleic acid described in (2) or the vector described in (3).

[0020] The fourth aspect of the invention provides the use of the substance described in the third aspect of the invention or the agent that promotes the acetylation of FH K80 in the preparation of a medicament for treating colorectal cancer.

[0021] A fifth aspect of the present invention provides a medicament for treating colorectal cancer, the medicament comprising the substance described in the third aspect of the present invention or an agent that promotes the acetylation of FH K80.

[0022] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0023] The sixth aspect of the present invention provides the use of FH K80 acetylation as a target in screening candidate drugs for the treatment of colorectal cancer.

[0024] Furthermore, the method for screening candidate drugs for treating colorectal cancer includes: treating a culture system expressing or containing FH K80 acetylation with a substance to be screened; and detecting the expression or activity of FH K80 acetylation in the system; wherein, when the substance to be screened promotes the expression level or activity of FH K80 acetylation, the substance to be screened is a candidate drug for treating colorectal cancer.

[0025] A seventh aspect of the present invention provides a method for screening candidate drugs for treating colorectal cancer, the method comprising: treating a culture system expressing or containing FH K80 acetylation with a substance to be screened; and detecting the expression or activity of FH K80 acetylation in the system; wherein, when the substance to be screened promotes the expression level or activity of FH K80 acetylation, the substance to be screened is a candidate drug for treating colorectal cancer.

[0026] The eighth aspect of the present invention provides a method for preparing a specific antibody for detecting FHK80 acetylation, the method comprising: synthesizing an FHK80 acetylated antigen polypeptide, and immunizing the synthesized polypeptide to obtain a specific FHK80 acetylated antibody.

[0027] Furthermore, the method also includes a step of purifying the synthesized peptide by immunization.

[0028] Furthermore, the method specifically includes:

[0029] (1) Synthesize FH K80 acetylated antigen polypeptide;

[0030] (2) Obtain polyclonal antiserum by immunizing with the polypeptide synthesized in step (1), determine the antibody titer in the serum, and purify the antibody to obtain the specific antibody.

[0031] The ninth aspect of the present invention provides a method for promoting apoptosis of colorectal cancer cells and inhibiting the proliferation / migration of colorectal cancer cells in vitro, the method comprising administering the substance described in the third aspect of the present invention or a reagent that promotes the acetylation of FH K80.

[0032] Furthermore, the colorectal cancer cells are selected from HCT116 cells and / or SW480 cells.

[0033] Furthermore, the method described is not for therapeutic purposes.

[0034] The tenth aspect of the present invention provides the application of FH K80 acetylation in the preparation of models / systems / devices for diagnosing colorectal cancer / predicting the prognosis of colorectal cancer.

[0035] Furthermore, the system / device includes:

[0036] Acquisition module: Used to acquire acetylation site data of FH protein in the sample to be tested;

[0037] Extraction module: used to extract acetylation data of the target site of FH protein in the sample to be tested, wherein the acetylation of the target site is FH K80 acetylation;

[0038] Prediction module: Based on the FH K80 acetylation data of the target site, classification prediction is performed to obtain the classification result of whether the test sample has colorectal cancer. If the FH K80 acetylation level is high, the classification result is that the test sample does not have colorectal cancer / colorectal cancer patients have high survival rate. If the FH K80 acetylation level is low, the classification result is that the test sample has colorectal cancer / colorectal cancer patients have low survival rate.

[0039] Advantages and beneficial effects of the present invention:

[0040] This application, through systematic screening of acetylated non-histone proteins and acetylation sites associated with CRC, has for the first time discovered the regulatory role of FH K80 acetylation modification levels on the malignant phenotype of CRC. FH K80 acetylation modification levels showed significant differences in CRC patients and were correlated with prognosis; a hyperacetylation state (FH K80Q) could inhibit CRC cell proliferation, migration, and invasion. This application provides a new direction for the diagnosis, prognostic prediction, and effective treatment of CRC, and has broad clinical application prospects. Attached Figure Description

[0041] Figure 1 This diagram shows the protein and site acetylation changes in cancerous tissue compared to adjacent normal tissue.

[0042] Figure 2 It is a volcano diagram of differentially acetylated proteins between cancerous tissue and adjacent normal tissue;

[0043] Figure 3 It is a clustering heatmap of differentially acetylated proteins;

[0044] Figure 4 This is a statistical graph showing the distribution of differentially acetylated proteins in the COG / KOG database. 4A shows acetylated proteins with downregulated T / N ratios, and 4B shows acetylated proteins with upregulated T / N ratios.

[0045] Figure 5 This is a differential protein interaction diagram with acetylation modification (arrow points to FH);

[0046] Figure 6 This is an acetylation map of FH and its sites, where 6A is the acetylation map of FH and 6B is the acetylation map of K80 and K221 sites;

[0047] Figure 7 This is a schematic diagram of the structure, site acetylation, and related sequences of FH. Among them, 7A is a schematic diagram of the structure of FH and a schematic diagram of acetylation at different sites, and 7B is a conserved sequence diagram of the protein sequence near K80 of FH in different species (red K represents lysine at position 80).

[0048] Figure 8This is a mass spectrum of the FH acetylation modification site;

[0049] Figure 9 These are graphs showing FH expression and FH K80 acetylation levels. 9A is a graph showing FH expression and FH K80 acetylation levels in CRC detected by Western blotting (WB); 9B is a graph showing FH expression and FH K80 acetylation levels in CRC detected by immunohistochemistry (40×); 9C is a graph showing the relationship between FH expression and CRC survival; and 9D is a graph showing the relationship between FH K80 acetylation levels and CRC survival.

[0050] Figure 10 This is a graph showing the effect of FH K80 acetylation levels on CRC cells. Among them, 10A shows the effect of different FH K80 acetylation levels on HCT116 cells, 10B shows the effect of different FH K80 acetylation levels on SW480 cells, 10C shows the effect of different FH K80 acetylation levels on cell colony formation ability, and 10D is a statistical graph showing the effect of different FH K80 acetylation levels on cell colony formation ability.

[0051] Figure 11 These are graphs showing the effect of FH K80 acetylation levels on CRC cell proliferation detected by the EdU method. 11A shows the effect of different FH K80 acetylation levels on HCT116 cell proliferation detected by the EdU method; 11B is a statistical graph showing the effect of different FH K80 acetylation levels on HCT116 cell proliferation detected by the EdU method; 11C is a graph showing the effect of different FH K80 acetylation levels on SW480 cell proliferation detected by the EdU method; and 11D is a statistical graph showing the effect of different FH K80 acetylation levels on SW480 cell proliferation detected by the EdU method.

[0052] Figure 12 These are graphs showing the effect of FHK80 acetylation level on CRC cell migration as detected by scratch assay. Among them, 12A is the graph showing the effect of FHK80 acetylation level on HCT116 cell migration, 12B is the statistical graph showing the effect of FHK80 acetylation level on HCT116 cell migration, 12C is the graph showing the effect of FHK80 acetylation level on SW480 cell migration, and 12D is the statistical graph showing the effect of FHK80 acetylation level on SW480 cell migration.

[0053] Figure 13These are Transwell assay results showing the effects of FH K80 acetylation levels on the migration and invasion abilities of CRC cells. 13A shows the effect of FH K80 acetylation levels on the migration and invasion abilities of HCT116 cells; 13B is a statistical graph showing the effect of FH K80 acetylation levels on the migration and invasion abilities of HCT116 cells; 13C shows the effect of FH K80 acetylation levels on the migration and invasion abilities of SW480 cells; and 13D is a statistical graph showing the effect of FH K80 acetylation levels on the migration and invasion abilities of SW480 cells.

[0054] Figure 14 This is a graph showing the relationship between FH K80 acetylation level and tumor growth and proliferation in vivo. Among them, 14A is a whole graph of mice in the control group and experimental group, 14B is a grouping and sorting graph of subcutaneous tumors in mice, 14C is a bar chart of tumor weight statistics, and 14D is a line graph of tumor growth.

[0055] Figure 15 This is an analysis of the acetylation level at the FH K80 site in mouse subcutaneous tumors. 15A shows the HE and IHC staining of mouse subcutaneous tumors, and 15B shows the IHC score of mouse subcutaneous tumors. Detailed Implementation

[0056] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0057] This invention provides the application of a reagent for detecting FH K80 acetylation levels in the preparation of products for diagnosing colorectal cancer / diagnosing colorectal cancer staging / predicting colorectal cancer prognosis.

[0058] In some implementations, FH includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed FH, as well as any form of FH derived from cell processing. The term encompasses naturally occurring variants of FH (e.g., splice variants or allelic variants). The term encompasses FH from, for example, human and any other vertebrate origin, including mammalian FH such as primates and rodents (e.g., mice and rats), gene ID: 2271.

[0059] The reagent also includes detectable markers.

[0060] In some embodiments, a detectable marker refers to a composition capable of generating a detectable signal indicating the presence of a target polynucleotide in the sample being measured. Suitable detectable markers include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent components, magnetic particles, and bioluminescent components. Therefore, a marker is any composition detectable by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrochemical, optical, chemical detection devices, or any other suitable device. In some embodiments, the marker can be detected visually without the aid of a device.

[0061] Among them, radioactive isotopes include but are not limited to 3 H, 14 C 35 S, 125 I, 131 I; Enzymes include, but are not limited to, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and acetylcholinesterase; Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors.

[0062] The products include chips, test strips, reagent kits, or nucleic acid membrane strips.

[0063] In some embodiments, the kit also includes instructions and buffer solutions. The instructions may include guidance on obtaining and processing samples. Additionally, the kit may contain bacterial genomic DNA as a positive control for PCR and sterile water as a negative control.

[0064] In some embodiments, the components of the kit may be packaged in an aqueous medium or in a lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container in which one component can be placed, and preferably, appropriately aliquoted. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container in which the additional components are placed separately. However, different combinations of components may be contained in a single vial. The kit of the present invention will also typically include a container for containing the reactants, sealed for commercial sale. Such a container may include injection-molded or blow-molded plastic containers in which the desired vials can be held.

[0065] The solid support of the kit may be, for example, plastic, silicon wafer, metal, resin, glass, membrane, particles, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate, or slide. The biological sample may be, for example, cell culture, cell line, tissue, oral tissue, gastrointestinal tissue, organ, organelle, biological fluid, plasma sample, urine sample, or skin.

[0066] In some embodiments, the nucleic acid membrane strip includes a substrate and probes fixed on the substrate; the substrate can be any substrate suitable for fixing the probes, including but not limited to nylon membranes, nitrocellulose membranes, polypropylene membranes, glass slides, silicone wafers, and micro-magnetic beads.

[0067] This invention provides any of the following substances:

[0068] (1) A polypeptide for treating colorectal cancer, wherein the 80th amino acid of the polypeptide is mutated from lysine to glutamine compared to wild-type FH protein;

[0069] (2) The nucleic acid encoding the polypeptide described in (1);

[0070] (3) A carrier comprising the nucleic acid described in (2);

[0071] (4) A host cell comprising the nucleic acid described in (2) or the vector described in (3).

[0072] In some embodiments, a vector refers to a medium into which a polynucleotide encoding a protein can be operatively inserted to induce expression of said protein. Vectors can be used to transform, transduce, or transfect host cells to express carried genetic elements within the host cells. Vectors may contain a variety of elements for controlling expression, including linker sequences, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication. An origin of replication is a sequence that initiates replication when present in the vector. An origin of replication may be recognized by a replication initiation factor or alternatively by a DNA helicase. Vectors may also include materials that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein envelopes.

[0073] In some embodiments, the host cell is a cell used to receive, maintain, replicate, and amplify the vector. The host cell can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. In one embodiment, the host cell is a genetic package that can be induced to express variant polypeptides on its surface. In another embodiment, the host cell is infected with the genetic package.

[0074] In one embodiment of this application, the host cell can actually be any cell available for the expression vector, including prokaryotic cells and eukaryotic cells.

[0075] The present invention provides a medicament for treating colorectal cancer, the medicament comprising the above-mentioned substance or a reagent that promotes the acetylation of FH K80.

[0076] The drug also includes pharmaceutically acceptable excipients.

[0077] In some embodiments, pharmaceutically acceptable excipients include all solvents, diluents, buffers (e.g., neutral buffered saline, or optionally Tris-HCl, acetate, or phosphate buffers), solubilizers (e.g., polysorbate 80), colloids, dispersion media, solvents, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, sweeteners, colorants, flavoring agents, aromatizers, thickeners, substances that provide storage effects, coating agents, antifungal agents, preservatives (e.g., Thimerosal™, benzyl alcohol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), tension control agents, absorption delay agents, adjuvants, extenders (e.g., lactose, mannitol), etc. The use of such media and reagents for formulating pharmaceuticals is well known in the art. Their use in pharmaceuticals may be considered unless any conventional media or reagent is incompatible with the active ingredient.

[0078] In some embodiments, the drug can be administered in a variety of ways, depending on whether local or systemic treatment is required and the area to be treated. Administration can be parenteral, including intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, such as intrathecal, intraventricular, or intracardiac administration. In some embodiments, the drug is administered intravenously, intraperitoneally, as a bolus, or directly to the target organ.

[0079] In some embodiments, excipients for systemic administration typically include at least one of the following: solvents, diluents, lubricants, binders, disintegrants, colorants, flavorings, sweeteners, antioxidants, preservatives, glidants, suspending agents, wetting agents, surfactants, combinations thereof, etc. All excipients in the drug are optional.

[0080] This invention provides the application of FH K80 acetylation in the preparation of models / systems / devices for diagnosing colorectal cancer / predicting the prognosis of colorectal cancer.

[0081] In some embodiments, the disclosed system / device can be implemented in other ways. For example, the system / device embodiments described above are merely illustrative. For instance, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be an indirect coupling or communication connection between devices or units through some interfaces, and may be electrical, mechanical, or other forms.

[0082] Those skilled in the art will understand that all or part of the steps in the various methods of the above embodiments can be implemented by a program instructing related hardware. The program can be stored in a computer-readable storage medium, which may include: read-only memory (ROM), random access memory (RAM), disk or optical disk, etc.

[0083] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0084] Example

[0085] 1. Experimental materials

[0086] 1) Tissue specimens and clinicopathological data

[0087] Between September and December 2021, seven pairs of surgically resected tumor tissue (T) and paired adjacent normal tissue (N) samples were collected from pathologically confirmed CRC patients consecutively at the Department of Gastrointestinal Surgery, Peking University People's Hospital. Adjacent normal tissue was defined as normal colorectal mucosa within 5 cm of the tumor margin. All patients had not received neoadjuvant therapy and had no distant metastases. Strict exclusion criteria were followed during sample collection, excluding cases with incomplete clinicopathological data, substandard total protein levels, or the presence of tumor thrombus formation, tumor-induced intestinal perforation, or intestinal obstruction.

[0088] After being collected according to standard procedures, tissue samples were divided into two aliquots. One aliquot was flash-frozen in liquid nitrogen 15 minutes after ex vivo to minimize thermal ischemia time, and then rapidly frozen in liquid nitrogen and stored at -80°C. The other aliquot was fixed in 4% formalin solution for paraffin embedding and sectioning. All specimens were pathologically confirmed as corresponding tumor tissues, and were reconfirmed using frozen sectioning before the formal experiment to ensure that the tissues used were CRC tissues or paired adjacent normal tissues. After the above screening process, a total of 7 patients were finally included in this study, all of whom had adenocarcinoma as their primary tumor lesion.

[0089] 2) Cell lines, strains, and plasmids

[0090] Human colon cancer cell lines SW480 and SW620, as well as tool cell line 293T, were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and preserved by the Surgical Oncology Research Laboratory of Peking University People's Hospital. SW480 was derived from a primary rectal adenocarcinoma lesion; SW620 from lymph node metastases of colon adenocarcinoma patients; HCT116, HT29, DLD1, RKO, and HCT8 were all derived from primary colon adenocarcinoma lesions; and LoVo was derived from metastases in the left supraclavicular region of colon adenocarcinoma patients.

[0091] Escherichia coli DH5α competent cells were purchased from Invitrogen Inc. (USA) and Tiangen Biotech (Beijing) Co., Ltd., respectively.

[0092] 3) Colorectal cancer tissue microarray

[0093] This study selected 120 patients with colorectal cancer (CRC) who underwent surgical resection at the Department of Gastrointestinal Surgery, Peking University People's Hospital in June 2020 as research subjects. Tumor tissue (T) and paired adjacent normal tissue (N) were collected. Adjacent normal tissue was defined as normal colorectal mucosa located 5 cm from the tumor margin. All patients had not received neoadjuvant therapy and had no distant metastases before surgery. Tissue samples obtained during surgery were collected according to standard procedures, then fixed in 4% formalin solution for subsequent paraffin embedding and sectioning.

[0094] 4) Main reagents and materials for tissue protein extraction and detection

[0095] Benzyl sulfonyl fluoride (PMSF) (Sigma, USA); Protease inhibitor (Sigma, USA); RIPA protein lysis buffer (Solepro China); BCA protein quantification kit (Solepro China); Sample loading buffer (5X) (Solepro China); Electrophoresis gel kit (Solepro China); GelCode Blue staining reagent (Bio-Rad, USA).

[0096] 5) Main reagents and materials for protein acetylation mass spectrometry detection

[0097] Sequencing-grade modified trypsin (Invitrogen, USA); acetonitrile (Invitrogen, USA); formic acid (Sinopharm Chemical Reagent Co., Ltd., China); iodoacetamide (Sigma-Aldrich, USA); dithiothreitol (Sigma-Aldrich, USA); triethylamine bicarbonate buffer (Sigma-Aldrich, USA); tetraethylamine bromide (TEAB) (Sigma-Aldrich, USA); TMT labeling kit (Thermo Fisher Scientific, USA); anti-acetylsine antibody magnetic beads (Hangzhou Jingjie Biotechnology Co., Ltd., China); tandem mass labeling kit (Thermo Fisher Scientific, USA); purified water (Thermo Fisher Scientific, USA).

[0098] 6) Main reagents and materials for cell culture

[0099] DMEM liquid culture medium (Gibco, USA); RPMI 1640 liquid culture medium (Gibco, USA); F12K liquid culture medium (Gibco, USA); Fetal bovine serum (FBS) (Gibco, USA); Trypsin-EDTA with phenol red (0.25%) (Gibco, USA); Penicillin-streptomycin mixture (100×, PS) (Gibco, USA); Phosphate-buffered saline (PBS) (Hyclone, USA); Dimethyl sulfoxide (DMSO) (Beijing Solarbio Science & Technology Co., Ltd., China); Canned compressed gaseous carbon dioxide (Peking University People's Hospital, China); Sterile water for injection (Shijiazhuang No. 4 Pharmaceutical Co., Ltd., China); Ultraviolet lamp (Guangzhou Langpu Optoelectronic Technology Co., Ltd., China); 75% medical disinfectant alcohol (Beijing Zhenyu Minsheng Pharmaceutical Co., Ltd., China); Sealing film (3M, USA); 1.8mL inner screw cap sterile cryovials (Thermo Fisher Scientific, USA); Centrifuge tubes (15 / 50mL) (NEST, China).

[0100] 7) Major instruments and equipment

[0101] Sterile surgical blades (Shanghai Lianhui Medical Supplies Co., Ltd., China); biological tissue embedding machine (Beijing Haifuda Technology Co., Ltd., China); paraffin microtome (Shenzhen Ruiwode Life Technology Co., Ltd., China); disposable pathology blades (Feather, Japan); biological tissue spreader (Shanghai Zhixin Instrument Co., Ltd., China); constant temperature drying oven (Tianjin Laiboteri Instrument Equipment Co., Ltd., China); fluorescence inverted microscope (Leica, Germany); -80℃ ultra-low temperature freezer (Thermo Fisher Scientific, USA); ordinary low temperature freezer (SIEMENS, Germany); mortar and pestle set (Jiangsu Boya Teaching Equipment Co., Ltd., China); ultrasonic cell disruptor (SONICS, USA); constant temperature water bath (Shanghai Lichen Bangxi Instrument Technology Co., Ltd., China); benchtop high-speed refrigerated centrifuge (Eppendorf, Germany); micropipette (Eppendorf, Germany); vortex mixer (Scientific Industries, USA); electrophoresis apparatus (Bio-Rad, USA); electrophoresis tank (Bio-Rad, USA); benchtop horizontal shaker (SCILOGEX). Chemiluminescence gel imaging system (Bio-Rad, USA); Ice maker (Jinan Oulaibo Scientific Instruments Co., Ltd., China); Dry thermostat (Hangzhou Aosheng Instruments Co., Ltd., China); Magnetic stirrer (Jiangsu Haimen Qilinbeier Instrument Manufacturing Co., Ltd., China); Vacuum freeze dryer (Thermo Fisher Scientific, USA); Microplate reader (Bio-Rad, USA); Strata XC18 solid phase extraction column (Phenomenex, USA); ZORBAX 300Extend-C18 column (Agilent, USA); EASY-nLC 1000 (Thermo Fisher Scientific, USA); Orbitrap Fusion™ Lumos™, Tribrid™ mass spectrometer (Thermo Fisher Scientific, USA); Nanospray Flex™ ion source (Thermo Fisher Scientific, USA).

[0102] 2. Experimental Methods

[0103] 1) Tissue homogenization treatment

[0104] Before extracting CRC tissue protein, first wipe the workbench with a clean cloth, then carefully wipe it with 75% alcohol to complete the disinfection process. Subsequent experiments can only be carried out after the alcohol has completely evaporated.

[0105] Pre-cool the mortar and pestle on ice, then add liquid nitrogen, repeating this process 4-5 times to ensure thorough pre-cooling. Remove the frozen tissue sample from the -80°C freezer and quickly cut it with a sterile, sharp blade. Accurately weigh approximately 500mg (about the size of a soybean) of tissue and place it in the pre-cooled mortar. Grind the tissue thoroughly using a grinding rod. During grinding, an assistant should intermittently add liquid nitrogen to the mortar to prevent complete evaporation. Once the tissue is ground into powder, transfer it to a sterile 50ml centrifuge tube. Add a pre-prepared mixture of pre-cooled 10% trichloroacetic acid / acetone, 50mM dithiothreitol, and 0.1% protease inhibitor to the centrifuge tube to precipitate the powder. Centrifuge the tube at 20,000g for 10 minutes at 4°C. Discard the supernatant after centrifugation.

[0106] 2) Protein extraction

[0107] Samples were removed from a -80°C cryogenic storage environment. An appropriate amount of tissue sample was accurately weighed and placed in a mortar pre-cooled with liquid nitrogen. Sufficient liquid nitrogen was then added to the mortar, and the tissue sample was thoroughly ground until it was completely pulverized. For each sample group, lysis buffer equivalent to four times the volume of the powder was added. This buffer consisted of 8M urea, 1% protease inhibitor, 3 μM TSA, and 50 mM NAM. After adding the lysis buffer, the samples were sonicated for lysis. After lysis, the samples were placed at 4°C and centrifuged at 12000g for 10 minutes. After centrifugation, the supernatant was carefully transferred to a new centrifuge tube, and the protein concentration in the supernatant was accurately determined using a BCA assay kit.

[0108] 3) Modification and enrichment

[0109] First, the peptides were dissolved in mobile phase A (i.e., 0.1% (v / v) formic acid aqueous solution) for liquid chromatography. The dissolved peptides were then separated using an EASY-nLC 1000 ultra-high performance liquid chromatography system. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid. The specific liquid phase gradient settings were as follows: from 0 to 42 minutes, the proportion of mobile phase B gradually increased from 7% to 23%; from 42 to 54 minutes, the proportion of mobile phase B further increased from 23% to 30%; from 54 to 57 minutes, the proportion of mobile phase B rapidly increased to 80%; and from 57 to 60 minutes, the proportion of mobile phase B was maintained at 80%, with the flow rate maintained at 450 nL / min throughout the process. After separation by the ultra-high performance liquid chromatography system, the peptides were injected into an NSI ion source for ionization and then analyzed by an Orbitrap Fusion Lumos mass spectrometer. During the analysis, the ion source voltage was set to 2.4 kV, and both the precursor ions and secondary fragments of the peptides were detected and analyzed using high-resolution Orbitrap. The primary mass spectrometry scan range was set to 350–1550 m / z with a scan resolution of 60,000; the secondary mass spectrometry scan range had a fixed starting point of 100 m / z and a secondary scan resolution of 15,000. Data acquisition employed a data-dependent scanning (DDA) procedure, where, after the primary scan, the top 20 peptide precursor ions with the highest signal intensity were selected and sequentially introduced into the HCD collision cell for fragmentation at 32% of the fragmentation energy, followed by secondary mass spectrometry analysis. To improve the efficiency of the mass spectrometry, automatic gain control (AGC) was set to 5E4, the signal threshold to 5000 ions / s, the maximum injection time to 100 ms, and the dynamic exclusion time for tandem mass spectrometry scans to 30 seconds to avoid repeated scanning of precursor ions.

[0110] 4) Liquid chromatography-mass spectrometry analysis

[0111] The peptides were dissolved in mobile phase A of the liquid chromatography system, which was an aqueous solution containing 0.1% formic acid and 2% acetonitrile. The peptides were then separated using an EASY-nLC 1200 ultra-high performance liquid chromatography system. Mobile phase A of this system was an aqueous solution containing 0.1% formic acid and 2% acetonitrile, and mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The specific liquid phase gradient was set as follows: from 0 to 38 minutes, the proportion of mobile phase B gradually increased from 6% to 22%; from 38.0 to 52.0 minutes, the proportion of mobile phase B increased from 22% to 32%; from 52.0 to 56.0 minutes, the proportion of mobile phase B rapidly increased from 32% to 80%; and from 56.0 to 60.0 minutes, the proportion of mobile phase B was maintained at 80%, with the flow rate maintained at 500 nL / min throughout the separation process. After separation by an ultra-high performance liquid chromatography (UHPLC) system, peptides were injected into an NSI ion source for ionization and then analyzed using a Q Exactive™ HF-X (ThermoFisherScientific) mass spectrometer. During analysis, the ion source voltage was set to 2.1 kV, and both the precursor ions and secondary fragments of the peptides were detected and analyzed using high-resolution orbitrap. The primary mass spectrometry scan range was set to 350–1400 m / z with a scan resolution of 60,000 m / s; the secondary mass spectrometry scan range had a fixed starting point of 100 m / z and a secondary scan resolution of 30,000 m / s. Data acquisition was performed using a data-dependent scanning (DDA) procedure, where the top 20 peptide precursor ions with the highest signal intensity after the primary scan were sequentially introduced into an HCD collision cell for fragmentation at 28% of the fragmentation energy, followed by subsequent secondary mass spectrometry analysis. To improve the efficiency of mass spectrometry, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 8.3E4 ions / s, the maximum injection time was set to 60 ms, and the dynamic exclusion time for tandem mass spectrometry scanning was set to 15 seconds to prevent repeated scanning of precursor ions.

[0112] 5) Tissue embedding and sectioning

[0113] a) Tissue sampling: In accordance with the standard procedures of this center, CRC and adjacent fresh tissue samples are taken from surgically removed specimens. The size of the tissue block is approximately 4×4×4mm, and the maximum diameter does not exceed 5mm.

[0114] b) Fixation: Place the cut tissue blocks in an appropriate amount of 4% paraformaldehyde tissue cell fixative and fix for 24-48 hours. The ratio of tissue blocks to fixative is approximately 1:10.

[0115] c) Clearing and Dehydration: After fixation, remove the tissue block and rinse it three times with 1×PBS, 5 min each time. Then, perform gradient dehydration with ethanol, immersing the tissue block sequentially in 75% ethanol for 4 h, 85% ethanol for 2 h, 90% ethanol for 2 h, 95% ethanol for 1 h, anhydrous ethanol I for 2 h, and anhydrous ethanol II for 30 min. Following this, perform clearing treatment, immersing the tissue block sequentially in 50% ethanol + 50% xylene for 1 h, xylene I for 30 min, xylene II for 20 min, and xylene III for 10 min. During clearing, the morphology of the tissue block should be observed in real time, and the time should be adjusted flexibly according to the actual situation, with appropriate clearing observed under light.

[0116] d) Paraffin impregnation: Melt the paraffin and maintain the temperature at around 55°C. Place the tissue block in 50% xylene + 50% paraffin for 2 hours, paraffin I for 2 hours, and paraffin II for 2 hours in sequence.

[0117] e) Embedding: Place the tissue block into a mold containing melted pure wax and fill it with wax. Then move the embedding frame to a low-temperature workbench. After the wax solidifies, remove the block, trim it, and place it in a 4°C refrigerator overnight.

[0118] f) Sectioning and spreading: Section using a paraffin microtome, with a thickness of 4-6µm. Carefully transfer the diced paraffin tissue fragments to clean water in a slide spreader to float and flatten them. Select high-quality slides and retrieve them using an adhesive slide.

[0119] g) Baking the slides: Place the glass slides in a 60℃ oven and remove them after the paraffin on the surface of the slides has melted. Store them at room temperature for later use.

[0120] 6) H&E staining

[0121] a) Dewaxing treatment: Place the sections in xylene I and xylene II in sequence, and soak each for 10 minutes. Take advantage of the good solubility of paraffin in xylene to fully remove the paraffin from the sections and prepare them for subsequent staining steps.

[0122] b) Hydration process: The sections were immersed in anhydrous ethanol I and anhydrous ethanol II for 5 minutes each to effectively remove residual xylene. Then, following a descending order of concentration, the sections were placed in 95%, 90%, 85%, and 75% ethanol, immersing for 5 minutes at each concentration, gradually reducing the ethanol concentration to acclimate the sections to the aqueous environment. Finally, the sections were rinsed three times with ddH2O for 5 minutes each time to thoroughly remove any residual ethanol.

[0123] c) Hematoxylin staining: Immerse the slide completely in the hematoxylin staining solution for 10 minutes. Hematoxylin stains the cell nuclei blue-purple, facilitating subsequent observation of nuclear morphology and structure. After staining, rinse the slide three times with ddH2O for 5 minutes each time to remove excess hematoxylin staining solution.

[0124] d) Color separation and blueing procedure: During color separation, use freshly prepared 1% hydrochloric acid-ethanol solution (1 mL concentrated hydrochloric acid + 99 mL 75% ethanol). Immerse the sections in the solution for a few seconds to quickly remove overstained hematoxylin and make the cell nuclei more clearly stained. Immediately after color separation, rinse the sections three times with ddH2O for 5 minutes each time to stop the color separation reaction. Then, immerse the sections in a 1% lithium carbonate aqueous solution for a few seconds to blue-restore the blue-violet color of the cell nuclei. After blue-restore, rinse the sections three more times with ddH2O for 5 minutes each time to ensure a complete reaction.

[0125] e) Eosin staining: Immerse the sections in eosin staining solution for 5 minutes. Eosin mainly stains the cytoplasm and extracellular matrix red, forming a sharp contrast with hematoxylin staining, making it easy to distinguish different tissue components. After staining, rinse the sections three times with ddH2O for 5 minutes each time to remove excess staining solution.

[0126] f) Dehydration and Clearing: During dehydration, the sections are sequentially immersed in 70%, 80%, 90%, 95% ethanol I, 95% ethanol II, anhydrous ethanol I, anhydrous ethanol II, and anhydrous ethanol III, for 5 minutes at each stage, gradually removing water from the sections to prepare for clearing. During clearing, the sections are sequentially immersed in xylene I, xylene II, and xylene III, for 5 minutes each. Xylene makes the sections transparent, facilitating light transmission during subsequent mounting and microscopic examination.

[0127] g) Mounting and Microscopic Examination: Mount the slides using neutral resin. Gently cover the slides with a coverslip, ensuring even distribution of the resin and avoiding air bubbles. After mounting, observe the slides under a microscope. Analyze the tissue morphology and pathological changes based on the staining of cell nuclei and cytoplasm.

[0128] 3. Experimental Results

[0129] 1) Differentially expressed acetylated proteins and acetylation sites

[0130] Differentially acetylated proteins and acetylation sites were analyzed in seven pairs of primary colorectal cancer lesion tissues (T) and paired adjacent normal tissues (N). Based on the inter-tissue differential expression screening criteria (fold change > 1.2-fold or < 0.833; P < 0.05), 263 significantly upregulated acetylated proteins and 403 acetylation sites were identified in the seven pairs of tissue specimens; 167 significantly downregulated acetylated proteins and 213 acetylation sites were identified. Figure 1 ). Figure 2 The volcano diagram shown summarizes the distribution of these differentially acetylated proteins.

[0131] A comprehensive analysis was performed on the differentially acetylated proteins selected above, and the cluster analysis results of the differentially acetylated proteins were obtained. Figure 3 A comparative observation of cancerous tissue (T1-T7) and adjacent normal tissue (N1-N7) revealed significant differences in the expression levels of differentially acetylated proteins. The heatmap uses variations in color intensity to visually represent the levels of protein acetylation.

[0132] 2) Functional classification of differentially modified proteins

[0133] Differentially acetylated proteins and sites between T and N were analyzed. Using the COG / KOG database for comparison, the differentially expressed modified proteins were functionally classified and statistically analyzed. Results are as follows: Figure 4 The data shows that for proteins with downacetylation: 23 proteins were identified in the "General Function Prediction" category; 7 proteins were identified in the "Unknown Function" category; and in the metabolism-related category, 19 proteins were identified in "Energy Production and Conversion," and 9 proteins were identified in "Carbohydrate Transport and Metabolism," among others. In the information storage and processing-related category, 10 proteins were identified in "Translation, Ribosome Structure, and Biogenesis." In the cellular processes and signal transduction-related category, 19 proteins were identified in "Post-translational Modification, Protein Turnover, and Molecular Chaperones," among others. For proteins with upacetylation: 33 proteins were identified in the "General Function Prediction" category; 5 proteins were identified in the "Unknown Function" category; and in the metabolism-related category, 10 proteins were identified in "Carbohydrate Transport and Metabolism," and 9 proteins were identified in "Lipid Transport and Metabolism," among others. In the information storage and processing-related category, 12 proteins were identified in "Translation, Ribosome Structure, and Biogenesis." In the cellular processes and signal transduction-related category, 44 proteins were identified in "Post-translational Modification, Protein Turnover, and Molecular Chaperones," and 38 proteins were identified in "Cytoskeleton," among others.

[0134] 3) Identification of FH acetylation sites

[0135] The database IDs or protein sequences of the obtained differentially modified proteins were compared with the STRING (v.11.0) protein interaction network database to obtain the interaction relationships between the differentially modified proteins. The results are as follows: Figure 5 As shown in the figure: Differentially modified proteins are represented by circles, and different colors are used to visually represent the differential expression of proteins. Green circles represent proteins upregulated by acetylation, and red circles represent proteins downregulated by acetylation. The intensity of the color is related to the fold change; the darker the color, the greater the fold change. Of particular note is the red arrow in the figure, which clearly points to FH, a key protein of focus in this study, and it may play an important role and have significant research value in the entire differentially modified protein interaction network.

[0136] Table 1 shows all FH sites detected by mass spectrometry. It can be seen that among all T / N differential acetylation sites, only K221 and K80 showed significant acetylation. To verify FH acetylation modification in a live cell system, this study ectopically expressed Flag-labeled FH protein in HEK293T cells and performed immunoprecipitation. Western blot experiments using a panacetylated lysine antibody confirmed that FH protein did indeed undergo acetylation modification. Furthermore, after the addition of nicotinamide (NAM, an inhibitor of the sirtuin (SIRT) deacetylase family), the degree of FH protein acetylation gradually increased over time. Figure 6 A).

[0137] To further investigate the acetylation modification of specific lysine sites in the FH protein, this study mutated lysine sites K80 and K221 in the FH protein to arginine (R), respectively. Since the amino acid remains positively charged after mutation from lysine (K) to arginine (R), but this prevents the acyl group in AcCoA (acetyl-CoA) from covalently binding to the amino group of the corresponding lysine residue, current research often uses KR mutations to simulate a low-acetylated state of the protein. This study detected acetylation modification in the FH wild-type (WT) and two KR mutants, K80R and K221R. The results showed that the acetylation modification level of the FH K80R mutant was significantly lower than that of the FH wild-type (WT). Figure 6 B).

[0138] PhosphoSitePlus (website: https: / / www.phosphosite.org / homeaction) is a well-known database of protein post-translational modification sites. Comparing FH acetylation modification sites in the database, this study found more evidence supporting acetylation at the K80 site. Figure 7 A). Furthermore, conservation analysis revealed that the FH K80 site is highly conserved in primates (humans, cynomolgus monkeys, even-toed ungulates, wild boars, rodents (brown rats and house mice), and a single-celled fungus, *Saccharomyces cerevisiae*. Figure 7 B).

[0139] To further confirm that the major acetylation site of FH is K80, Fourier transform mass spectrometry (FTMS) was used to detect the acetylation modification of the FH protein. Mass spectrometry analysis identified a peptide containing acetylation of lysine (K) at position 80 of the protein (acK), with the amino acid sequence TMNF-acK-IGGVTER. In the mass spectrum, the horizontal axis represents the mass-to-charge ratio (m / z), and the vertical axis represents the relative abundance of ions. Based on the peptide fragmentation pattern, the b-ion series generated from N-terminal fragmentation and the y-ion series generated from C-terminal fragmentation are marked in blue and red, respectively, in the figure. Figure 8 Analysis of the mass-to-charge ratio and relative abundance of each ion peak showed that the mass-to-charge ratio of the b-ion and y-ion series peaks was consistent with the theoretically derived mass-to-charge ratio of the peptide cleavage ions, and it was also determined that the acetylation modification site was located at the 80th lysine residue.

[0140] Table 1. All acetylation sites detected by FH in mass spectrometry.

[0141]

[0142] Note: * indicates the location of lysine (K) in the peptide segment.

[0143] 4) Relationship between FH K80 acetylation level and survival in colon cancer tissue

[0144] Western blot analysis was performed on frozen samples of colon cancer tissue and adjacent normal tissue collected clinically using an FH K80 site acetylation antibody. The results showed... Figure 9 The results shown in Figure A indicate that FH expression levels did not differ between tumor tissue (T) and adjacent normal tissue (N), while the acetylation level of FH K80 was significantly higher in normal tissue than in tumor tissue. This suggests that the expression levels of FH protein in colon cancer tissue and normal tissue were similar in this study, but the degree of acetylation modification at its 80th lysine residue differed significantly.

[0145] Next, IHC staining was performed on paraffin sections of 120 cases of CRC and paired normal colorectal mucosal tissue collected from our unit. The average optical density of the stained areas was used to score and analyze the immunohistochemistry. The results are as follows: Figure 9 B showed that there were no differences in FH protein expression levels between colorectal cancer tissues at different stages and normal tissues, and FH expression had no effect on the prognosis of CRC patients. Figure 9 C). However, the results showed that the acetylation level of FH was highest in normal tissues, and gradually decreased with tumor progression. Figure 9 B). Kaplan-Meier curves showed a significant correlation between FH K80 acetylation levels and overall survival (OS). Figure 9 (D), which corroborates the result expressed in CRC.

[0146] 5) Effects of FH K80 acetylation levels on the proliferation and clonal capacity of colorectal cancer cells.

[0147] To investigate the effect of FH K80 acetylation modification level regulation on CRC cell proliferation, this study conducted a CCK-8 cell proliferation assay. The results showed that in both HCT116 and SW480 cell lines, FH K80Q significantly inhibited cell proliferation; while FH K80R significantly enhanced the proliferation of both HCT116 and SW480 cells. Figure 10 A, B).

[0148] Subsequently, the effect of FHK80 acetylation modification level on the proliferation capacity of CRC single cells was further verified by plate colony formation assay. The results showed that when the acetylation modification level of FHK80 increased, the proliferation capacity of both HCT116 and SW480 single cells decreased significantly (HCT116: wild-type (WT) colony number 265±9.54, FHK80R group 306.33±7.10, FHK80Q group 173.67±8.62, P<0.05; SW480: WT colony number 282.67±10.60, FHK80R group 343.67±9.50, FHK80Q group 197±16.82, P<0.05). Figure 10 C, D).

[0149] To further investigate the effect of FHK80 acetylation modification level on the proliferation of colon cancer cells, this study conducted an EdU experiment, using two colon cancer cell lines, HCT116 and SW480, and set up a WT control group, a stable FHK80R cell line group simulating low acetylation, and an FHK80Q group simulating the high acetylation state of lysine at position 80.

[0150] The experimental results showed that in the HCT116 cell line, the proportion of EdU-positive cells was (24.44±2.54)% (n=3) in the WT group, (44.26±4.42)% (n=3) in the FH K80R group, and (14.83±2.92)% (n=3) in the FH K80Q group. Statistical analysis showed that compared with the WT group, the proportion of EdU-positive cells in the FH K80R group was significantly increased (P<0.05), indicating that FH K80R, which simulates low acetylation, can promote HCT116 cell proliferation; while the proportion of EdU-positive cells in the FH K80Q group was significantly decreased (P<0.05), suggesting that FH K80Q, which simulates high acetylation, has an inhibitory effect on HCT116 cell proliferation. Figure 11 A, B).

[0151] In the SW480 cell line, the proportion of EdU-positive cells was (20.27±3.16)% (n=3) in the WT group, (39.65±4.47)% (n=3) in the FH K80R group, and (9.29±0.92)% (n=3) in the FH K80Q group. Similarly, compared with the WT group, the proportion of EdU-positive cells in the FH K80R group was significantly increased (P<0.05), indicating that FH K80R can enhance the proliferation ability of SW480 cells; the proportion of EdU-positive cells in the FH K80Q group was significantly decreased (P<0.05), showing that FH K80Q has an inhibitory effect on the proliferation of SW480 cells. Figure 11 C, D).

[0152] In summary, the EdU experimental results indicate that the acetylation modification level of FH K80 has a significant impact on the proliferation ability of colon cancer cells. The low acetylation state (FH K80R) promotes cell proliferation, while the high acetylation state (FH K80Q) inhibits cell proliferation. These results provide important evidence for further elucidating the role of FH K80 acetylation modification in the occurrence and development of CRC.

[0153] 6) Effects of FH K80 acetylation levels on the invasion and migration abilities of colorectal cancer cells

[0154] To investigate the effect of different acetylation states at the FH K80 site on the migration ability of colon cancer cells, this study conducted scratch assays on two colon cancer cell lines, HCT116 and SW480. In the scratch assay of the HCT116 cell line, the mean scratch healing rate was (54.00±4.36)% (n=3) in the WT group, (70.67±5.03)% (n=3) in the FH K80R group, and (20.67±3.79)% (n=3) in the FH K80Q group. Statistical analysis showed that the scratch healing rate was significantly higher in the FH K80R group compared to the WT group (P<0.05), indicating that the simulated low-acetylation FH K80R significantly promoted the migration of HCT116 cells; while the scratch healing rate was significantly lower in the FH K80Q group (P<0.05), indicating that the simulated high-acetylation FH K80Q inhibited the migration of HCT116 cells. Figure 12 A, B).

[0155] For the SW480 cell line, the mean scratch healing rate was (47.00±9.07)% (n=3) in the WT group, (77.00±11.00)% (n=3) in the FH K80R group, and (22.33±3.61)% (n=3) in the FH K80Q group. Similarly, compared with the WT group, the scratch healing rate was significantly increased in the FH K80R group (P<0.05), indicating that FH K80R can enhance the migration ability of SW480 cells; the scratch healing rate was significantly decreased in the FH K80Q group (P<0.05), meaning that FH K80Q has an inhibitory effect on the migration of SW480 cells. Figure 12 C, D).

[0156] To further investigate the effect of FHK80 acetylation level on the migration and invasion ability of CRC cells, this study conducted Transwell assays using two colon cancer cell lines, HCT116 and SW480. In the HCT116 cell line assay, the mean number of migrating cells was (234.0±21.08) in the WT group (n=3), (370.33±14.02) in the FHK80R group (n=3), and (125.00±7.21) in the FHK80Q group (n=3). Statistical analysis showed that compared with the WT group, the number of migrating cells in the FHK80R group was significantly increased (P<0.05), indicating that the simulated low-acetylation FHK80R promoted the migration of HCT116 cells; the number of migrating cells in the FHK80Q group was significantly decreased (P<0.05), indicating that the simulated high-acetylation FHK80Q inhibited the migration of HCT116 cells.

[0157] In terms of invasive ability detection, the mean number of invasive cells in the HCT116 cell line was (222.67±9.72) cells (n=3), in the FH K80R group (KR) it was (332.33±18.92) cells (n=3), and in the FH K80Q group (KQ) it was (111.67±8.02) cells (n=3). Similarly, compared with the WT group, the number of invasive cells in the FH K80R group was significantly increased (P<0.05), indicating that FH K80R enhanced the invasive ability of HCT116 cells; the number of invasive cells in the FH K80Q group was significantly decreased (P<0.05), showing that FH K80Q had an inhibitory effect on HCT116 cell invasion. Figure 13 A, B).

[0158] For the SW480 cell line, migration ability assays showed that the mean number of migrating cells in the WT group was (214.00±11.66) (n=3), in the FH K80R group it was (309.00±13.87) (n=3), and in the FH K80Q group it was (108.33±3.06) (n=3). Compared with the WT group, the number of migrating cells in the FH K80R group was significantly increased (P<0.05), indicating that FH K80R promoted the migration of SW480 cells; the number of migrating cells in the FH K80Q group was significantly decreased (P<0.05), meaning that FH K80Q inhibited the migration of SW480 cells.

[0159] Regarding invasive ability, the mean number of invasive cells in the WT group of the SW480 cell line was (200.33±11.76) (n=3), in the FH K80R group it was (302.33±16.44) (n=3), and in the FH K80Q group it was (98.33±13.01) (n=3). Compared with the WT group, the FH K80R group showed a significantly increased number of invasive cells (P<0.05), indicating that FH K80R enhanced the invasive ability of SW480 cells; the FH K80Q group showed a significantly decreased number of invasive cells (P<0.05), indicating that FH K80Q inhibited the invasion of SW480 cells. Figure 13 C, D).

[0160] In summary, the experimental results of this study indicate that the acetylation level of FH K80 has a significant impact on the migration and invasion capabilities of CRC cells. The low acetylation state (FH K80R) promotes cell migration and invasion, while the high acetylation state (FH K80Q) inhibits cell migration and invasion, providing important evidence for revealing the mechanism of action of FH K80 acetylation modification in the CRC metastasis process.

[0161] 7) The relationship between FH K80 acetylation levels and tumor growth and proliferation in vivo

[0162] To further investigate the effect of hypoacetylation of lysine 80 (K80) in the FH protein on tumor growth in vivo, this study conducted a subcutaneous tumorigenesis experiment in nude mice. A WT control group was established, inoculated with wild-type HCT116 cells; simultaneously, an FH K80R experimental group was established, inoculated with a stable cell line simulating K80 hypoacetylation.

[0163] Based on grouped comparison data ( Figure 14(A, B, C) The tumor weight in the FH K80R group was significantly higher than that in the WT group. The mean tumor weight in the WT group was 145.97 ± 33.26 mg (n=5), while the mean tumor weight in the FH K80R group was 298.44 ± 72.19 mg (n=5), and the difference between the two groups was statistically significant (independent samples t-test, P=0.0014). Notably, the highest tumor weight in the FH K80R group reached 379.87 mg, approximately 2.13 times the highest value in the WT group (178.38 mg), suggesting that FH K80R significantly enhances the in vivo tumorigenic capacity of tumor cells.

[0164] Tumor volume monitoring over 18 consecutive days ( Figure 14 (D) The tumor growth rate in the FH K80R group was significantly higher than that in the WT group. The mean tumor volume in the WT group on day 18 post-inoculation was 181.40±58.21 mm³ (n=5), while that in the FH K80R group reached 502.67±20.32 mm³ (n=5), a highly significant difference (P<0.0001). Dynamic analysis showed that in the early stage (days 3-9): the tumor volume in the WT group increased slowly (from 1.82±1.18 mm³ to 17.66±5.38 mm³), while the FH K80R group showed a rapid proliferation trend (from 9.48±0.70 mm³ to 59.09±9.82 mm³), and the difference between the two groups was significant from day 6 onwards (P=0.003). In the mid-to-late stages (days 12-18): the tumor volume in the FH K80R group increased exponentially, reaching 2.77 times higher than that in the WT group on day 18 (repeated measures ANOVA, time × treatment interaction P < 0.001), indicating that the FH K80R mutation may accelerate tumor progression by continuously activating tumor-promoting signaling pathways.

[0165] Subcutaneous tumors in mice were completely dissected, embedded in paraffin, and stained with hematoxylin and eosin (HE) and chromogenic iodine (IHC). The results showed that the IHC intensity of FH K80R tumors (stained with FH K80Ac) was significantly lower than that of the control group. Figure 15 This proves the reliability of the conclusions of this study.

[0166] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of reagents for detecting FH K80 acetylation levels in the preparation of products for diagnosing colorectal cancer, staging colorectal cancer, or predicting the prognosis of colorectal cancer.

2. The application according to claim 1, characterized in that, The reagents include a specific antibody against FH K80.

3. The application according to claim 2, characterized in that, The reagents also include those used to detect FH K80 acetylation levels by ELISA, Western Blot, immunoprecipitation, mass spectrometry, or immunocytochemistry.

4. The application according to claim 3, characterized in that, The reagent also includes detectable markers.

5. The application according to claim 4, characterized in that, The detectable markers include radioactive isotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent components, magnetic particles, or bioluminescent components.

6. The use of any of the following substances or agents that promote the acetylation of FH K80 in the preparation of drugs for treating colorectal cancer: (1) A polypeptide for treating colorectal cancer, characterized in that, Compared to the wild-type FH protein, the polypeptide has a mutation at amino acid position 80, where lysine is replaced by glutamine. (2) The nucleic acid encoding the polypeptide described in (1); (3) A carrier comprising the nucleic acid described in (2); (4) A host cell comprising the nucleic acid described in (2) or the vector described in (3).

7. Application of FH K80 acetylation as a target in screening candidate drugs for the treatment of colorectal cancer.

8. The application according to claim 7, characterized in that, A method for screening candidate drugs for the treatment of colorectal cancer includes: treating a culture system expressing or containing FH K80 acetylation with a substance to be screened; and detecting the expression or activity of FH K80 acetylation in the system; wherein, when the substance to be screened promotes the expression level or activity of FH K80 acetylation, the substance to be screened is a candidate drug for the treatment of colorectal cancer.

9. A method for screening candidate drugs for the treatment of colorectal cancer, characterized in that, The method includes: treating a culture system expressing or containing FH K80 acetylation with a substance to be screened; and detecting the expression or activity of FH K80 acetylation in the system; wherein, when the substance to be screened promotes the expression level or activity of FH K80 acetylation, the substance to be screened is a candidate drug for the treatment of colorectal cancer.

10. A method for promoting apoptosis of colorectal cancer cells in vitro, or inhibiting the proliferation or migration of colorectal cancer cells, characterized in that, The method includes applying any of the following substances or agents that promote the acetylation of FH K80: (1) A polypeptide for treating colorectal cancer, characterized in that, compared with wild-type FH protein, the 80th amino acid of the polypeptide is mutated from lysine to glutamine; (2) The nucleic acid encoding the polypeptide described in (1); (3) A carrier comprising the nucleic acid described in (2); (4) A host cell comprising the nucleic acid described in (2) or the vector described in (3).

11. The method according to claim 10, characterized in that, The colorectal cancer cells were selected from HCT116 cells and / or SW480 cells.

12. Application of FH K80 acetylation in the preparation of models, systems or devices for diagnosing colorectal cancer or predicting the prognosis of colorectal cancer.

13. The application according to claim 12, characterized in that, The system or apparatus includes: Acquisition module: Used to acquire acetylation site data of FH protein in the sample to be tested; Extraction module: used to extract acetylation data of the target site of FH protein in the sample to be tested, wherein the acetylation of the target site is FH K80 acetylation; Prediction module: Based on the FH K80 acetylation data of the target site, classification prediction is performed to obtain the classification result of whether the test sample has colorectal cancer. If the FH K80 acetylation level is high, the classification result is that the test sample does not have colorectal cancer or that the survival rate of colorectal cancer patients is high. If the FH K80 acetylation level is low, the classification result is that the test sample has colorectal cancer or that the survival rate of colorectal cancer patients is low.

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