Use of reagents for detecting RNF111 S255 phosphorylation in the preparation of products for the diagnosis of colorectal cancer

Abstract

The present application provides the use of a reagent for detecting RNF111 S255 phosphorylation in the preparation of a product for diagnosing colorectal cancer. The use of a reagent for detecting RNF111 S255 phosphorylation in the preparation of a product for evaluating the prognosis of a colorectal cancer patient and a product for predicting precancerous lesions of colorectal cancer is also provided. The use of the present application provides a breakthrough scientific basis for early diagnosis and prognosis stratification of colorectal cancer.

Description

Technical Field

[0001] This invention relates to the field of tumor molecular biology, and more specifically to the use of a reagent for detecting RNF111 S255 phosphorylation in the preparation of products for the diagnosis of colorectal cancer. Background Technology

[0002] Cancer is one of the leading causes of death and impact on human health worldwide. The number of cancer cases globally is projected to increase by 60% from 2018 to 2040, with approximately 29.4 million new cases expected in 2040. Therefore, in-depth research into the molecular mechanisms of cancer development and progression, and the identification of key regulatory molecules, is of significant scientific importance for developing precision oncology treatment strategies.

[0003] Chromosomal instability (CIN) is a hallmark of cancer, commonly found in refractory solid tumors, such as approximately 70% of colorectal cancers and 60% of non-small cell lung cancers. CIN is not only a concomitant event of cancer but can also drive its development. CIN is primarily caused by the continuous misseparation of chromosomes during cell division, leading to abnormalities in chromosome number and structure, often accompanied by micronucleus formation. While CIN is detrimental to the survival of healthy cells, tumor cells have evolved adaptive mechanisms, such as the STING signaling pathway. These mechanisms not only promote tumor cell proliferation and immune evasion but also drive tumor evolution through cell division, increasing the genetic diversity and heterogeneity of tumor cells and enabling them to acquire tolerance and resistance to drug treatment. Therefore, cancer patients with CIN are considered to have high invasiveness and poor prognosis.

[0004] However, persistent and excessive CIN can lead to catastrophic DNA damage and genomic instability in cancer cells, resulting in tumor cell death. In fact, cancer cells are far more sensitive to CIN than healthy, normal cells. Recent research shows that two FDA-approved drugs can achieve good therapeutic effects by increasing CIN levels in chromosomally unstable tumors, suggesting that targeting CIN has great clinical therapeutic potential.

[0005] However, since previous studies were mostly qualitative and lacked analysis of the quantitative regulatory mechanisms of CIN, there is a lack of biomarkers that can be directly assessed and quantified to guide clinical diagnosis and treatment.

[0006] In recent years, advancements in molecular biology have provided powerful tools for identifying clinical tumor markers. Genome-wide unbiased RNAi screening has identified the APC / C protein complex as a core regulatory molecule promoting excessive CIN (cancer intraepithelial neoplasia). However, while APC / C has multiple functions and its activity is tightly regulated in cells, the underlying regulatory mechanisms remain unclear. Although existing technologies have made preliminary progress at the molecular level, they have not yet addressed practical clinical needs.

[0007] Assessing CIN levels in clinical oncology patients has become a current research hotspot, and peripheral issues in cancer treatment have also become major social medical problems. Therefore, identifying molecular markers that can assess CIN levels in cancer patients is crucial and urgent for guiding clinical medication. Summary of the Invention

[0008] The technical problem to be solved by this invention is how to assess chromosomal instability, diagnose colorectal cancer, and evaluate its prognosis.

[0009] As described above, the APC / C protein complex is a core regulatory molecule promoting excessive colorectal intestinal cancer (CIN). Based on extensive preliminary experiments, we identified RNF111, a selective regulator of APC / C, as a potential novel molecular marker for CIN. The inventors therefore focused on the phosphorylation site of RNF111 (S255) and confirmed its use as a diagnostic marker for colorectal cancer using clinical samples, thus completing this invention. Accordingly, the technical solution adopted by this invention to solve its technical problem is as follows.

[0010] The first aspect of the present invention provides the use of a reagent for detecting the phosphorylation of RNF111 S255 in the preparation of products for the diagnosis of colorectal cancer.

[0011] A second aspect of the invention provides the use of a reagent for detecting RNF111 S255 phosphorylation in the preparation of products for assessing the prognosis of patients with colorectal cancer.

[0012] A third aspect of the invention provides the use of a reagent for detecting RNF111 S255 phosphorylation in the preparation of products for predicting precancerous lesions of colorectal cancer.

[0013] In some implementations, the reagent detects the expression level of RNF111 S255 phosphorylation in the sample.

[0014] In some implementations, the reagent includes an antibody that specifically binds to the phosphorylated protein RNF111 S255.

[0015] In some implementations, the antibody is an antibody that specifically binds to the peptide shown in SEQ ID NO.2.

[0016] In some embodiments, the antibody is a polyclonal antibody or antiserum obtained by immunizing animals with the peptide shown in SEQ ID NO.2 as an immunogen.

[0017] The present invention has at least the following beneficial effects:

[0018] This invention uses RNF111 phosphorylation modification as a target for tumor subtyping and diagnosis. The RNF111 phosphorylation modification antibody can specifically recognize the phosphorylation modification at the S255 site of RNF111. This has important clinical significance for in-depth research and large-scale verification of the correlation between RNF111 phosphorylation modification and chromosomal unstable tumors, for predicting tumor prognosis and providing precise treatment plans for individual patients.

[0019] On the one hand, the RNF111 S255 phosphorylation site can serve as a biomarker for the diagnosis of colorectal cancer in clinical samples.

[0020] On the other hand, the RNF111 S255 phosphorylation site can serve as a biomarker for prognostic assessment of colorectal cancer.

[0021] On the other hand, the RNF111 S255 phosphorylation site can serve as a biomarker for predicting precancerous lesions of colorectal cancer. Attached Figure Description

[0022] Figure 1 This diagram illustrates partial results of the preparation and verification of phosphorylated antibodies targeting the S255 site of the RNF111 protein in embodiments of the present invention. In the diagram, A represents the antigenic polypeptide used to prepare the antibody, which is conserved across species; B shows the AlphaFold prediction of the RNF111 protein structure, displaying the structural information of the S255 site; and C represents the in vitro kinase experiment, followed by Western blot analysis to evaluate the specificity of the antibody targeting the phosphorylated S255 site of RNF111 in cells.

[0023] Figure 2This is a schematic diagram showing partial results of the detection of novel biomarkers for metastasis, prognosis, and chromosomal instability in colorectal cancer patients using an antibody modified by phosphorylation at the S255 site of the RNF111 protein in an embodiment of the present invention. In this table, A shows the results of immunohistochemical detection of phosphorylation modification levels at the RNF111 S255 site in cancerous and adjacent tissues of colorectal cancer patients; B shows the analysis results of the degree of phosphorylation modification at the RNF111 S255 site and the metastasis status of colorectal cancer patients; C shows the KM curve analysis results of the degree of phosphorylation modification at the RNF111 S255 site and the prognosis of colorectal cancer patients; D shows the ROC curve analysis results of phosphorylation modification at the RNF111 S255 site in normal tissues and TNM stage 1 cancer tissues; E shows the WB analysis results of phosphorylation modification at the RNF111 S255 site in normal intestinal tissues, adenomas, and colorectal cancer tissues; F shows the WB analysis results of phosphorylation modification at the RNF111 S255 site and proteins related to chromosomal instability in colorectal cancer tissues; G, H, and I show the correlation analysis results of phosphorylation modification at the RNF111 S255 site and the expression of chromosomal instability-related proteins KIF20A and histone H3 in colorectal cancer tissues.

[0024] Figure 3 This diagram illustrates partial results of the effect of phosphorylation modification level of RNF111 protein at S255 site on sensitivity to drugs promoting chromosomal instability in embodiments of the present invention. A represents the effect of WEE1 inhibitors on cell proliferation in normal and RNF111 phosphorylation-deficient tumor cells; B represents the effect of WEE1 inhibitors on RNF111 S255 site phosphorylation modification and pyroptosis in normal and RNF111 phosphorylation-deficient tumor cells; C and D represent the effect of WEE1 inhibitors on mouse xenograft growth in tumor cells with high RNF111 phosphorylation modification. Detailed Implementation

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood in the technical field to which this application pertains. The following definitions are supplementary to those definitions in the art and relate to this application, but are not extrapolated to any relevant or unrelated situation, such as any conventionally used patent or application. While any methods and materials similar to or equivalent to those described herein may be used in practical testing of this application, the materials and methods described herein are preferred. Therefore, the terminology used herein is intended to describe specific embodiments only and is not intended to limit the invention.

[0026] The terms “including,” “comprising,” or “having”, when used before a step or element, indicate the addition of a further step or element, which is optional and not excluded.

[0027] As used herein, the RNF111 protein enhances the transcription of TGF-β target genes by promoting negatively regulated ubiquitination and proteasome degradation, such as the interaction of Smad protein with the transforming growth factor (TGF)-β / Nodal signaling pathway. As a modulator of the node signaling cascade, this protein plays a crucial role in the induction of the mesoderm during embryonic development. The amino acid sequence of the RNF111 protein is shown in SEQ ID NO.1 (NCBI accession number NP_001257459.1).

[0028] SEQ ID NO.1:

[0029] MSQWTPEYNELYTLKVDMKSEIPSDAPKTQESLKGILLHPEPIGAAKSFPAGVEMINSKVGNEFSHLCDDSQKQEKEMNGNQQEQEKSLVVRKKRKSQQAGPSYVQNCVKENQGILGLRQHLGTPSDEDNDSSFSDCLSSPSSSLHFGDSDTVTSDEDKEVSVRHSQTILNAKSRSHSARSHKWPRTETESVSGLLMKRPCLHGSSLRRLPCRKRFVKNNSSQRTQKQKERILMQRKKREVLARRKYALLPSSSSSSENDLSSESSSSSSTEGEEDLFVSASENHQNNPAVPSGSIDEDVVVIEASSTPQVTANEEINVTSTDSEVEIVTVGESYRSRSTLGHSRSHWSQGSSSHASRPQEPRNRSRISTVIQPLRQNAAEVVDLTVDEDEPTVVPTTSARMESQATSASINNSNPSTSEQASDTASAVTSSQPSTVSETSATLTSNSTTGTSIGDDSRRTTSSAVTETGPPAMPRLPSCCPQHSPCGGSSQNHHALGHPHTSCFQQHGHHFQHHHHHHHTPHPAVPVSPSFSDPACPVERPPQVQAPCGANSSSGTSYHEQQALPVDLSNSGIRSHGSGSFHGASAFDPCCPVSSSRAAIFGHQAAAAAPSQPLSSIDGYGSSMVAQPQPQPPPQPSLSSCRHYMPPPYASLTRPLHHQASACPHSHGNPPPQTQPPPQVDYVIPHPVHAFHSQISSHATSHPVAPPPPTHLASTAAPIPQHLPPTHQPISHHIPATAPPAQRLHPHEVMQRMEVQRRRMMQHPTRAHERPPPHPHRMHPNYGHGHHIHVPQTMSSHPRQAPERSAWELGIEAGVTAATYTPGALHPHLAHYHAPPRLHHLQLGALPLMVPDMAGYPHIRYISSGLDGTSFRGPFRGNFEELIHLEERLGNVNRGASQGTIERCTYPHKYKKVTTDWFSQRKLHCKQDGEEGTEEDTEEKCTICLSILEEGEDVRRLPCMHLFHQVCVDQWLITNKKCPICRVDIEAQLPSES

[0030] In one aspect of the invention, the reagent for detecting RNF111 S255 phosphorylation can be used to prepare a product for diagnosing colorectal cancer. As demonstrated in the examples below, the degree of phosphorylation modification at the RNF111 S255 site is significantly increased in colorectal cancer patient tissues. ROC curves were plotted to obtain sensitivity and specificity data, and the AUC (Area Under Curve) value was calculated to be 0.896, thereby establishing the association between RNF111 S255 phosphorylation and colorectal cancer.

[0031] In another aspect of the invention, the reagent for detecting RNF111 S255 phosphorylation can be used to prepare a product for assessing the prognosis of colorectal cancer patients. As demonstrated in the examples below, patients with high levels of phosphorylation modification at the S255 site of the RNF111 protein have a low cumulative survival probability, thereby allowing the detection of RNF111 S255 phosphorylation to be applied to the prognostic assessment of colorectal cancer patients.

[0032] In another aspect of the invention, the reagent for detecting RNF111 S255 phosphorylation can be used to prepare products for predicting precancerous lesions of colorectal cancer. As demonstrated in the examples below, the degree of phosphorylation modification at the S255 site of the RNF111 protein gradually increases in normal intestinal tissue, adenoma, and colorectal cancer in the same patient, thereby allowing the detection of RNF111 S255 phosphorylation to be applied to predict precancerous lesions of colorectal cancer.

[0033] In some implementations, the reagent detects the expression level of RNF111 S255 phosphorylation in the sample.

[0034] In some implementation schemes, immunohistochemistry is used for detection.

[0035] In some implementations, the reagent includes an antibody that specifically binds to the phosphorylated protein RNF111 S255.

[0036] In some implementations, the antibody is an antibody that specifically binds to the peptide shown in SEQ ID NO.2.

[0037] In some embodiments, the antibody is a polyclonal antibody or antiserum obtained by immunizing animals with the peptide shown in SEQ ID NO.2 as an immunogen.

[0038] In other aspects of the invention, the reagent for detecting RNF111 S255 phosphorylation can be used to prepare products for assessing the degree of chromosomal instability. As demonstrated in the examples below, phosphorylation modification at the S255 site of the RNF111 protein is significantly positively correlated with the expression of the chromosomal instability marker KIF20A, thus the phosphorylation modification at the S255 site of the RNF111 protein can be used to assess the degree of chromosomal instability.

[0039] In other aspects of the invention, the reagent for detecting RNF111 S255 phosphorylation can be used to prepare products for evaluating the therapeutic effects of drugs for chromosomal instability. As demonstrated in the examples below, the small molecule inhibitor Adavasertib can effectively inhibit the growth of tumors with high RNF111 phosphorylation expression, thereby phosphorylation modification of the RNF111 protein at the S255 site can be used to evaluate the therapeutic effects of drugs for chromosomal instability.

[0040] In this article, the product may be in the form of a kit, for example.

[0041] In some embodiments, the kit further includes labeled antibodies, including one or more of horseradish peroxidase-labeled antibodies, alkaline phosphatase-labeled antibodies, biotin-labeled antibodies, or fluorescent (such as FITC)-labeled antibodies, for example, horseradish peroxidase-labeled antibodies.

[0042] In some implementations, the kit may include, in addition to reagents for detecting RNF111 S255 phosphorylation, one or more of the following: processing solution, staining solution, buffer solution, washing solution, positive control, and negative control.

[0043] In some implementations, the detection methods that the kit can employ include one or more of enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blotting, and immunohistochemistry.

[0044] In some implementation schemes, when different detection methods are used, the instruments that can be used to observe the results include fluorescence microscopes, microplate readers, chemiluminescence analyzers, flow cytometers, and mass spectrometers.

[0045] In the specific operation process of this invention, the corresponding labeled antibody, processing solution, staining solution, buffer solution, and washing solution are selected according to the specific detection method of the kit.

[0046] In another aspect, the present invention provides an RNF111 protein-specific antibody, which is a polyclonal antibody phosphorylated at the S255 site of the RNF111 protein, the sequence of the S255 site of the RNF111 protein being shown in SEQ ID NO.2.

[0047] The following describes preferred embodiments of the present invention, but the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. All reagents used, unless otherwise specified, are commercially available conventional products.

[0048] Example 1: Design and preparation of a phosphorylation-modified antibody targeting the S255 site of the RNF111 protein

[0049] Materials and methods

[0050] 1. Materials

[0051] Physiological saline, Freund's complete adjuvant, Freund's incomplete adjuvant, two healthy 6-week-old New Zealand white rabbits (approximately 2 kg), and immunogen.

[0052] 2. Method

[0053] (1) A peptide was designed and synthesized based on the covalent binding of a phosphate group to the S255 site of the RNF111 protein. The specific sequence of the peptide is shown below (SEQ ID No. 2), where the phosphate group in parentheses is the modified phosphate group on the 4th serine residue: SSSS(-PO4). 3- )SSENDLSS. This peptide targets the phosphate group at the S255 site of the RNF111 protein and can be used as an immunogen for antibody preparation, i.e., an antigen for detecting antibody titer.

[0054] (2) Select two healthy 6-week-old New Zealand white rabbits, let them adapt to the new living environment, and stabilize for a few days before taking the first blood sample as a negative control.

[0055] (3) Dilute the immunogen with physiological saline, then mix it with the appropriate adjuvant in a 1:1 volume ratio. The antigen and adjuvant should be completely mixed to form a stable emulsion. This emulsion is administered subcutaneously around the rabbit's shoulders and intramuscularly in the hind thigh. Approximately 1 / 4 of the immunogen is used for each area. The initial immunization is given with 400 μg of antigen using Freund's complete adjuvant; subsequent booster immunizations are given with 100 μg of antigen each time, using Freund's incomplete adjuvant.

[0056] (4) Blood Collection: The rabbit was placed supine on a rabbit rack with its head fixed and its limbs secured with gauze. The head was slightly lowered to expose the neck. The fur was shaved and the skin was disinfected. A scalpel was used to make an incision about 10 cm along the midline of the neck, separating the subcutaneous connective tissue until the sternocleidomastoid muscles on both sides of the trachea were exposed. The loose tissue in the cervical triangle between the sternocleidomastoid muscles and the trachea was separated using straight hemostats to expose the common carotid artery. The artery was then freed using curved ophthalmic forceps, and the nerves and connective tissue were dissected. Two black silk threads were inserted under the artery, one at the distal end and the other at the proximal end. The silk thread at the distal end was ligated. The artery at the proximal end was clamped with a vascular clamp. The little finger was placed under the blood vessel, and a small incision was made in the arterial wall between the two silk threads using pointed ophthalmic scissors. A plastic drainage vessel was inserted. The silk thread at the proximal end was then ligated and fixed to the drainage vessel to prevent slippage. The hemostats were released to allow the blood to flow into the container. 100 mL of blood was collected from each rabbit.

[0057] (5) Separation of serum: Place the container in an oven at 37°C for 2 hours, transfer it to 4°C for precipitation overnight, and use a pipette to extract the serum the next morning. Add NaN3 to the serum to a final concentration of 1g / mL, aliquot and store at -20°C.

[0058] (6) ELISA titer measurement

[0059] Antigen coating: First, prepare 0.05M Na2CO3-NaHCO3 buffer (pH 9.6), then dilute the antigen (peptide shown in SEQ ID No. 2) to a concentration of 1:100 with freshly prepared 0.05M Na2CO3-NaHCO3 buffer. Take out a 96-well microplate and add 100 μL of the diluted antigen to each of 2×10 wells using a pipette. Label the wells without antigen coating and incubate overnight at 4°C.

[0060] Washing: Remove the coated microplate from 4°C, shake off the coating solution, add 100 μL of PBS containing 0.05% Tween-20 to each well and wash 5 times for 5 min each time. After the last wash, pat dry on absorbent paper.

[0061] Blocking: Add 100 μL of PBS-T (0.05% Tween-20-PBS) containing 5% normal bovine serum to each well using a pipette, and incubate at 37°C for 1 hour to block non-specific binding sites.

[0062] Washing: Remove the sealed microplate, shake off the blocking solution, add 100 μL of PBS containing 0.05% Tween-20 to each well and wash 5 times for 5 minutes each time. After the last wash, pat dry on absorbent paper.

[0063] Sample addition: The prepared rabbit antiserum was serially diluted 3-fold from 1:2K (2000) to 1:4374K using PBS-T. Then, rabbit serum before immunization was added to the first pair of wells in the two rows of wells coated with coating solution as a negative control. The remaining wells were added with samples in the rabbit antiserum concentration gradient, 100 μL per well, and incubated in a constant temperature incubator at 37℃ for 1 hour.

[0064] Washing: Remove the plate containing the sample, shake off the sample serum, add 100 μL of PBS containing 0.05% Tween-20 to each well and wash 5 times for 5 minutes each time. After the last wash, pat dry on absorbent paper.

[0065] Add enzyme-labeled secondary antibody: Dilute horseradish peroxidase-labeled goat anti-rabbit antibody to 1:10000 with PBS-T, add 100 μL to each well, and incubate at 37°C for 1 hour.

[0066] Washing: Remove the ELISA plate containing the enzyme-labeled secondary antibody, shake off the horseradish peroxidase-labeled goat anti-rabbit antibody, add 100 μL of PBS containing 0.05% Tween-20 to each well and wash 5 times for 5 minutes each time. After the last wash, pat dry on absorbent paper.

[0067] Add substrate and colorimetric solution: Prepare 0.4 mg / mL OPD colorimetric solution containing 0.03% H2O2 dissolved in phosphate-citric acid buffer (7 mL of 0.1 M Na2HPO4 and 3 mL of 0.1 M citric acid). Add 100 μL of freshly prepared OPD colorimetric solution to each well and let it stand at room temperature in the dark for 40 min.

[0068] Add stop solution: After the color development is uniform (the solution turns pale yellow), add 50 μL of 12% H2SO4 to each well to stop the reaction.

[0069] Microplate reader reading: Place the microplate with stop solution into the microplate reader and adjust the wavelength to 490nm to read the data.

[0070] (7) Prepare affinity columns for antibody affinity purification.

[0071] Prepare a protein A affinity column: Select 5 mL or 10 mL of protein A packing material, mix equal volumes of packing material and PBS buffer solution, stir, and remove air bubbles from the packing material by vacuuming.

[0072] Slowly add protein A packing material into the glass column to prepare the chromatography column. Avoid letting the column dry out during this process. After perfusion, equilibrate the column with 10 volumes of pre-cooled PBS buffer.

[0073] Protein A affinity chromatography:

[0074] After filtering the serum, it is loaded onto a pre-equilibrated protein A chromatography column. To detect the binding efficiency between the antiserum and the packing material, the eluent should be retained.

[0075] The column was washed with PBS buffer and then eluted with 150 mM glycine buffer. The eluent was collected and neutralized with neutralizing buffer to adjust the pH to 7.

[0076] Enrichment of target antibodies: The crude pure IgG obtained after purifying protein A is loaded onto a well-balanced antigen-peptide affinity chromatography column to specifically enrich the target antibodies.

[0077] Removal of non-specific antibodies: The target antibody obtained in the previous step is loaded onto a balanced negative peptide affinity chromatography column, and the eluent is collected directly to remove non-specific antibody components.

[0078] After obtaining the eluted antibody, it is concentrated with sucrose or polyethylene glycol and then dialyzed in PBS to remove salt. The antibody OD value is measured at a wavelength of 280 nm using a UV-Vis spectrophotometer. The obtained OD value divided by 1.35 is the concentration of the antibody. An equal volume of glycerol is added and the antibody can be stored at -20℃ for a long time.

[0079] (8) Western blot analysis of antibody efficacy and specificity

[0080] To verify whether the prepared antibody could specifically recognize the phosphorylation modification at the S255 site of human RNF111 protein, an in vitro kinase reaction system was constructed. The intracellular CDK1 kinase complex was purified using an in vitro immunoassay (IP) assay. Simultaneously, Flag-tagged wild-type RNF111 protein and the S255A mutant protein were also purified. The specific procedures are as follows:

[0081] Cells transfected with different plasmids were lysed using HEPES lysis buffer, followed by thorough cell lysis by sonication on ice. After centrifugation at 14,000 rpm for 10 min, the supernatant was collected, and immunoprecipitation was performed with the corresponding tag antibody. After incubation for 4 hours, protein A / G agarose beads were added, followed by another 6 hours of incubation. The cells were washed three times with HEPES lysis buffer, and then eluted with elution reagent. The eluted CDK1 kinase complex was co-incubated with RNF111 wild-type protein and S255A mutant protein, with or without the CDK1 inhibitor Ro-3306 added to verify the specificity of the reaction. After adding 100 μM ATP, the cells were incubated at 30°C for 1 hour for biochemical reactions. After the in vitro reaction, 5× loading solution was added, and the cells were boiled in a water bath. Proteins were collected for Western blot analysis. Protein samples were loaded onto an SDS gel for electrophoresis. After successful electrophoresis, the samples were transferred to a membrane at 90 mA for 3 hours, then blocked in 5.0% skim milk at room temperature for 1 hour, followed by the addition of primary antibody and incubation at 4°C overnight. The following day, the membrane was washed three times with TBST reagent, 10 minutes each time. After incubating the secondary antibody at room temperature for 1 hour, the membrane was washed three times with TBST reagent and then developed.

[0082] result

[0083] The structure of the peptide segment shown in SEQ ID No. 2 is as follows: Figure 1 As shown in Figure A, antibodies with good titers were prepared by immunizing rabbits using this peptide. Subsequently, the protein structure of RNF111 was predicted using AlphaFold, and its spatial structural features at the S255 position were displayed. Figure 1 The results of the Western blot were as follows: Figure 1 As shown in Figure C, both the prepared RNF111 protein S255 phosphorylation modification antibody and the phosphorylated serine pan-antibody can detect the phosphorylation modification of human RNF111 protein at the S255 site, and both can be inhibited by Ro-3306. These results indicate that the antibody has good specificity and can accurately identify the phosphorylation modification of RNF111 protein at the S255 site in cells. Therefore, the antibody prepared in this embodiment can be used for subsequent experiments.

[0084] Example 2: Changes in the degree of phosphorylation modification of RNF111 S255 site in colorectal cancer tissue and adjacent normal tissue

[0085] Materials and methods

[0086] 1. Materials

[0087] Human colorectal cancer tumor chip (Xinchao Biotechnology Co., Ltd., colorectal cancer COC1601, containing pathological information of cancer tissue and adjacent tissue of 80 clinical colorectal cancer patients), and the RNF111 protein S255 site phosphorylation modified antibody prepared in Example 1.

[0088] 2. Method

[0089] (1) Place the slices in a 60℃ oven and bake for 20 minutes to melt the wax;

[0090] (2) Dewaxing and hydration: Xylene 1 → Xylene 2 → Anhydrous ethanol 1 → Anhydrous ethanol 2 → 95% ethanol → 90% ethanol → 80% ethanol → 70% ethanol → water, 5 min each;

[0091] (3) Wash with PBS for 5 min;

[0092] (4) Microwave antigen retrieval: Preheat the commercially available sodium citrate retrieval solution (Beyotime, P0081) to boiling point in the microwave. Immerse the slides in the sodium citrate retrieval solution and microwave on medium heat for 15 minutes. After retrieval, allow the beaker to cool to room temperature before removing the slides. (Alternatively, allow them to cool to room temperature at 4°C).

[0093] (5) Wash with PBS for 5 min, repeat 3 times;

[0094] (6) Hydrogen peroxide blocking: Dilute 30% hydrogen peroxide solution with PBS to a concentration of 3% hydrogen peroxide solution, and block in the dark for 20 min;

[0095] (7) Wash with PBS for 5 min, 3 times;

[0096] (8) Blocking: Wipe the untissued part of the slide with filter paper, shake off excess water on the tissue, draw a circle around the tissue with an immunohistochemistry pen, quickly add 10% BSA-PBS, put it in a humidified chamber, and block at 37°C for 30 min.

[0097] (9) Primary antibody incubation: Dilute the phosphorylation-modified antibody at the S255 site of RNF111 protein prepared in Example 1 with 5% BSA-PBS at a ratio of 1:100. After blocking, remove the slide, shake off the blocking solution on the tissue, quickly add the diluted antibody, and place it in a humidified chamber overnight at 4°C.

[0098] (10) Wash with PBS for 5 min, repeat 5 times;

[0099] (11) Secondary antibody incubation: Dilute rabbit secondary antibody with 5% BSA-PBS at a ratio of 1:200. After removing water from the tissue, quickly add the diluted secondary antibody and incubate at 37°C for 45 min.

[0100] (12) Wash with PBS for 5 min, repeat 5 times;

[0101] (13) DAB color development: Prepare DAB color development solution with 1 drop of solution A and 1 mL of solution B. Shake off the water on the tissue, quickly add DAB color development solution, develop for 5 minutes, observe under a microscope during color development, and immerse in pure water after staining to stop color development.

[0102] (14) Wash with pure water for 25 minutes;

[0103] (15) Staining the nucleus: Remove excess water from the tissue, add hematoxylin staining solution and stain for 5 minutes, then separate the color with separation solution for 2 seconds until it turns pink. Then immerse it in pure water and observe the staining effect under a microscope. Wash with tap water for 15 minutes or use blue-return solution to restore the nucleus color to blue.

[0104] (16) Mounting: pure water → 70% ethanol → 80% ethanol → 90% ethanol → 95% ethanol → anhydrous ethanol → anhydrous ethanol → xylene → xylene for 5 minutes each. Remove the slide, wipe off the excess liquid with filter paper, drop mounting resin on one end of the tissue on the slide, and gently cover the tissue with the slide, making sure there are no air bubbles on the tissue.

[0105] result

[0106] Following staining and scanning, Image Pro Plus software was used to analyze the IOD (accumulated optical density) levels of the RNF111 S255 site phosphorylation-modified antibody staining, followed by data processing and statistical analysis. Results of colorectal cancer patient tissues and their corresponding adjacent normal tissues are shown below. Figure 2 As shown in Figure AC, the phosphorylation level of RNF111 at S255 is significantly increased in colorectal cancer tissues compared to adjacent normal tissues. Furthermore, a comparative analysis of expression levels between patients without metastasis and those with metastasis yielded the following results: Figure 2 As shown in Figure D, the degree of phosphorylation modification at the S255 site of RNF111 was found to be closely related to the transfer.

[0107] To further verify the correlation between RNF111 (S255) and colorectal cancer, this invention conducted a statistical evaluation of its diagnostic performance in 11 patients with stage I AJCC, and plotted ROC curves as follows. Figure 2 As shown in Figure D, the sensitivity (true positive rate) was 100, the specificity (true negative rate) was 75, and the calculated AUC (Area Under Curve) value was 0.896, indicating that RNF111 (S255) can be used as a biomarker for the diagnosis of colorectal cancer.

[0108] Example 3: KM curve analysis of the effect of RNF111 S255 phosphorylation modification on the prognosis of colorectal cancer patients

[0109] Materials and methods

[0110] 1. Materials

[0111] Human colorectal cancer tumor microarray (same as Example 2), with the RNF111 protein S255 site phosphorylation modified antibody prepared in Example 1.

[0112] 2. Method

[0113] The prognostic value of RNF111(S255) expression in colorectal cancer was assessed based on microarray-associated clinical information. The microarray was processed using immunohistochemistry, as in Example 2.

[0114] result

[0115] Using GraphPad Prism 5 software, the Kaplan-Meier test was employed to analyze the correlation between the phosphorylation level of RNF111 protein at site S255 and the cumulative survival rate of colorectal cancer patients. Results are as follows: Figure 2 As shown in Figure E (where protein levels are defined by the median relative expression), patients with high levels of phosphorylation at the S255 site of the RNF111 protein have a lower cumulative survival probability, and vice versa. This suggests that the degree of phosphorylation at the S255 site of the RNF111 protein can serve as a prognostic molecular marker for colorectal cancer.

[0116] Example 4: Analysis of changes in phosphorylation modification at the S255 site of RNF111 in early colorectal cancer tissues

[0117] Materials and methods

[0118] 1. Materials

[0119] Four normal mesenteric tissues, adenomas, and colorectal cancer tissues from clinical colorectal cancer patients were selected (patient tissue microarrays were purchased from Shanghai Zhuohan Pharmaceutical Technology Co., Ltd., serial number COC1601, and four of these cases were selected in this example). The RNF111 protein S255 site phosphorylation modification antibody prepared in Example 1 was used.

[0120] 2. Method

[0121] Western blot, the specific steps are as follows:

[0122] (1) Extraction of tissue protein: Cut tissue pieces the size of mung beans and add 200 μL of RIPA lysis buffer (containing protease inhibitors). Grind the tissue using a tissue homogenizer, centrifuge at 4°C to collect the supernatant, add loading and boil in water for 10 minutes.

[0123] (2) Electrophoresis: After loading the sample protein and marker onto the SDS gel, electrophoresis is performed by turning on the current. The electrophoresis is stopped after the sample is fully separated in the separating gel.

[0124] (3) Transfer: The SDS adhesive is bonded to the NC membrane in the transfer solution, placed in the transfer tank, and after adding sufficient transfer solution, the transfer is started with a current of 90 mA and the membrane is transferred in ice water for 3 hours.

[0125] (4) Sealing: Place the transferred NC membrane into 5% skim milk and incubate at room temperature for 1 hour.

[0126] (5) Primary antibody: Dilute the primary antibody (the phosphorylation-modified antibody at the S255 site of the RNF111 protein prepared in Example 1) according to the corresponding ratio, put the membrane into the primary antibody solution, and incubate overnight at 4°C.

[0127] (6) Washing the membrane: After recovering the primary antibody solution, put the membrane into TBST solution and wash it three times on a shaker for 10 minutes each time.

[0128] (7) Secondary antibody: Place the washed membrane into the goat anti-rabbit secondary antibody solution according to the corresponding resistance and incubate at room temperature for 1 hour.

[0129] (8) Membrane washing: After recovering the secondary antibody solution, put the membrane into TBST solution and wash it three times on a shaker for 10 minutes each time.

[0130] (9) Development: Add substrate color developing solution to NC membrane, incubate for 5 minutes, then place in dark box and develop in dark room.

[0131] result

[0132] Western blot results are as follows Figure 2 As shown in Figure F, the analysis revealed that the phosphorylation level of the S255 site of the RNF111 protein gradually increased in normal intestinal tissue, adenoma, and colorectal cancer of the same patient, indicating that phosphorylation modification of the S255 site of the RNF111 protein can effectively predict precancerous lesions.

[0133] Example 5: Analysis of the relationship between the degree of phosphorylation modification at the S255 site of RNF111 in colorectal cancer tissue and chromosomal instability

[0134] Materials and methods

[0135] 1. Materials

[0136] In addition, 9 clinical colorectal cancer tissues (patient tissues were obtained from fresh tumor tissue specimens collected from the Cancer Hospital of Chinese Academy of Medical Sciences and Peking Union Medical College, with ethics number NCC2018-047; 9 cases were selected in this example) were used to prepare the RNF111 protein S255 site phosphorylation modification antibody in Example 1.

[0137] 2. Method

[0138] Western blot was used to detect the phosphorylation level of RNF111 protein at S255 and the expression of the chromosomal instability marker KIF20A in different tumor tissues. Subsequently, ImageJ software was used to analyze the correlation between the phosphorylation level of RNF111 protein at S255 and KIF20A / histone H3 expression. The Western blot method was the same as in Example 4.

[0139] result

[0140] like Figure 2 China G to Figure 2 As shown in Figure I, phosphorylation modification at site S255 of RNF111 protein was found to be significantly positively correlated with KIF20A expression and significantly negatively correlated with histone H3 expression, indicating that phosphorylation modification at site S255 of RNF111 protein can effectively assess the degree of chromosomal instability and serve as a novel biomarker for tumor chromosomal instability.

[0141] Example 6: Analysis of the relationship between the degree of phosphorylation modification at the S255 site of RNF111 and the therapeutic effect of drugs that promote chromosome instability on tumors.

[0142] Materials and methods

[0143] 1. Materials

[0144] A stable CT26 tumor cell line expressing wild-type RNF111 and phosphorylation modification site mutations was constructed using the Beyotime CCK8 kit. BALBA / c mice (n=5 per group, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) and the WEE1 inhibitor adavasertib were used.

[0145] 2. Method

[0146] The CCK8 experiment was performed according to the kit instructions.

[0147] Western blot, the method is the same as in Example 4.

[0148] Mouse xenograft experiment

[0149] (1) Cell counting: CT26 tumor cells stably expressing wild-type RNF111 and phosphorylation modification site mutations were digested into single-cell suspensions for cell counting and diluted to 1×10⁻⁶ cells per cell. 6 cells / 100 μL.

[0150] (2) Subcutaneous injection: 100 μL of cell suspension was injected subcutaneously into the groin of each mouse.

[0151] (3) After one week of treatment, observe the tumor formation. When the tumor volume is less than 150 mm... 3 Intervention was initiated by administering a placebo and a WEE1 inhibitor (by gavage, 50 mg / kg, every two days for 10 doses).

[0152] (4) Measure the tumor volume every 3 days. Tumor volume = major diameter × minor diameter. 2 / 2, and plot the volume change curve.

[0153] result

[0154] like Figure 3 As shown in Figure A, the proliferation of cells in each group was detected using CCK8 assay. It was found that Adavosertib effectively inhibited the proliferation of RNF111 wild-type cells, but could not inhibit the proliferation of cells with phosphorylation modification site mutations. Figure 3 As shown in Figure B, downstream cellular pathways were examined using Western blot. Adavosertib specifically promoted GSDME-related pyroptosis in RNF111 wild-type cells, but GSDME cleavage activation was not detected in cells with phosphorylation modification sites. Figure 3 As shown in C and D, a mouse xenograft model was constructed, and the effect of adavasertib on the proliferation of RNF111 wild-type cells in vivo was analyzed. It was found that adavasertib can effectively inhibit the growth of tumors with high expression of RNF111 phosphorylation, indicating that the degree of phosphorylation modification at the S255 site of RNF111 can be used to evaluate the therapeutic effect of chromosomal instability.

[0155] This document uses specific embodiments to illustrate the principles and implementation methods of the present invention. The descriptions of these embodiments are merely illustrative of the method and its central idea, and are not intended to limit the process. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall under the protection of the claims of the present invention.

Claims

1. Use of a reagent for detecting RNF111 S255 phosphorylation in the preparation of products for the diagnosis of colorectal cancer.

2. The use according to claim 1, characterized in that, The reagent was used to detect the expression level of RNF111 S255 phosphorylation in the sample.

3. The use according to claim 1, characterized in that, The reagent includes an antibody that specifically binds to the phosphorylated RNF111 S255 protein.

4. The use according to claim 3, characterized in that, The antibody is an antibody that specifically binds to the peptide SSSSSSENDLSS, wherein the 4th serine residue of the peptide is modified with a phosphate group.

5. The use according to claim 3, characterized in that, The antibody is a polyclonal antibody obtained by immunizing rabbits with the peptide SSSSSSENDLSS as an immunogen, wherein the 4th serine residue of the peptide is modified with a phosphate group.

6. Use of reagents for detecting RNF111 S255 phosphorylation in the preparation of products for assessing the prognosis of patients with colorectal cancer.

7. The use according to claim 6, characterized in that, The reagent includes an antibody that specifically binds to the phosphorylated RNF111 S255 protein.

8. The use according to claim 7, characterized in that, The antibody is an antibody that specifically binds to the peptide SSSSSSENDLSS, wherein the 4th serine residue of the peptide is modified with a phosphate group.

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