Application of the biomarker PLXNC1 in the diagnosis, treatment and prognostic prediction of colorectal cancer

Abstract

This invention discloses the application of the biomarker PLXNC1 in the diagnosis, treatment, and prognosis prediction of colorectal cancer. The biomarker PLXNC1 has good diagnostic efficacy for colorectal cancer, with high accuracy, sensitivity, and specificity. Furthermore, this invention experimentally verifies for the first time that reagents that inhibit PLXNC1 expression levels can significantly inhibit the proliferation, migration, and invasion of colorectal cancer cells, indicating that the biomarker PLXNC1 described in this invention can be applied to the diagnosis, treatment, and prognosis prediction of colorectal cancer and has good clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of the biomarker PLXNC1 in the diagnosis, treatment and prognosis prediction of colorectal cancer. Background Technology

[0002] Colorectal carcinoma (CRC) is one of the most common malignant tumors of the digestive system that seriously affect human health and survival. The 2018 World Cancer Statistics report indicates that among 36 common cancers, colorectal cancer had over 1.8 million new cases, accounting for approximately 10.2% of all new cancer cases, ranking third; and approximately 880,000 cancer deaths, accounting for approximately 9.2% of all cancer deaths, ranking second. Among 185 countries and regions worldwide, my country's incidence and mortality rates for malignant tumors are at a moderately high level (Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer JClin, 2018, 68(6):394-424.). The latest research data shows that the number of newly diagnosed colorectal cancer patients exceeded 1.8 million in 2020, and the incidence rate is showing an increasing trend year by year.

[0003] The high mortality rate of colorectal cancer is due to the lack of obvious clinical symptoms in the early stages, often leading to diagnosis at an advanced stage. Reports indicate that the 5-year survival rate for patients diagnosed early is approximately 90%, while the rate drops to 5%-10% for those diagnosed late (Lee PY, Chin SF, Low TY, et al. Probing the colorectal cancer proteome for biomarkers: Current status and perspectives[J]. J Proteomics, 2018, 187:93-105.). Therefore, early screening and diagnosis of colorectal cancer can significantly reduce its incidence and mortality. Currently, widely used screening methods include fecal testing, imaging examinations, endoscopy, and tumor marker testing. However, fecal testing has low specificity, imaging examinations are expensive, and endoscopy has poor compliance. Tumor markers, on the other hand, offer advantages in effectiveness, cost-effectiveness, and safety, improving overall acceptance of screening and making them an important tool for colorectal cancer screening.

[0004] With the completion of the Human Genome Project and the rapid development of high-throughput sequencing technology, gene screening technology has become a new diagnostic method for colorectal cancer, showing significant advantages in the early diagnosis of colorectal cancer. However, because colorectal cancer often presents with no obvious symptoms in its early stages, potential patients still require further diagnosis through colonoscopy. Therefore, providing an accurate and effective biomarker for colorectal cancer in its early diagnosis and screening to assist in early diagnosis and later treatment is of great significance in this field. In this regard, this invention has found that PLXNC1 can be used for the early diagnosis and prognostic prediction of colorectal cancer. Further experimental studies have shown that reducing the expression level of PLXNC1 can significantly reduce the proliferation, migration, and invasion of colorectal cancer cells, and can be used in the treatment of colorectal cancer. Currently, there are no reports on the application of PLXNC1 in the diagnosis, treatment, and prognostic prediction of colorectal cancer. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide an application of the biomarker PLXNC1 in the diagnosis, treatment and prognosis prediction of colorectal cancer, so as to achieve early diagnosis, early intervention and early treatment of colorectal cancer, and further improve the quality of life and survival rate of patients.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides the use of a reagent for detecting the expression level of the biomarker PLXNC1 in a sample in the preparation of a product for the early diagnosis of colorectal cancer.

[0008] Furthermore, the reagents include those used to detect the expression level of the biomarker PLXNC1 in the sample using sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, chromatography technology, and mass spectrometry technology.

[0009] Furthermore, the reagents for detecting the expression level of the biomarker PLXNC1 in the sample include reagents for detecting the expression level of the biomarker PLXNC1 mRNA in the sample, and / or reagents for detecting the expression level of the protein and / or peptide encoded by the biomarker PLXNC1 in the sample;

[0010] Preferably, the reagent for detecting the expression level of biomarker PLXNC1 mRNA in the sample includes a probe that specifically recognizes the biomarker PLXNC1 and / or primers that specifically amplify the biomarker PLXNC1;

[0011] Preferably, the reagent for detecting the expression level of the protein and / or peptide encoded by the biomarker PLXNC1 in the sample includes an antibody that specifically binds to the biomarker PLXNC1, and / or an antibody fragment, and / or an affinity protein.

[0012] More preferably, the sequences of the primers for the specific amplification of the biomarker PLXNC1 are shown in SEQ ID NO:3-SEQ ID NO:4.

[0013] Furthermore, the sequencing technologies include (but are not limited to): first-generation sequencing, second-generation sequencing, and third-generation sequencing. First-generation sequencing, also known as Sanger sequencing, is a sequencing technology that utilizes DNA polymerase synthesis reactions. First-generation sequencing is a sequencing technology based on the Sanger method. Second-generation sequencing is based on massive parallel analysis (MPS), which can simultaneously complete the synthesis of complementary strands of sequencing templates and the acquisition of sequence data. Third-generation sequencing is based on single-molecule sequencing and massive parallel sequencing technologies.

[0014] Furthermore, the nucleic acid hybridization technology refers to the process by which complementary nucleotide sequences (DNA and DNA, DNA and RNA, RNA and RNA, etc.) form non-covalent bonds through Watson-Crick base pairing, thereby forming stable homologous or heterologous double-stranded molecules, also known as nucleic acid hybridization.

[0015] Furthermore, the aforementioned nucleic acid amplification technology refers to a general term for a large class of technical methods. Currently, nucleic acid amplification technologies include conventional PCR, real-time fluorescence PCR, and isothermal nucleic acid amplification technology, which can specifically amplify trace amounts of target DNA by millions of times, thereby greatly improving the ability to analyze and detect DNA molecules. It can detect single DNA molecules or samples containing only one target DNA molecule per 100,000 cells.

[0016] Furthermore, the protein immunoassay technique refers to a class of methods that include radioimmunoassay, direct, indirect or comparative enzyme-linked immunosorbent assay, enzyme immunoassay, fluorescence immunoassay, Western blotting, immunoprecipitation, and immunoassay based on any particle (e.g., using gold particles, silver particles or latex particles, magnetic particles or quantum dots) to detect target analytes, and can be performed in the form of microtiter plates or strips.

[0017] Furthermore, the chromatographic technique refers to a method for separating and analyzing various components in a complex mixture. It utilizes the fact that different substances have different partition coefficients in a system composed of a stationary phase and a mobile phase. When the two phases move relative to each other, these substances move together with the mobile phase and undergo repeated partitioning between the two phases, thereby achieving separation of the substances.

[0018] Furthermore, the mass spectrometry technique refers to a method that uses electric and magnetic fields to separate and detect moving ions (charged atoms, molecules or molecular fragments, molecular ions, isotopic ions, fragment ions, rearranged ions, multiply charged ions, metastable ions, negative ions, and ions generated by ion-molecule interactions) according to their mass-to-charge ratio. Measuring the accurate mass of the ions allows for the determination of their compound composition.

[0019] Furthermore, the sample is selected from the subject's blood or tissue.

[0020] A second aspect of the invention provides the use of a reagent for detecting the expression level of the biomarker PLXNC1 in a sample in the preparation of products for predicting the prognosis of colorectal cancer.

[0021] A third aspect of the present invention provides a product for the early diagnosis of colorectal cancer or for predicting the prognosis of colorectal cancer.

[0022] Furthermore, the product includes reagents for detecting the expression level of the biomarker PLXNC1 in the sample;

[0023] Preferably, the product includes a chip, a reagent kit, a test strip, and a high-throughput sequencing platform;

[0024] Preferably, the reagent for detecting the expression level of biomarker PLXNC1 in the sample includes reagents for detecting the expression level of biomarker PLXNC1 mRNA in the sample, and / or reagents for detecting the expression level of protein and / or peptide encoded by biomarker PLXNC1 in the sample;

[0025] More preferably, the reagent for detecting the expression level of biomarker PLXNC1 mRNA in the sample includes a probe that specifically recognizes biomarker PLXNC1 and / or primers that specifically amplify biomarker PLXNC1;

[0026] More preferably, the reagent for detecting the expression level of the protein and / or peptide encoded by the biomarker PLXNC1 in the sample includes an antibody that specifically binds to the biomarker PLXNC1, and / or an antibody fragment, and / or an affinity protein.

[0027] Most preferably, the primer sequences for the specific amplification of the biomarker PLXNC1 are shown in SEQ ID NO:3-SEQ ID NO:4.

[0028] Furthermore, the chip can be prepared using conventional biochip preparation methods known to those skilled in the art, including (but not limited to): using a solid-phase support of a modified glass slide or silicon wafer, the 5' end of the probe containing an amino-modified polydT string, preparing the oligonucleotide probe into a solution, and then using a spotting instrument to spot it onto the modified glass slide or silicon wafer, arranging it into a predetermined sequence or array, and then fixing it by leaving it overnight, thus obtaining the chip described in this invention.

[0029] Furthermore, the kit also includes instructions for use or a label, a positive control, a negative control, a buffer, an adjuvant, or a solvent; the instructions for use or the label details how to use the kit to test samples and how the kit is used to detect colorectal cancer.

[0030] Furthermore, the kit may also contain a variety of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed in this invention. Any reagent that is based on the detection of the biomarker PLXNC1 to diagnose colorectal cancer is included within the scope of protection of this invention.

[0031] The primers included in the products described in this invention can be prepared by chemical synthesis. They can be appropriately designed using methods well known to those skilled in the art and referenced in known information, and are prepared by chemical synthesis, and are not limited to the primers shown in SEQ ID NO:3-SEQ ID NO:4 as described in this invention. The antibodies in the products described in this invention can be antibodies or fragments thereof of any structure, size, immunoglobulin class, origin, etc., as long as they bind to the target protein. The antibodies or fragments thereof included in the products of this invention can be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody that retains the binding activity of the antibody against the antigen, or a peptide containing a portion of an antibody. Antibody fragments may include F(ab′)2, Fab′, Fab, single-chain Fv (scFv), disulfide-bonded Fv (dsFv) or polymers thereof, dimerized V region (biantibody), or peptides containing CDR. Antibodies can be obtained by methods well known to those skilled in the art. For example, a mammalian cell expression vector that retains the whole or part of the target protein or integrates the polynucleotides encoding them can be prepared as an antigen. After immunizing an animal with the antigen, immune cells are obtained from the immunized animal and fused with cancer cells to obtain a hybridoma. Antibodies are then collected from hybridoma cultures. Finally, monoclonal antibodies against the marker protein can be obtained by antigen-specific purification of the obtained antibodies using the marker protein or a portion thereof, which is used as the antigen.

[0032] A fourth aspect of the invention provides the use of the biomarker PLXNC1 in the preparation of pharmaceutical compositions for the treatment and / or prevention of colorectal cancer.

[0033] Furthermore, the pharmaceutical composition includes an inhibitor that reduces PLXNC1 expression levels;

[0034] Preferably, the sequence of the inhibitor that reduces PLXNC1 expression level is shown in SEQ ID NO:1-SEQ ID NO:2.

[0035] Furthermore, the inhibitors for reducing PLXNC1 expression levels described in this invention are not limited to those shown in SEQ ID NO:1-SEQ ID NO:2. Any reagent that can inhibit or reduce PLXNC1 expression levels is within the scope of protection of this invention.

[0036] A fifth aspect of the present invention provides a pharmaceutical composition for treating and / or preventing colorectal cancer.

[0037] Furthermore, the pharmaceutical composition includes an inhibitor that reduces PLXNC1 expression levels;

[0038] Preferably, the sequence of the inhibitor that reduces PLXNC1 expression level is shown in SEQ ID NO:1-SEQ ID NO:2.

[0039] Furthermore, the pharmaceutical composition may also include one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending primarily on the route of administration and the designed dosage form.

[0040] Therapeutic inert inorganic or organic carriers known to those skilled in the art include (but are not limited to): lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyhydroxy compounds such as polyethylene glycol, water, sucrose, ethanol, glycerin, and the like. Various preservatives, lubricants, dispersants, flavoring agents, humectants, antioxidants, sweeteners, colorants, stabilizers, salts, buffers, and the like may also be added as needed to aid in the stability of the formulation or to help improve its activity or bioavailability, or to produce an acceptable taste or odor when taken orally. The formulations that can be used in such compositions may be in the form of the original compound itself, or optionally in the form of its pharmaceutically acceptable salts. The compositions thus formulated can be administered by any suitable method known to those skilled in the art as needed. When using the pharmaceutical composition, a safe and effective amount of the drug of the present invention is administered to a human, and the specific dosage should also take into account factors such as the route of administration and the patient's health condition, all of which are within the scope of a skilled physician's skill.

[0041] A sixth aspect of the present invention provides a method for screening candidate drugs for the treatment and / or prevention of colorectal cancer.

[0042] Furthermore, the method includes the following steps:

[0043] (1) Contact the analyte with a system containing or expressing PLXNC1;

[0044] (2) Detect the expression level of PLXNC1 in the system;

[0045] (3) Select substances that can reduce PLXNC1 expression levels as candidate drugs for the treatment and / or prevention of colorectal cancer.

[0046] Furthermore, the system includes (but is not limited to): cellular system, subcellular system, solution system, tissue system, organ system, or animal system.

[0047] Furthermore, the substances to be tested include (but are not limited to): interfering molecules, nucleic acid inhibitors, and small molecule compounds designed to target the biomarker PLXNC1 or its upstream or downstream genes.

[0048] A seventh aspect of the invention provides the use of the biomarker PLXNC1 in screening candidate drugs for the treatment and / or prevention of colorectal cancer.

[0049] Unless otherwise defined, all technical and scientific terms used in the context of this invention have the same meaning as understood by one of ordinary skill in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention; furthermore, some terms are explained below.

[0050] As used herein, the term "expression level" refers to the degree to which a specific biomarker sequence is transcribed from its genomic locus, i.e., the concentration of the biomarker in one or more analyzed blood samples. In a specific embodiment of the invention, the specific biomarker is PLXNC1.

[0051] As used herein, the term "biomarker" refers to a molecular indicator possessing specific biological, biochemical, or other characteristics that can be used to determine the presence or absence of a particular disease or condition and / or the severity of a particular disease or condition. In a specific embodiment of the invention, the particular disease or condition refers to colorectal cancer. In this invention, the term "biomarker" specifically refers to a compound, preferably a gene, that is differentially present (i.e., increased or decreased) in a biological sample from a subject or group of subjects with a first phenotype (e.g., having the disease), compared to a biological sample from a subject or group of subjects with a second phenotype (e.g., no disease). This term generally refers to the presence / concentration / content of one gene or the presence / concentration / content of two or more genes; in a specific embodiment of the invention, the biomarker is PLXNC1.

[0052] Biomarkers can be present differentially at any level, but are generally present at levels that are increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, and at least 140%. %, at least 150%, or more; or generally present at levels reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% (i.e., absent), preferably, the biomarker is statistically significant (P < 0.05).

[0053] As used herein, the term "sample" refers to a composition obtained from or derived from a subject (e.g., an individual of interest) containing cells and / or other molecular entities to be characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological characteristics. For example, a sample refers to any sample derived from a subject of interest that is expected or known to contain cells and / or molecular entities to be characterized. Samples include, but are not limited to, tissue samples (e.g., tumor tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, tissue culture fluid, tissue extracts, homogenized tissue, tumor tissue, cell extracts, and combinations thereof. As a preferred embodiment, the sample is selected from blood, serum, and plasma; as another preferred embodiment, the sample is selected from tissue.

[0054] As used herein, the term "subject" refers to any animal, including both human and non-human animals. Non-human animals include all vertebrates, such as mammals like non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; as well as non-mammals such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human.

[0055] As used herein, the term "treatment" refers to any treatment involving humans or animals (e.g., applied by a veterinarian) that achieves certain desired therapeutic effects, such as inhibiting the development of a disease (including slowing its progression, stopping its development), improving the condition, and curing the disease. It also includes treatment as a preventative measure (e.g., prevention). Use in patients who have not yet developed the disease but are at risk of developing it is also included in the term "treatment."

[0056] As used herein, the term "differential expression" refers to a change in the expression level of a biomarker in a target sample compared to a control sample, which may be a sample from a healthy individual. This change can be either upregulated (i.e., an increase in the concentration of the biomarker in the target sample) or downregulated (i.e., a decrease in the concentration of the biomarker or its disappearance in the target sample). In a specific embodiment of the invention, the biomarker is PLXNC1.

[0057] As used in this article, the term "inhibitor" or "inhibitor that reduces the expression level of a biomarker" refers to a chemically modified inhibitor specifically targeting a specific biomarker in the cell, thereby specifically targeting and knocking out the biomarker.

[0058] The biomarker PLXNC1 (plexin C1) described in this invention has a Gene ID of 10154 in NCBI (https: / / www.ncbi.nlm.nih.gov / ) and is located on band 2 of region 2 on the long arm of chromosome 12.

[0059] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0060] (1) This invention first discovered that PLXNC1 can be used as a biomarker for early diagnosis and prognosis prediction of colorectal cancer. It shows significant differential expression between colorectal cancer tissue and normal adjacent tissue, and has high diagnostic efficacy with an AUC value as high as 0.90. It can be used for early diagnosis or auxiliary diagnosis of colorectal cancer, as well as prognosis prediction.

[0061] (2) This invention is the first to discover that inhibiting the expression of PLXNC1 can significantly inhibit the proliferation, migration and invasion of colorectal cancer cells, indicating that the reagent that inhibits the expression level of the biomarker PLXNC1 can be used in the treatment of colorectal cancer and has a very good clinical application prospect in this field. Attached Figure Description

[0062] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0063] Figure 1 The graph shows the differential expression of PLXNC1 mRNA between the control group and the colorectal cancer group;

[0064] Figure 2 The figure shows the results of differential expression of PLXNC1 mRNA between cancerous tissue and normal adjacent tissue in patients with primary colorectal cancer;

[0065] Figure 3 This image shows the staining results of microarrays of cancerous tissue and normal adjacent tissue from a patient with primary colorectal cancer.

[0066] Figure 4 The figure shows the results of differential expression of PLXNC1 protein between cancerous tissue and normal adjacent tissue in patients with primary colorectal cancer;

[0067] Figure 5 The ROC curve results of PLXNC1 mRNA level as a biomarker for diagnosing colorectal cancer are shown.

[0068] Figure 6 The ROC curve results of PLXNC1 protein level as a biomarker for diagnosing colorectal cancer are shown.

[0069] Figure 7 The results show the relationship between PLXNC1 and colorectal cancer patients in different datasets, where A: TCGA, B: GSE39582, C: GSE17536, and D: GSE37892;

[0070] Figure 8 The graph shows the results of qPCR detection of the relative expression levels of PLXNC1 in LOVO cells in the NC-siRNA group and the PLXNC1-siRNA group;

[0071] Figure 9 A statistical graph showing the results of the LOVO CCK-8 cell proliferation experiment;

[0072] Figure 10 The results and statistical graphs of the LOVO cell migration experiment are shown in Figure A: Results Graph, and Figure B: Statistical Result Graph.

[0073] Figure 11 The results and statistical graphs of the LOVO cell invasion experiment are shown in Figure A: Results Graph, and Figure B: Statistical Result Graph.

[0074] Figure 12 The figure shows the results of differential expression of PLXNC1 between CMS1-3 and CMS4 colorectal cancer tissues. In the figure, A: TCGA, B: GSE17536, C: GSE37892, D: GSE35896.

[0075] Figure 13The results of PLXNC1's diagnostic efficacy for molecular subtyping of CMS1-3 and CMS4 colorectal cancer are shown in the figure. Figure A: TCGA, Figure B: GSE17536, Figure C: GSE37892, and Figure D: GSE35896. Detailed Implementation

[0076] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations.

[0077] Example 1: Screening and Validation Analysis of Biomarkers Associated with Colorectal Cancer

[0078] 1. Screening Method

[0079] (1) Patient cohort

[0080] mRNA sequencing data and clinical information from the TCGA colorectal cancer cohort were downloaded from the GDC website. All expression data were converted to TPM units before use. After removing normal, duplicate, and missing samples, a total of 590 samples were used for survival and subsequent analysis. In this embodiment, four gene chip datasets (GSE39582, GSE37892, GSE17536, and GSE37182) were selected as validation sets, and expression matrices and clinical information were downloaded from the GEO database.

[0081] (2) Database query

[0082] PLXNC1 mRNA differential expression and ROC curve analysis data were obtained from GSE37182; PLXNC1 protein differential expression analysis data were obtained from CPTAC.

[0083] (3) Tissue chip

[0084] The inventors of this application obtained tissue microarrays of 30 pairs of colon cancer and adjacent normal colon tissue samples from Superbiotek in Shanghai, China. This study used a commercial TMA for a retrospective analysis and was for scientific research purposes only, without involving sensitive clinical information of patients.

[0085] This embodiment utilizes TMA for immunohistochemical analysis. The TMA was dewaxed, hydrated, and incubated with 3% H2O2 for 10 minutes to remove endogenous peroxidase. The sample was then boiled in citrate buffer (pH 6.0) for 90 seconds. The sample was then blocked in 10% goat serum for 30 minutes and incubated overnight at 4°C with rabbit anti-human primary PLXNC1 antibody (HPA066899, Sigma-Aldrich, Darmstadt, Germany). The sample was then incubated with goat anti-rabbit horseradish peroxidase (HRP)-labeled secondary antibody (BST19013894, Bostek, Wuhan, China) at 37°C for 30 minutes. Finally, it was incubated with 3,3'-diaminobenzidine (DAB) and stained with hematoxylin.

[0086] The staining results were digitally analyzed using APERIO ScanScope (Leica Biosystems, Germany) and evaluated using a pixel-counting algorithm in APERIO ImageScope (Leica Biosystems, Germany), which categorized staining as negative, weakly positive, positive, or strongly positive. The histological score (h score) for each specimen was calculated using the following formula: 1 × (% weakly positive) + 2 × (% positive) + 3 × (% strongly positive). These specimens were independently scored by two experienced pathologists who were unaware of the clinical parameters.

[0087] (4) Survival Analysis

[0088] This embodiment first assesses the prognostic impact of PLXNC1 on overall survival (OS) using Kaplan-Meier analysis. Patients were divided into two groups based on the optimal cutoff point for PLXNC1 expression.

[0089] 2. Statistical methods

[0090] The Wilcoxon signed-rank test was used to assess the differential expression of PLXNC1 in TMA-paired samples. Survival analysis was performed using the Log-rank test. The AUC value of the ROC curve was used to assess the predictive power of PLXNC1. Other data were analyzed using Student's t-test (for normally distributed variables) or the Wilcoxon rank-sum test (for non-normally distributed variables). All statistical tests were performed using R (version 3.6.3), with a significance threshold of 0.05.

[0091] 3. Experimental Results

[0092] The results showed that, compared with normal tissues, the expression level of PLXNC1 was significantly upregulated in colorectal cancer tissues (p<0.001) (see...). Figure 1Further investigation was conducted on the differential expression of PLXNC1 protein levels in CPTAC data, and validation was performed on tissue microarrays containing paired samples from 30 colorectal cancer patients. The results showed that the expression level of PLXNC1 protein in primary colorectal cancer tumor tissues was significantly higher than that in normal tissues (p = 0.005) (see...). Figure 2 Tissue microarray results showed that the expression level of PLXNC1 protein in colorectal cancer tumor tissue was significantly higher than that in adjacent normal tissue (p<0.001) (see...). Figure 3 and Figure 4 );

[0093] The results showed that the AUC values ​​of PLXNC1 mRNA and protein levels as biomarkers for the diagnosis of colorectal cancer were as high as 0.90 and 0.76, respectively (see...). Figure 5 and Figure 6 The results showed high accuracy, indicating that PLXNC1 can be used for the early diagnosis of colorectal cancer.

[0094] To assess the relationship between PLXNC1 and prognosis, this embodiment divided TCGA-COREAD patients into two groups based on PLXNC1 expression levels. Kaplan-Meier curves showed that patients with high PLXNC1 expression had poorer overall survival (see...). Figure 7 A), further validation on three GEO datasets showed that in all three validation sets, patients with high PLXNC1 expression had lower overall survival rates. Figure 7 (B, 7C, and 7D) The above results demonstrate that PLXNC1 has good predictive performance for colorectal cancer prognosis and can be used for accurate prediction of colorectal cancer prognosis.

[0095] Example 2: Study on the relationship between PLXNC1 expression and colorectal cancer

[0096] 1. Cell culture and siRNA transfection

[0097] LoVo cell line (human colon cancer cell line) was purchased from BNBIO (Beijing, China) and cultured in F-12K medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Gibco, Carlsbad, CA, USA) at 37°C in an incubator with 5% CO2. LoVo cells were seeded in 12-well plates using... (Polyplus transfection company, Illkirch, France) and 50 nM siRNA transfection for 48 hours;

[0098] The siRNA sequence targeting PLXNC1 is as follows:

[0099] The justice chain is 5'-GAAACAACUCUUGCAUGUAAATT-3' (SEQ ID NO:1);

[0100] The antisense chain is 5'-UUUACAUGCAAGAGUUGUUUCTT-3' (SEQ ID NO:2).

[0101] 2. Detection of PLXNC1 expression level in cells using qPCR

[0102] Total RNA was extracted from LoVo cells using the RNeasy kit (Beyotime, Shanghai, 456 China, R0027) according to the manufacturer's instructions. 1 μg of total RNA was reverse transcribed using SuperScript II reverse transcriptase (TaKaRa, Japan, RR047). Real-time quantitative PCR analysis was performed using SYBR Green Mix (TaKaRa, Japan, RR820) and an ABI 7900HT Real-Time PCR system, following the manufacturer's instructions. The two-step PCR amplification procedure consisted of a pre-denaturation step (98℃ for 30 seconds) and a second PCR amplification step (95℃ for 5 seconds, 60℃ for 30 seconds, 40 cycles). -ΔΔCT Perform relative quantification;

[0103] First, we designed the amplification primers for qPCR. The specific primer sequences are as follows:

[0104] PLXNC1:

[0105] The forward primer is 5'-GGTCTGGTCCCCATTGAAGG-3' (SEQ ID NO:3);

[0106] The reverse primer is 5'-TTGGGCAACTCTCCTACCCT-3' (SEQ ID NO:4);

[0107] Internal reference gene GAPDH:

[0108] The forward primer is 5'-CTGACTTCAACAGCGACACC-3' (SEQ ID NO:5);

[0109] The reverse primer is 5'-TGAGCTTGACAAAGTGGTCGT-3' (SEQ ID NO:6); 3. CCK-8 cell proliferation assay

[0110] In this example, a reagent kit was used to count cells (CCK-8; DOJINDO, Kumamoto, Japan). LoVo cells were seeded in 96-well plates (1×10⁶ cells / well). 4 Cells / wells were prepared and transfected with siRNA. 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 4 h. The absorbance was measured at 450 nm using a microplate reader (PerkinElmer EnVision, MA, USA).

[0111] 4. Cell migration and cell invasion assays

[0112] This embodiment will use 2×10 4 LoVo cells were seeded in the upper chamber of a Transwell (24 wells, 8 μm, Corning, NY, USA) to study migration responses; 2 × 10⁶ LoVo cells were seeded in the upper chamber of a Transwell apparatus. 4 LoVo cells were seeded in the upper chamber of a Transwell (BD Biosciences, San Jose, CA, USA) to study the invasion response. After siRNA transfection, 500 μL of medium supplemented with 10% fetal bovine serum was added to the lower chamber as an inducer. The Transwell chambers were incubated at 37°C and 5% CO2 for 48 hours. Then, 4% paraformaldehyde (PFA) was added to both the top and bottom surfaces of each Transwell chamber to fix the cells for 20 minutes. After staining with 0.1% crystal violet for 10 minutes, the upper layer of cells was removed, and the cell count was performed under an optical microscope.

[0113] 5. Statistical methods

[0114] The Wilcoxon signed-rank test was used to assess the differential expression of PLXNC1 in TMA-paired samples. Survival analysis was performed using the Log-rank test. The AUC value of the ROC curve was used to assess the predictive power of PLXNC1. Other data were analyzed using Student's t-test (for normally distributed variables) or the Wilcoxon rank-sum test (for non-normally distributed variables). All statistical tests were performed using R (version 3.6.3), with a significance threshold of 0.05.

[0115] 6. Experimental Results

[0116] To further elucidate the role of PLXNC1 in colorectal cancer, this study knocked down the expression level of PLXNC1 in tumor cells by transfecting PLXNC1 siRNA. PCR results showed that, compared with the control group transfected with NC-siRNA, the expression level of PLXNC1 in LOVO cells transfected with PLXNC1 siRNA was significantly reduced (see [link to study]). Figure 8 );

[0117] The results of the CCK-8 cell proliferation assay showed that in the LOVO colorectal cancer cell line, the OD450 value of the experimental group transfected with PLXNC1siRNA was significantly lower than that of the control group transfected with NC-siRNA (see [link to CCK-8 cell proliferation assay]). Figure 9 This indicates that PLXNC1 can significantly affect the proliferative activity of colorectal cancer cells, and reducing the expression level of PLXNC1 can significantly reduce the proliferative capacity of colorectal cancer cells.

[0118] The results of cell migration assays showed that in the LOVO colorectal cancer cell line, the number of migrating cells in the experimental group transfected with PLXNC1siRNA was significantly lower than that in the control group transfected with NC-siRNA (see [link to assay]). Figure 10 This indicates that PLXNC1 can significantly affect the migration ability of colorectal cancer cells, and reducing the expression level of PLXNC1 can significantly reduce the migration ability of colorectal cancer cells.

[0119] The results of cell invasion assays showed that in the LOVO colorectal cancer cell line, the number of invasive cells in the experimental group transfected with PLXNC1siRNA was significantly lower than that in the control group transfected with NC-siRNA (see [link to assay]). Figure 11 This indicates that PLXNC1 can significantly affect the invasive ability of colorectal cancer cells, and reducing the expression level of PLXNC1 can significantly reduce the invasive ability of colorectal cancer cells.

[0120] In conclusion, reducing the expression level of PLXNC1 can significantly reduce the proliferation, migration, and invasion of colorectal cancer cells, suggesting that agents that reduce the expression level of PLXNC1 can be used in the treatment of colorectal cancer.

[0121] Example 3: Study of PLXNC1 as a molecular subtyping marker for colorectal cancer

[0122] 1. Analytical and Validation Methods

[0123] (1) Patient cohort

[0124] mRNA sequencing data and clinical information from the TCGA colorectal cancer cohort were downloaded from the GDC website. All expression data were converted to TPM units before use, and normal, duplicate, and missing samples were removed. In this embodiment, three gene chip datasets (GSE17536, GSE37892, and GSE35896) were selected. Among them, the TCGA colorectal cancer dataset and the GEO datasets GSE17536, GSE37892, and GSE35896 were used as validation sets. The expression matrix and clinical information were downloaded from the GEO database.

[0125] In the validation set TCGA, there were 353 patients with CMS1-3 colorectal cancer and 136 patients with CMS4 colorectal cancer.

[0126] In the validation set GSE17536, there were 120 patients with CMS1-3 colorectal cancer and 40 patients with CMS4 colorectal cancer.

[0127] In the validation set GSE37892, there were 79 patients with CMS1-3 colorectal cancer and 41 patients with CMS4 colorectal cancer.

[0128] In the validation set GSE35896, there were 70 patients with CMS1-3 colorectal cancer and 22 patients with CMS4 colorectal cancer.

[0129] The Consensus molecular subtype (CMS) for colorectal cancer was developed in 2015 by the CRC Subtyping Consortium (CRCSC) using an integrated subtyping algorithm based on network biology, which synthesizes data from six CRC subtyping datasets. The standard molecular subtyping derived from this algorithm is as follows:

[0130] CMS1 type: also known as MSI immune type, characterized by high MSI and CIMP, low CIN and strong immunogenicity, nearly 70% of BRAF mutation patients are concentrated in this type;

[0131] CMS2 type: also known as the classic type, it is characterized by high CIN and low CIMP features. Therefore, the tumor has typical epithelial differentiation features and a large number of somatic copy number alterations (SCNAs).

[0132] CMS3 type: Also known as the metabolic type, it presents with moderate CIN and CIMP, with 30% of individuals also exhibiting MSI characteristics. Patients with KRAS mutations are also relatively enriched in this type. The most prominent feature of this type of patient is the altered cellular metabolic profile and metabolic reprogramming, with active metabolism of various sugars, lipids, amino acids, and nucleotides, especially abnormal activation of glutamine breakdown and lipogenesis pathways. CMS3 type tumors also have lower immunogenicity, but the overall prognosis of patients is better.

[0133] CMS4 type: also known as mesenchymal type, is characterized by high CIN, low MSI and CIMP. Its main difference from CMS2 type is the presence of a large number of stromal cells in the adjacent tissue. CMS4 type is characterized by changes in mesenchymal phenotype and immune microenvironment, and has the worst prognosis among the four CMS types.

[0134] (2) Differential expression analysis

[0135] The student's test in R software was used to perform differential analysis on the expression levels of PLXNC1 in the TCGA, GSE17536, GSE37892, and GSE35892 datasets.

[0136] (3) Validation of the diagnostic efficacy of PLXNC1 for molecular subtyping of colorectal cancer

[0137] The receiver operating characteristic (ROC) analysis was performed using the R package "pROC". ROC curves were plotted for four validation sets. In these sets, the biomarker PLXNC1 was used as the detection variable to analyze its AUC value, sensitivity, and specificity as a molecular subtyping marker for colorectal cancer, thus determining the diagnostic efficacy of PLXNC1 for colorectal cancer molecular subtyping. The analysis used PLXNC1 expression levels. First, the pROC package was called, then the PLXNC1 expression matrix was read in, and the command to plot the ROC curve was run. This command used a for loop and included commands to add AUC, Thres (threshold), and Smooth (curve fitting).

[0138] 2. Experimental Results

[0139] The results showed that, in all four validation sets, the expression level of PLXNC1 in CMS4 colorectal cancer tissue was significantly higher than that in CMS1-3 colorectal cancer tissue (see [link to validation set]). Figure 12 The results (A-12D) indicated a significant differential expression of PLXNC1 between CMS1-3 and CMS4 colorectal cancer (p < 0.001).

[0140] The results showed that, in the four validation sets, PLXNC1 had high accuracy in differentiating between CMS1-3 and CMS4 colorectal cancer subtypes, with AUC values ​​greater than 0.80 (see [link to validation set]). Figure 13 The results (A-13D) demonstrate that PLXNC1 can be used for accurate differential diagnosis of colorectal cancer molecular subtypes.

[0141] Although the foregoing invention has been described in great detail with reference to illustrations and examples for clarity, it is apparent that certain changes and modifications may be made within the scope of the claims, and such changes and modifications will also fall within the protection scope of the claims of this invention.

Claims

1. Application of reagents for detecting the expression level of the biomarker PLXNC1 in samples in the preparation of products for early diagnosis of colorectal cancer; The reagents used to detect the expression level of the biomarker PLXNC1 in the sample are probes that specifically recognize the biomarker PLXNC1, primers that specifically amplify the biomarker PLXNC1, antibodies that specifically bind to the biomarker PLXNC1, antibody fragments that specifically bind to the biomarker PLXNC1, or affinity proteins that specifically bind to the biomarker PLXNC1.

2. The application according to claim 1, characterized in that, The sequences of the primers for the specific amplification of the biomarker PLXNC1 are shown in SEQ ID NO:3-SEQ ID NO:

4.

3. A product for the early diagnosis of colorectal cancer, characterized in that, The product contains a reagent for detecting the expression level of the biomarker PLXNC1 in samples; The reagents used to detect the expression level of the biomarker PLXNC1 in the sample are probes that specifically recognize the biomarker PLXNC1, primers that specifically amplify the biomarker PLXNC1, antibodies that specifically bind to the biomarker PLXNC1, antibody fragments that specifically bind to the biomarker PLXNC1, or affinity proteins that specifically bind to the biomarker PLXNC1.

4. The product according to claim 3, characterized in that, The sequences of the primers for the specific amplification of the biomarker PLXNC1 are shown in SEQ ID NO:3-SEQ ID NO:

4.

5. The product according to claim 3, characterized in that, The products mentioned are chips, reagent kits, test strips, or high-throughput sequencing platforms.

6. The use of inhibitors that reduce PLXNC1 expression levels in the preparation of pharmaceutical compositions for the treatment and / or prevention of colorectal cancer; The sequences of the inhibitors that reduce PLXNC1 expression levels are shown in SEQ ID NO:1-SEQ ID NO:

2.

7. A pharmaceutical composition for treating and / or preventing colorectal cancer, characterized in that, The pharmaceutical composition contains an inhibitor that reduces PLXNC1 expression levels; The sequences of the inhibitors that reduce PLXNC1 expression levels are shown in SEQ ID NO:1-SEQ ID NO:2.

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