A colorectal cancer molecular subtyping marker, and related products and uses thereof

Abstract

The application discloses a colorectal cancer molecular typing marker, and related products and uses thereof, wherein the colorectal cancer molecular typing marker is PLXNC1; the product comprises a reagent for detecting the expression level of PLXNC1, and the product can be used for accurately diagnosing and distinguishing the molecular typing of colorectal cancer of a subject; and the colorectal cancer molecular typing marker has extremely high clinical application value, and provides an important reference for clinicians to select individualized treatment schemes and realize precise individualized treatment.

Description

Technical Field

[0001] This invention belongs to the field of cancer diagnosis and molecular biology. Specifically, this invention relates to a molecular subtyping marker for colorectal cancer and its related products and uses. More specifically, the molecular subtyping marker for colorectal cancer involved in this invention is PLXNC1. Background Technology

[0002] Colorectal carcinoma (CRC) is one of the major malignant tumors threatening human health, and its incidence and mortality rates are increasing year by year. Colorectal cancer is currently the third most common malignant tumor in men and the second most common in women worldwide. New cases of colorectal cancer account for about 10% of all new cases of malignant tumors, and deaths caused by colorectal cancer account for about 8% of all malignant tumor deaths (Siegel RL, Miller KD, Jemal A. Cancerstatistics, 2015. CA Cancer J Clin, 2015, 65(1):5-29.). Increasing evidence suggests that colorectal cancer is not a single, uniform disease type, but rather a molecularly heterogeneous disease composed of a series of gene alterations (Bae JM, Kim JH, Kang GH. Molecular subtypes of colorectal cancer and their clinicopathologic features, with an emphasis on the serrated neoplasia pathway[J]. Archives of pathology & laboratory medicine, 2016, 140(5):406-412.). Traditional pathological staging, such as TNM staging or Dukes staging, is based on the depth of tumor invasion into the intestinal wall, lymph node metastasis, and distant metastasis, without considering tumor heterogeneity, and thus has certain limitations. Therefore, further understanding of colorectal cancer from a molecular pathological perspective, from variable clinical course to characteristic molecular subtypes, is crucial for precise individualized treatment.

[0003] With the rapid development of high-throughput omics, various omics technologies such as whole-genome sequencing, epigenetics, and proteomics have been applied to colorectal cancer research. Multiple independent groups worldwide have studied the subtypes of colorectal cancer, but none of the classification methods have gained widespread acceptance. Currently, the Consensus Molecular Subtypes (CMS) classification is the most widely recognized and convincing colorectal cancer classification system, and many researchers have conducted targeted therapy studies for colorectal cancer based on it. The CMS classification was proposed at the transcriptomic level by the International Colorectal Cancer Subtyping Consortium. There are four subtypes of CMS: microsatellite instability (MSI) immune activation type (CMS1), accounting for 14%, characterized by hypermutation, MSI, and high immune activation, commonly seen in the right colon of women, with a high incidence of BRAF mutations, and very low survival rate after recurrence; classic colorectal cancer type (CMS2), accounting for 37%, characterized by epithelial cell activation, significant activation of Wnt and Myc signaling pathways, mainly seen in the left colon, with high survival rate after recurrence; metabolic type (CMS3), accounting for 13%, with significant metabolic disorders and a high incidence of KRAS mutations; and mesenchymal type (CMS4), accounting for 23%, characterized by significant activation of transforming growth factor β (TGF-β), mesenchymal invasion, and angiogenesis, with the lowest overall survival and recurrence-free survival rates.

[0004] Colorectal cancer is a biologically highly heterogeneous tumor. Molecular subtyping helps determine a patient's sensitivity to treatment, identify the histological origin of the tumor, predict the risk of tumor progression or metastasis and recurrence, and is a necessary prerequisite for personalized precision treatment. However, current research on molecular subtyping of colorectal cancer is still in its early stages, and only a small number of biomarkers can assist in the diagnosis, clinical staging, prognosis assessment, and guidance of clinical treatment of colorectal cancer. Therefore, there is still an urgent need in this field for new biomarkers that can accurately diagnose and differentiate molecular subtypes of colorectal cancer. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a molecular subtyping biomarker for colorectal cancer and its related products and uses, so as to achieve precise individualized treatment and provide important reference for clinicians to select individualized treatment plans.

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

[0007] A first aspect of the present invention provides a product for diagnosing and differentiating molecular subtypes of colorectal cancer.

[0008] Furthermore, the product contains a reagent for detecting the expression level of the biomarker PLXNC1 in subject samples.

[0009] Furthermore, the product is used to diagnose and differentiate between CMS1-3 and CMS4 colorectal cancer.

[0010] Furthermore, the expression level of the biomarker PLXNC1 was significantly upregulated in CMS4 colorectal cancer.

[0011] Furthermore, the products include reagent kits, chips, test strips, and formulations.

[0012] Furthermore, the kit includes reagents for detecting the expression level of the biomarker PLXNC1 mRNA in the subject sample, and / or reagents for detecting the expression level of the protein and / or peptide encoded by the biomarker PLXNC1 in the subject sample.

[0013] Furthermore, the reagents for detecting the expression level of the biomarker PLXNC1 mRNA in the subject sample include probes that specifically recognize the biomarker PLXNC1 and / or primers that specifically amplify the biomarker PLXNC1.

[0014] Furthermore, the reagents for detecting the expression levels of proteins and / or peptides encoded by the biomarker PLXNC1 in the subject samples include antibodies, and / or antibody fragments, and / or affinity proteins that specifically bind to the biomarker PLXNC1.

[0015] Furthermore, the kit includes a qPCR kit, an immunoblotting detection kit, an immunochromatographic detection kit, a flow cytometry analysis kit, an immunohistochemistry detection kit, an ELISA kit, and an electrochemiluminescence detection kit;

[0016] Preferably, the kit also includes instructions on how to diagnose and differentiate colorectal cancer molecular subtypes based on the test results.

[0017] Furthermore, the kit also includes instructions for use or a label, a positive control, a negative control, a buffer, an adjuvant, or a solvent.

[0018] Furthermore, the instruction manual or label indicates that the kit is used for diagnosing and differentiating molecular subtypes of colorectal cancer.

[0019] Furthermore, the chip includes a solid support and a probe for the specific recognition biomarker PLXNC1 attached to the solid support.

[0020] Furthermore, the chip can be prepared using conventional methods for preparing biochips known in the art.

[0021] Furthermore, as an alternative implementation, the kit includes one or more probes that specifically bind to the biomarker PLXNC1. As a further implementation, the kit also includes a washing solution. As a further implementation, the kit also includes reagents for hybridization experiments, tools for nucleic acid isolation or purification, detection tools, and positive and negative controls. As a still further implementation, the kit also includes instructions for using the kit, describing how to use the kit for detection, how to use the detection results to determine and differentiate the molecular subtype of colorectal cancer in subjects, and how to select treatment regimens. Such a kit can be, for example, a test strip, membrane, chip, disc, test strip, filter, microsphere, slide, multiwell plate, or optical fiber. The solid support of the kit can be plastic, silicon wafer, metal, resin, glass, membrane, particles, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate, or slide.

[0022] The biomarkers described in this invention refer to any detectable compound present in or derived from a sample, such as proteins, peptides, proteoglycans, glycoproteins, lipoproteins, carbohydrates, lipids, nucleic acids (e.g., DNA, such as cDNA or amplified DNA, or RNA, such as mRNA), organic or inorganic chemicals, natural or synthetic polymers, small molecules (e.g., metabolites), or differentiating molecules or differentiating fragments of any of the foregoing. As used herein, "derived from" means a compound that indicates the presence of a specific molecule in the sample upon detection. For example, the detection of a specific cDNA can indicate the presence of a specific RNA transcript in the sample. As another example, the detection of a specific antibody or binding to a specific antibody can indicate the presence of a specific antigen (e.g., protein) in the sample. The differentiating molecule or fragment is a molecule or fragment that indicates the presence or abundance of the compounds identified above upon detection. Biomarkers can, for example, be isolated from the sample, measured directly in the sample, or detected or determined in the sample. Biomarkers can, for example, be functional, partially functional, or non-functional. In a specific embodiment of the invention, the biomarker is PLXNC1;

[0023] Furthermore, 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.

[0024] The expression level or level mentioned in this invention refers to the absolute or relative amount of the biomarker described in this invention. The expression level of any one of the biomarkers described in this invention can be determined by various techniques. In particular, the absolute or relative amount of the biomarker described in this invention can be detected by methods well known to those skilled in the art.

[0025] The primers described in this invention refer to nucleic acid fragments containing 5-100 nucleotides, preferably 15-30 nucleotides that can initiate an enzymatic reaction (e.g., an enzymatic amplification reaction).

[0026] The probe described in this invention refers to a nucleic acid sequence comprising at least 5 nucleotides, for example, containing 5-100 nucleotides, which can hybridize with the expression product of a target gene or the amplification product of the expression product under specified conditions to form a complex. The hybridization probe may also include a marker for detection, including (but not limited to) markers for quantitative real-time PCR or fluorescence in situ hybridization.

[0027] The antibody described in this invention refers to a specific immunoglobulin targeting an antigen site. The antibody in this invention specifically binds to the PLXNC1 peptide and / or protein described in this invention. Antibodies can be manufactured according to conventional methods in the art. The forms of antibodies include polyclonal or monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (e.g., bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigen-binding site, and any other modified immunoglobulin molecule containing an antigen-binding site, provided that the antibody exhibits the desired biobinding activity.

[0028] The polypeptide described in this invention refers to a compound composed of amino acids linked by peptide bonds, including the full length of the polypeptide or amino acid fragments. The expression level of the polypeptide encoded by the gene can be standardized based on the amount of total protein in the sample or the amount of polypeptide encoded by the housekeeping gene.

[0029] Furthermore, the sample is selected from the subject's blood or tissue.

[0030] The subject referred to in this invention refers to any animal, including both human and non-human animals. The term "non-human animal" includes all vertebrates, such as mammals, including 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.

[0031] A second aspect of the invention provides the use of a reagent for detecting the expression level of the biomarker PLXNC1 in a subject sample in the preparation of a product for diagnosing and differentiating molecular subtypes of colorectal cancer.

[0032] Furthermore, the reagent used to detect the expression level of the biomarker PLXNC1 in the subject's sample is selected from:

[0033] Reagents for detecting the expression level of the biomarker PLXNC1 mRNA in subject samples; or

[0034] A reagent for detecting the expression levels of proteins and / or peptides encoded by the biomarker PLXNC1 in subject samples.

[0035] Furthermore, the reagents for detecting the expression level of the biomarker PLXNC1 mRNA in the subject sample include probes that specifically recognize the biomarker PLXNC1, and / or primers that specifically amplify the biomarker PLXNC1;

[0036] Preferably, the reagent for detecting the expression level of the protein and / or peptide encoded by the biomarker PLXNC1 in the subject sample includes an antibody, and / or antibody fragment, and / or affinity protein that specifically binds to the biomarker PLXNC1.

[0037] Furthermore, the sequences of the primers for the specific amplification of the biomarker PLXNC1 are shown in SEQ ID NO:3-SEQ ID NO:4.

[0038] Furthermore, the sample is selected from the subject's blood or tissue.

[0039] Furthermore, the reagents for detecting the expression level of the biomarker PLXNC1 in the subject samples also include reagents for detecting the expression level of the biomarker PLXNC1 in the subject samples using sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, and protein immunoassay technology.

[0040] Furthermore, the sequencing technology is nucleic acid sequencing technology, including Sanger sequencing and dye-terminated sequencing. Those skilled in the art will recognize that because RNA is less stable in cells and more susceptible to nuclease attack in experiments, it is typically reverse transcribed into DNA before sequencing. In addition, the sequencing technology also includes next-generation sequencing (i.e., deep sequencing / high-throughput sequencing). High-throughput sequencing is a single-cluster sequencing-by-synthesis technique based on a proprietary reversible termination chemical reaction principle. During sequencing, random fragments of genomic DNA are attached to an optically transparent glass surface. After extension and bridging amplification, these DNA fragments form hundreds of millions of clusters on the glass surface. Each cluster is a single-molecule cluster with thousands of copies of the same template. Then, using four special deoxyribonucleotides with fluorescent groups, the template DNA to be tested is sequenced through reversible sequencing-by-synthesis technology.

[0041] Furthermore, the nucleic acid hybridization techniques include (but are not limited to) in situ hybridization (ISH), microarrays, and Southern or Northern blotting.

[0042] Furthermore, the nucleic acid amplification technology is selected from polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA).

[0043] Furthermore, the protein immunoassay techniques include sandwich immunoassays, such as sandwich ELISA, in which two antibodies recognizing different epitopes on the biomarker PLXNC1 are used to detect the biomarker PLXNC1; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), Western blotting, immunoprecipitation, and immunoassays based on any particle (such as those using gold particles, silver particles or latex particles, magnetic particles or quantum dots).

[0044] A third aspect of the present invention provides a system or apparatus for diagnosing and differentiating molecular subtypes of colorectal cancer.

[0045] Furthermore, the system or apparatus includes:

[0046] (1) Analysis unit: The analysis unit is suitable for detecting the expression level of the biomarker PLXNC1 in the subject sample;

[0047] (2) Evaluation unit: The evaluation unit includes a stored reference and a data processor. The data processor has implemented an algorithm for comparing the expression level of the biomarker PLXNC1 in the subject sample obtained by the analysis unit with the stored reference, thereby diagnosing and differentiating the molecular subtype of colorectal cancer in the subject.

[0048] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0049] This invention is the first to discover that PLXNC1 can be used as a molecular subtyping marker for colorectal cancer, and it has high accuracy in the diagnosis and differentiation of molecular subtyping of colorectal cancer. The molecular subtyping marker for colorectal cancer has extremely high clinical application value and provides an important reference for clinicians to select individualized treatment plans and achieve precise individualized treatment. Attached Figure Description

[0050] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0051] Figure 1 The graph shows the differential expression of PLXNC1 mRNA between the control group and the colorectal cancer group;

[0052] Figure 2 The figure shows the results of differential expression of PLXNC1 protein between cancerous tissue and normal adjacent tissue in patients with primary colorectal cancer;

[0053] Figure 3 This image shows the staining results of microarrays of cancerous tissue and normal adjacent tissue from a patient with primary colorectal cancer.

[0054] 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;

[0055] Figure 5 The ROC curve results of PLXNC1 mRNA level as a biomarker for diagnosing colorectal cancer are shown.

[0056] Figure 6 The ROC curve results of PLXNC1 protein level as a biomarker for diagnosing colorectal cancer are shown.

[0057] Figure 7 The following graphs show the relationship between PLXNC1 and the prognosis of colorectal cancer patients in different datasets: A: TCGA, B: GSE39582, C: GSE17536, D: GSE37892.

[0058] Figure 8The 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;

[0059] Figure 9 A statistical graph showing the results of the LOVO CCK-8 cell proliferation experiment;

[0060] 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.

[0061] 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.

[0062] 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.

[0063] Figure 13 The 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

[0064] 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.

[0065] Example 1: Screening and Validation Analysis of Biomarkers Associated with Colorectal Cancer

[0066] 1. Screening Method

[0067] (1) Patient cohort

[0068] 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.

[0069] (2) Database query

[0070] PLXNC1 mRNA differential expression and ROC curve analysis data were obtained from GSE37182; PLXNC1 protein differential expression analysis data were obtained from CPTAC.

[0071] (3) Tissue chip

[0072] 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.

[0073] 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.

[0074] 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.

[0075] (4) Survival Analysis

[0076] 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.

[0077] 2. Statistical methods

[0078] 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.

[0079] 3. Experimental Results

[0080] 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 1 Further 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 );

[0081] 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.

[0082] 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.

[0083] Example 2: Study on the relationship between PLXNC1 expression and colorectal cancer

[0084] 1. Cell culture and siRNA transfection

[0085] 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 nMsiRNA transfection for 48 hours;

[0086] The siRNA sequence targeting PLXNC1 is as follows:

[0087] The justice chain is 5'-GAAACAACUCUUGCAUGUAAATT-3' (SEQ ID NO:1);

[0088] The antisense chain is 5'-UUUACAUGCAAGAGUUGUUUCTT-3' (SEQ ID NO:2).

[0089] 2. Detection of PLXNC1 expression level in cells using qPCR

[0090] 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;

[0091] First, we designed the amplification primers for qPCR. The specific primer sequences are as follows:

[0092] PLXNC1:

[0093] The forward primer is 5'-GGTCTGGTCCCCATTGAAGG-3' (SEQ ID NO:3);

[0094] The reverse primer is 5'-TTGGGCAACTCTCCTACCCT-3' (SEQ ID NO:4);

[0095] Internal reference gene GAPDH:

[0096] The forward primer is 5'-CTGACTTCAACAGCGACACC-3' (SEQ ID NO:5);

[0097] The reverse primer was 5'-TGAGCTTGACAAAGTGGTCGT-3' (SEQ ID NO:6); 3. CCK-8 cell proliferation assay

[0098] 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 the cells were incubated at 37°C for 4 h. The absorbance was measured at 450 nm using a microplate reader (PerkinElmer EnVision, MA, USA).

[0099] 4. Cell migration and cell invasion assays

[0100] 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 to study migration responses. 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.

[0101] 5. Statistical methods

[0102] 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.

[0103] 6. Experimental Results

[0104] 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 );

[0105] 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.

[0106] 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 PLXNC1 siRNA 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.

[0107] 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 PLXNC1 siRNA 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.

[0108] 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.

[0109] Example 3: Study of PLXNC1 as a molecular subtyping marker for colorectal cancer

[0110] 1. Analytical and Validation Methods

[0111] (1) Patient cohort

[0112] 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.

[0113] In the validation set TCGA, there were 353 patients with CMS1-3 colorectal cancer and 136 patients with CMS4 colorectal cancer.

[0114] In the validation set GSE17536, there were 120 patients with CMS1-3 colorectal cancer and 40 patients with CMS4 colorectal cancer.

[0115] In the validation set GSE37892, there were 79 patients with CMS1-3 colorectal cancer and 41 patients with CMS4 colorectal cancer.

[0116] In the validation set GSE35896, there were 70 patients with CMS1-3 colorectal cancer and 22 patients with CMS4 colorectal cancer.

[0117] 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:

[0118] 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;

[0119] 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).

[0120] 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.

[0121] 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.

[0122] (2) Differential expression analysis

[0123] 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.

[0124] (3) Validation of the diagnostic efficacy of PLXNC1 for molecular subtyping of colorectal cancer

[0125] 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).

[0126] 2. Experimental Results

[0127] 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).

[0128] 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.

[0129] 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. sequence list <110> Institute of Materia Medica, Chinese Academy of Medical Sciences <120> A molecular subtyping biomarker for colorectal cancer and its related products and uses <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty three <212> RNA <213> Artificial Sequence <400> 1 gaaacaacuc uugcauguaa auu 23 <210> 2 <211> twenty three <212> RNA <213> Artificial Sequence <400> 2 uuuacaugca agaguuguuu cuu 23 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 ggtctggtcc ccattgaagg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 ttgggcaact ctcctaccct 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 ctgacttcaa cagcgacacc 20 <210> 6 <211> twenty one <212> DNA <213> Artificial Sequence <400> 6 tgagcttgac aaagtggtcg t 21

Claims

1. The application of a reagent for detecting the expression level of the biomarker PLXNC1 in subject samples in the preparation of products for diagnosing and differentiating molecular subtypes of colorectal cancer, characterized in that, The molecular subtypes of colorectal cancer are CMS1-3 and CMS4. The expression level of PLXNC1 in CMS4 colorectal cancer was significantly higher than that in CMS1-3 colorectal cancer.

2. The application according to claim 1, characterized in that, The reagents used to detect the expression level of the biomarker PLXNC1 in subject samples were selected from: Reagents for detecting the expression level of the biomarker PLXNC1 mRNA in subject samples; or A reagent for detecting the expression level of the protein encoded by the biomarker PLXNC1 in subject samples.

3. The application according to claim 2, characterized in that, The reagents used to detect the expression level of the biomarker PLXNC1 mRNA in the subject samples include probes that specifically recognize the biomarker PLXNC1 and / or primers that specifically amplify the biomarker PLXNC1.

4. The application according to claim 2, characterized in that, The reagents used to detect the expression level of the protein encoded by the biomarker PLXNC1 in the subject sample include antibodies that specifically bind to the biomarker PLXNC1, and / or antibody fragments, and / or affinity proteins.

5. The application 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.

6. The application according to claim 1, characterized in that, The sample was selected from the subject's tissue.

7. A system or apparatus for diagnosing and differentiating molecular subtypes of colorectal cancer, characterized in that, The system or apparatus includes: (1) Analysis unit: The analysis unit is adapted to detect the expression level of the biomarker PLXNC1 in the subject sample; (2) Evaluation unit: The evaluation unit includes a stored reference and a data processor. The data processor has implemented an algorithm for comparing the expression level of the biomarker PLXNC1 in the subject sample obtained by the analysis unit with the stored reference, thereby diagnosing and differentiating the molecular subtype of colorectal cancer in the subject. The molecular subtypes of colorectal cancer are CMS1-3 and CMS4. The expression level of PLXNC1 in CMS4 colorectal cancer was significantly higher than that in CMS1-3 colorectal cancer.

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