Use of smox in the manufacture of a product for diagnosing or prognosticating colorectal cancer

Abstract

The application discloses application of SMOX in preparation of a colorectal cancer diagnosis or prognosis prediction product, and relates to the field of biotechnology.It is proved that the expression of SMOX in colon cancer and rectal cancer tissues is higher than that in normal tissues, and high expression of SMOX is related to poor prognosis of colon cancer.The application applies SMOX to diagnosis or prognosis prediction of colorectal cancer, thereby providing a new source for preparation of a colorectal cancer diagnosis or prognosis prediction product, and discovering new medical value of SMOX.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of SMOX in the preparation of colorectal cancer diagnosis or prognosis prediction products. BACKGROUND

[0002] Colorectal cancer (CRC) is a malignant tumor of the epithelial cells of the colon or rectal segment, most of which are adenocarcinoma, and a small number of which are squamous cell carcinoma or mucinous carcinoma. It is one of the most common malignant tumors, ranking third in all cancer types and second in cancer mortality. Globally, the incidence of colorectal cancer is higher in developed countries than in underdeveloped countries, while the mortality rate is higher in underdeveloped countries. The incidence and mortality rates increase with age, but there has been a trend towards younger patients in recent years. There is no significant difference in the incidence between men and women. The risk of colon cancer is about half that of rectal cancer. The occurrence of colorectal cancer is related to a variety of risks, including activation of oncogenes, inactivation of tumor suppressor genes, population aging, and lifestyle. Therefore, its occurrence, development, and treatment are complex, and traditional methods mainly based on surgery and chemotherapy have been difficult to meet the needs, and people are exploring more new molecular treatment options to provide new and practical tumor diagnosis and treatment methods for colorectal cancer patients.

[0003] Polyamines are small cationic molecules that play an important role in DNA transcription, translation, and gene regulation, and are essential for the growth and development of normal cells. However, when polyamine metabolism is disturbed, it can promote the transformation of cells into cancer cells. Studies have shown that the malignant proliferation of cancer cells depends on high levels of polyamines, so polyamines are potential targets for chemotherapy and chemical prevention. Abnormal polyamine metabolism is mainly due to abnormal expression of enzymes involved in the metabolic pathway. The rate-limiting enzyme involved in the polyamine metabolic pathway, spermine oxidase (SMOX), is a FAD-dependent enzyme that specifically oxidizes spermine (SPM) to produce spermidine (SPD), hydrogen peroxide (H2O2), and aldehyde 3-aminopropanal (3-AP), and plays a dominant role in mammalian polyamine catabolism. As a metabolic enzyme, SMOX can regulate cell proliferation, survival, apoptosis, stress response, and tumor microenvironment reconstruction through post-translational acetylation modification and other mechanisms. However, there is currently no research on the relationship between SMOX and the occurrence of colorectal cancer in the field of tumor research. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art and provide the application of SMOX in the preparation of colorectal cancer diagnosis or prognosis prediction products.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present application is: the application of SMOX in the preparation of products for the diagnosis of colorectal cancer.

[0006] Polyamines are a class of ubiquitous, biologically active low-molecular-weight compounds present in various cells, mainly including biological amines such as spermidine and spermine. Various diseases such as autoimmune diseases and chronic inflammatory diseases bronchial asthma are closely related to polyamine metabolic disorders. SMOX is a polyamine metabolic enzyme that has attracted more attention in recent years in diseases such as nervous system diseases and diabetes. Its main function is to maintain the balance of SPD and SPM, and its expression will change to a certain extent with the level of inflammation. The inventors of the present application have found that the expression of SMOX in colon cancer and rectal cancer tissues is higher than that in normal tissues, and the diagnosis of colorectal cancer can be made by detecting the expression of SMOX in tissues.

[0007] As a preferred embodiment of the application, the product comprises a reagent for detecting the expression level of SMOX in the sample.

[0008] As a preferred embodiment of the application, the expression level of SMOX in the sample is up-regulated.

[0009] As a preferred embodiment of the application, the reagent comprises a reagent for detecting the expression level of SMOX by an immunological method.

[0010] The application also provides the use of SMOX in the preparation of a product for predicting the prognosis of colon cancer.

[0011] The inventors of the present application have found that high expression of SMOX is related to poor prognosis of colon cancer, and colon cancer patients with high expression of SMOX (ICB) have poor efficacy and short survival period after receiving ICB treatment.

[0012] As a preferred embodiment of the application, SMOX is highly expressed in colon cancer tissue, and the colon cancer patient has poor prognosis.

[0013] The application also provides a kit for diagnosing colorectal cancer, which comprises a reagent for detecting the expression level of SMOX.

[0014] The application also provides a kit for predicting the prognosis of colon cancer, which comprises a reagent for detecting the expression level of SMOX.

[0015] The application also provides the use of a reagent for inhibiting the expression of SMOX in the preparation of a drug for improving the effect of immunotherapy of colorectal cancer.

[0016] The inventors of the present application have found that the expression of SOMX in colorectal cancer has certain correlation with immune cells, and the abundance of immune cells in colorectal cancer patients with high expression of SMOX is lower than that in normal tissues. Meanwhile, the expression of SMOX gene affects the effect of immunotherapy of colorectal cancer.

[0017] The application provides application of SMOX in preparation of a colorectal cancer diagnosis or prognosis prediction product. The application proves that expression of SMOX in colon cancer and rectal cancer tissues is higher than that in normal tissues, and high expression of SMOX is related to poor prognosis of colon cancer. The application of SMOX in diagnosis or prognosis prediction of colorectal cancer not only provides a new source for preparation of the colorectal cancer diagnosis or prognosis prediction product, but also discovers new medical value of SMOX. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 FIG. 1 is a diagram showing expression of SMOX in colorectal tissues; wherein A-D are diagrams showing analysis of expression difference of SMOX in cancer tissues and normal tissues in CRC from a GEO data set; and E is a diagram showing analysis of expression difference of SMOX in cancer tissues and normal tissues in TCGA-COAD and TCGA-READ by GEPIA data.

[0019] Figure 2 FIG. 2 is a diagram showing gene mutation landscape of SMOX in colorectal tissues; wherein A is a diagram showing gene mutation landscape of SMOX in colon cancer, and the mutation rate is 2.28%; B is a diagram showing gene mutation landscape of SMOX in rectal cancer, and the mutation rate is 0.76%; the blue circle is Missense_Mutation, the light blue circle is Frame_Shift_Del, and the green circle is Frame_Shift_Ins.

[0020] Figure 3A-B is the survival analysis of SMOX high and low expression in colon and rectal cancer using OncoLnc database; C-D is the survival analysis of SMOX high and low expression in colon and rectal cancer using TCGAportal database.

[0021] Figure 4 A-B is the survival analysis of SMOX high and low expression in colon and rectal cancer using OncoLnc database; C-D is the survival analysis of SMOX high and low expression in colon and rectal cancer using TCGAportal database.

[0022] Figure 5 A-B is the survival analysis of SMOX high and low expression in colon and rectal cancer using OncoLnc database; C-D is the survival analysis of SMOX high and low expression in colon and rectal cancer using TCGAportal database.

[0023] Figure 6: AB shows the expression of SMOX protein in adjacent normal tissue and colorectal cancer tissue (B) by immunohistochemical staining; C shows the statistical analysis of SMOX expression in cancer tissue and adjacent normal epithelial cells (p<0.05).

[0024] Figure 7 Image A shows HE staining of poorly differentiated colorectal cancer tissue; Image B shows SMOX expression in poorly differentiated colorectal cancer tissue; Image C shows HE staining of moderately differentiated colorectal cancer tissue; Image D shows SMOX expression in moderately differentiated colorectal cancer tissue; Image E shows HE staining of well-differentiated colorectal cancer tissue; Image F shows SMOX expression in well-differentiated colorectal cancer tissue; Image G shows statistical analysis of SMOX expression in poorly, moderately, and well-differentiated cancer tissues, with statistically significant differences between poorly and moderately differentiated tissues and well-differentiated tissues.

[0025] Figure 8 A is a volcano plot of co-expressed genes of SMOX in colorectal cancer; B is a heatmap of the top 50 positively correlated co-expressed genes of SMOX in colorectal cancer; C is a heatmap of the top 50 negatively correlated co-expressed genes of SMOX in colorectal cancer; D is a GO biological function enrichment analysis of co-expressed genes of SMOX in colorectal cancer; E is a KEEG enrichment analysis of co-expressed genes of SMOX in colorectal cancer.

[0026] Figure 9 The results of the correlation analysis between SMOX and immune cell infiltration in the TIMER database are shown in the figure.

[0027] Figure 10 TIMER scores for high and low SMOX expression in (A) colon cancer and (B) rectal cancer.

[0028] Figure 11 The study investigated the impact of high and low SMOX expression on the efficacy of immunotherapy in colorectal cancer. A represents the difference in immune checkpoint molecules between the high and low SMOX expression groups in colon cancer; B represents the difference in immune checkpoint molecules between the high and low SMOX expression groups in rectal cancer; and CD represent the TIDE scores of the high and low SMOX expression groups in colon cancer (C) and rectal cancer (D).

[0029] Figure 12 AC represents the expression of CD68 (B) and CD45RO (C) in cancer tissue with weak SMOX expression (A); DF represents the expression of CD68 (E) and CD45RO (F) in cancer tissue with strong SMOX expression (D). Detailed Implementation

[0030] The foregoing will be further clarified by a specific example, which is set forth to demonstrate the present application. It should be understood that the following example is given by way of example only and should not be construed as limiting the subject matter of the present application.

[0031] Example 1

[0032] In this example, SMOX mRNA expression data was retrieved from GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ), and four datasets were obtained for analysis, including GSE87211 (203 cases of colorectal cancer and 160 normal paired mucosa tissues were analyzed), GSE74602 (30 cases of colorectal cancer and 30 normal tissues were analyzed), GSE35279 (74 cases of colorectal cancer and 5 normal tissues were analyzed), and GSE21815 (132 cases of colorectal cancer and 9 normal tissues were analyzed). The detailed information of the datasets is shown in Table 1. The expression value of SMOX gene is the relative expression value.

[0033] Table 1 Detailed information of GEO datasets

[0034]

[0035] GEPIA database (http: / / gepia.cancer-pku.cn / ) is composed of RNA sequencing expression data of 9736 tumor and 8587 normal samples from TCGA and GTEx databases. The expression level of SMOX in colorectal cancer tissues and normal tissues was analyzed by using GEPIA database. P<0.05 was considered statistically significant. The analysis used the default settings, with |Log2FC|>1 and p-value critical value <0.01 as the critical value standard to determine the differentially expressed genes, Log Scale using log2(TPM+1), Jitter Size 0.4, and cancer selection of colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ). Wilcoxon rank-sum test was used to assess the significance of the observed differences.

[0036] Ualcan (http: / / ualcan.path.uab.edu / ) is a comprehensive website for analyzing cancer omics data. In this study, it was used to analyze the expression differences of SMOX in colorectal cancer with different clinicopathological features. Wilcoxon rank-sum test was used to assess the significance of the observed differences.

[0037] The RNAseq data (level 3), mutation maf data and corresponding clinical information of colon and rectal cancer were obtained from The Cancer Genome Atlas (TCGA) dataset (https: / / portal.gdc.com). The somatic mutations of colon and rectal cancer patients were downloaded and visualized by using the maftools package in R software.

[0038] The relationship between the expression of SMOX and OS in colorectal cancer was retrieved from OncoLnc and TCGAportal online databases, and a survival analysis graph was constructed.

[0039] The RNAseq data (level 3) of colon cancer and corresponding clinical information were obtained from The Cancer Genome Atlas (TCGA) database (https: / / portal.gdc.com). The logrank was used to test the KM survival analysis to compare the survival differences between two or more groups, and the timeROC analysis was performed to compare the prediction accuracy of SMOX gene.

[0040] The results, as shown in Figures 1-4 , the expression of SMOX in colorectal cancer tissue was significantly higher than that in the adjacent normal tissue (p<0.01) Figure 1 and Table 1). The mutation landscape of SMOX gene in colorectal cancer was obtained by TCGA database, and the results showed that the mutation rate of SMOX in colon cancer was higher than that in rectal cancer Figure 2 . At the same time, the expression of SMOX in different clinical pathological parameters of colon cancer COAD and rectal cancer READ was analyzed by UALCAN, including race, gender, age, body weight, tumor type, tumor stage and lymph node metastasis status, which consistently showed that the transcription level of SMOX was increased Figure 3 . The high expression of SMOX was related to poor prognosis of colon cancer Figure 4 , but had little relationship with the prognosis of rectal cancer (OncoLnc analysis colon cancer COAD log-rank p=0.03, while rectal cancer READ log-rank p=0.4; TCGAportal analysis colon cancer COAD log-rank p=0.0074, while rectal cancer READ log-rank p=0.69).

[0041] As can be seen from Figure 5 , SMOX gene significantly affected the prognosis of colon cancer (p<0.05), and the ROC curve and AUC value showed that SMOX gene had good accuracy as a diagnostic marker for the prognosis of colon cancer.

[0042] Example 2 Immunohistochemical Staining

[0043] 1. Specimen source

[0044] The colorectal cancer tissue specimens of the present embodiment were derived from 94 patients diagnosed with colorectal cancer in Dongguan Binhaiwan Central Hospital from 2012 to 2021, including 83 cases of adenocarcinoma, 11 cases of adenocarcinoma with signet ring cell carcinoma, and normal tissue adjacent to the cancer as control. Clinical data included age, gender, tumor location, tumor differentiation degree and tumor stage. The specimens came from tumor embedded paraffin blocks stored in the pathology room. The specific steps are as follows: (1) cut into 3 μm sections and place on poly cysteine glass slides; (2) deparaffinization and rehydration: melt the paraffin by placing the paraffin sections in a 60°C oven for 2h; deparaffinize in xylene for 10 min three times, gradient alcohol for 1 min each, PBS buffer for 3 times; (3) antigen retrieval: use epitope retrieval solution (EDTA buffer with pH 8.0 from Aperio) in a high pressure pot for 3 min, and cool naturally to room temperature; (4) block endogenous peroxidase: place the sections in 3% hydrogen peroxide solution, incubate at room temperature for 25 min in the dark, and wash with distilled water; (5) incubate the sections with SMOX (1:200) monoclonal antibody, CD68 antibody (Aperio ready-to-use antibody, IM043) and CD45RO antibody (Aperio ready-to-use antibody, IM039) at 4°C overnight, and then wash with PBS for 3 times for a total of 5 min after rewarming the next day; (6) add secondary antibody: incubate at room temperature for 30 min; (7) DAB color development: freshly prepared DAB color developing solution, control the color development time under a microscope, and stop the color development by immersing the sections in tap water; (8) counterstaining: counterstain the sections with hematoxylin, and wash with running water for 10 min, and mount with neutral resin.

[0045] SMOX protein is mainly expressed on the cell cytoplasm and cell membrane, and also partly expressed in the nucleus. Non-specific small brownish yellow particles with higher staining intensity than the background are positive cells. Semi-quantitative method is used to randomly select 10 different angles under a 400-fold magnification ordinary optical microscope. Calculate the number of positive cells per 1000 cells, and grade each section according to the number of positive cells and staining intensity: the percentage of immunoreactive positive cells is divided into 4 categories: 1 (0-25%), 2 (26-50%), 3 (51-75%) and 4 (76-100%). The immunoreaction intensity is scored as follows: deep red-brown color is strong immunoreaction, scored as 3; yellow color is moderate, scored as 2; weak is light yellow, scored as 1; and negative is blue, scored as 0. Two pathologists review the pathological sections without knowing the clinical and pathological diagnosis of the patients, and further evaluate the activity level of the protein by immunoreaction score (IRS). The calculation method of IRS is the product of reaction intensity and percentage of positive cells. According to IRS, the immunoreaction mode of the protein is defined as: negative (NE; IRS0), weak (We; IRS1-4), moderate (Mo; IRS6-8) and strong (ST; IRS9-12).

[0046] The results are as follows Figures 6-7 As shown in Table 2, positive immunohistochemical staining results show a yellow or brownish-yellow staining signal, indicating that SMOX is mainly expressed in the cytoplasm and cell membrane. Figure 6 In adjacent normal tissue, staining is mostly negative or weakly positive. Figure 6 A), while in colorectal cancer tissue, the staining is mostly moderately positive or strongly positive (A). Figure 6 B), staining comprehensive score ( Figure 6 C) indicates that SMOX expression in colorectal cancer tissues was higher than that in adjacent normal tissues, with a statistically significant difference (p < 0.05). This is consistent with the results obtained from the database analysis above. Simultaneously, the immunohistochemical results of SMOX protein in colorectal cancer patients with different clinical parameters and pathological stages and grades were analyzed (Table 2). The results showed that SMOX protein expression differed among different pathological grades of colorectal cancer, mainly between low-to-moderate and high-differentiation cancers (p < 0.05). Figure 7 There were no significant differences in age, gender, location, TNM-T staging, and TNM-N staging.

[0047] Table 2. Immunohistochemical analysis of SMOX protein in different clinicopathological parameters of colorectal cancer patients.

[0048]

[0049] Example 3: Related genes co-expressed with SMOX in colorectal cancer

[0050] The LinkedOmics database (http: / / linkedomics.org / login.php) contains multi-omics and clinical data for 32 cancer types, as well as data from 11,158 patients in the TCGA project. It is also a multi-omics database integrating mass spectrometry (MS) proteomics data from selected TCGA tumor samples. This study used the database to identify genes associated with SMOX expression and performed enrichment analysis.

[0051] The results are as follows Figure 8 As shown in Table 3, GO enrichment biological functions are mainly in "extracellular structural tissues", "endoderm development", "collagen metabolism", "heart valve development", and "mesenchymal development". KEEG pathways are mainly in "complement and coagulation", "ECM receptor response", "cell adhesion molecules", and "hypertrophic cardiomyopathy".

[0052] Table 3 shows the top 50 genes positively correlated with SMOX, analyzed using LinkedOmics.

[0053]

[0054]

[0055] Example 4: SMOX and Immune Relevance

[0056] TIMER (https: / / cistrome.shinyapps.io / timer / ) is a database for systematically analyzing immune infiltration in various cancer types. The TIMER 2.0 database consists of 10,897 samples from 32 cancer types in the TCGA database, used to assess the abundance of immune infiltrates. Here, TIMER was used to analyze SMOX expression and its interaction with six immune cell types (B cells, CD4+, CD4+, CD4+). + T cells, CD8 + The correlation between T cells, neutrophils, macrophages, and dendritic cells was investigated. Gene expression levels were detected using log2TPM. Immunohistochemical staining was performed on CD68 (a macrophage marker) and CD45RO (an activated T cell marker) in colorectal cancer tissues, and statistical analysis was conducted.

[0057] The level of immune cells in the tumor microenvironment is closely related to the proliferation and development of cancer cells. SMOX, as a metabolic enzyme, may play a regulatory role in the infiltration of immune cells in the tumor microenvironment. Furthermore, KEEG enrichment analysis of co-expressed genes of SMOX in colorectal cancer suggests that SMOX may be involved in immune processes. This study utilizes the TIMER database to explore the correlation between SMOX and immune cell infiltration (e.g., Figures 9-10 (As shown). SMOX expression is correlated with the immune microenvironment in colorectal cancer. Specifically, colorectal cancer patients with high SMOX expression have higher levels of B cells and CD4+. + T cells, CD8 + The abundance of T cells, macrophages, neutrophils, and myeloid dendritic cells was lower than in normal tissues. Figure 10 A) In rectal cancer patients with high SMOX expression, B cells and CD8... + The abundance of T cells, macrophages, and myeloid dendritic cells was lower than in normal tissues. Figure 10 B). These results indicate that the SMOX gene plays a role in the immune microenvironment of colorectal cancer.

[0058] Immune checkpoint molecules are regulatory molecules that play an inhibitory role in the immune system, which is essential for maintaining self-tolerance, preventing autoimmune reactions, and minimizing tissue damage by controlling the time and intensity of immune responses. Immune checkpoint molecules are expressed on immune cells, which inhibit immune cell function, making the body unable to produce an effective anti-tumor immune response, and tumors form immune escape. This embodiment analyzes the differences between immune checkpoints in colorectal cancer with high and low SMOX expression, and the results show that in colon cancer, there are significant differences in PD1 and SIGLEC15 between SMOX high expression and low expression patients, and PD1 and SIGLEC15 in SMOX high expression patients are significantly higher than those in SMOX low expression patients Figure 11 A, p<0.05), while in rectal cancer, there is no difference in immune checkpoint molecules between high and low expression groups of SMOX Figure 11 B, p>0.05). The effect of SMOX gene expression on the ICB treatment effect of colorectal cancer was analyzed by TIDE score, and the results showed that the TIDE score of colorectal cancer patients with high SMOX expression was higher than that of patients with low SMOX expression Figure 11 C-D, p<0.05), indicating that colorectal cancer patients with high SMOX expression have poor ICB efficacy and short survival time after receiving ICB treatment. These results indicate that the SMOX gene affects the immune therapy effect of colorectal cancer patients. Immunohistochemical analysis of macrophage marker CD68 and activated T cell marker CD45RO showed Figure 12 , Tables 4-5, the expression of CD68 is correlated with the expression of SMOX (p<0.05, rho=0.248), and the expression of CD45RO has no great correlation with the expression of SMOX (p>0.05).

[0059] Table 4

[0060]

[0061] Table 5

[0062]

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and not to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. Use of an agent that inhibits SMOX expression in the manufacture of a medicament for improving the effectiveness of immunotherapy for colon cancer.

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