Biomarker for prognosis of colorectal cancer and application of biomarker

Abstract

The invention provides a biomarker for prognosis of colorectal cancer and application thereof, and belongs to the technical field of biomedicine, the biomarker comprises a TRIM27 gene and a CERS1 gene; it is found that the TRIM27 gene is remarkably high in expression, the CERS1 gene is remarkably low in expression, the TRIM27 gene and the CERS1 gene are positively correlated with tumor enlargement, high grading and more far-end metastatic focus points of colorectal cancer patients, the prognosis of the corresponding patients is poor, and the five-year survival rate is low. Therefore, joint detection of the expression levels of the TRIM27 gene and the CERS1 gene has reliable value in prognosis evaluation of the colorectal cancer patient.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a biomarker for colorectal cancer prognosis and application thereof. Background Art

[0002] Colorectal cancer, or colorectal malignancy, is a malignant tumor that originates in the epithelial cells lining the colon or rectum. Those that develop in the colon are called colon cancer, while those that develop in the rectum are called rectal cancer. The two share certain similarities in pathological features and clinical symptoms, so they are often collectively referred to as colorectal cancer. Adenocarcinoma is the most common pathological type of colorectal cancer, accounting for over 90% of all colorectal cancers. Other less common types include undifferentiated carcinoma and squamous cell carcinoma.

[0003] Under normal circumstances, the epithelial cells of the colorectal mucosa continuously proliferate, renew, and die, maintaining a dynamic balance. However, when cells are affected by genetic factors, environmental factors (such as long-term high-fat and low-fiber diet, lack of exercise, smoking, alcoholism, long-term exposure to chemical carcinogens, etc.), lifestyle, and certain chronic intestinal diseases (such as ulcerative colitis, Crohn's disease, etc.), the genes in the cells mutate, and this balance is broken, leading to abnormal cell proliferation and the gradual formation of tumors. Early colorectal cancer may be confined to the intestinal wall. As the disease progresses, tumor cells can break through the intestinal wall, infiltrate the surrounding tissues, and metastasize to distant sites through lymphatic vessels and blood vessels, invading important organs such as the liver, lungs, and bones, seriously threatening the patient's life and health.

[0004] Colorectal cancer is a common malignant tumor with a continuously rising incidence and mortality rate. Early diagnosis and treatment are effective ways to reduce the mortality rate of colorectal cancer. A full analysis of the molecular mechanisms of the occurrence and development of colorectal cancer is of great significance for the early diagnosis and treatment of colorectal cancer. With the deepening of understanding of the disease and the continuous emergence of evidence, the detection and application of molecular markers have begun to enter the public's field of vision. Precision diagnosis and treatment based on the molecular level are changing the current status of clinical practice of colorectal cancer. However, there are currently no molecular targets and products on the market for predicting colorectal cancer metastasis and effective prognosis assessment. In-depth exploration of new prognostic targets for colorectal cancer is of great clinical significance. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a biomarker for the prognosis diagnosis of colorectal cancer and its application; the present invention's research found that patients with high expression of TRIM27 and low expression of CERS1 have a lower five-year survival rate, and the combined detection of the expression levels of TRIM27 and CERS1 genes has reliable value in the prognosis evaluation of colorectal cancer patients.

[0006] The present invention provides a biomarker for the prognosis and diagnosis of colorectal cancer, comprising the TRIM27 gene and the CERS1 gene.

[0007] Preferably, patients with significantly high expression of the TRIM27 gene and significantly low expression of the CERS1 gene have a poor prognosis.

[0008] Preferably, the prognosis includes survival rate, such as five-year survival rate or ten-year survival rate.

[0009] The present invention provides the use of a reagent for detecting the expression level of the biomarker in preparing a kit for colorectal cancer prognosis diagnosis.

[0010] Preferably, the reagent comprises an anti-TRIM27 antibody and an anti-CERS1 antibody.

[0011] Preferably, the prognostic risk is assessed based on the expression levels of the TRIM27 gene and the CERS1 gene; the prognostic risk assessment model is as follows: Riskscore = (0.605)*TRIM27+(0.8862)*CERS1.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention combines the information of the colorectal cancer clinical database to explore the expression of TRIM27 gene and CERS1 gene at the molecular level and clinical tissue sample levels, and finds and confirms that the expression of CERS1 gene is negatively correlated with TRIM27 gene in colorectal cancer patients. Through the correlation analysis of the expression of TRIM27 gene and CERS1 gene with the pathological grade, metastasis and prognosis of colorectal cancer patients, it is found that high expression of TRIM27 gene and low expression of CERS1 gene are positively correlated with the enlargement of tumor, higher grade and more distal metastatic foci in colorectal cancer patients. Finally, the relationship between the high and low expression of TRIM27 gene and CERS1 gene and the prognosis of colorectal cancer patients is statistically analyzed, and it is found that patients with high expression of TRIM27 gene and low expression of CERS1 gene have a lower five-year survival rate. According to the statistical results, it is determined that the combined detection of the expression levels of TRIM27 gene and CERS1 gene has reliable value in the prognosis assessment of colorectal cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The Riskscore, survival time, and survival status of the CRC dataset. The top scatter plot represents the risk score from low to high; different colors represent different groups; the scatter plot distribution represents the Riskscore of different samples corresponding to the survival time and survival status, and the bottom heat map shows the expression of CERS1 and TRIM27.

[0015] Figure 2Kaplan-Meier survival analysis was performed based on the risk model obtained from the analysis of the CRC dataset. The log-rank test was used to compare different groups. HR (High groups) represents the hazard ratio of low-expression samples to high-expression samples. HR>1 indicates that TRIMN27 and CERS1 are joint risk factors. HR (95% CI) represents the median survival time (LT50) of different groups, expressed in years.

[0016] Figure 3 ROC curves and AUCs for TRIM27 and CERS1 genes;

[0017] Figure 4 To detect the expression of TRIM27 and CERS1 in cancerous tissues and adjacent tissues of 120 colorectal cancer patients by immunohistochemical staining;

[0018] Figure 5 The relationship between TRIM27 gene and CERS1 gene expression was that CERS1 gene expression was increased in colorectal cancer patients with decreased TRIM27 gene expression;

[0019] Figure 6 The relationship between TRIM27 gene and CERS1 gene expression is shown. CERS1 gene is highly expressed in colorectal cancer tissues of patients with low TRIM27 gene expression.

[0020] Figure 7 Person correlation analysis was conducted to analyze the relationship between TRIM27 gene and CERS1 gene expression in cancer tissues of colorectal cancer patients;

[0021] Figure 8 Kaplan-Meier analysis was performed to determine the five-year survival of patients with different TRIM27 and CERS1 gene expressions. DETAILED DESCRIPTION

[0022] The present invention provides a biomarker for the prognosis and diagnosis of colorectal cancer, comprising the TRIM27 gene and the CERS1 gene.

[0023] The present invention found that patients with significantly high expression of the TRIM27 gene and significantly low expression of the CERS1 gene have a poor prognosis.

[0024] In the present invention, the prognosis includes survival rate, which can be selected as five-year survival rate or ten-year survival rate; the poor prognosis means that the five-year or ten-year survival rate is significantly lower than that of other groups.

[0025] The present invention provides the use of a reagent for detecting the expression level of the biomarker in preparing a kit for colorectal cancer prognosis diagnosis.

[0026] The present invention has no particular limitation on the type of the reagent, as long as it can detect the expression levels of the TRIM27 gene and the CERS1 gene. In the specific implementation of the present invention, the reagent preferably includes an anti-TRIM27 antibody and an anti-CERS1 antibody.

[0027] The present invention preferably assesses prognostic risk based on the expression levels of the TRIM27 gene and the CERS1 gene; the prognostic risk assessment model is as follows: Riskscore = (0.605) * TRIM27 + (0.8862) * CERS1. The AIC of this prognostic risk assessment model is 1744.8976.

[0028] In the present invention, the risk model Kaplan-Meier survival analysis was derived from a CRC dataset, and the log-rank test was used for comparisons between different groups. HR (High groups) represents the hazard ratio of low-expression samples to high-expression samples. HR>1 indicates that high TRIMN27 expression and low CERS1 expression are combined risk factors. It was found that patients in the high-risk group had a poor prognosis. The ROC curves and AUCs of the TRIM27 and CERS1 genes showed that the combined TRIM27-CERS1 gene had an AUC value of 0.71, which had a good ability to predict the patient's 10-year survival.

[0029] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] 1) The colorectal cancer tissue chip used in the present invention was purchased from Hunan Aifang Biotechnology Co., Ltd. with the catalog number AF-CocSur2201. 40 cancer and adjacent tissues were obtained from patients diagnosed with colorectal cancer during colonoscopy at the Department of Gastroenterology, Affiliated Hospital of Guangdong Medical University.

[0031] 2) Hematoxylin stain was purchased from Wuhan Sewell Biotechnology Co., Ltd., product number G1004-100ML.

[0032] 3) 1× phosphate buffered saline (1× PBS buffer) was purchased from VICMED, China, with the catalog number VC2001P.

[0033] 4) Rabbit two-step detection kit was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number PV9001.

[0034] 5) Anti-TRIM27 antibody (Anti-TRIM27 Polyclonal Antibody 12205-1-AP) was purchased from Wuhan Tri-Taiwan Biotechnology Co., Ltd.

[0035] 6) Anti-CERS1 antibody PS09092 ) was purchased from Abimate Pharmaceutical Technology (Shanghai) Co., Ltd.

[0036] 7) DAB color development kit (20×) was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. with the catalog number ZLI-9018.

[0037] 8) Anhydrous ethanol was purchased from Shanghai Hushi Chemical Co., Ltd.

[0038] 9) Isopropyl alcohol was purchased from Shanghai Hushi Chemical Co., Ltd.

[0039] Example 1

[0040] Analysis of the relationship between TRIM27-CERS1 expression and prognosis of colorectal cancer patients using the TCGA database

[0041] 1. Experimental Procedure

[0042] Download STAR-counts data and clinical information of colorectal cancer tumors from the TCGA dataset library (https: / / portal.gdc.cancer.gov). Extract the data in TPM format and then normalize it to log2. (TPM+1) , and finally retained the samples with RNAseq data and clinical information for subsequent analysis. Log-rank was used to test KM survival analysis to compare the survival differences between the above two or more groups, and timeROC analysis was performed to discriminate the accuracy of the prediction model. Lasso: The least absolute shrinkage and selection operator (LASSO) regression algorithm was used for feature selection, and 10-fold cross-validation was used. The above analysis was performed using the R software glmnet package. COX: Multivariate cox regression analysis was used to construct a prognostic model, and the above analysis was performed using the R software survival package. Step: First, multivariate cox regression analysis was performed and then iterative analysis was performed using the step function to select the optimal model as the final model. For the Kaplan-Meier curve, the P value and the hazard ratio (HR) with 95% confidence interval (CI) were obtained by log-rank test and univariate Cox regression. All the above analysis methods and R software packages were performed using R software version v4.0.3 (RFoundation for Statistical Computing, 2020). P<0.05 was considered statistically significant.

[0043] 2. Experimental Results

[0044] Riskscore, survival time and survival status of CRC dataset are as follows Figure 1As shown, the top scatter plot represents risk scores from low to high. Different colors represent different groups. The scatter plot distribution shows the Riskscore corresponding to survival time and survival status for different samples. The bottom heat map shows the expression of CERS1 and TRIM27. This shows that low TRIM27 expression in the gene expression heat map of colorectal cancer patients may be accompanied by high CERS1 expression, and these patients can be classified as high-risk.

[0045] The risk model Kaplan-Meier survival analysis was derived from the CRC data set, and the log-rank test was used for comparison between different groups. HR (High groups) represents the hazard ratio of low-expression samples to high-expression samples. When HR>1, it indicates that TRIMN27 and CERS1 are joint risk factors, and HR=1.511 indicates that TRIMN27 and CERS1 are joint risk factors. HR (95% Cl), median survival time (LT50) of different groups, in years, were 1.044 (High-risk group) and 2.187 (low-risk group), respectively. It was found that patients in the High-risk group had a poor prognosis ( Figure 2 ).

[0046] The ROC curves and AUCs of TRIM27 and CERS1 are shown in Figure 2 Figure 3 As shown in the figure, the combined AUC value of TRIM27-CERS1 was 0.71, indicating that it has a good ability to predict the patient's 10-year survival.

[0047] Example 2

[0048] Immunohistochemical staining to detect the expression of TRIM27 and CERS1 in colorectal cancer tissues

[0049] 1. Experimental Procedure

[0050] To further explore the expression of TRIM27 and CERS1 at the protein level, as well as their expression correlation and clinical significance, the present invention used immunohistochemical staining to detect the expression of TRIM27 and CERS1 in 120 colorectal cancer tissue chip samples.

[0051] Baking: Place the slides containing the tissue in a 60°C oven overnight. Dewaxing and hydration: Place the sections in xylene (I) and (II) for 30 minutes each, then place them in anhydrous ethanol (I), (II), 95% ethanol, 90% ethanol, 85% ethanol, 80% ethanol, 70% ethanol, and distilled water for 5 minutes each. Removal of endogenous peroxidase: Cover the tissue with 3% H2O2, place in a humidified chamber, and incubate at room temperature in the dark for 10 minutes. Wash with 1× PBS three times, 5 minutes each time.

[0052] Antigen retrieval: Using high temperature and high pressure retrieval - Pour enough citric acid (pH 6.0) retrieval solution into the pressure cooker to ensure that the slices are covered. After the liquid boils, add the slices and cover the lid. After the pressure cooker starts to vent, count down for 3 minutes and perform antigen retrieval. After retrieval is complete and the pressure drops to normal pressure, open the lid and let the slices warm at room temperature. Then wash them three times with 1× PBS for 5 minutes each.

[0053] Blocking: Cover the sections with 1% goat serum blocking solution and incubate in a humidified box at room temperature for 30 minutes in the dark.

[0054] Primary antibody overnight: Dilute TRIM27 and CERS1 with PBS at 1:250, cover each section with approximately 50 μL, and place in a humidified chamber at 4°C overnight; the next day, place the humidified chamber at room temperature for 30 minutes, wash three times with 1× PBS, each time for 5 minutes, and wash off excess primary antibody; incubate with secondary antibody: select the corresponding secondary antibody according to the primary antibody, and refer to the secondary antibody instructions for use - incubate the sections with enhancement solution, incubate in a humidified chamber at room temperature for 20 minutes, wash three times with 1× PBS, each time for 5 minutes, incubate with antibody solution, incubate in a humidified chamber at room temperature for 20 minutes, and wash three times with 1× PBS, each time for 5 minutes;

[0055] DAB color development: Prepare DNA color development solution, add 1 drop of solution B to 1 mL of solution A, mix well, protect from light, prepare immediately before use, add the color development solution to the slices, incubate in the dark for several minutes depending on the condition of the slices, until a brown precipitate appears on the slices, rinse the slices with distilled water to terminate the color development reaction;

[0056] Hematoxylin counterstaining: After developing the color, rinse the tissue sections thoroughly with distilled water and then soak in hematoxylin for 3–5 minutes. Hydrochloric acid acidification and lithium carbonate debluing: Acidify with 1% hydrochloric acid, draw the sample several times, rinse the remaining liquid with distilled water, and place in saturated lithium carbonate for 1–2 minutes to debluing. Observe the staining under a microscope. Dehydrate and mount: Dehydrate in the reverse order of dewaxing, mount with gum, observe under a light microscope, and photograph and analyze.

[0057] 2. Experimental Results

[0058] Immunohistochemical staining was used to detect the expression of TRIM27 and CERS1 in cancer tissues of 120 patients with colorectal cancer. Figure 4 As shown in the figure, there may be a negative correlation between TRIM27 expression and CERS1 expression.

[0059] Example 3

[0060] IHC grading and verification of the relationship between TRIM27 and CERS1 expression in colorectal cancer tissues

[0061] 1. Experimental Procedure

[0062] Immunohistochemistry (IHC) staining was graded by three professionals in a double-blind manner: brownish-yellow granular precipitates in the cytoplasm or cell membrane were considered positive. The percentage of positive cells was first scored as 0 to 4, with none, 1-25%, 26-50%, 51-75%, and 76-100% respectively. The staining intensity was then graded from 0 to 3, with no staining (negative) receiving 0, yellow (weakly positive) receiving 1, brownish-yellow (positive) receiving 2, and yellowish-brown (strongly positive) receiving 3. The product of the two scores was combined to give a maximum total score of 12, with higher scores indicating stronger positivity. TRIM27 expression was scored as low (0 to 4) and high (6 to 12). CERS1 expression was scored as low (0 to 4) and high (6 to 12).

[0063] 2. Experimental Results

[0064] The experimental results are as follows Figure 5-7 As shown, CERS1 expression was increased in colorectal cancer patients with decreased TRIM27 expression ( Figure 5 ), and CERS1 was highly expressed in colorectal cancer tissues of patients with low TRIM27 expression ( Figure 6 These data suggest that the expression of CERS1 in colorectal cancer tissues may be negatively correlated with the expression of TRIM27. Person correlation analysis was also verified ( Figure 7 ).

[0065] Example 4

[0066] Relationship between TRIM27 and CERS1 expression and overall survival in patients with colorectal cancer

[0067] 1. Experimental Procedure

[0068] According to IHC scores (TRIM27: 0-4 for low expression, 6-12 for high expression; CERS1: 0-4 for low expression, 6-12 for high expression), 120 colorectal cancer patients were divided into four groups: TRIM27 High CERS1 Low TRIM27 High CERS1 High TRIM27 Low CERS1 Low TRIM27 Low CERS1 High The five-year survival of the four groups of patients was analyzed using Kaplan-Meier method, and survival curves were drawn.

[0069] 2. Experimental Results

[0070] Kaplan-Meier analysis of the five-year survival of patients with different TRIM27 and CERS1 expressions Figure 8 As shown in the results, patients with high TRIM27 expression and low CERS1 expression showed the worst survival. These clinical data suggest that the expression of TRIM27 and CERS1 in colorectal cancer patients is negatively correlated, and the combined detection of TRIM27-CERS1 has reliable prognostic value in colorectal cancer patients.

[0071] The above examples demonstrate that CERS1 and TRIM27 gene expression are negatively correlated in colorectal cancer patients. High TRIM27 expression and low CERS1 expression are positively correlated with larger tumors, higher tumor grade, and more distal metastases, leading to lower five-year survival rates. These statistical results confirm that combined detection of TRIM27 and CERS1 gene expression levels is valuable for prognostic assessment in colorectal cancer patients.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A biomarker for the prognosis of colorectal cancer, characterized in that: Including the TRIM27 gene and the CERS1 gene.

2. The biomarker according to claim 1, characterized in that Patients with significantly high expression of the TRIM27 gene and significantly low expression of the CERS1 gene have a poor prognosis.

3. The biomarker according to claim 2, characterized in that The prognosis includes survival rate.

4. Use of a reagent for detecting the expression level of the biomarker according to claim 1 in the preparation of a colorectal cancer prognosis diagnostic kit.

5. The use according to claim 4, characterized in that The reagents include anti-TRIM27 antibodies and anti-CERS1 antibodies.

6. The use according to claim 4 or 5, characterized in that The prognostic risk was assessed based on the expression levels of TRIM27 and CERS1 genes; the prognostic risk assessment model was as follows: Riskscore = (0.605)*TRIM27 + (0.8862)*CERS1.

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