TIMP1 and FOXP3 biomarker combination for diagnosing colorectal cancer and application of TIMP1 and FOXP3 biomarker combination

Abstract

The invention discloses a TIMP1 and FOXP3 biomarker combination for diagnosing colorectal cancer and application of the TIMP1 and FOXP3 biomarker combination, and belongs to the technical field of biological medicine detection. The invention provides an effective colorectal cancer early diagnosis and disease staging method based on the detection of the expression quantities of TIMP1 and FOXP3, the levels of the two markers can be accurately determined in blood or tumor samples through technologies such as ELISA or immunohistochemistry, the two biomarkers are jointly detected, and the sensitivity and specificity of diagnosis are remarkably improved. And an effective means is provided for early screening and prognosis evaluation of cancers. The method has the advantages of simplicity, convenience, rapidness and high repeatability, and is suitable for early diagnosis, disease staging and prognosis prediction of colorectal cancer. The technical support is provided for further expanding the sample scale and verifying the application potential of the TIMP1 and the FOXP3 in diagnosis and prognosis evaluation of other gastrointestinal tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, and in particular to a combination of TIMP1 and FOXP3 as biomarkers for diagnosing colorectal cancer and an application thereof. Background Art

[0002] Colorectal cancer is one of the cancers with the highest morbidity and mortality rates worldwide, especially in developed countries. Early detection and treatment of colorectal cancer can significantly improve the survival rate of patients. However, existing diagnostic methods such as colonoscopy, imaging tests and histopathological analysis have the limitations of high cost, high invasiveness and insufficient sensitivity for early cancer detection.

[0003] In tumor biology, the detection of biomarkers provides a new direction for non-invasive, highly sensitive and specific early cancer diagnosis. TIMP1 (inhibitor of matrix metalloproteinases) is widely involved in tumor invasion and metastasis. Studies have shown that TIMP1 is upregulated in a variety of cancers and is associated with poor prognosis. FOXP3 (forkhead box P3) is an important immune regulatory molecule that regulates the function of T cells, especially the development and function of regulatory T cells (Tregs), and plays a key role in the tumor immune microenvironment. The expression of FOXP3 is closely related to the mechanism of tumor immune escape.

[0004] There is currently no systematic study on the diagnostic value of TIMP1 and FOXP3 in colorectal cancer, and their combined detection may provide new ideas for early diagnosis and prognosis of cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a combination of TIMP1 and FOXP3 as biomarkers for diagnosing colorectal cancer and their application, so as to solve the problems existing in the above-mentioned prior art. The combined detection of the expression levels of TIMP1 and FOXP3 in serum or tumor tissue significantly improves the sensitivity and specificity of diagnosing colorectal cancer or evaluating the disease stage.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a biomarker combination for diagnosing colorectal cancer, wherein the biomarker combination consists of TIMP1 and FOXP3.

[0008] The present invention also provides the use of a reagent for detecting the expression amount of the biomarker combination in serum in the preparation of a product for diagnosing colorectal cancer.

[0009] Preferably, the expression level of TIMP1 in serum is significantly increased compared with that of the healthy control group, indicating that the subject is more likely to suffer from colorectal cancer;

[0010] The expression level of FOXP3 in serum was significantly higher than that of the healthy control group, indicating that the subjects were more likely to suffer from colorectal cancer.

[0011] The present invention also provides a product for diagnosing colorectal cancer, comprising a reagent for detecting the expression level of the above-mentioned biomarker combination in serum.

[0012] Preferably, the reagent comprises an antibody that specifically binds to the TIMP1 and an antibody that specifically binds to the FOXP3.

[0013] The present invention also provides the use of a reagent for detecting the expression amount of the biomarker combination in colorectal cancer in the preparation of a product for evaluating the disease stage of colorectal cancer patients.

[0014] Preferably, the expression level of TIMP1 in colorectal cancer tumor tissue is significantly increased, indicating that the colorectal cancer patient is likely to have advanced colorectal cancer;

[0015] The expression level of FOXP3 in colorectal cancer tumor tissue is significantly increased, indicating that the colorectal cancer patient is likely to have advanced colorectal cancer.

[0016] The present invention also provides a product for evaluating the disease stage of colorectal cancer patients, including a reagent for detecting the expression amount of the biomarker combination in colorectal cancer tumor tissue.

[0017] Preferably, the reagent comprises an antibody that specifically binds to the TIMP1 and an antibody that specifically binds to the FOXP3.

[0018] Preferably, the product is an ELISA kit

[0019] The present invention discloses the following technical effects:

[0020] The present invention provides an effective method for early diagnosis and disease staging of colorectal cancer based on the detection of TIMP1 and FOXP3 expression levels. Through ELISA or immunohistochemistry and other techniques, the levels of the two markers can be accurately measured in blood or tumor tissue samples. The combined detection of the two biomarkers significantly improves the sensitivity and specificity of diagnosis. It provides an effective means for early screening and prognosis evaluation of cancer. The method has the advantages of simplicity, rapidity, and strong repeatability, and is suitable for early diagnosis and disease staging of colorectal cancer. It provides technical support for further expanding the sample size and verifying the application potential of TIMP1 and FOXP3 in the diagnosis and prognosis evaluation of other digestive tract tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The bar graph shows the difference in expression levels of TIMP1 and FOXP3 in patients with early-stage and advanced colorectal cancer and healthy controls; Control is the healthy control group, Early-Stage is the early-stage colorectal cancer patients, and Advanced-Stage is the advanced-stage colorectal cancer patients;

[0023] Figure 2 This is the ROC curve diagram of TIMP1, FOXP3 and the combined detection of TIMP1 and FOXP3 in the diagnosis of colorectal cancer; wherein, a is TIMP1, b is FOXP3, and c is TIMP1 combined with FOXP3;. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The kit information used in the present invention is as follows:

[0030] TIMP1 ELISA Kit:

[0031] Human TIMP1 ELISA Kit (Thermo Fisher Scientific, Catalog No. EH456RB) is suitable for the detection of TIMP1 in serum, plasma and cell culture supernatant, with a detection sensitivity of 40 pg / mL, and is suitable for the diagnostic method proposed in the present invention.

[0032] FOXP3 ELISA Kit:

[0033] Human FOXP3 ELISA Kit (purchased from Abcam, catalog number ab289644), using SimpleStep The technology has a detection time of 90 minutes and a sensitivity of 0.325ng / mL, which is suitable for rapid clinical testing and routine laboratory testing.

[0034] Example 1 ELISA-based combined detection method of TIMP1 and FOXP3

[0035] This example provides a method for the combined detection of TIMP1 and FOXP3 based on ELISA technology, and the specific steps are as follows:

[0036] 1. Sample collection: Serum samples were collected from 50 colorectal cancer patients (including 25 early-stage and 25 advanced-stage patients) and 50 healthy controls.

[0037] 2. Marker detection: Use commercial TIMP1 ELISA kit and FOXP3 ELISA kit according to the kit instructions to quantitatively detect the levels of TIMP1 and FOXP3 in each sample.

[0038] 3. Data analysis: The MannWhitney U test was used to compare the differences in TIMP1 and FOXP3 expression levels between patients with different stages of colorectal cancer and the healthy control group.

[0039] The experimental results are shown in Table 1 and Figure 1 shown.

[0040] Table 1 Statistics of TIMP1 and FOXP3 levels in samples

[0041] Group TIMP1 average value (ng / mL) FOXP3 average value (ng / mL) Healthy control group 8.1±1.3 4.2±1.1 Early colorectal cancer patients <![CDATA[10.5±1.5 * ]]> <![CDATA[5.7±1.2 * ]]> Advanced colorectal cancer patients <![CDATA[13.9±2.0 **# ]]> <![CDATA[8.6±1.4 **# ]]>

[0042] Note: * indicates P<0.05 compared with the healthy control group, ** indicates P<0.01 compared with the healthy control group, # indicates P<0.05 compared with the early colorectal cancer patient group.

[0043] Experimental data showed that TIMP1 and FOXP3 showed significant differences among the healthy control group, early colorectal cancer patient group and advanced patient group.

[0044] 4. ROC curve analysis: By drawing the ROC curve, the AUC, sensitivity and specificity are calculated, and the diagnostic efficacy of single detection and combined detection is compared.

[0045] The experimental results are as follows Figure 2 As shown in Tables 2 and 3 (the meanings of the predictive variables in Tables 2 and 3 are the same), the AUC of combined detection of TIMP1 and FOXP3 was 0.92, which was significantly better than the single detection of TIMP1 (AUC = 0.85) or FOXP3 (AUC = 0.85).

[0046] Table 2 AUC results of TIMP1 and FOXP3 alone or in combination

[0047] Predictor variables Predicting the outcome Area under the curve (AUC) Confidence Interval (CI) a(TIMP1) Reverse 0.851 0.771-0.932 b(FOXP3) Reverse 0.852 0.776-0.927 c(TIMP1 combined with FOXP3) Reverse 0.919 0.866-0.972

[0048] Table 3 AUC test table of TIMP1 and FOXP3 alone or in combination

[0049] Variable 1 Variable 2 Statistics p-value Inspection methods Trend Direction a b -0.0071788 0.9943 DeLong's test Consistency a c -2.2217 0.0263 DeLong's test Consistency b c -1.9858 0.0471 DeLong's test Consistency

[0050] Experimental data showed that TIMP1 and FOXP3 showed significant differences in the healthy control group, early colorectal cancer patient group and advanced patient group. The expression levels of TIMP1 and FOXP3 in patients with early colorectal cancer were significantly higher than those in the healthy control group, while the expression levels in advanced patients were further increased. This shows that the expression levels of the two can not only be used for early diagnosis, but also as biomarkers for cancer progression. The role of TIMP1 in matrix remodeling and tumor invasion has been verified, and its high expression in advanced tumors suggests that it is related to cancer invasion and metastasis. As a key factor of regulatory T cells, FOXP3 is involved in the regulatory mechanism of tumor immune escape, and its high expression is also closely related to cancer progression.

[0051] The present invention provides an effective method for early diagnosis and prognosis evaluation of colorectal cancer, which significantly improves the sensitivity and specificity of diagnosis based on the combined detection of TIMP1 and FOXP3. Through techniques such as ELISA, doctors can accurately measure the levels of two markers in blood or tissue samples, thereby providing a reliable basis for clinical decision-making. The combined detection of TIMP1 and FOXP3 greatly improves the sensitivity and specificity of diagnosis, and is significantly better than the diagnostic efficacy of detecting TIMP1 or FOXP3 alone. The AUC value of TIMP1 alone is 0.85, while the AUC value of FOXP3 alone is 0.85. The AUC value of the combined detection of the two reaches 0.92, indicating that it has a strong diagnostic ability, especially in the diagnosis of early cancer. The combined detection not only shows significant advantages in early diagnosis, but also can help doctors accurately determine the tumor stage through the differences in expression levels of patients at different stages.

[0052] The detection method proposed in the present invention not only has significant clinical application value in the early diagnosis of colorectal cancer, but can also be used to evaluate disease progression and staging. By regularly testing patients' TIMP1 and FOXP3, doctors can monitor the patient's disease dynamics and adjust treatment plans according to changes in marker levels.

[0053] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A biomarker combination for diagnosing colorectal cancer, characterized in that: The biomarker combination consists of TIMP1 and FOXP3.

2. Use of a reagent for detecting the expression level of the biomarker combination as described in claim 1 in serum in the preparation of a product for diagnosing colorectal cancer.

3. The use according to claim 2, characterized in that The expression level of TIMP1 in serum was significantly higher than that in the healthy control group, indicating that the tested person is more likely to suffer from colorectal cancer; The expression level of FOXP3 in serum was significantly higher than that of the healthy control group, indicating that the subjects were more likely to suffer from colorectal cancer.

4. A product for diagnosing colorectal cancer, characterized in that: The invention comprises a reagent for detecting the expression level of the biomarker combination as claimed in claim 1 in serum.

5. The product according to claim 4, characterized in that The reagents include an antibody that specifically binds to the TIMP1 and an antibody that specifically binds to the FOXP3.

6. Use of a reagent for detecting the expression level of the biomarker combination as claimed in claim 1 in colorectal cancer tumor tissue in the preparation of a product for evaluating the disease stage of colorectal cancer patients.

7. The use according to claim 6, characterized in that The expression level of TIMP1 in colorectal cancer tumor tissue is significantly increased, indicating that the colorectal cancer patient is likely to have advanced colorectal cancer; The expression level of FOXP3 in colorectal cancer tumor tissue is significantly increased, indicating that the colorectal cancer patient is likely to have advanced colorectal cancer.

8. A product for evaluating the disease stage of colorectal cancer patients, characterized in that: It comprises a reagent for detecting the expression level of the biomarker combination as claimed in claim 1 in colorectal cancer tumor tissue.

9. The product according to claim 8, characterized in that The reagents include an antibody that specifically binds to the TIMP1 and an antibody that specifically binds to the FOXP3.

10. The product according to claim 8, characterized in that The product is an ELISA kit.

Images