CN109696547B

CN109696547B - Marker for judging colorectal cancer prognosis and application thereof

Info

Publication number: CN109696547B
Application number: CN201710999399.2A
Authority: CN
Inventors: 蔡剑平; 李瑾
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-10-23
Filing date: 2017-10-23
Publication date: 2021-10-26
Anticipated expiration: 2037-10-23
Also published as: CN109696547A

Abstract

The invention relates to a marker for judging colorectal cancer prognosis and application thereof, belonging to the technical field of medical biotechnology and medical inspection. Use of a MutT-related protein as a biomarker for the prognosis of a patient with colorectal cancer. The MutT related protein can effectively distinguish the postoperative survival time of a colorectal cancer patient, thereby providing a new way for colorectal cancer prognosis diagnosis and providing reference basis for a clinician to analyze the colorectal cancer condition.

Description

Marker for judging colorectal cancer prognosis and application thereof

Technical Field

The invention relates to a marker for judging colorectal cancer prognosis and application thereof, belonging to the technical field of medical biotechnology and medical inspection.

Background

Colorectal cancer (CRC) is a malignant cancer caused by various carcinogenic factors such as environment and heredity of colonic mucosal epithelium, is one of the most common digestive tract malignant tumors, has a third-place morbidity and a fourth-place mortality in the world, has about 120 thousands of new cases every year, and seriously threatens human health. Colorectal cancer is high in incidence in asia, and is mainly related to risk factors such as age, family history, unhealthy diet and living habits in China. Colon cancer is hidden, has no obvious clinical symptoms in the early stage, and has slow disease development, most colorectal cancer patients are in the tumor progression stage when being diagnosed, the optimal treatment time is lost, the five-year survival rate is less than 20 percent, and the prognosis is poor. Therefore, the search for an evaluation method for the rapid diagnosis of colorectal cancer, which improves the diagnosis and treatment level and the survival rate, is a hot point and a difficult point of the current research.

MTH1(MutT homolog 1) is also called NUDT1(Nudix hydro-lase 1), MTH2(MutT homolog 2) is also called NUDT15(Nudix hydro-lase 15), MTH3(MutT homolog 3) is also called NUDT18 (Nudix hydro-lase 18) and NUDT5(Nudix hydro-lase 5), and the four proteins belong to the mammalian Nudix hydrolase superfamily. These proteins have structural and functional similarities to the E.coli MutT protein and are also known as MutT-related proteins.

In the invention, the expression of MutT related proteins including MTH1, MTH2, MTH3 and NUDT5 in colorectal cancer tissues (CRC) and paracarcinoma tissues is detected, the correlation between the expression quantity of the proteins and clinical pathological data (such as sex, age, position, tumor size, clinical stage, differentiation degree and the like) is analyzed, and the overall survival prognosis judgment value of the proteins for patients after colorectal cancer radical treatment is researched.

Disclosure of Invention

The present invention is made based on the following findings of the present inventors:

cells produce large amounts of ROS, either during normal metabolic activity or upon exogenous stimuli, which can damage DNA, RNA, and free nucleotides. Guanine, because of its lowest oxidation potential, its oxidation product, 8-oxoG, is the most abundant oxidized base in the cell. Nucleotides containing 8-oxoG can be incorporated into DNA or RNA, and as 8-oxoG pairs with A and C with equal efficiency, mismatches at the replication and transcription levels are caused.

The MutT protein found in E.coli (E.coli) has the ability to hydrolyze 8-oxoGTP and 8-oxoGTP to monophosphates. The more precise mechanism involved in the clearance of nucleotide oxide in nucleotide precursor pool is possessed in mammalian cells, and MTH1, MTH2, NUDT5 and MTH3, etc. found in mammalian cells are homologous proteins of MutT. The substrate specificity of these proteins is different, MTH1 can degrade 8-oxodGTP and 8-oxoGTP, but cannot act on 8-oxodGP and 8-oxoGDP; MTH2 can degrade 8-oxodGTP, 8-oxoGTP, 8-oxoGDP and 8-oxoGDP, but has weak activity; MTH3 can degrade 8-oxoGDP and 8-oxoGDP, but cannot act on 8-oxoGTP and 8-oxoGTP; NUDT5 has activity of hydrolyzing 8-oxo-dGDP, and also has wider substrate specificity, and can hydrolyze 8-oxo-dADP, 2-oxo-dADP, and 5-CHO-dUDP.

We found for the first time that proteins MTH2, MTH3 and NUDT5 were expressed in increased amounts in CRC tissues and correlated with CRC progression and prognosis.

The expression levels of MTH1, MTH2, MTH3 and NUDT5 proteins are related to colorectal cancer AJCC stage and lymph node metastasis. The CRC patients in MTH1, MTH2 and NUDT5 high expression groups have lower overall survival rate after surgical treatment, and the survival rate after surgery of the patients with all four proteins high expression groups is the worst. Therefore, the MutT-related protein can be used as a novel marker for judging the prognosis of colorectal cancer patients. Meanwhile, multi-factor analysis indicates that the NUDT5 protein is an independent factor influencing the prognosis of CRC patients.

In one aspect of the invention, there is provided the use of a MutT-related protein as a biomarker for the prognosis of a patient with colorectal cancer.

The MutT related protein is one or more of MTH1 protein, MTH2 protein, MTH3 protein and NUDT5 protein.

Preferably, the MutT-related protein is one or more of MTH2 protein, MTH3 protein and NUDT5 protein.

The expression level of any one of the above proteins can be used to detect the presence of colorectal cancer, preferably by measuring the expression level of multiple proteins.

In a second aspect of the invention, there is provided the use of a MutT-related protein for the manufacture of a product for detecting, predicting or diagnosing the progression and/or prognosis of colorectal cancer.

The product refers to a product for diagnosing or predicting colorectal cancer metastasis, colorectal cancer clinical stage and colorectal cancer patient prognosis. The products include, but are not limited to, chemical reagents, biological reagents, diagnostic kits, colloidal gold test strips, protein chips, monoclonal antibodies, polyclonal antibodies, and the like.

The MutT related protein is one or more of MTH2 protein, MTH3 protein and NUDT5 protein.

The "individual" as the test subject may be any human or non-human mammal. Non-human mammals include primates, livestock animals (e.g., horses, cows, sheep, pigs), laboratory test animals (e.g., mice, rats, rabbits, guinea pigs), companion animals (e.g., dogs, cats) and captive wild animals (e.g., deer).

Preferably, the mammal is a human.

The assay products of the invention, and the assay methods using these products, can be performed on any suitable biological sample. For this purpose, the sample may be any sample of biological material that is derived from an animal, such as, but not limited to, cellular material, biological fluid (e.g., blood), stool, tissue biopsy specimen, surgical specimen, or fluid that has been introduced into the animal's body and subsequently removed (e.g., solution withdrawn from an enema wash). The samples tested according to the methods of the invention may be tested directly or may require some form of processing prior to testing. For example, a biopsy sample or surgical sample may require homogenization prior to testing or it may require sectioning for testing qualitative expression levels of individual genes in situ. In addition, if the biological sample is not in liquid form, it may be necessary to add reagents, such as buffers, to mobilize the sample.

In a third aspect of the invention, a novel human colon cancer biomarker MutT-related protein is provided for preparing a preparation for diagnosing, predicting, detecting or screening human colorectal cancer; in particular for preparing a kit for diagnosing, predicting, detecting or screening human colorectal cancer. The MutT-related protein of the human colon cancer protein marker can also be used for preparing preparations for diagnosing, predicting, detecting or screening human colorectal cancer cell diffusion, lymph node metastasis, clinical staging and patient prognosis; in particular to the preparation of diagnostic reagent for diagnosing, predicting, detecting or screening the human colorectal cancer cell diffusion, colorectal cancer lymph node metastasis, colorectal cancer clinical stage and colorectal cancer patient prognosis.

A kit for detecting the progression and/or prognosis of colorectal cancer, which comprises a reagent for detecting the expression level of MutT-related protein.

The MutT related protein is one or more of MTH1 protein, MTH2 protein, MTH3 protein and NUDT5 protein. The expression level of any one of the proteins can be used for diagnosing the existence of colorectal cancer.

The MutT related protein is MTH1 protein, MTH2 protein and NUDT5 protein. The expression level of the histone can be used for predicting the prognosis of colorectal cancer.

The MutT related protein is MTH1 protein, MTH2 protein, MTH3 protein and NUDT 5. The expression level of the histone can be used for predicting the prognosis of colorectal cancer.

The reagent is an antibody, and is a monoclonal antibody and/or a polyclonal antibody. The protein antibody may be a commercially available antibody or an antibody to the corresponding protein prepared according to conventional procedures in the art.

Has the advantages that: the MutT related protein can effectively distinguish the postoperative survival time of a colorectal cancer patient, thereby providing a new way for colorectal cancer prognosis diagnosis and providing reference basis for a clinician to analyze the colorectal cancer condition. The product and the detection method using the product can be used for diagnosis, prognosis, classification, prediction, detection of disease relapse, selection of therapy and course monitoring of disease risks.

The various aspects and advantages of the disclosure will become more readily apparent and may be understood in more detail with reference to the following detailed description and the accompanying drawings, which are not to be taken in a limiting sense. Equivalents of the art made in accordance with the disclosure are intended to be covered by the present invention.

Drawings

FIG. 1 shows the expression of MutT-related proteins in colorectal cancer tissues and paired paraneoplastic tissues, FIG. 1A shows representative Western blotting bands for the expression of 4 MutT-related proteins in 6 pairs of colorectal cancer tissues and paired paraneoplastic tissues, and FIGS. 1B to 1E show the quantitative results of the expression of proteins MTH1, MTH2, MTH3 and NUDT5 in 20 pairs of colorectal cancer tissues and paired paraneoplastic tissues, respectively, and the statistical method is Student's t-test.

FIG. 2 is a representation of the immunohistochemistry of MutT-related proteins in colorectal cancer tissues and paired paracarcinoma tissues; FIGS. 2A-2D represent the expression of proteins MTH1, MTH2, MTH3 and NUDT5 in normal paracancer tissues, respectively, FIGS. 2E-2H represent the low expression of proteins MTH1, MTH2, MTH3 and NUDT5 in colorectal cancer tissues, respectively, and FIGS. 2I-2L represent the high expression of proteins MTH1, MTH2, MTH3 and NUDT5 in colorectal cancer tissues, respectively.

FIG. 3 is a Kaplan-Meier survival curve; FIG. 3A is a survival curve showing the difference between the amount of expression of MTH1 protein and the prognosis of a patient with colorectal cancer, FIG. 3B is a survival curve showing the difference between the amount of expression of MTH2 protein and the prognosis of a patient with colorectal cancer, FIG. 3C is a survival curve showing the difference between the amount of expression of MTH3 protein and the prognosis of a patient with colorectal cancer, FIG. 3D is a survival curve showing the difference between the amount of expression of NUDT5 protein and the prognosis of a patient with colorectal cancer, and FIG. 3E is a survival curve showing the difference between the amounts of expression of the four proteins and the prognosis of a patient with colorectal cancer.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1

Materials and methods

1. Experimental Material

(1)44 colorectal cancer tissue and corresponding paracancerous normal tissue (provided by the first Hospital affiliated with the university of Jia Musi)

(2) Colon cancer tissue chip-87 cancer tissues and corresponding paracancer normal tissues (Shanghai core Biotechnology Co., Ltd.) before radical treatment of colorectal cancer

(3) anti-MTH 1 antibody (Abcam), anti-MTH 3 antibody (Abcam), anti-NUDT 5 antibody (Abcam), anti-MTH 2 antibody (Abclonal), anti-GAPDH antibody (Abcam)

2. Experimental methods

(1) The expression levels of MTH1, MTH2, MTH3 and NUDT5 in the colorectal cancer tissues and the corresponding paracancerous normal tissues are detected by Western Blotting 44, and the correlation between the expression levels of the proteins and clinical pathological data (such as sex, age, position, tumor size, clinical stage, differentiation degree and the like) is analyzed. The method comprises the following specific steps:

the preparation method comprises the following steps of grinding tissues by liquid nitrogen, adding RIPA lysate (Beijing Solebao science and technology Co., Ltd.), wherein the RIPA lysate contains 1X PMSF (Solebao), 1X protease and phosphatase inhibitor (CST, USA), standing on ice for 30min, centrifuging at 4 ℃ for 20min at 12000g, and taking supernatant and transferring to a new 1.5ml EP tube.

② the BCA method (Thermo fisher, USA) to determine the total protein concentration.

③ 12 percent SDS-PAGE electrophoresis, the sample loading for detecting the expression of GAPDH is 10ug, the sample loading for detecting the expression of MTH1 and NUDT5 is 20ug, and the sample loading for detecting the expression of MTH2 and MTH3 is 40 ug.

(iv) transferring the membrane, and electrotransfering the protein in the PAGE to a PVDF membrane (Millipore, USA).

Fifthly, sealing the mixture by immunity, and sealing the mixture for 2 hours by using 5 percent of skimmed milk.

Sixthly, primary antibody incubation, wherein the dilution ratio of the anti-MTH 1 antibody, the anti-MTH 3 antibody, the anti-NUDT 5 antibody and the anti-MTH 2 antibody is 1: 1000, the dilution ratio of the anti-GAPDH antibody is 1: 2000, the mixture is kept overnight at 4 ℃, and TBST is washed for 5 times, and each time is 5 min.

And seventhly, incubating the goat anti-rabbit IgG-HRP (Biyunshi Biotechnology Co., Ltd., 1: 2000) or the goat anti-mouse IgG-HRP (Biyunshi, 1: 2000), incubating at room temperature for 2h, and washing for 5 times (5 min each time) by TBST.

Exposing, mixing display liquid (Millipore, USA) 1: 1, dripping onto the strip, and exposing and developing.

Ninthly, quantifying the results by using Image J, and statistically analyzing the expression of the four proteins in the colorectal cancer tissues and the tissues beside the cancer by using Student's t-test.

(2) The expression levels of MTH1, MTH2, MTH3 and NUDT5 in 87 cases of cancer tissues and corresponding paracancer normal tissues (tissue chips) are detected by immunohistochemistry, the cancer tissues are divided into a high expression group and a low expression group according to the degree of staining and the staining area, the correlation between the expression levels of the proteins and clinical pathological data (such as sex, age, position, tumor size, clinical stage, differentiation degree and the like) is analyzed, and the overall survival prognosis judgment value of the proteins for patients after radical colorectal cancer treatment is researched. The method comprises the following specific steps:

the tissue chip is dewaxed by xylene for 2d, and is subjected to gradient hydration for 2min by 100%, 95%, 85% and 75% ethanol and is hydrated for 10min by PBS.

② the antigen repair (pH6.0 citric acid repair liquid) microwave repair for 30min, and cooling to room temperature.

③ sealing the goat serum (Beijing Zhongshirt Jinqiao Biotech Co., Ltd.) for 30 min.

Fourthly, primary antibody incubation, the dilution ratio of the anti-MTH 1 antibody, the anti-MTH 2 antibody, the anti-MTH 3 antibody and the anti-NUDT 5 antibody is 1: 300, 1: 100, 1: 500 and 1: 1000 respectively, the mixture is washed by PBS for 5min and X3 times at 4 ℃.

Fifthly, secondary antibody incubation, using PV-6001 goat anti-rabbit IgG/HRP polymer or PV-6002 goat anti-mouse IgG/HRP polymer (Zhongxiu gold bridge), incubating for 20 minutes at room temperature, and washing for 5min X3 times with PBS.

DAB (Chinese shirt and gold bridge) coloration

Seventhly, counterstaining hematoxylin, gradient dewatering and transparent sealing.

3. The MutT related protein expression high-low scoring standard in the tissue chip:

(1) dyeing strength: 0 (none); 1 (weak); 2 (middle); 3 (Strong)

(2) Dyeing area: 0 (0%); 1 (1-25%); 2 (26-50%); 3 (51-75%); 4 (76-100%);

(3) the final score is the staining intensity X stained area: low expression (0-6); high expression (7-12)

4. Statistical method

Software SPSS statistics version 19.

(1) Student's t-test compares the two sets of means;

(2) pearson 'X2 or Fisher's exact test comparing the correlation of the MutT-related protein to the CRC clinical pathology data;

(3) Kaplan-Meier analysis calculates the overall survival rate (OS), the log-rank test compares the two groups of OS;

(4) cox clinical and pathological parameters and the influence of MutT-related protein expression on the survival rate and prognosis of CRC patients are clarified by Cox clinical and pathological models, and single-factor analysis is performed on all parameters, and then multi-factor analysis is performed on the parameters with significant meaning.

Second, experimental results

1. Increased expression of MutT-related protein in colorectal cancer tissue

(1) We firstly carried out Western blotting detection 20 to detect the protein expressions of MTH1, MTH2, MTH3 and NUDT5 in human colorectal cancer tissues and paired paracancer normal tissues, and the results show that the protein expressions in the four kinds of human colorectal cancer tissues are all obviously higher than those in normal tissues (Student' st-test, P < 0.001, figure 1).

(2) We then performed immunohistochemical detection of MTH1, MTH2, MTH3 and NUDT5 protein expression in tissue chips (87 human colorectal cancer tissue and matched paracancerous tissue), again demonstrating upregulation of these four proteins in cancer tissue (fig. 2). The four proteins of the paracancer normal tissue are weakly immunostained, and the four proteins of the cancer tissue are different in staining degree, namely weak, medium and strong. According to the intensity and the range of immunostaining of the cancer tissues, the proteins MTH1, MTH2, MTH3 and NUDT5 in 87 cancer tissues are highly expressed by 54, 49, 45 and 42 respectively.

Correlation of MutT-related protein expression with colorectal cancer clinicopathological parameters

(1) According to the results of immunohistochemical staining of proteins MTH1, MTH2, MTH3 and NUDT5, 87 individual colorectal cancer tissues in a tissue chip were divided into a high expression group and a low expression group, and the correlation of the expression of the proteins with age, sex, position, tumor size, AJCC stage, T stage, N stage, M stage, differentiation degree and vascular metastasis was analyzed by Pearson 'X2 or Fisher's exact test, and the results showed that the expression levels of the proteins were significantly correlated with the AJCC stage and the N stage (lymph node metastasis) (P < 0.05, Table 1).

Table 1 correlation of MutT-related protein expression with colorectal cancer clinicopathologic parameters (immunohistochemistry, n ═ 87)

^aChi-square test

^bFisher′s exact test

^*P＜0.05

(2) Western blotting examined the relative expression of proteins MTH1, MTH2, MTH3 and NUDT5 in 44 human colorectal tissues, and Student's T-test was used to analyze the clinical pathological correlation of the protein expression and CRC, and the results show that the protein expression level is significantly related to AJCC stage, T stage (tumor infiltration degree) and N stage (lymph node metastasis) (P < 0.05, Table 2).

TABLE 2 correlation of MutT-related protein expression with colorectal cancer clinicopathologic parameters (Western Blotting, n ═ 44)

^*P＜0.05

Effect of MutT-related protein expression on prognosis of patients with colorectal cancer

(1) According to the results of immunohistochemical staining of proteins MTH1, MTH2, MTH3 and NUDT5, 87 individual colorectal cancer tissues in a tissue chip are divided into a high expression group and a low expression group respectively, the total survival rate (OS) of CRC patients after surgical treatment is calculated by using Kaplan-Meier analysis, and log-rank test is used to compare the OS of the high expression group and the low expression group, so that the results show that the survival rates of the high expression group and the low expression group of MTH1, MTH2 and NUDT5 are lower (P is 0.005, 0.021 and 0.003), and the survival rates of the high expression group and the low expression group of MTH3 are not significantly different (P is 0.089). In addition, considering the co-expression of these four proteins in cancer tissues, 87 cancer tissues were divided into three groups: first, four proteins were underexpressed; second, one or two or three proteins are highly expressed; in the third group, four proteins were highly expressed. The third group had the lowest survival rate, followed by the second group (P ═ 0.005). (FIG. 3)

(2) As shown in Table 3, single factor analysis of CRC clinical pathology data and levels of MTH1, MTH2, MTH3 and NUDT5 proteins using a Cox presentation hazards model showed that the following factors affect the survival of CRC patients: AJCC staging (P < 0.001), N staging (P ═ 0.001), M staging (P ═ 0.006), degree of differentiation (P ═ 0.003), expression level of MTH1 (P ═ 0.008), expression level of MTH2 (P ═ 0.026), and expression level of NUDT5 (P ═ 0.005). Then, multifactorial analysis was performed to find that the expression level of NUDT5 was an independent factor affecting the prognosis of CRC patients (HR 2.282; 95% CI 1.152-4.517, P ═ 0.018).

TABLE 3 Cox Single and Multi-factor analysis

^*P＜0.05

Conclusion III

In the present study, the MTH2, MTH3 and NUDT5 proteins are found to be expressed in CRC tissues in an increased amount for the first time, and are related to CRC progression and prognosis.

Example 2 kit for detecting the progression and prognosis of colorectal cancer

Kit composition

1. anti-MTH 1 antibody (Abcam)

2. anti-MTH 2 antibody (Abclonal)

3. anti-MTH 3 antibody (Abcam)

4. anti-NUDT 5 antibody (Abcam)

Second, use method

See example 1 "detection of expression levels of MTH1, MTH2, MTH3, and NUDT5 in 87 cases of cancer tissues and corresponding paraneoplastic normal tissues (tissue chips) by immunohistochemistry" or "detection of expression levels of MTH1, MTH2, MTH3, and NUDT5 in 44 cases of colorectal cancer tissues and corresponding paraneoplastic normal tissues by Western Blotting".

Example 3 kit for detecting prognosis of colorectal cancer

Kit composition

1. anti-MTH 1 antibody (Abcam)

2. anti-MTH 2 antibody (Abclonal)

3. anti-NUDT 5 antibody (Abcam)

Second, use method

Claims

The application of the MutT related protein in preparing products for detecting, predicting or diagnosing colorectal cancer is disclosed, wherein the MutT related protein is one or more of MTH2 protein, MTH3 protein and NUDT5 protein.
2. Use according to claim 1, characterized in that: the product is a product for diagnosing or predicting colorectal cancer metastasis, colorectal cancer clinical stage and colorectal cancer patient prognosis; the product is a chemical reagent, a biological reagent, a diagnostic kit, colloidal gold test paper or a protein chip.
3. Use according to claim 2, characterized in that: the biological reagent is a monoclonal antibody or a polyclonal antibody.
4. A kit for detecting the prognosis and/or progression of colorectal cancer, characterized by: the kit contains a reagent for detecting the expression quantity of MutT related protein, wherein the MutT related protein is one or more of MTH2 protein, MTH3 protein and NUDT5 protein; or the MutT related protein is MTH2 protein and NUDT5 protein; or the MutT related protein is MTH2 protein, MTH3 protein and NUDT5 protein.
5. The kit of claim 4, wherein: the reagent is an antibody, and is a monoclonal antibody and/or a polyclonal antibody.