A molecular marker for colorectal cancer

Abstract

The present invention discloses a molecular marker for colorectal cancer, which relates to the technical field of colorectal cancer diagnosis. The molecular marker is TAB2. The present invention firstly discovers that the mRNA level expression of TAB2 in HcoEpic cells, which are colon epithelial cells derived from normal people, is significantly higher than that in two human colon cancer cells, namely HCT116 and LoVo. The molecular marker TAB2 of the present invention for colorectal cancer can be applied to the preparation of products for diagnosing colorectal cancer and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of colorectal cancer diagnosis, and particularly to a molecular marker for colorectal cancer. Background Art

[0002] The occurrence and development of colorectal cancer usually follow the "adenoma-carcinoma" sequence. It takes 5 to 10 years for precancerous lesions to evolve into cancer, providing a time window for early diagnosis and intervention. Its prognosis is closely related to the diagnostic stage. The 5-year relative survival rate for stage I is as high as 90%, while for stage IV it is only 14%. The staging of colon cancer is based on the depth of invasion of the primary focus, lymph node and distant organ metastasis. Without regional lymph node metastasis and the depth of invasion limited within the muscular layer is stage I; reaching the serosa layer and invading adjacent organs outside the serosa is stage II; the presence of pericolonic lymph node metastasis is stage III; the presence of distant organ metastasis is stage IV.

[0003] Currently, the screening methods for colorectal cancer mainly include fecal occult blood test and colonoscopy. Among them, the fecal occult blood test has low sensitivity and specificity; although colonoscopy has high accuracy, it is an invasive test and patients have low compliance. In the early stage of colorectal cancer, there are no specific symptoms, so it is easily ignored by patients. When they go to the hospital for examination after obvious symptoms appear and are finally diagnosed with colorectal cancer, the cancer cells have spread and the best treatment opportunity has been missed. Currently, with the completion of the Human Genome Project and the development of high-throughput sequencing technology, gene screening technology has become a new method for diagnosing colorectal cancer and has significant advantages in the early diagnosis of colorectal cancer. Summary of the Invention

[0004] To solve the above problems, the present invention provides a molecular marker for colorectal cancer to achieve early diagnosis of colorectal cancer.

[0005] In a first aspect, the present invention provides the use of a reagent for detecting the expression level of a molecular marker in a biological sample in the preparation of a product for diagnosing colorectal cancer in a subject, wherein the molecular marker is TAB2.

[0006] The molecular marker TAB2 may also be referred to as CHTD2, TGF-beta-activated kinase 1 and MAP3K7-binding protein 2, FLJ21885, KIAA0733, DKFZp781G0741, MAP3K7IP2, and its HGNC ID is 17075.

[0007] Preferably, the product includes a kit, a chip, and a nucleic acid membrane strip.

[0008] Preferably, the reagent is a reagent for detecting the mRNA expression level or the protein expression level of the molecular marker.

[0009] Preferably, the reagents for detecting the mRNA expression level of the molecular marker include the reagents used in the following methods: polymerase chain reaction, reverse transcription polymerase chain reaction, transcription-mediated amplification, ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, rolling circle amplification, in situ hybridization, microarray, Southern blot, Northern blot, high-throughput sequencing platform method.

[0010] Preferably, the reagents for detecting the mRNA expression level of the molecular marker include specific nucleic acid probes that bind to the target sequence, specific primers for amplifying the target sequence, non-specific fluorescent dyes, or combinations thereof.

[0011] Preferably, the nucleic acid probe is a single-labeled or double-labeled nucleic acid probe, and the nucleic acid probe includes a labeled probe, a biotin-labeled probe, a horseradish peroxidase-labeled probe, a digoxin-labeled probe, or a fluorescent group-labeled probe.

[0012] Preferably, the reagents for detecting the mRNA expression level of the molecular marker include mRNA expression level auxiliary detection reagents: reagents for visualizing amplicons by agarose gel electrophoresis, enzyme-linked gel method, chemiluminescence method, in situ hybridization method, fluorescence detection method; RNA extraction reagents; reverse transcription reagents; cDNA amplification reagents; standards for preparing standard curves; positive control products.

[0013] Preferably, the reagents for detecting the protein expression level of the molecular marker include the reagents used in the following methods: immunohistochemical staining method, hematoxylin-eosin staining method, safranin O-fast green staining, Western blot, enzyme-linked immunosorbent assay, radioimmunoassay, mass spectrometry, immunoprecipitation analysis, flow cytometry fluorescence technique, and protein chip method.

[0014] Preferably, the reagents for detecting the protein expression level of the molecular marker include the reagents required for immunological detection.

[0015] Preferably, the immunological detection includes Elispot detection, ELISA detection, Western blot, or surface plasmon resonance method.

[0016] Preferably, the reagents for detecting the protein expression level of the molecular marker include protein expression level auxiliary detection reagents.

[0017] Preferably, the protein expression level auxiliary detection reagents include blocking solution, antibody diluent, washing buffer, color development termination solution, standards for preparing standard curves.

[0018] Preferably, the device includes a PCR instrument, a high-throughput sequencing platform, a detection chip, and a chip signal reader;

[0019] The chip includes probes for detecting the expression level of a biomarker;

[0020] The chip includes internal reference probes;

[0021] The internal reference includes GAPDH or β-Actin;

[0022] The chip includes a protein chip and / or a gene chip.

[0023] Preferably, the sequencing technology includes: first-generation sequencing, second-generation sequencing, and third-generation sequencing. Among them, first-generation sequencing, also known as Sanger sequencing, is a sequencing technology that utilizes DNA polymerase synthesis reactions. First-generation sequencing is a sequencing technology based on the Sanger method; second-generation sequencing is based on massive parallel sequencing technology (MPS), which can simultaneously complete the synthesis of complementary strands of sequencing templates and the acquisition of sequence data; third-generation sequencing is based on single-molecule sequencing and massive parallel sequencing technology.

[0024] The biological sample described in the present invention refers to a composition obtained from or derived from a subject (or a test subject), which contains cells and / or other molecular entities to be characterized and / or identified according to, for example, physical, biochemical, chemical, and / or physiological characteristics. Specifically, the sample includes, but is not limited to: tissue, peripheral blood, primary or cultured cells or cell lines, cell supernatant, cell lysate, platelets, blood, serum, plasma, whole blood, sweat, mucus, vitreous humor, follicular fluid, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, lymph fluid, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid, and bronchoalveolar lavage fluid, tumor lysate, tissue culture medium, tissue extract, and combinations thereof.

[0025] Preferably, the biological sample is tissue. More specifically, biopsy tissue.

[0026] In the present invention, the nucleic acid membrane strip includes a substrate and oligonucleotide probes fixed to the substrate; the substrate can be any substrate suitable for fixing oligonucleotide probes, such as nylon membrane, nitrocellulose membrane, polypropylene membrane, glass slide, silica wafer, microscale magnetic beads, etc.

[0027] In the present invention, the "chip" is also referred to as an "array", which refers to a solid support containing linked nucleic acid or peptide probes. An array usually contains a variety of different nucleic acid or peptide probes connected to the surface of the substrate at different known positions. These arrays, also known as "microarrays", can usually be produced by mechanical synthesis methods or light-directed synthesis methods, and the light-directed synthesis method combines a combination of photolithography methods and solid-phase synthesis methods. The array can include a flat surface, or can be nucleic acid or peptide on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate.

[0028] In the present invention, the kit may include reagents for detecting biomarkers in a sample derived from a subject. For example, in one embodiment, the kit may include reagents that specifically hybridize with the biomarker; specifically, nucleic acid molecules suitable for detecting the form of the biomarker, such as probes or primers. The kit may include reagents for performing assays to detect one or more biomarkers, for example, reagents that can be used to detect one or more biomarkers in a qPCR reaction. The kit may also include a microarray for detecting one or more biomarkers.

[0029] On the other hand, the present invention provides a system for diagnosing colorectal cancer, the system comprising:

[0030] (1) A computing device for determining whether a subject has colorectal cancer based on the detection results of molecular biomarkers;

[0031] (2) An input device for inputting the expression levels of molecular biomarkers of the subject;

[0032] (3) An output device for outputting the diagnosis result of colorectal cancer;

[0033] The molecular biomarker includes TAB2.

[0034] Preferably, the system may further include a detection device for detecting the expression levels of molecular biomarkers.

[0035] Preferably, when the expression level of the molecular biomarker is the mRNA expression level, the detection device may be a PCR instrument, and an amplification program for amplifying the molecular biomarker with specific primers is run in the PCR instrument.

[0036] Preferably, the PCR instrument simultaneously runs an amplification program for amplifying the internal reference with specific primers.

[0037] Preferably, when the expression level of the molecular biomarker is the protein expression level, the detection device may be the device used in immunohistochemical staining, wherein a specific antibody of the molecular biomarker is used to label the molecular biomarker, and the protein expression level of the molecular biomarker can be determined according to the staining situation.

[0038] As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, non-mammals, etc., that will be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein when referring to human subjects.

[0039] On the other hand, the present invention provides the application of the above system for diagnosing colorectal cancer in the preparation of products for diagnosing colorectal cancer.

[0040] On the other hand, the present invention provides a method for diagnosing colorectal cancer, the method comprising determining whether a subject is a patient with colorectal cancer according to the expression level of a molecular marker, and the molecular marker includes TAB2.

[0041] More specifically, the method is to detect the expression level of the molecular marker in a sample from a subject, and when the measured expression level is higher than a threshold (cutoff value), the subject is a healthy person, and when the measured expression level is lower than the threshold, the subject is a patient.

[0042] As described in the present invention, the method for measuring the threshold is well known in the art, and under different expression level detection methods, the threshold changes accordingly.

[0043] On the other hand, the present invention provides a computer-readable medium for diagnosing colorectal cancer, and the foregoing method for diagnosing colorectal cancer is recorded or run on the computer-readable medium.

[0044] Preferably, the computer-readable medium includes but is not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tapes; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROM; and mixtures of these categories, such as magnetic / optical storage media. Those skilled in the art can easily understand how to use any currently known computer-readable medium to create a manufacture containing the information records of this database. "Recording" refers to the process of storing information on a computer-readable medium using any such method known in the art. Depending on the means for accessing the stored information, any convenient data storage structure can be selected.

[0045] The various data processor programs and formats can be used for storage, such as word processing text files, database formats, etc.

[0046] The implementation of the method and / or system provided by the present invention may include performing or completing the selected tasks manually, automatically, or in combination thereof. Moreover, according to the actual instruments and devices of the implementation manners of the method and / or system of the present invention, multiple selected tasks can be implemented by hardware, by software, by firmware, or by a combination of them using an operating system. For example, the hardware for performing the selected tasks according to the implementation manners of the present invention can be implemented as a chip or a circuit. As software, the selected tasks according to the implementation manners of the present invention can be implemented as multiple software instructions executed by a computer using any suitable operating system.

[0047] Based on the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention. The reagents and raw materials used in the present invention are all commercially available.

[0048] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings

[0049] Figure 1 It is the expression difference of TAB2 between LOVO cells, HCT116 cells and HcoEpic cells in transcriptome data.

[0050] Figure 2 To verify the diagnostic ability of TAB2 using the GSE20842 dataset, where part A is the analysis of the expression difference of TAB2 in normal and cancer tissues, and part B is the ROC analysis of TAB2 in normal and cancer tissues.

[0051] Figure 3 To verify the diagnostic ability of TAB2 using the GSE44076 dataset, where part A is the analysis of the expression difference of TAB2 in normal and cancer tissues, and part B is the ROC analysis of TAB2 in normal and cancer tissues.

[0052] Figure 4 To verify the diagnostic ability of TAB2 for colon cancer using a self-owned clinical cohort, where part A is the expression difference of TAB2 in 23 pairs of normal and colon cancer tissues, and part B is the diagnostic ROC of TAB2 in 23 pairs of normal and colon cancer tissues.

[0053] Figure 5 To verify the diagnostic ability of TAB2 for rectal cancer using a self-owned clinical cohort, where part A is the expression difference of TAB2 in 23 pairs of normal and rectal cancer tissues, and part B is the diagnostic ROC of TAB2 in 23 pairs of normal and rectal cancer tissues.

[0054] Figure 6 It is the diagnostic ability of TAB2 for the combined samples of the self-owned clinical cohort, where part A is the expression difference of TAB2 in 46 pairs of normal and colorectal cancer tissues, and part B is the diagnostic ROC of TAB2 in 46 pairs of normal and colorectal cancer tissues.

[0055] Figure 7 To verify the diagnostic ability of TAB2 using a colon cancer tissue microarray containing 30 patients, where part A is the immunohistochemical staining of the colon cancer tissue microarray (scale bar: 100μm), part B is the diagnostic ROC, and part C is the analysis of the expression difference of TAB2.

[0056] Figure 8 To verify the diagnostic ability of TAB2 using a rectal cancer tissue microarray containing 30 patients, where part A is the immunohistochemical staining of the rectal cancer tissue microarray (scale bar: 100μm), part B is the diagnostic ROC, and part C is the analysis of the expression difference of TAB2.

[0057] Figure 9 To verify the diagnostic ability of TAB2 using a combined colorectal cancer tissue microarray containing 60 patients, where part A is the diagnostic ROC and part B is the differential expression of TAB2. Detailed implementation

[0058] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clearly understood, the following further details the embodiments of the present invention in conjunction with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not used to limit the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts belong to the scope of protection of this application.

[0059] Example 1

[0060] Two human colon cancer cell lines, LoVo and HCT116, and normal colon epithelial cells HcoEpic were selected for transcriptome sequencing on the DNBSEQ platform. Subsequently, differential analysis was performed on the transcriptome levels of the HCT116 and LoVo colon cancer cell lines compared with the transcriptome of HcoEpic normal colon epithelial cells, and a diagnostic marker TAB2 with good discrimination ability for colorectal cancer was found. The mRNA level expression of TAB2 in HcoEpic cells was significantly higher than that in HCT116 and LoVo cells, as Figure 1 shown.

[0061] Example 2

[0062] Two gene chip datasets, GSE20842 (65 pairs of cancer and adjacent cancer) and GSE44076 (98 cases of cancer and 148 cases of normal), were used as validation sets, and the expression matrix and clinical information were both downloaded from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo). The diagnostic ability of TAB2 was verified using these two GEO datasets.

[0063] The differential analysis in the GEO dataset is as shown in part A of Figure 2 and part A of Figure 3 , the expression of TAB2 in normal tissues / adjacent cancer tissues was significantly higher than that in cancer tissues; as shown in part B of Figure 2 and part B of Figure 3 , the ROC analysis showed that the AUC of TAB2 for diagnosing CRC in the GSE20842 dataset was 0.833, and the AUC of TAB2 for diagnosing CRC in the GSE44076 dataset was 0.911. It can be seen from the results that TAB2 has good diagnostic ability in both GEO datasets.

[0064] Example 3

[0065] A new clinical dataset was established, and the samples were from the Affiliated Hospital of Qingdao University. The inclusion criteria for patients were as follows: diagnosed with colorectal cancer by tissue biopsy, without radiotherapy or chemotherapy before tissue sample collection, the collected tissue samples were quickly frozen in liquid nitrogen after rinsing with pre-cooled PBS, and then transferred to an -80°C refrigerator for storage. A total of 46 pairs of colorectal cancer (23 pairs of colon cancer and 23 pairs of rectal cancer) and adjacent cancer samples were collected in this cohort.

[0066] The RT-qPCR method was used to detect the expression level of TAB2. First, the total RNA of tissue samples was extracted using the RNeasy kit (Beyotime, Shanghai, R0027), the RNA concentration was measured, 1 μg of RNA was used to remove the DNA with DNase, and then the RNA was reverse transcribed into cDNA using SuperScript II reverse transcriptase (TaKaRa, Japan, RR047). Then, the SYBRGreen kit (TaKaRa, Japan, RR820) was used for quantitative PCR reaction. The relative expression level of TAB2 was calculated with the expression of human GAPDH as a control. The ROC curve was plotted with the TAB2 expression level for disease or not, the AUC value was obtained, and the expression difference of TAB2 between cancer and adjacent cancer was statistically analyzed and a box plot was drawn.

[0067] As Figure 4 shown in A, the differential analysis in colon cancer samples showed that the expression of TAB2 in adjacent cancer tissues was significantly higher than that in cancer tissues; as Figure 5 shown in A, the differential analysis in rectal cancer samples showed that the expression of TAB2 in adjacent cancer tissues was significantly higher than that in cancer tissues. As Figure 4 shown in B, the AUC of TAB2 for diagnosing colon cancer was 0.992; as Figure 5 shown in B, the AUC of TAB2 for diagnosing rectal cancer was 0.911.

[0068] The combined CRC samples were analyzed. As Figure 6 shown in A, the expression of TAB2 in adjacent cancer tissues was significantly higher than that in cancer tissues; as Figure 6 shown in B, the ROC curve showed that the AUC of TAB2 for diagnosing CRC was 0.951. Thus, it can be seen that TAB2 has good diagnostic ability in colon cancer, rectal cancer, and CRC.

[0069] Example 4

[0070] Commercially available human colon cancer tissue microarrays (Shanghai CoreStar Biotech Co., Ltd., Shanghai, HCol-Ade090PG-01-M-055) were purchased. This microarray contains 30 pairs of colon cancer and paired adjacent tissue samples; human rectal cancer tissue microarrays (Shanghai CoreStar Biotech Co., Ltd., Shanghai, HRec-Ade060-PG-01) were also purchased, which contain 30 pairs of rectal cancer and paired adjacent tissue samples. The expression levels of TAB2 protein in cancer and adjacent tissues were analyzed by immunohistochemistry. The anti-human TAB2 antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd. (Wuhan Sanying Biotechnology Co., Ltd., Wuhan, 14410-1-AP).

[0071] By means of manual reading of slides, the expression level of TAB2 was scored according to the staining intensity and area. The staining intensity of the tissue was divided into four grades, represented by 0 if none, 1 for weak positive, 2 for medium positive, and 3 for strong positive; the tissue staining area was divided into five grades, where 0 represents none, 1 represents 1% - 25% of the tissue being positive, 2 represents 25% - 50% of the tissue being positive, 3 represents 50% - 75% of the tissue being positive, and 4 represents 75% - 100% of the tissue being positive. The immunohistochemical score of TAB2 for each sample is equal to the staining intensity multiplied by the staining area. Therefore, the immunohistochemical score of TAB2 is a series of integers ranging from 0 to 12. This score was used for statistical analysis of the expression differences of TAB2 in cancer and adjacent tissues, and the ROC curve for diagnosing colorectal cancer with TAB2 was plotted.

[0072] The immunohistochemical staining results of the colon cancer tissue microarray are as Figure 7 shown in A of Figure 8 the figure, and the immunohistochemical staining of the rectal cancer tissue microarray is as

[0073] shown in A of Figure 7 the figure. Figure 8 As shown in B of

[0074] the figure and Figure 7 B of Figure 8 the figure, the diagnostic AUCs of TAB2 for colon cancer patients and rectal cancer patients are 0.858 and 0.951 respectively.

[0075] The colon cancer microarray and the rectal cancer microarray were analyzed together. As Figure 9 shown in A of Figure 9Analysis of the difference in B showed that the expression of TAB2 in adjacent tissues of CRC was significantly higher than that in cancer tissues. From the results of immunohistochemical staining experiments, at the protein level, TAB2 had good diagnostic ability in colon cancer, rectal cancer, and CRC.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of a reagent for detecting the expression level of a molecular marker in a biological sample in the preparation of a product for diagnosing colorectal cancer in a subject, characterized in that: The molecular marker is TAB2.

2. The use according to claim 1, characterized in that: The products include kits, systems, devices, and computer-readable media.

3. The use according to claim 1, characterized in that: The reagent is a reagent for detecting the mRNA expression amount of a molecular marker or the protein expression amount of a molecular marker.

4. The use according to claim 3, characterized in that: The reagents for detecting the mRNA expression of molecular markers include reagents used in the following methods: polymerase chain reaction, reverse transcription polymerase chain reaction, transcription-mediated amplification, ligase chain reaction, strand displacement amplification and nucleic acid sequence-based amplification, rolling cycle amplification, in situ hybridization, microarray, Southern blot, Northern blot, high-throughput sequencing platform method; Or the reagent for detecting the mRNA expression of the molecular marker includes a specific nucleic acid probe that binds to the target sequence, a specific primer that amplifies the target sequence, a non-specific fluorescent dye, or a combination thereof; The nucleic acid probe is a single-labeled or double-labeled nucleic acid probe, and the nucleic acid probe includes a biotin-labeled probe, a horseradish peroxidase-labeled probe, a digoxigenin-labeled probe, or a fluorescent group-labeled probe; Or the reagent for detecting the mRNA expression of the molecular marker includes an auxiliary detection reagent for mRNA expression: a reagent for visualizing the amplicon by agarose gel electrophoresis, enzyme-linked gel electrophoresis, chemiluminescence, in situ hybridization, and fluorescence detection; an RNA extraction reagent; a reverse transcription reagent; a cDNA amplification reagent; a standard substance used to prepare a standard curve; and a positive control substance.

5. The use according to claim 3, characterized in that: The reagents for detecting the protein expression of molecular markers include reagents used in the following methods: immunohistochemical staining method, hematoxylin-eosin staining method, safranin O-fast green staining, protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, mass spectrometry, immunoprecipitation analysis, flow cytometry fluorescence technology and protein chip method; Or the reagents for detecting the protein expression of the molecular markers include reagents required for immunological detection; the immunological detection includes Elispot detection, ELISA detection, Western blotting or surface plasmon resonance method; Or the reagent for detecting the protein expression level of the molecular marker includes a protein expression level auxiliary detection reagent; the protein expression level auxiliary detection reagent includes a blocking solution, an antibody diluent, a washing buffer solution, a color development stop solution, and a standard substance used to prepare a standard curve.

6. The use according to claim 2, characterized in that: The device includes a PCR instrument, a high-throughput sequencing platform, a detection chip and a chip signal reader; The chip includes a probe for detecting the expression amount of the marker; The chip includes an internal reference probe; The internal reference includes GAPDH or β-Actin; The chip includes a protein chip and / or a gene chip.

7. The use according to claim 2, characterized in that: The system comprises: (1) A computing device for determining whether a subject has colorectal cancer based on the results of a molecular marker test; (2) an input device for inputting the expression level of the subject's molecular marker; (3) An output device for outputting the diagnosis result of colorectal cancer.

8. The use according to claim 7, characterized in that: The system also includes a detection device for detecting the expression amount of the molecular marker; The detection device includes a device for running a PCR program or a device used when performing immunohistochemical staining.

9. The use according to claim 2, characterized in that: The computer-readable medium records or runs a method for diagnosing colorectal cancer, which includes determining whether the subject is a colorectal cancer patient based on the expression level of the molecular marker.

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