Molecular marker for colorectal cancer

Abstract

The invention discloses a molecular marker for colorectal cancer, and relates to the technical field of colorectal cancer diagnosis, and the molecular marker is TAB2. According to the invention, it is found for the first time that the mRNA level expression of TAB2 in a colonic epithelial cell HcoEpic cell derived from a normal person is significantly higher than the expression of TAB2 in two human colon cancer cells HCT116 and LoVo. The colorectal cancer molecular marker and the molecular marker TAB2 can be applied to preparation of products for diagnosing colorectal cancer and have wide application prospects.

Description

Technical Field

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

[0002] The development of colorectal cancer usually follows the "adenoma-cancer" 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 of stage I is as high as 90%, while that of stage IV is only 14%. The staging of colon cancer is based on the depth of invasion of the primary lesion, lymph node and distant organ metastasis. Stage I is when there is no regional lymph node metastasis and the depth of invasion is limited to the muscle layer; stage II is when it reaches the serosa and invades adjacent organs outside the serosa; stage III is when there is periintestinal lymph node metastasis; and stage IV is when there is distant organ metastasis.

[0003] At present, the screening methods for colorectal cancer mainly include fecal occult blood test and colonoscopy. Among them, the sensitivity and specificity of fecal occult blood test are low; although colonoscopy is highly accurate, it is an invasive test and patient compliance is low. In the early stages of colorectal cancer, there is a lack of specific symptoms, so it is easy to be ignored by patients. When patients go to the hospital for examination only after obvious symptoms appear and are finally diagnosed with colorectal cancer, the cancer cells have spread and the best time for treatment has been missed. At present, with the completion of the Human Genome Project and the development of Qualcomm's sequencing technology, genetic screening technology has become a new method for diagnosing colorectal cancer, which has significant advantages in the early diagnosis of colorectal cancer. Summary of the invention

[0004] In order 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 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 can also be called CHTD2, TGF-beta-activated kinase 1andMAP3K7-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 of a molecular marker or the protein expression level of a molecular marker.

[0009] Preferably, 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 blotting, Northern blotting, and high-throughput sequencing platform methods.

[0010] Preferably, 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.

[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 digoxigenin-labeled probe, or a fluorescent group-labeled probe.

[0012] Preferably, the reagents for detecting the mRNA expression of molecular markers include auxiliary detection reagents for mRNA expression: reagents for visualizing amplicons by agarose gel electrophoresis, enzyme-linked gel electrophoresis, chemiluminescence, in situ hybridization, and fluorescence detection; RNA extraction reagents; reverse transcription reagents; cDNA amplification reagents; standard substances used to prepare standard curves; and positive controls.

[0013] Preferably, the reagents for detecting the protein expression amount of the molecular marker 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.

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

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

[0016] Preferably, the reagent for detecting the protein expression level of the molecular marker includes a protein expression level auxiliary detection reagent.

[0017] Preferably, the protein expression level auxiliary detection reagent includes a blocking solution, an antibody diluent, a washing buffer, a color development stop solution, and a standard substance used for preparing a standard curve.

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

[0019] The chip includes a probe for detecting the expression amount of the marker;

[0020] The chip includes an internal reference probe;

[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, wherein first-generation sequencing, also known as Sanger sequencing, is a sequencing technology that utilizes DNA polymerase synthesis reaction, and first-generation sequencing is a sequencing technology based on the Sanger method; second-generation sequencing is based on massively parallel sequencing technology (Massive parallel analysis, MPS), which can simultaneously complete the synthesis of complementary chains of sequencing templates and the acquisition of sequence data; third-generation sequencing is based on single-molecule sequencing and massively parallel sequencing technology.

[0024] The biological sample of the present invention refers to a composition obtained from or derived from a subject (or a subject to be tested), 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 supernatants, cell lysates, platelets, blood, serum, plasma, whole blood, sweat, mucus, vitreous fluid, follicular fluid, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, lymph, ascites, pleural effusion, amniotic fluid, bladder washing fluid and bronchoalveolar lavage fluid, tumor lysates, tissue culture fluid, tissue extracts and combinations thereof.

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

[0026] In the present invention, the nucleic acid membrane strip comprises a substrate and an oligonucleotide probe fixed on the substrate; the substrate can be any substrate suitable for fixing the oligonucleotide probe, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass sheet, a silica gel wafer, a micro-magnetic bead, etc.

[0027] In the present invention, the "chip", also referred to as an "array", refers to a solid support comprising connected nucleic acid or peptide probes. The array typically comprises a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known positions. These arrays, also referred to as "microarrays", can typically be produced using mechanical synthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid phase synthesis methods. The array may comprise a flat surface, or may be a nucleic acid or peptide on a bead, gel, polymer surface, fiber such as an optical fiber, 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 biomarkers; specifically, nucleic acid molecules in a form suitable for detecting biomarkers, 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] In another aspect, 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 results of a molecular marker test;

[0031] (2) an input device for inputting the expression level of a subject's molecular marker;

[0032] (3) an output device for outputting colorectal cancer diagnosis results;

[0033] The molecular markers include TAB2.

[0034] Preferably, the system may also include a detection device for detecting the expression level of the molecular marker.

[0035] Preferably, when the expression level of the molecular marker is the expression level of mRNA, the detection device may be a PCR instrument, in which an amplification program of the molecular marker is run using specific primers.

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

[0037] Preferably, when the expression level of the molecular marker is the protein expression level, the detection device can be a device used for immunohistochemical staining, wherein the molecular marker is labeled with a specific antibody of the molecular marker, and the protein expression level of the molecular marker can be determined based on the staining condition.

[0038] The term "subject" as used herein refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, non-mammals, etc., that is to 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 use of the above-mentioned system for diagnosing colorectal cancer in the preparation of a product 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 colorectal cancer patient based on the expression level of a molecular marker, wherein the molecular marker comprises TAB2.

[0041] More specifically, the method is to detect the expression level of molecular markers in a sample from a subject. 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 the threshold changes accordingly under different expression detection methods.

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

[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. A person skilled in the art can easily understand how to use any currently known computer readable medium to create a record containing the information in the database. "Recording" refers to the process of storing information on a computer readable medium using any such method known in the art. Any convenient data storage structure may be selected depending on the means used to access the stored information.

[0045] The various data processor programs and formats described may be used for storage, such as word processing text files, database formats, and the like.

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

[0047] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention. The reagents and raw materials used in the present invention are all commercially available.

[0048] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is the difference in TAB2 expression between LOVO cells and HCT116 cells and HcoEpic cells in the transcriptome data;

[0050] Figure 2 To validate the diagnostic ability of TAB2 using the GSE20842 dataset, Part A is the differential expression analysis 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 validate the diagnostic ability of TAB2 using the GSE44076 dataset, Part A is the differential expression analysis of TAB2 in normal and cancer tissues, and Part B is the ROC analysis of TAB2 in normal and cancer tissues;

[0052] Figure 4 In order to verify the diagnostic ability of TAB2 for colon cancer using our own clinical cohort, 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 In order to verify the diagnostic ability of TAB2 for rectal cancer using our own clinical cohort, 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 The diagnostic ability of TAB2 for the combined samples of the own clinical cohort, of which 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 chip containing 30 patients, Part A is the immunohistochemical staining of colon cancer tissue chips, Part B is the differential expression analysis of TAB2, and Part C is the diagnostic ROC (scale bar: 100 μm);

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

[0057] Fig. 9 To verify the diagnostic ability of TAB2 using a combined colorectal cancer tissue chip containing 60 patients, Part A is the TAB2 expression difference, and Part B is the diagnostic ROC. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention more clearly understood, the embodiments of the present invention are further described in detail below in conjunction with the accompanying 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 intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0059] Embodiment 1

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

[0061] Embodiment 2

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

[0063] The difference analysis in the GEO dataset is as follows Figure 2 Middle A and Figure 3 As shown in Figure A, the expression of TAB2 in normal tissues / paracancerous tissues is significantly higher than that in cancer tissues; Figure 2 ROC analysis of B and 3B showed that the AUC of TAB2 for diagnosing CRC was 0.833 in the GSE20842 dataset and 0.911 in the GSE44076 dataset. The results show that TAB2 has good diagnostic ability in both GEO datasets.

[0064] Embodiment 3

[0065] A new clinical data set was established. The samples were collected from the Affiliated Hospital of Qingdao University. The inclusion criteria for patients were: colorectal cancer was confirmed by tissue biopsy, no radiotherapy or chemotherapy was performed before tissue sample collection, and the collected tissue samples were rinsed with pre-cooled PBS and then quickly frozen with liquid nitrogen and then transferred to a -80℃ 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 expression level of TAB2 was detected by RT-qPCR. First, the total RNA of the tissue sample was extracted using the RNeasy kit (Biyuntian, Shanghai, R0027), the RNA concentration was determined, 1 μg of RNA was taken and the DNA was removed with DNase, and then the RNA was reverse transcribed into cDNA using SuperScript II reverse transcriptase (TaKaRa, Japan, RR047), and 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 the control, and the ROC curve was drawn with the TAB2 expression level versus the disease to obtain the AUC value, and the expression difference of TAB2 in cancer and adjacent cancer was statistically analyzed and a box plot was drawn.

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

[0068] The combined CRC samples were analyzed. Figure 6 As shown in Figure A, the expression of TAB2 in paracancerous tissues was significantly higher than that in cancer tissues; Figure 6 In Figure B, the ROC curve shows that the AUC of TAB2 for diagnosing CRC is 0.951. This shows that TAB2 has good diagnostic ability in colon cancer, rectal cancer, and CRC.

[0069] Embodiment 4

[0070] Commercial human colon cancer tissue microarray (Shanghai Xinchao Biotechnology Co., Ltd., Shanghai, HCol-Ade090PG-01-M-055) was purchased, which contained 30 pairs of colon cancer and paired adjacent tissue samples; human rectal cancer tissue microarray (Shanghai Xinchao Biotechnology Co., Ltd., Shanghai, HRec-Ade060-PG-01) was purchased, which contained 30 pairs of rectal cancer and paired adjacent tissue samples. Immunohistochemistry was used to analyze the expression level of TAB2 protein in cancer and adjacent tissues, and anti-human TAB2 antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd. (Wuhan Sanying Biotechnology Co., Ltd., Wuhan, 14410-1-AP).

[0071] The expression level of TAB2 was scored according to the staining intensity and area by manual film reading. The staining intensity of the tissue was divided into four levels, with 0 representing none, 1 representing weak positive, 2 representing moderately positive, and 3 representing strongly positive; the tissue staining area was divided into five levels, with 0 representing none, 1 representing 1% to 25% of the tissue being positive, 2 representing 25% to 50% of the tissue being positive, 3 representing 50% to 75% of the tissue being positive, and 4 representing 75% to 100% of the tissue being positive. The TAB2 histochemical score of each sample was equal to the staining intensity multiplied by the staining area. Therefore, the histochemical score of TAB2 is a series of integers ranging from 0 to 12. This score was used to statistically analyze the expression difference of TAB2 in cancer and adjacent tissues, and to draw the ROC curve of TAB2 in diagnosing colorectal cancer.

[0072] Results of immunohistochemical staining of colon cancer tissue microarray Figure 7 As shown in A, immunohistochemical staining of rectal cancer tissue microarray Figure 8 As shown in A.

[0073] like Figure 7 Medium B and Figure 8 As shown in B, the diagnostic AUC of TAB2 for patients with colon cancer and rectal cancer were 0.858 and 0.951, respectively.

[0074] like Figure 7 Middle C and Figure 8 As shown in C, the expression of TAB2 in paracancerous tissues of colon cancer is significantly higher than that in cancer tissues, and the expression of TAB2 in paracancerous tissues of rectal cancer is significantly higher than that in cancer tissues.

[0075] Combined analysis of colon cancer chip and rectal cancer chip. Fig. 9 As shown in A, the AUC of TAB2 for the diagnosis of colorectal cancer patients is 0.896; Fig. 9The difference analysis showed that the expression of TAB2 in CRC paracancerous tissues was significantly higher than that in cancer tissues. According to the results of immunohistochemical staining experiments, TAB2 has good diagnostic ability in colon cancer, rectal cancer and CRC at the protein level.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution 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 labeled probe, 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 a 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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