Colorectal cancer molecular marker and application thereof

Abstract

The invention discloses a colorectal cancer molecular marker and application thereof, and belongs to the technical field of biological medicine. The invention finds that ATP5MK has significant differential expression in colorectal cancer patients for the first time, can be used as a biomarker for colorectal cancer diagnosis, has the advantages of high accuracy, good specificity, high sensitivity and the like, provides a brand new idea and strategy for early diagnosis of colorectal cancer, and has good clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a molecular marker for colorectal cancer and its application. Background Technology

[0002] Early diagnosis and intervention for colorectal cancer can improve prognosis; therefore, improving diagnostic capabilities is crucial for enhancing patients' quality of life.

[0003] Currently, first-line methods for colorectal cancer screening mainly include colonoscopy, fecal immunochemical testing (FIT), and carcinoembryonic antigen (CEA) testing, but all three have significant limitations. While colonoscopy is the gold standard for diagnosis and boasts high accuracy, it is an invasive procedure with cumbersome bowel preparation, a high risk of complications, and high costs, resulting in generally low patient acceptance and compliance. FIT, as a non-invasive primary screening method, can increase patient participation, but its sensitivity in identifying precancerous lesions is limited. Regular CEA testing, while providing indicators of colorectal cancer, still falls short of clinical needs in terms of specificity and sensitivity. Furthermore, other commonly used screening methods, such as imaging assessments and histopathological examinations, also suffer from high costs, invasiveness, or insufficient diagnostic efficacy. It is noteworthy that most colorectal cancers have a development cycle of 10 to 15 years, providing a critical time window for early detection and intervention. Therefore, developing a non-invasive, highly accurate, and easily accessible early detection technology for colorectal cancer has become an urgent need in the clinical and public health fields.

[0004] In recent years, molecular biology techniques have demonstrated the great potential of gene biomarkers in the early diagnosis of colorectal cancer, and numerous studies have confirmed that screening, early diagnosis, and early treatment of colorectal cancer can effectively reduce its mortality rate. Therefore, developing a novel biomarker and applying it to the diagnosis and prognosis prediction of colorectal cancer is of significant research importance. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular marker for colorectal cancer and its application, aiming to overcome the deficiencies of existing technologies, achieve non-invasive early diagnosis of colorectal cancer, and thus improve patient prognosis, quality of life and survival rate.

[0006] To achieve the above objectives, the present invention provides a molecular marker for colorectal cancer, wherein the molecular marker is ATP5MK, and the Gene ID of ATP5MK in NCBI is 84833; The expression level of ATP5MK in the colorectal cancer tissue was significantly lower than that in normal tissue.

[0007] The application of the above-mentioned colorectal cancer molecular marker in the preparation of reagents for the diagnosis or auxiliary diagnosis of colorectal cancer, wherein the colorectal cancer includes colon cancer and rectal cancer; the reagent is a reagent for detecting the expression level of the molecular marker ATP5MK.

[0008] Preferably, the reagent is a reagent for detecting the mRNA expression level of ATP5MK.

[0009] Preferably, the reagents used to detect the mRNA expression level of ATP5MK include those 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 level of ATP5MK 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 reagent for detecting the mRNA expression level of ATP5MK further includes an auxiliary reagent for detecting mRNA expression levels, which includes: reagents for visualizing amplicones by agarose gel electrophoresis, enzyme-linked gel electrophoresis, chemiluminescence, in situ hybridization, or fluorescence detection; RNA extraction reagents; reverse transcription reagents; cDNA amplification reagents; standards for preparing standard curves; and positive controls.

[0012] Preferably, the nucleic acid probe includes a labeled probe, a biotin-labeled probe, a horseradish peroxidase-labeled probe, a digoxigenin-labeled probe, or a fluorescently labeled probe.

[0013] Preferably, the nucleic acid probe is a single-labeled or double-labeled nucleic acid probe.

[0014] Preferably, the reagent is a reagent for detecting the protein expression level of ATP5MK; Preferably, the reagents used to detect the protein expression level of ATP5MK include those used in the following methods: immunohistochemical staining, hematoxylin-eosin staining, safranin O-fast green staining, Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, mass spectrometry, immunoprecipitation analysis, flow cytometry, and protein chip analysis.

[0015] Preferably, the reagents for detecting the protein expression level of ATP5MK include reagents required for immunological detection, which includes ELISA, Elispot assay, Western blotting, or surface plasmon resonance assay.

[0016] Preferably, the reagent for detecting the protein expression level of ATP5MK further includes a protein expression level auxiliary detection reagent, which includes: blocking solution, antibody dilution solution, washing buffer, colorimetric stop solution, and ATP5MK standard used to prepare the standard curve.

[0017] The use of the above-mentioned reagent in the preparation of products for the diagnosis or auxiliary diagnosis of colorectal cancer, said products including reagent kits, systems, devices and computer-readable media.

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

[0019] Preferably, the detection chip includes probes for detecting the expression levels of biomarkers.

[0020] Preferably, the detection chip includes an internal reference probe.

[0021] Preferably, the internal reference includes GAPDH or β-Actin.

[0022] Preferably, the detection chip includes a protein chip and / or a gene chip.

[0023] Preferably, the method for detecting ATP5MK expression is recorded or executed on the computer-readable medium.

[0024] The application of the above-mentioned product in the preparation of a diagnostic or auxiliary diagnostic system for colorectal cancer, the system comprising: (1) A computing device for determining whether a subject has colorectal cancer based on the results of molecular marker detection; (2) An input device for inputting the expression levels of molecular markers in the subject; (3) Output device for outputting colorectal cancer diagnosis results.

[0025] Therefore, the present invention provides a molecular biomarker for colorectal cancer and its application, which has the following beneficial effects: The present invention is the first to discover that ATP5MK is significantly differentially expressed in colorectal cancer patients, and can be used as a biomarker for the diagnosis of colorectal cancer. It has the advantages of high accuracy, good specificity and high sensitivity, providing a new idea and strategy for the early diagnosis of colorectal cancer in this field, and has good clinical application value.

[0026] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The difference in ATP5MK expression between normal and cancerous tissues in the GSE44076 dataset and the diagnostic ROC; where A represents the difference in ATP5MK expression between normal and cancerous tissues, and B represents the diagnostic ROC. Figure 2 The difference in ATP5MK expression between normal and cancerous tissues in the GSE20842 dataset and the diagnostic ROC; where A represents the difference in ATP5MK expression between normal and cancerous tissues, and B represents the diagnostic ROC. Figure 3 The study aimed to identify differences in ATP5MK expression and diagnostic ROCs among 30 pairs of normal and colorectal cancer tissues in our own clinical cohort; where A represents differences in ATP5MK expression between normal and cancerous tissues, and B represents diagnostic ROCs. Figure 4 The differences in ATP5MK expression and diagnostic ROCs between 22 pairs of normal and rectal cancer tissues in our own clinical cohort were analyzed; where A represents the differences in ATP5MK expression between normal and cancer tissues, and B represents the diagnostic ROCs. Figure 5 The study aimed to determine the differences in ATP5MK expression and the diagnostic ROC among 58 normal tissues and 55 colorectal cancer tissues in our own clinical cohort; where A represents the differences in ATP5MK expression between normal and cancer tissues, and B represents the diagnostic ROC. Figure 6 The differential expression and diagnostic ROC curve of ATP5MK in adjacent and cancerous tissues in colorectal cancer tissue microarrays; where A is the immunohistochemical staining result, B is the ATP5MK histochemical score, C is the relative abundance analysis of ATP5MK in adjacent and cancerous tissues, and D is the ROC curve. Figure 7 The differential expression and diagnostic ROC curve of ATP5MK in adjacent and cancerous tissues in rectal cancer tissue microarrays; where A is the immunohistochemical staining result, B is the ATP5MK histochemical score, C is the relative abundance analysis of ATP5MK in adjacent and cancerous tissues, and D is the ROC curve. Figure 8 The differential expression and diagnostic ROC curve of ATP5MK in adjacent and cancerous tissues in colorectal cancer tissue microarrays were analyzed. Among them, A is the ATP5MK histochemical score, B is the relative abundance analysis of ATP5MK in adjacent and cancerous tissues, and C is the ROC curve. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0030] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0031] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0032] Example 1 Two gene chip datasets, GSE44076 (98 cancer cases, 148 normal cases) and GSE20842 (65 cancer cases, 65 normal cases), were used as validation sets. Expression matrices and clinical information were downloaded from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo). These two GEO datasets were used to validate the ability of ATP5MK (ATP synthase membrane subunit K) as a molecular marker for colorectal cancer. The Gene ID of ATP5MK in NCBI (https: / / www.ncbi.nlm.nih.gov / ) is 84833.

[0033] Differences in ATP5MK expression between normal and cancerous tissues in the GEO dataset and diagnostic ROCs. Figure 1 and Figure 2 As shown. Figure 1 For the GSE44076 dataset, Figure 1 In section A, the difference in ATP5MK expression between normal and cancerous tissues is represented. Figure 1 B in the diagram represents the diagnostic ROC. Figure 2 For the GSE20842 dataset, Figure 2 In section A, the difference in ATP5MK expression between normal and cancerous tissues is represented. Figure 2 B in the diagram represents the diagnostic ROC.

[0034] Depend on Figure 1 China A and Figure 2 As shown in Figure A, the expression of ATP5MK in cancer tissues is significantly lower than that in normal tissues.

[0035] Depend on Figure 1 China B and Figure 2As shown in B, the AUC of ATP5MK for diagnosing CRC is 0.875 in the GSE44076 dataset and 0.808 in the GSE20842 dataset.

[0036] It is evident that ATP5MK demonstrates good diagnostic capabilities in both GEO datasets.

[0037] Example 2 This embodiment used a novel clinical cohort. Samples were obtained from the Affiliated Hospital of Qingdao University. Inclusion criteria included: diagnosis of colorectal cancer or rectal cancer via tissue biopsy; no prior radiotherapy or chemotherapy administered before sample collection; and collection of tissue samples followed by rinsing with pre-cooled PBS, flash freezing in liquid nitrogen, and storage at -80°C. This cohort collected 58 cases of colorectal cancer and 55 adjacent normal tissue samples.

[0038] The clinical samples involved in this embodiment were obtained with the informed consent of the patients before acquisition and were approved by the Research Ethics Committee of the Affiliated Hospital of Qingdao University. Furthermore, the implementation process complied with the 1964 and later versions of the Declaration of Helsinki.

[0039] RT-qPCR was used to detect ATP5MK expression. First, total RNA was extracted from tissue samples using the SPARKeasy Tissue / Cell RNA Rapid Extraction Kit (CiscoJet, Shandong, AC0202). RNA concentration was measured, and 1 μg of RNA was used to remove DNA with DNase. Then, RNA was reverse transcribed into cDNA using SPARKscript II All-in-one RT SuperMix for qPCR (CiscoJet, Shandong, AG0305). Quantitative PCR was then performed using 2×SYBR Green qPCR Mix (CiscoJet, Shandong, AH0104). The relative expression level of ATP5MK was calculated using GAPDH expression as an internal control. The expression differences of ATP5MK in cancerous and adjacent normal tissues were statistically analyzed and box plots were generated. Additionally, ROC curves were plotted against disease status using ATP5MK expression levels to obtain AUC values.

[0040] Differences in ATP5MK expression and diagnostic ROCs among 30 pairs of normal and colon cancer tissues in a clinical cohort. Figure 3 As shown, ATP5MK expression in cancerous tissues is significantly lower than in adjacent normal tissues. Furthermore, the ROC curve indicates that the AUC for ATP5MK in diagnosing colorectal cancer is 0.7783. The RT-qPCR results demonstrate that ATP5MK possesses good diagnostic capabilities for colorectal cancer at the mRNA level.

[0041] Differences in ATP5MK expression and diagnostic ROC between 22 pairs of normal and rectal cancer tissues in our own clinical cohort. Figure 4 As shown, ATP5MK expression in cancerous tissues is significantly lower than in adjacent normal tissues; simultaneously, the ROC curve shows that the AUC of ATP5MK for diagnosing colorectal cancer is 0.7389. The RT-qPCR results demonstrate that ATP5MK possesses good diagnostic ability for rectal cancer at the mRNA level.

[0042] Differences in ATP5MK expression and diagnostic ROC among 58 normal tissues and 55 colorectal cancer tissues in our own clinical cohort. Figure 5 As shown, ATP5MK expression in cancerous tissues was significantly lower than in adjacent normal tissues; simultaneously, the ROC curve showed that the AUC of ATP5MK for diagnosing colorectal cancer was 0.742. The RT-qPCR results indicate that ATP5MK possesses good diagnostic ability for colorectal cancer at the mRNA level.

[0043] Example 3 Commercially available human colorectal cancer and rectal cancer tissue microarrays were purchased from Shanghai Xinchao Biotechnology Co., Ltd., Shanghai, with catalog numbers HClAde090PG01 for colorectal cancer and HREcAde060PG01 for rectal cancer. The colorectal cancer microarray contained 60 adjacent normal tissue samples and 30 cancer tissue samples, while the rectal cancer microarray contained 30 pairs of rectal cancer and paired adjacent normal tissue samples. Immunohistochemistry was used to analyze the expression level of ATP5MK protein in cancer and adjacent normal tissues. After three cycles of xylene dewaxing and graded ethanol rehydration, the tissue microarrays were washed with tap water and distilled water. After high-pressure antigen retrieval and cooling, the microarrays were edged with an immunohistochemical pen, incubated in a humidified chamber with 3% H2O2 to remove enzymes, washed with PBS, blocked with 5% BSA, and then incubated overnight at 4°C with 1:100 diluted ATP5MK primary antibody. The next day, after equilibration at room temperature, the microarrays were washed with PBS, incubated with secondary antibody at 37°C, and then developed with DAB in the dark. Once the target was reached, the staining was stopped with tap water. After hematoxylin counterstaining, hydrochloric acid alcohol differentiation, and running water blueing, the microarrays were finally dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and air-dried in a fume hood.

[0044] Anti-human ATP5MK antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd., catalog number: Cat: 17716-1-AP. The antibody was diluted 1:100. The expression level of ATP5MK protein was evaluated manually by slide reading. Scoring was based on staining intensity and the proportion of positive cells. Tissue staining intensity was divided into four grades: 0 represents none, 1 represents weak positive, 2 represents moderate positive, and 3 represents strong positive. Tissue stained area was divided into five grades: 0 represents none, 1 represents 1-25% positive, 2 represents 25-50% positive, 3 represents 50-75% positive, and 4 represents 75-100% positive. The ATP5MK histochemical score was calculated using the following formula: .

[0045] The ATP5MK histochemical score is a series of integers ranging from 0 to 12. This score was used to statistically analyze the differences in ATP5MK expression between colorectal cancer and adjacent non-cancerous tissues, and to plot the ROC curve for ATP5MK in diagnosing colorectal cancer. First, the results of microarray analysis of colon and rectal cancer tissues were analyzed separately, and then the two results were combined for analysis. Statistical analysis of ATP5MK expression in adjacent and cancerous tissues was performed, and box plots were plotted. Then, based on the median score, ATP5MK expression levels were divided into high and low groups. The percentage of high / low ATP5MK expression in the adjacent and cancerous groups was analyzed using a chi-square test. Finally, the ability of ATP5MK expression level to diagnose colorectal cancer was analyzed by plotting ROC curves.

[0046] Differential expression and diagnostic ROC curves of ATP5MK in adjacent and cancerous tissues in colorectal cancer tissue microarrays. Figure 6 As shown, A represents the immunohistochemical staining results, B represents the ATP5MK histochemical score, C represents the relative abundance analysis of ATP5MK in adjacent and cancerous tissues, and D represents the ROC curve.

[0047] The results showed that the expression level of ATP5MK in adjacent normal tissues was significantly higher than that in cancerous tissues, and the percentage of ATP5MK protein with high expression in adjacent normal tissues was significantly higher than that in tumor tissues. P The value was less than 0.001. Further evaluation of its diagnostic efficacy using ROC curves showed that ATP5MK had an AUC value of 0.9433 for diagnosing colorectal cancer patients.

[0048] Differential expression and diagnostic ROC curves of ATP5MK in adjacent and cancerous tissues in rectal cancer tissue microarrays. Figure 7 As shown, A represents the immunohistochemical staining results, B represents the ATP5MK histochemical score, C represents the relative abundance analysis of ATP5MK in adjacent and cancerous tissues, and D represents the ROC curve.

[0049] The results showed that the expression of ATP5MK in adjacent normal tissues was significantly higher than that in cancerous tissues, and the difference in the expression distribution of ATP5MK between the two groups was highly statistically significant. P <0.001). ROC curve analysis showed that the AUC value of ATP5MK for the diagnosis of rectal cancer was 0.9528.

[0050] To further validate the diagnostic value of ATP5MK in overall colorectal cancer, tissue microarray data from colorectal and rectal cancer were integrated and analyzed. The differential expression and diagnostic ROC of ATP5MK in adjacent and cancerous tissues in colorectal cancer tissue microarrays are shown in the figure below. Figure 8 As shown, A represents the ATP5MK histochemical score, B represents the relative abundance analysis of ATP5MK in adjacent and cancerous tissues, and C represents the ROC curve.

[0051] The results showed that ATP5MK expression in adjacent normal tissues was significantly higher than in cancerous tissues, and the percentage of high ATP5MK expression in adjacent normal tissues was also significantly higher than in tumor tissues. ROC curve analysis showed that the AUC value of ATP5MK for the comprehensive diagnosis of colorectal cancer was 0.9376.

[0052] The above series of immunohistochemical experiments confirmed at the protein level that ATP5MK showed good diagnostic ability in colon cancer, rectal cancer, and overall colorectal cancer.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of a colorectal cancer molecular marker in the preparation of reagents for the diagnosis or auxiliary diagnosis of colorectal cancer, characterized in that: The molecular marker is ATP5MK, and the Gene ID of ATP5MK in NCBI is 84833. The expression level of ATP5MK in the colorectal cancer tissue was significantly lower than that in normal tissue. The colorectal cancers mentioned include colon cancer and rectal cancer; The reagent is used to detect the expression level of the molecular marker ATP5MK.

2. The application according to claim 1, characterized in that, The reagent is used to detect the mRNA expression level of ATP5MK; The reagents used to detect the mRNA expression level of ATP5MK include those 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. The reagents for detecting the mRNA expression level of ATP5MK include specific nucleic acid probes that bind to the target sequence, specific primers that amplify the target sequence, non-specific fluorescent dyes, or combinations thereof.

3. The application according to claim 2, characterized in that, The reagents for detecting the mRNA expression level of ATP5MK also include mRNA expression level auxiliary detection reagents, which include: reagents for visualizing amplicones by agarose gel electrophoresis, enzyme-linked gel electrophoresis, chemiluminescence, in situ hybridization, and fluorescence detection; RNA extraction reagents; reverse transcription reagents; cDNA amplification reagents; standards for preparing standard curves; and positive controls.

4. The application according to claim 2, characterized in that: The nucleic acid probes include labeled probes, biotin-labeled probes, horseradish peroxidase-labeled probes, digoxigenin-labeled probes, or fluorescently labeled probes. The nucleic acid probe is a single-labeled or double-labeled nucleic acid probe.

5. The application according to claim 1, characterized in that, The reagent is used to detect the protein expression level of ATP5MK; The reagents used to detect the protein expression level of ATP5MK include those used in the following methods: immunohistochemical staining, hematoxylin-eosin staining, safranin O-fast green staining, Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, mass spectrometry, immunoprecipitation analysis, flow cytometry, and protein chip analysis. The reagents for detecting the protein expression level of ATP5MK include reagents required for immunological assays, which include ELISA, Elispot assay, Western blotting, or surface plasmon resonance assay.

6. The application according to claim 5, characterized in that, The reagents for detecting the protein expression level of ATP5MK also include auxiliary reagents for detecting protein expression levels, which include: blocking solution, antibody dilution solution, washing buffer, colorimetric stop solution, and ATP5MK standard used to prepare the standard curve.

7. The use of a reagent as described in any one of claims 1-6 in the preparation of products for diagnosing or assisting in the diagnosis of colorectal cancer, characterized in that: The products include reagent kits, systems, devices, and computer-readable media.

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