A colorectal cancer molecular marker and application thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a colorectal cancer molecular marker and application thereof. BACKGROUND

[0002] Early diagnosis and intervention of colorectal cancer can improve the prognosis, therefore, improving the diagnosis level is crucial to improve the quality of life of patients.

[0003] At present, the first-line means of colorectal cancer screening mainly includes colonoscopy, fecal immunochemical test (FIT) and carcinoembryonic antigen (CEA) detection, but all of them have obvious limitations. Among them, colonoscopy is the gold standard for diagnosis and has high accuracy, but it is an invasive operation, which has problems such as complicated intestinal preparation, easy to cause complications and high cost, resulting in generally low patient acceptance and compliance; FIT, as a non-invasive primary screening method, can improve patient participation, but its sensitivity for identifying precancerous lesions is limited; regular detection of CEA can provide indications related to colorectal cancer, but its specificity and sensitivity still cannot meet the clinical needs. In addition, other commonly used screening methods such as imaging evaluation and histopathological examination also have problems such as high cost, invasiveness or insufficient diagnostic efficiency. It is worth noting that the formation cycle of most colorectal cancer is 10 to 15 years, which provides a key time window for early detection and intervention of the disease. Therefore, developing a non-invasive, highly accurate and well-accessible early detection technology for colorectal cancer has become an urgent need in the current clinical and public health fields.

[0004] In recent years, molecular biology technology has shown great potential in the early diagnosis of colorectal cancer by gene biomarkers, and a large number of studies have confirmed that screening and early diagnosis and treatment of colorectal cancer can effectively reduce its mortality. Based on this, it is of great research significance to develop a new biomarker and apply it to the diagnosis and prognosis prediction of colorectal cancer. SUMMARY

[0005] The purpose of the present application is to provide a colorectal cancer molecular marker and application thereof, aiming to make up for the defects of the prior art, realize non-invasive early diagnosis of colorectal cancer, and thus improve the prognosis of patients, improve the quality of life and survival rate of patients.

[0006] To achieve the above purpose, the present application provides a colorectal cancer molecular marker, which is ATP5MK, and the Gene ID of ATP5MK in NCBI is 84833.

[0007] The expression amount of ATP5MK in the colorectal cancer tissue is significantly lower than that in the normal tissue.

[0008] Use of the above-mentioned colorectal cancer molecular marker in the preparation of a reagent for diagnosing or aiding in the diagnosis of colorectal cancer, including colon cancer and rectal cancer; the reagent is a reagent for detecting the expression level of the molecular marker ATP5MK.

[0009] Preferably, the reagent is a reagent for detecting the mRNA expression level of ATP5MK.

[0010] Preferably, the reagent for detecting the mRNA expression level of ATP5MK includes 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 circle amplification, in situ hybridization, microarray, Southern blotting, Northern blotting, high-throughput sequencing platform method.

[0011] Preferably, the reagent for detecting the mRNA expression level of ATP5MK includes specific nucleic acid probes that bind to target sequences, specific primers that amplify target sequences, non-specific fluorescent dyes, or combinations thereof.

[0012] Preferably, the reagent for detecting the mRNA expression level of ATP5MK further includes mRNA expression amount auxiliary detection reagents, which include: 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 samples.

[0013] Preferably, 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.

[0014] Preferably, the nucleic acid probe is a single-labeled or double-labeled nucleic acid probe.

[0015] Preferably, the reagent is a reagent for detecting the protein expression level of ATP5MK;

[0016] Preferably, the reagent for detecting the protein expression level of ATP5MK includes reagents used in the following methods: immunohistochemical staining method, hematoxylin-eosin staining method, safranin O-fast green staining, Western blotting, enzyme-linked immunosorbent assay, radioimmunoassay, mass spectrometry, immunoprecipitation analysis method, flow cytometry fluorescence technology, and protein chip method.

[0017] Preferably, the reagent for detecting the protein expression level of ATP5MK comprises reagents required for immunological detection, including ELISA detection, Elispot detection, Western blotting or surface plasmon resonance method.

[0018] Preferably, the reagent for detecting the protein expression level of ATP5MK further comprises protein expression amount auxiliary detection reagents, including blocking solution, antibody diluent, washing buffer, color development termination solution, and ATP5MK standard for preparing a standard curve.

[0019] The reagent described above is used for preparing a product for diagnosing or assisting in diagnosing colorectal cancer, which comprises a kit, a system, a device, and a computer readable medium.

[0020] Preferably, the device comprises a PCR instrument, a high-throughput sequencing platform, a detection chip, and a chip signal reader.

[0021] Preferably, the detection chip comprises probes for detecting the expression amount of a marker.

[0022] Preferably, the detection chip comprises an internal reference probe.

[0023] Preferably, the internal reference comprises GAPDH or β-Actin.

[0024] Preferably, the detection chip comprises a protein chip and / or a gene chip.

[0025] Preferably, the computer readable medium records or runs a method for detecting the expression amount of ATP5MK.

[0026] The product described above is used for preparing a system for diagnosing or assisting in diagnosing colorectal cancer, which comprises:

[0027] (1) a computing device for determining whether a subject has colorectal cancer according to the detection result of a molecular marker;

[0028] (2) an input device for inputting the expression amount of a molecular marker of a subject;

[0029] (3) an output device for outputting a colorectal cancer diagnosis result.

[0030] Therefore, the colorectal cancer molecular marker and the application thereof have the following beneficial effects: the present application first discovers that ATP5MK is significantly differentially expressed in patients with colorectal cancer, and can be used as a biomarker for diagnosing colorectal cancer, and has the advantages of high accuracy, good specificity, high sensitivity, etc., provides a new idea and strategy for early diagnosis of colorectal cancer in the field, and has good clinical application value.

[0031] The technical solutions of the present application will be further described in detail below with the aid of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required to be used in the description of the embodiments of the present application will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative labor on the basis of these drawings.

[0033] Figure 1 ATP5MK expression difference between normal and cancer tissues and diagnostic ROC in GSE44076 data set; wherein A is the ATP5MK expression difference between normal and cancer tissues, and B is the diagnostic ROC;

[0034] Figure 2 ATP5MK expression difference between normal and cancer tissues and diagnostic ROC in GSE20842 data set; wherein A is the ATP5MK expression difference between normal and cancer tissues, and B is the diagnostic ROC;

[0035] Figure 3 ATP5MK expression difference between normal and cancer tissues and diagnostic ROC in 30 pairs of normal and colon cancer tissues in self-owned clinical cohort; wherein A is the ATP5MK expression difference between normal and cancer tissues, and B is the diagnostic ROC;

[0036] Figure 4 ATP5MK expression difference between normal and cancer tissues and diagnostic ROC in 22 pairs of normal and rectal cancer tissues in self-owned clinical cohort; wherein A is the ATP5MK expression difference between normal and cancer tissues, and B is the diagnostic ROC;

[0037] Figure 5 ATP5MK expression difference between normal and cancer tissues and diagnostic ROC in 58 normal and 55 colorectal cancer tissues in self-owned clinical cohort; wherein A is the ATP5MK expression difference between normal and cancer tissues, and B is the diagnostic ROC;

[0038] Figure 6 ATP5MK expression difference between paracancer and cancer tissues in colon cancer tissue chip and diagnostic ROC; wherein A is the result of immunohistochemical staining, B is the ATP5MK histochemical score, C is the relative abundance analysis of ATP5MK in paracancer and cancer tissues, and D is the ROC curve.

[0039] Figure 7Figure 6 is a ROC of the differential expression and diagnosis of ATP5MK in the colorectal cancer tissue chip; wherein A is the immunohistochemical staining result, B is the ATP5MK group score, C is the relative abundance analysis of ATP5MK in the cancer and cancer tissue, and D is the ROC curve.

[0040] Figure 8 Figure 6 is a ROC of the differential expression and diagnosis of ATP5MK in the colorectal cancer tissue chip; wherein A is the immunohistochemical staining result, B is the ATP5MK group score, C is the relative abundance analysis of ATP5MK in the cancer and cancer tissue, and D is the ROC curve. DETAILED DESCRIPTION

[0041] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0042] In order to make the purpose, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present application are described clearly and completely below by means of the accompanying drawings and examples. The following detailed description is a description of the examples, which aims to provide further detailed description of the present application. Unless otherwise specified, all technical terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0043] The instrument equipment and reagent materials used in the examples are obtained through commercial channels.

[0044] Example 1

[0045] GSE44076 (cancer 98, normal 148), GSE20842 (cancer 65, normal 65) two gene chip data sets were used as verification sets, and the expression matrix and clinical information were downloaded from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo). The two GEO data sets were used to verify the ability of ATP5MK (ATP synthase membrane subunit k) as a colon cancer molecular marker. The Gene ID of ATP5MK in NCBI (https: / / www.ncbi.nlm.nih.gov / ) is 84833.

[0046] The ATP5MK expression difference between normal and cancer tissues in the GEO data set and the diagnostic ROC are shown in Figure 1 and Figure 2 . Figure 1 For the GSE44076 data set, Figure 1 A is the ATP5MK expression difference between normal and cancer tissues, Figure 1 B is the diagnostic ROC. Figure 2 For the GSE20842 data set,Figure 2 ATP5MK expression difference between normal and cancer tissues, Figure 2 ROC for diagnosis.

[0047] ATP5MK expression difference between normal and cancer tissues, Figure 1 ATP5MK expression difference between normal and cancer tissues, Figure 2 ATP5MK expression difference between normal and cancer tissues, ATP5MK expression difference between normal and cancer tissues,

[0048] ATP5MK expression difference between normal and cancer tissues, Figure 1 ATP5MK expression difference between normal and cancer tissues, Figure 2 ATP5MK expression difference between normal and cancer tissues, ATP5MK expression difference between normal and cancer tissues,

[0049] ATP5MK expression difference between normal and cancer tissues,

[0050] Example 2

[0051] In this example, a new clinical cohort was used, and the samples were collected from the Affiliated Hospital of Qingdao University. The inclusion criteria were: diagnosed as colon cancer or rectal cancer by tissue biopsy, and no radiotherapy or chemotherapy before tissue sample collection. The collected tissue samples were washed with pre-cooled PBS, then quickly frozen in liquid nitrogen, and then transferred to a -80°C refrigerator for storage. This cohort collected 58 cases of colorectal cancer and 55 cases of cancer adjacent samples.

[0052] The clinical samples involved in this example have obtained the informed consent of the patients before acquisition, and have obtained the approval of the Research Ethics Committee of the Affiliated Hospital of Qingdao University, and in the implementation process, the Declaration of Helsinki in 1964 and later versions are followed.

[0053] RT-qPCR was used to detect the expression of ATP5MK. First, the total RNA of the tissue sample was extracted by SPARKeasy Tissue / Cell RNA Fast Extraction Kit (Scoth, Shandong, AC0202), and the RNA concentration was determined. 1 μg of RNA was used to remove DNA using DNAase, and then the RNA was reverse transcribed into cDNA using SPARKscript II All-in-one RT SuperMix for qPCR (Scoth, Shandong, AG0305). Then, 2xSYBR Green qPCR Mix (Scoth, Shandong, AH0104) was used for quantitative PCR reaction. The expression of GAPDH was used as an internal control to calculate the relative expression level of ATP5MK. The expression difference of ATP5MK in cancer and adjacent cancer was statistically analyzed and box plots were drawn. In addition, the ROC curve was drawn with the expression of ATP5MK to determine whether the disease was present or not, and the AUC value was obtained.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] Example 3

[0058] 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.

[0059] 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.

[0060] Anti-human ATP5MK antibody was purchased from Wuhan Three Yike Biological Technology Co., Ltd., Cat: 17716-1-AP, the antibody was diluted at 1:100, the ATP5MK protein expression level was evaluated by manual reading, and the staining intensity and the proportion of positive cells were scored, the staining intensity of the tissue was divided into four grades, of which 0 represented no, 1 represented weak positive, 2 represented moderate intensity positive, and 3 represented strong positive; the tissue staining area was divided into five grades, of which 0 represented no, 1 represented 1-25% of the tissue was positive, 2 represented 25-50% of the tissue was positive, 3 represented 50-75% of the tissue was positive, and 4 represented 75-100% of the tissue was positive, ATP5MK group chemical score was calculated, and the calculation formula was:

[0061] .

[0062] The ATP5MK group chemical score was a series of integers ranging from 0 to 12, and the score was used to statistically analyze the expression difference of ATP5MK in colorectal cancer and paracancerous tissues, and to draw the ROC curve of ATP5MK for diagnosing colorectal cancer. First, the results of colon cancer and rectal cancer tissue chips were analyzed respectively, and then the two results were combined for analysis. The expression of ATP5MK in paracancerous and cancerous tissues was statistically analyzed and box plots were drawn, then according to the median of the score, the expression of ATP5MK was divided into high and low two groups, the chi-square test was performed on the percentage of high / low expression of ATP5MK in paracancerous and cancerous groups, finally, the ROC curve was drawn to analyze the resolution ability of ATP5MK expression for diagnosing colorectal cancer.

[0063] The differential expression of ATP5MK in paracancerous and cancerous tissues in colon cancer tissue chip and the diagnostic ROC are shown in Figure 6 , wherein A is the result of immunohistochemical staining, B is the ATP5MK group chemical score, C is the relative abundance analysis of ATP5MK in paracancerous and cancerous tissues, and D is the ROC curve.

[0064] The results showed that the expression level of ATP5MK in paracancerous tissue was significantly higher than that in cancer tissue, and the percentage of high expression of ATP5MK protein in paracancerous tissue was significantly higher than that in tumor tissue, P with a p value less than 0.001. Further evaluation of its diagnostic performance by ROC curve showed that the AUC value of ATP5MK for diagnosing colon cancer patients was 0.9433.

[0065] The differential expression of ATP5MK in paracancerous and cancerous tissues in rectal cancer tissue chip and the diagnostic ROC are shown in Figure 7As shown in the figure, A is the result of immunohistochemical staining, B is the ATP5MK group histological score, C is the relative abundance analysis of ATP5MK in paracancerous and cancerous tissues, and D is the ROC curve.

[0066] The results show that the expression of ATP5MK in paracancerous tissues is also significantly higher than that in cancer tissues, and the difference in expression distribution of ATP5MK between the two groups has a high statistical significance (P<0.001). P <0.001). ROC curve analysis shows that the AUC value of ATP5MK for the diagnosis of colorectal cancer is 0.9528.

[0067] To further verify the diagnostic value of ATP5MK in the whole colorectal cancer, the colon cancer and rectal cancer tissue chip data were integrated for analysis. In the colorectal cancer tissue chip, the differential expression of ATP5MK in paracancerous and cancerous tissues and the diagnostic ROC are as shown in the figure. Figure 8 As shown in the figure, A is the ATP5MK group histological score, B is the relative abundance analysis of ATP5MK in paracancerous and cancerous tissues, and C is the ROC curve.

[0068] The results show that the expression of ATP5MK in paracancerous tissues is still significantly higher than that in cancer tissues, and the percentage of high expression of ATP5MK in paracancerous tissues is also significantly higher than that in tumor tissues. ROC curve analysis shows that the AUC value of ATP5MK for the comprehensive diagnosis of colorectal cancer is 0.9376.

[0069] The above series of immunohistochemical experiments confirm that ATP5MK has good diagnostic ability in colon cancer, rectal cancer and whole colorectal cancer at the protein level.

[0070] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application and not to limit it, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: it can still modify or equivalently replace the technical solutions of the present application, and these modifications or equivalent replacements also cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. The application of a reagent for specifically detecting the expression levels of ATP5MK mRNA and / or protein in the preparation of diagnostic or auxiliary diagnostic reagents for colorectal cancer, characterized in that: 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 reagents are probes, primers, or antibodies that specifically detect the expression levels of ATP5MK mRNA and / or protein.

2. The application of a reagent for specifically detecting the expression level of ATP5MK mRNA and / or protein in the preparation of products for diagnosing or assisting in the diagnosis of colorectal cancer, characterized in that: The product is a reagent kit; The Gene ID of ATP5MK in NCBI is 84833; The reagents are probes, primers, or antibodies that specifically detect the expression levels of ATP5MK mRNA and / or protein.

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