Biomarker composition for diagnosing gastric cancer and use method thereof

Abstract

The invention relates to the field of biological medicine, in particular to a biomarker composition for diagnosing gastric cancer and a use method, and the biomarker composition comprises the following biomarkers: a biomarker A, a biomarker B and a biomarker C; the biomarker A is a protein highly expressed in gastric cancer cells; the biomarker B belongs to a class of small molecule RNA; the biomarker C is a newly found metabolite. The biomarker combination disclosed by the invention contains multiple types of biomarkers such as protein, small molecule RNA and metabolites, so that the diagnosis accuracy is improved; according to the detection method, body fluid samples such as serum and gastric juice are mainly adopted, and compared with invasive operation such as gastroscopy, the non-invasive or minimally invasive advantage is achieved; the biomarker composition and the use method are simple and convenient to operate, relatively low in cost and suitable for wide popularization and application in clinical laboratories, and have good clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a biomarker combination for diagnosing gastric cancer and a method of using the same. Background Art

[0002] Gastric cancer is one of the most common malignant tumors in the world, which seriously threatens human health. According to statistics, there are many new cases of gastric cancer in the world every year, and the mortality rate is high. Patients with early gastric cancer usually have no obvious symptoms, and most patients are already in the middle and late stages when diagnosed. At this time, the treatment effect is often poor, and the 5-year survival rate is low; therefore, early diagnosis of gastric cancer is crucial to improve the survival rate and prognosis of patients.

[0003] At present, the diagnostic methods of gastric cancer mainly include gastroscopy, pathological biopsy, and imaging examinations (such as CT, MRI, etc.); gastroscopy and pathological biopsy are the gold standard for diagnosing gastric cancer, but gastroscopy is an invasive operation, with low patient acceptance and a certain risk of complications; imaging examinations have limited diagnostic sensitivity for early gastric cancer; serological marker detection, as a non-invasive diagnostic method, has the advantages of simple operation and good patient compliance, and has always been a research hotspot in the field of gastric cancer diagnosis; however, the existing single serological markers (such as carcinoembryonic antigen CEA, carbohydrate antigen CA19-9, etc.) have unsatisfactory sensitivity and specificity in the diagnosis of gastric cancer and cannot meet clinical needs.

[0004] Therefore, those skilled in the art provide a biomarker combination for diagnosing gastric cancer and a method of use to solve the problems raised in the above background technology. Summary of the invention

[0005] To solve the above technical problems, the present invention provides a biomarker combination for diagnosing gastric cancer and a method of use;

[0006] include

[0007] A biomarker combination for diagnosing gastric cancer

[0008] The following biomarkers were included: biomarker A, biomarker B, and biomarker C;

[0009] The biomarker A is a protein highly expressed in gastric cancer cells;

[0010] The biomarker B belongs to a class of small molecule RNA and is abnormally expressed in gastric cancer tissue and serum;

[0011] The biomarker C is a newly discovered metabolite, and its content in the body fluids (such as serum, gastric juice, etc.) of gastric cancer patients is significantly higher than that in healthy people.

[0012] Preferably, the biomarker A is pepsinogen II, which is a protein highly expressed in gastric cancer cells; its amino acid sequence consists of 375 amino acid residues, has a unique folding structure, and contains multiple functional domains, among which the catalytic domain is essential for its proteolytic activity; this protein is involved in biological processes such as proliferation, migration and invasion of gastric cancer cells; in the occurrence and development of gastric cancer, the high expression of pepsinogen II will promote the degradation of the extracellular matrix, create conditions for the migration and invasion of gastric cancer cells, and thus play an important role in the occurrence and development of gastric cancer.

[0013] Preferably, the biomarker B is microRNA-21, which belongs to a class of small molecule RNA; it is abnormally highly expressed in gastric cancer tissue and serum; it can regulate the expression of related genes such as programmed cell death protein 4 through complementary pairing with the target mRNA, inhibit the apoptosis of gastric cancer cells, and promote their proliferation, migration and invasion and other biological behaviors, and is closely related to the occurrence, development and prognosis of gastric cancer.

[0014] Preferably, the biomarker C is kynurenine, which is a newly discovered metabolite; its content in the body fluids (such as serum, gastric juice, etc.) of gastric cancer patients is significantly higher than that in healthy people; in normal cells, kynurenine is mainly metabolized through the kynurenine pathway to generate a series of downstream products with functions such as neural regulation; while in gastric cancer cells, its metabolic pathway has undergone significant changes, the metabolic flux has increased, and the enzyme activity of some metabolic branch points has changed, resulting in the accumulation of kynurenine in cells and body fluids, which can be used as a potential biomarker for the diagnosis of gastric cancer.

[0015] A method for using a biomarker combination for diagnosing gastric cancer comprises the following steps:

[0016] Step 1: Set the normal reference ranges for biomarker A, biomarker B, and biomarker C;

[0017] The reference range is determined by testing a large number of samples from healthy people and statistically analyzing the normal reference range of each biomarker;

[0018] Step 2: Detecting the content and expression level of biomarker A, biomarker B and biomarker C in the individual sample to be diagnosed;

[0019] Step 3: Compare the test results with the normal reference range:

[0020] If the content of biomarker A is higher than the upper limit of the normal reference, and the expression level of biomarker B is higher than the upper limit of the normal reference, and the content of biomarker C is higher than the upper limit of the normal reference, it is judged that the individual to be diagnosed is more likely to have gastric cancer;

[0021] If two or more of the indicators of biomarker A, biomarker B, and biomarker C are beyond the normal reference range, further relevant examinations (such as gastroscopy, pathological biopsy, etc.) are recommended to confirm the diagnosis;

[0022] If biomarker A, biomarker B, and biomarker C are all within the normal reference range, it indicates that the individual to be diagnosed is less likely to have gastric cancer, but a comprehensive judgment still needs to be made based on clinical symptoms and other examination results.

[0023] Preferably, the detection method of the biomarker A is as follows:

[0024] For biomarker A, enzyme-linked immunosorbent assay, protein immunoblotting or immunohistochemistry can be used for detection; taking enzyme-linked immunosorbent assay as an example, the specific steps are as follows:

[0025] Sa1. Prepare specific antibodies against biomarker A and coat them on an ELISA plate:

[0026] Sa2, adding the sample to be tested (such as serum, tissue homogenate, etc.) to allow the biomarker A in the sample to bind to the coated antibody;

[0027] Sa3, washing to remove unbound substances, adding enzyme-labeled secondary antibodies to react with biomarker A bound to the coated antibody;

[0028] Sa4. After washing again, add substrate for color development, measure the absorbance value by enzyme marker, and calculate the content of biomarker A in the sample according to the standard curve.

[0029] Preferably, the detection method of the biomarker B is as follows:

[0030] For biomarker B, reverse transcription polymerase chain reaction, real-time fluorescence quantitative PCR or in situ hybridization can be used for detection; taking real-time fluorescence quantitative PCR as an example, the specific steps are as follows:

[0031] Sb1, extract total RNA from samples (such as serum, tissue, etc.);

[0032] Sb2, using total RNA as a template, synthesize cDNA by reverse transcriptase;

[0033] Sb3 and cDNA were used as templates to design specific primers for qRT-PCR amplification;

[0034] Sb4. During the PCR reaction, the amount of amplified product is monitored in real time through the change of the fluorescence signal, and the relative expression amount of biomarker B in the sample is calculated according to the standard curve.

[0035] Preferably, the detection method of the biomarker C is as follows:

[0036] For biomarker C, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry or nuclear magnetic resonance technology can be used for detection; taking liquid chromatography-mass spectrometry as an example, the specific steps are as follows:

[0037] Sc1. Pre-treat samples (such as serum, gastric juice, etc.) such as protein precipitation, solid phase extraction, etc. to remove impurities and enrich biomarker C;

[0038] Sc2, inject the pretreated sample into the liquid chromatograph and separate different compounds through the chromatographic column;

[0039] Sc3. The separated compounds enter the mass spectrometer and the content and structure of biomarker C are determined by detecting their mass-to-charge ratio (m / z).

[0040] Technical effects and advantages of the present invention:

[0041] Improve diagnostic accuracy: The biomarker combination of the present invention includes various types of biomarkers such as proteins, small molecule RNAs and metabolites, which reflect the biological characteristics of gastric cancer from different levels; by jointly detecting these biomarkers, the sensitivity and specificity of gastric cancer diagnosis can be significantly improved, reducing the occurrence of misdiagnosis and missed diagnosis.

[0042] Achieve early diagnosis: Biomarker A, biomarker B, and biomarker C may show abnormal expression or content changes in the early stages of gastric cancer. Therefore, the biomarker combination and detection method of the present invention are helpful to achieve early diagnosis of gastric cancer and strive for the best treatment opportunity for patients.

[0043] Non-invasive or minimally invasive testing: The detection method of the present invention mainly uses body fluid samples such as serum and gastric juice. Compared with invasive operations such as gastroscopy, it has the advantages of being non-invasive or minimally invasive, with high patient acceptance and good repeatability.

[0044] Broad prospects for clinical application: The biomarker combination and method of use of the present invention are easy to operate and relatively low in cost, suitable for widespread promotion and application in clinical laboratories, and have good prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a flow chart of a biomarker combination and a method of use for diagnosing gastric cancer provided in an embodiment of the present application;

[0046] Figure 2 This is a flow chart of a method for detecting biomarker A in a biomarker combination and a method for using the combination for diagnosing gastric cancer provided in an embodiment of the present application;

[0047] Figure 3 This is a flow chart of a method for detecting biomarker B in a biomarker combination and a method for using the combination for diagnosing gastric cancer provided in an embodiment of the present application;

[0048] Figure 4 This is a flow chart of a method for detecting biomarker C in a biomarker combination and a method for using the combination for diagnosing gastric cancer provided in an embodiment of the present application; DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The embodiments of the present invention are provided for the purpose of illustration and description, and are not intended to be exhaustive or to limit the present invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention, and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

[0050] Example 1

[0051] See also Figures 1 to 4 In this embodiment, a biomarker combination for diagnosing gastric cancer and a method of using the biomarker combination are provided, including:

[0052] A biomarker combination for diagnosing gastric cancer

[0053] The following biomarkers were included: biomarker A, biomarker B, and biomarker C;

[0054] The biomarker A is a protein highly expressed in gastric cancer cells;

[0055] The biomarker B belongs to a class of small molecule RNA and is abnormally expressed in gastric cancer tissue and serum;

[0056] The biomarker C is a newly discovered metabolite, the content of which in the body fluids of gastric cancer patients is significantly higher than that in healthy people.

[0057] The biomarker A is pepsinogen II, a protein highly expressed in gastric cancer cells; its amino acid sequence consists of 375 amino acid residues, has a unique folding structure, and contains multiple functional domains, among which the catalytic domain is essential for its proteolytic activity; this protein is involved in biological processes such as proliferation, migration and invasion of gastric cancer cells; in the occurrence and development of gastric cancer, the high expression of pepsinogen II will promote the degradation of the extracellular matrix, create conditions for the migration and invasion of gastric cancer cells, and thus play an important role in the occurrence and development of gastric cancer.

[0058] The biomarker B is microRNA-21, which belongs to a class of small molecule RNA; it is abnormally highly expressed in gastric cancer tissues and serum; it can regulate the expression of related genes such as programmed cell death protein 4 through complementary pairing with target mRNA, inhibit the apoptosis of gastric cancer cells, and promote their proliferation, migration and invasion and other biological behaviors, and is closely related to the occurrence, development and prognosis of gastric cancer.

[0059] The biomarker C is kynurenine, a newly discovered metabolite. Its content in the body fluids of gastric cancer patients is significantly higher than that in healthy people. In normal cells, kynurenine is mainly metabolized through the kynurenine pathway to generate a series of downstream products with functions such as neural regulation. In gastric cancer cells, its metabolic pathway has undergone significant changes, the metabolic flux has increased, and the enzyme activity of some metabolic branch points has changed, resulting in the accumulation of kynurenine in cells and body fluids. It can be used as a potential biomarker for the diagnosis of gastric cancer.

[0060] A method for using a biomarker combination for diagnosing gastric cancer comprises the following steps:

[0061] Step 1: By testing a large number of samples from healthy people, statistical analysis is performed to obtain the normal reference ranges of biomarkers A, biomarker B, and biomarker C;

[0062] Step 2: Detecting the content and expression level of biomarker A, biomarker B and biomarker C in the individual sample to be diagnosed;

[0063] Among them, the detection method of biomarker A is as follows:

[0064] For biomarker A, enzyme-linked immunosorbent assay, protein immunoblotting or immunohistochemistry can be used for detection; taking enzyme-linked immunosorbent assay as an example, the specific steps are as follows:

[0065] Sa1. Prepare specific antibodies against biomarker A and coat them on an ELISA plate:

[0066] Sa2, adding the sample to be tested (such as serum, tissue homogenate, etc.) to allow the biomarker A in the sample to bind to the coated antibody;

[0067] Sa3, washing to remove unbound substances, adding enzyme-labeled secondary antibodies to react with biomarker A bound to the coated antibody;

[0068] Sa4, after washing again, adding substrate for color development, measuring the absorbance value by ELISA instrument, and calculating the content of biomarker A in the sample according to the standard curve;

[0069] Among them, the detection method of biomarker B is as follows:

[0070] For biomarker B, reverse transcription polymerase chain reaction (RT-PCR), real-time fluorescence quantitative PCR (qRT-PCR) or in situ hybridization (ISH) can be used for detection; taking real-time fluorescence quantitative PCR (qRT-PCR) as an example, the specific steps are as follows:

[0071] Sb1, extract total RNA from samples (such as serum, tissue, etc.);

[0072] Sb2, using total RNA as a template, synthesize cDNA by reverse transcriptase;

[0073] Sb3 and cDNA were used as templates to design specific primers for qRT-PCR amplification;

[0074] Sb4. During the PCR reaction, the amount of amplified product is monitored in real time by the change of the fluorescence signal, and the relative expression amount of biomarker B in the sample is calculated according to the standard curve;

[0075] Wherein, the detection method of the biomarker C is as follows:

[0076] For biomarker C, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry or nuclear magnetic resonance technology can be used for detection; taking liquid chromatography-mass spectrometry as an example, the specific steps are as follows:

[0077] Sc1. Pre-treat samples (such as serum, gastric juice, etc.) such as protein precipitation, solid phase extraction, etc. to remove impurities and enrich biomarker C;

[0078] Sc2, inject the pretreated sample into the liquid chromatograph and separate different compounds through the chromatographic column;

[0079] Sc3, the separated compounds enter the mass spectrometer, and the content and structure of biomarker C are determined by detecting their mass-to-charge ratio (m / z);

[0080] Step 3: Compare the test results with the normal reference range:

[0081] If the content of biomarker A is higher than the upper limit of the normal reference, and the expression level of biomarker B is higher than the upper limit of the normal reference, and the content of biomarker C is higher than the upper limit of the normal reference, it is judged that the individual to be diagnosed is more likely to have gastric cancer;

[0082] If two or more of the indicators of biomarker A, biomarker B, and biomarker C are beyond the normal reference range, further relevant examinations (such as gastroscopy, pathological biopsy, etc.) are recommended to confirm the diagnosis;

[0083] If biomarker A, biomarker B, and biomarker C are all within the normal reference range, it indicates that the individual to be diagnosed is less likely to have gastric cancer, but a comprehensive judgment still needs to be made based on clinical symptoms and other examination results.

[0084] Example 2: Application of biomarker combination in gastric cancer diagnosis

[0085] Sample collection

[0086] Serum samples from 100 patients with pathologically confirmed gastric cancer and 100 healthy subjects were collected; informed consent was obtained from the patients or subjects before all samples were collected.

[0087] Biomarker testing

[0088] Detection of biomarker A: Enzyme-linked immunosorbent assay was used to detect the content of biomarker A in serum.

[0089] Detection of biomarker B: The relative expression of biomarker B in serum was detected by real-time fluorescence quantitative PCR; total RNA in serum was extracted, reverse transcribed into cDNA, and then amplified by real-time fluorescence quantitative PCR; the primer sequence was designed according to the gene sequence of biomarker B.

[0090] Detection of biomarker C: Liquid chromatography-mass spectrometry was used to detect the content of biomarker C in serum; after pretreatment, the serum samples were injected into the liquid chromatography-mass spectrometry system for analysis.

[0091] Diagnosis result judgment

[0092] The test results were judged according to the set normal reference ranges of biomarker A, biomarker B and biomarker C; the results showed that among 100 gastric cancer patients, 85 patients had two or more indicators of biomarker A, biomarker B and biomarker C exceeding the normal reference range; among 100 healthy physical examination subjects, only 5 had similar situations. This example shows that the biomarker combination of the present invention has high sensitivity and specificity in the diagnosis of gastric cancer.

[0093] Example 3: Biomarker combination for gastric cancer monitoring

[0094] Sample collection

[0095] Twenty gastric cancer patients who underwent surgical treatment were selected, and serum samples were collected before surgery, 1 week after surgery, and 1 month after surgery.

[0096] Biomarker testing

[0097] Similar to Example 2, the contents or expression levels of biomarker A, biomarker B and biomarker C in serum were detected by enzyme-linked immunosorbent assay, real-time fluorescence quantitative PCR and liquid chromatography-mass spectrometry, respectively.

[0098] Disease monitoring and analysis

[0099] The results showed that the content or expression level of biomarker A, biomarker B and biomarker C in the serum of patients before surgery was significantly higher than the normal reference range; one week after surgery, the levels of these biomarkers decreased; one month after surgery, the biomarker levels of most patients were close to the normal reference range. This example shows that the biomarker combination of the present invention can be used to monitor the condition of gastric cancer patients and evaluate the effect of surgical treatment.

[0100] Example 4: Biomarker combination for gastric cancer prognosis assessment

[0101] Sample collection

[0102] Serum samples were collected from 50 gastric cancer patients, and these patients were followed up for 3 years to record the survival and recurrence of the patients.

[0103] Biomarker testing

[0104] As in Example 1, the contents or expression levels of biomarker A, biomarker B and biomarker C in serum were detected.

[0105] Prognostic evaluation analysis

[0106] According to the test results, the patients were divided into a high-expression group and a low-expression group of biomarkers. The follow-up results showed that the survival rate of patients in the high-expression group of biomarkers was significantly lower than that in the low-expression group, and the recurrence rate was significantly higher than that in the low-expression group. This example shows that the biomarker combination of the present invention can be used for the prognosis assessment of gastric cancer patients and provide a reference for the formulation of clinical treatment plans.

[0107] In addition, when the biomarker combination and detection method of the present invention are actually applied, the relevant operating procedures should be strictly followed to ensure the accuracy and reliability of the detection results.

[0108] The biomarker combination and detection method of the present invention are only used as auxiliary means for the diagnosis of gastric cancer. The final diagnosis result still needs to be comprehensively judged in combination with clinical symptoms, signs and other examination results (such as gastroscopy, pathological biopsy, etc.).

[0109] With the continuous development of technology and in-depth research, it may be necessary to adjust and optimize the normal reference range of biomarkers in a timely manner to improve the accuracy of diagnosis.

[0110] During sample collection, transportation and storage, care should be taken to avoid sample contamination and degradation to ensure that sample quality meets testing requirements.

[0111] The electrical components appearing in this article are all electrically connected to an external main controller and 220V AC power, and the main controller can be a conventional known device that controls a computer, etc. The specific implementation method of the present disclosure omits the detailed description of known functions and known components. To ensure the compatibility of the equipment, the operating methods used are consistent with the parameters of marketed equipment.

[0112] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field and related fields without creative work should fall within the scope of protection of the present invention. The structures, devices and operating methods not specifically described and explained in the present invention are implemented according to the conventional means in the field unless otherwise specified and limited.

Claims

1. A biomarker combination for diagnosing gastric cancer, characterized in that: Includes biomarker A, biomarker B, and biomarker C; The biomarker A is a protein highly expressed in gastric cancer cells; The biomarker B belongs to a class of small molecule RNA and is abnormally expressed in gastric cancer tissue and serum; The biomarker C is a newly discovered metabolite, the content of which in the body fluids of gastric cancer patients is significantly higher than that in healthy people.

2. A biomarker combination for diagnosing gastric cancer according to claim 1, characterized in that: The biomarker A is pepsinogen II, and the amino acid sequence of pepsinogen II consists of 375 amino acid residues.

3. A biomarker combination for diagnosing gastric cancer according to claim 1, characterized in that: The biomarker B is microRNA-21, which belongs to a class of small molecule RNA; the microRNA-21 is abnormally highly expressed in gastric cancer tissue and serum.

4. A biomarker combination for diagnosing gastric cancer according to claim 1, characterized in that: The biomarker C is kynurenine, a newly discovered metabolite; its content in the body fluids of gastric cancer patients is significantly higher than that in healthy people.

5. The method for using a biomarker combination for diagnosing gastric cancer according to claim 1, characterized in that: The following steps are involved: Step 1: Set the normal reference ranges for biomarker A, biomarker B, and biomarker C; The reference range is determined by testing a large number of samples from healthy people and statistically analyzing the normal reference range of each biomarker; Step 2: Detecting the content and expression level of biomarker A, biomarker B and biomarker C in the individual sample to be diagnosed; Step 3: Compare the test results with the normal reference range: If the content of biomarker A is higher than the upper limit of the normal reference, and the expression level of biomarker B is higher than the upper limit of the normal reference, and the content of biomarker C is higher than the upper limit of the normal reference, it is judged that the individual to be diagnosed is more likely to have gastric cancer; If two or more of the indicators of biomarker A, biomarker B, and biomarker C are beyond the normal reference range, further relevant examinations are recommended to confirm the diagnosis; If biomarker A, biomarker B, and biomarker C are all within the normal reference range, it indicates that the individual to be diagnosed is less likely to have gastric cancer, but a comprehensive judgment still needs to be made based on clinical symptoms and other examination results.

6. The method for using a biomarker combination for diagnosing gastric cancer according to claim 5, characterized in that: The detection method of biomarker A in step 2 is as follows: For biomarker A, enzyme-linked immunosorbent assay, protein immunoblotting or immunohistochemistry can be used for detection; taking enzyme-linked immunosorbent assay as an example, the specific steps are as follows: Sa1. Prepare specific antibodies against biomarker A and coat them on an ELISA plate: Sa2, adding the sample to be tested to allow the biomarker A in the sample to bind to the coated antibody; Sa3, washing to remove unbound substances, adding enzyme-labeled secondary antibodies to react with biomarker A bound to the coated antibody; Sa4. After washing again, add substrate for color development, measure the absorbance value by enzyme marker, and calculate the content of biomarker A in the sample according to the standard curve.

7. The method for using a biomarker combination for diagnosing gastric cancer according to claim 5, characterized in that: The detection method of biomarker B in step 2 is as follows: For biomarker B, reverse transcription polymerase chain reaction, real-time fluorescence quantitative PCR or in situ hybridization can be used for detection; taking real-time fluorescence quantitative PCR as an example, the specific steps are as follows: Sb1, total RNA from extracted samples; Sb2, using total RNA as a template, synthesize cDNA by reverse transcriptase; Sb3 and cDNA were used as templates to design specific primers for real-time fluorescence quantitative PCR amplification; Sb4. During the PCR reaction, the amount of amplified product is monitored in real time through the change of the fluorescence signal, and the relative expression amount of biomarker B in the sample is calculated according to the standard curve.

8. The method for using a biomarker combination for diagnosing gastric cancer according to claim 5, characterized in that: The detection method of biomarker C in step 2 is as follows: For biomarker C, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry or nuclear magnetic resonance technology can be used for detection; taking liquid chromatography-mass spectrometry as an example, the specific steps are as follows: Sc1, pre-treat the sample to remove impurities and enrich biomarker C; Sc2, inject the pretreated sample into the liquid chromatograph and separate different compounds through the chromatographic column; Sc3. The separated compounds enter the mass spectrometer and the content and structure of biomarker C are determined by detecting their mass-to-charge ratio.

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