Marker for gastric cancer diagnosis and treatment target and application thereof

Abstract

The invention discloses a marker for gastric cancer diagnosis and treatment targets and application of the marker, and relates to the technical field of biomedicine. The marker is miRNA (micro Ribonucleic Acid), and the miRNA comprises at least one of miR-192-5p, miR-4488, miR-127-3p and miR-93-3p. The miRNA molecule (the marker) secreted by the gastric cancer stem cells can be used as a biomarker for diagnosis and treatment of gastric cancer for the first time. The marker provided by the invention is highly expressed in secretion of gastric cancer stem cells, the expression quantity of the marker is increased along with the growth of the gastric cancer stem cells, further, the marker is highly expressed in the gastric cancer cells and is lowly expressed in normal gastric epithelial cells, and the expressions of the marker in tumor cells and normal cells are remarkably different; it is indicated that the molecular marker has high diagnosis sensitivity and specificity on gastric cancer as a gastric cancer diagnosis molecule, has great potential in screening of early gastric cancer, and has important practical significance in early diagnosis or prognosis prediction of gastric cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a marker for gastric cancer diagnosis and treatment target and application thereof. Background Art

[0002] Malignant tumors are high-morbidity and high-mortality diseases that seriously endanger human health. Among them, gastric cancer (GC) is the most common malignant tumor of the digestive system, and its morbidity and mortality rates are both among the top five malignant tumors. my country is a high-incidence area for gastric cancer, with high morbidity and mortality rates. The number of new cases ranks fifth in the world, while the mortality rate ranks third among malignant tumors. Early detection and diagnosis of tumors are crucial to improving the survival rate of patients. Studies have found that if gastric cancer is discovered in the early stage, its 5-year survival rate can be close to 95%, while in the late stage, this indicator is only 20-60%.

[0003] Currently, there are many methods for early cancer screening, including tumor marker testing, imaging examinations, tissue biopsies, liquid biopsy technology, etc. Among them, liquid biopsy is a molecular diagnosis, which has become a development trend in early cancer screening methods because of its non-invasiveness and convenience. Liquid biopsy is a test of liquid samples such as blood and urine to measure the expression levels of biomarkers such as circulating tumor cells (CTC), circulating tumor DNA (ctDNA), and micro-molecule RNA (miRNA). Once these biomarkers are abnormally expressed, it indicates that cancer may be present and is progressing.

[0004] miRNA is widely present in eukaryotic cells. It is a type of non-coding RNA with a length of 18 to 25 nucleotides that acts on downstream target genes to regulate tumor cell functions and is involved in the occurrence and development of tumors. miRNA plays a core role in the process of cell evolution and can inhibit or degrade the translation of mRNAs by binding to various targeted mRNAs. In the early stages of tumor development, the expression level or self-modification state of related miRNAs changes. Accurate monitoring of these changes is expected to become a diagnostic method for early cancer.

[0005] Studies have shown that miRNA plays an important role in the diagnosis, treatment and prognosis monitoring of tumors, especially for early malignant tumors that do not have typical clinical symptoms, have no specific examinations and are difficult to detect. As an innovative tumor marker, miRNA has good stability and relatively high abundance, making its detection more advantageous than traditional methods such as ctDNA and protein.

[0006] Cancer stem cells (CSCs) are a small number of cell subpopulations in tumors that have stem cell characteristics, namely, the ability to self-renew and produce tumor cells of different degrees of differentiation. They are considered to be the "seed" cells in tumors and the root cause of tumor occurrence, metastasis, recurrence and resistance to radiotherapy and chemotherapy. However, the research on gastric cancer stem cells (GCSCs) started relatively late. In 2009, Takaishi et al. first proved the existence of GCSCs. Compared with other tumor types, the current understanding of them is still very limited. The research on gastric cancer stem cells at home and abroad is not in-depth. Related research reports mainly focus on finding surface markers of gastric cancer stem cells, isolating gastric cancer stem cells and studying the biological characteristics of gastric cancer stem cells. So far, the important molecules and mechanisms regulating the survival of gastric cancer stem cells are not clear, and there is a serious lack of detection and target molecules. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide new markers for gastric cancer diagnosis and treatment targets and their applications.

[0008] In order to solve the above problems, the present invention proposes the following technical solutions:

[0009] The present invention provides a marker for gastric cancer diagnosis and treatment target, wherein the marker is miRNA, and the miRNA includes at least one of miR-192-5p, miR-4488, miR-127-3p, and miR-93-3p.

[0010] Furthermore, the miRNA is derived from serum or plasma extracellular secretions.

[0011] The present invention also provides the use of the above-mentioned marker for gastric cancer diagnosis and treatment target in preparing a product for gastric cancer diagnosis and treatment target by the following method, wherein the method comprises the following steps:

[0012] Measuring the expression level of the marker according to claim 1 or 2 in the sample; and

[0013] The diagnosis, treatment and prognosis of gastric cancer are evaluated based on the expression level of the marker.

[0014] The present invention also provides a reagent for auxiliary diagnosis of gastric cancer, wherein the reagent can detect the expression level of miRNA in a sample, wherein the miRNA includes at least one of miR-192-5p, miR-4488, miR-127-3p, and miR-93-3p.

[0015] The present invention also provides a kit for auxiliary diagnosis of gastric cancer, the kit comprising a reagent capable of detecting the expression level of miRNA in a sample, wherein the miRNA comprises at least one of miR-192-5p, miR-4488, miR-127-3p, and miR-93-3p.

[0016] The present invention also provides a chip for auxiliary diagnosis of gastric cancer, wherein the chip comprises a reagent capable of detecting the expression level of miRNA in a test sample, wherein the miRNA comprises at least one of miR-192-5p, miR-4488, miR-127-3p, and miR-93-3p.

[0017] Compared with the prior art, the technical effects achieved by the present invention include:

[0018] The present invention first discovered that the miRNA molecules (miR-192-5p, miR-4488, miR-127-3p, miR-93-3p) secreted by gastric cancer stem cells can be used as biomarkers for gastric cancer diagnosis and treatment. The above markers provided by the present invention are highly expressed in the secretion fluid of gastric cancer stem cells, and their expression increases with the growth of gastric cancer stem cells. Furthermore, the above markers are highly expressed in gastric cancer cells and lowly expressed in normal gastric epithelial cells. There is a significant difference in their expression in tumor cells and normal cells, indicating that they have high diagnostic sensitivity and specificity for gastric cancer as gastric cancer diagnostic molecules, have great potential in the screening of early gastric cancer, and have important practical significance for the early diagnosis or prognosis prediction of gastric cancer.

[0019] Detecting the expression levels of the above-mentioned markers in samples and combining them with imaging examinations can help improve the specificity of gastric cancer diagnosis and reduce false positives, which has important practical significance for the early diagnosis, treatment and prognosis prediction of gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0021] Figure 1 Shows the expression differences of secretion-related miRNAs in the culture medium of human gastric cancer stem cells at different culture time periods.

[0022] Figure 2 The real-time PCR validation results of secretion-related miRNAs in the culture medium of human gastric cancer stem cells at different culture time periods are shown.

[0023] Figure 3 The expression of miR-192-5P secreted in the culture medium of human normal gastric epithelial cells (GES-1) and different gastric cancer cell lines (AGS, HGC-7, MGC-803) is shown.

[0024] Figure 4 The expression of miR-4488 secreted in the culture medium of human normal gastric epithelial cells (GES-1) and different gastric cancer cell lines (AGS, HGC-7, MGC-803) is shown.

[0025] Figure 5 The expression of miR-127-3P secreted in the culture medium of human normal gastric epithelial cells (GES-1) and different gastric cancer cell lines (AGS, HGC-7, MGC-803) is shown.

[0026] Figure 6 The expression of miR-93-3P secreted in the culture medium of human normal gastric epithelial cells (GES-1) and different gastric cancer cell lines (AGS, HGC-7, MGC-803) is shown. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0029] It should also be understood that the terms used in this description of the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. As used in the description of the embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0030] It should be noted that, in order to compare the differences in the expression levels of secreted miRNAs in different samples, miR-16 was used as an internal reference in the embodiments of the present invention to facilitate normalization of the data.

[0031] In the embodiments of the present invention, the source and culture of gastric cancer stem cells refer to the articles Cell Research 2012; Int.J.Biol.Sci.2019; Cell Death Dis.2023; Acta Pharmacol Sin.2024, etc. The specific culture process is as follows: After the gastric cancer stem cells are revived, they are inoculated in a serum-free culture medium containing EGF and bFGF, cultured in an environment of 5% CO2 and 37°C, and the culture medium is added once after 3 days. The expression levels of different miRNA molecules secreted in the culture medium after 1h of culture (D0) and the expression levels of different miRNA molecules secreted in the culture medium after 6 days of culture (D6) were detected respectively. The results are shown in Figure 1-Figure 2 .

[0032] Figure 1 The results of RNA-seq sequencing differential expression of secreted miRNAs in the culture medium of human gastric cancer stem cells derived from gastric cancer patients at different culture times are shown. It can be seen that the expression of different miRNA molecules secreted in the culture medium of gastric cancer stem cells after 6 days of culture (D6) is significantly upregulated compared with that in the culture medium of 1h (D0) (the numbers in each bar graph interval represent the corresponding miRNA upregulation ratio) (***P<0.001, ****P<0.0001, where the expression value of each miRNA in D0 is set to 1).

[0033] Figure 2 The real-time PCR validation results of secreted related miRNAs in the culture medium of gastric cancer stem cells at different times of culture are shown. Different miRNA molecules secreted in the culture medium of gastric cancer stem cells cultured for 6 days (D6) and 1 hour (D0) were confirmed by real-time PCR that the expression of related miRNAs in the secretion fluid was significantly upregulated during the culture of gastric cancer stem cells (the numbers in each bar graph interval represent the upregulation ratio of the corresponding miRNA) (*P<0.05, **P<0.01, where the expression value of each miRNA in D0 was set to 1, and miR-16 was used as the internal reference).

[0034] Normal gastric epithelial cells and different gastric cancer cell lines (AGS, HGC-7, MGC-803) were cultured separately to observe the expression of miR-192-5P, miR-4488, miR-127-3P and miR-93-3P secreted in each culture medium. Figure 3-Figure 6 .

[0035] Figure 3The expression of miR-192-5P secreted in the culture medium of normal gastric epithelial cells (GES-1) and different gastric cancer cell lines (AGS, HGC-7, MGC-803) is shown. The secreted miR-192-5P molecules in the culture medium of gastric epithelial cells (GES-1) and different gastric cancer cell lines (AGS, HGC-7, MGC-803) cultured for 6 days (D6) were analyzed by real-time PCR, confirming that miR-192-5P was highly expressed in gastric cancer cells and lowly expressed in normal gastric epithelial cells (the numbers in each bar graph interval represent the corresponding miRNA up-regulation ratio) (*P<0.05, **P<0.01, where the expression value of GES-1 was set to 1 and miR-16 was used as an internal reference).

[0036] Figure 4 The expression of miR-4488 secreted in the culture medium of normal gastric epithelial cells (GES-1) and different gastric cancer cell lines (AGS, HGC-7, MGC-803) is shown. The secreted miR-4488 molecules in the culture medium of gastric epithelial cells (GES-1) and different gastric cancer cell lines (AGS, HGC-7, MGC-803) cultured for 6 days (D6) were analyzed by real-time PCR, confirming that miR-4488 was highly expressed in gastric cancer cells and lowly expressed in normal gastric epithelial cells (the numbers in each bar graph interval represent the corresponding miRNA up-regulation ratio) (*P<0.05, **P<0.01, where the expression value of GES-1 was set to 1 and miR-16 was used as an internal reference).

[0037] Figure 5 The expression of miR-127-3P secreted in the culture medium of normal gastric epithelial cells (GES-1) and different gastric cancer cell lines (AGS, HGC-7, MGC-803) is shown. The secreted miR-127-3P molecules in the culture medium of gastric epithelial cells (GES-1) and different gastric cancer cell lines (AGS, HGC-7, MGC-803) cultured for 6 days (D6) were analyzed by real-time PCR, confirming that miR-127-3P was highly expressed in gastric cancer cells and lowly expressed in normal gastric epithelial cells (the numbers in each bar graph interval represent the corresponding miRNA up-regulation ratio) (*P<0.05, ****P<0.0001, where the expression value of GES-1 was set to 1 and miR-16 was used as an internal reference).

[0038] Figure 6The expression of miR-93-3P secreted in the culture medium of normal gastric epithelial cells (GES-1) and different gastric cancer cell lines (AGS, HGC-7, MGC-803) is shown. The secreted miR-93-3P molecules in the culture medium of gastric epithelial cells (GES-1) and different gastric cancer cell lines (AGS, HGC-7, MGC-803) cultured for 6 days (D6) were analyzed by real-time PCR, confirming that miR-93-3P was highly expressed in gastric cancer cells and lowly expressed in normal gastric epithelial cells (the numbers in each bar interval represent the up-regulation ratio of the corresponding miRNA) (*P<0.05, ****P<0.0001, where the expression value of GES-1 was set to 1 and miR-16 was used as an internal reference).

[0039] From the above results, we can see that miR-192-5p, miR-4488, miR-127-3p, and miR-93-3p are highly expressed in gastric cancer cells, but lowly expressed in normal gastric epithelial cells. There are significant differences in their expression between tumor cells and normal cells, indicating that they have high diagnostic sensitivity and specificity for gastric cancer as diagnostic molecules for gastric cancer.

[0040] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0041] The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A marker for gastric cancer diagnosis and treatment target, characterized in that: The marker is miRNA, and the miRNA includes at least one of miR-192-5p, miR-4488, miR-127-3p, and miR-93-3p.

2. The marker according to claim 1, characterized in that The miRNA is derived from serum or plasma extracellular secretions.

3. Use of the marker for gastric cancer diagnosis and treatment target according to claim 1 or 2 in preparing a product for gastric cancer diagnosis and treatment target by the following method, characterized in that: The method comprises the following steps: Measuring the expression level of the marker according to claim 1 or 2 in the sample; and The diagnosis, treatment and prognosis of gastric cancer are evaluated based on the expression level of the marker.

4. A reagent for auxiliary diagnosis of gastric cancer, characterized in that: The reagent can detect the expression level of miRNA in a sample, and the miRNA includes at least one of miR-192-5p, miR-4488, miR-127-3p, and miR-93-3p.

5. A kit for auxiliary diagnosis of gastric cancer, characterized in that: The kit includes a reagent capable of detecting the expression level of miRNA in a sample, wherein the miRNA includes at least one of miR-192-5p, miR-4488, miR-127-3p, and miR-93-3p.

6. A chip for auxiliary diagnosis of gastric cancer, characterized in that: The chip includes a reagent capable of detecting the expression level of miRNA in a test sample, wherein the miRNA includes at least one of miR-192-5p, miR-4488, miR-127-3p, and miR-93-3p.

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