SYK as marker for diagnosing gastric cancer and preparation method of application of SYK

Abstract

According to the invention, a molecular interference sequence library G-003105-01 Human siGENOME siRNA Library-Tyrosine Kinase containing human tyrosine kinase is adopted for the first time, and a CCK-8 proliferation experiment is adopted to screen out a differential molecule SYK obviously related to proliferation from the molecular interference sequence library G-003105-01 Human siGENOME siRNA Library-Tyrosine Kinase. The invention provides application of SYK as a diagnostic marker for diagnosing gastric cancer. Only an immunohistochemical method is needed for detecting SYK in a tissue sample of a gastric cancer patient, operation is simple and easy, and clinical application and popularization feasibility is high; the SYK provided by the invention can be applied to gastric cancer diagnosis and rapid judgment of curative effect. The invention provides application of SYK in preparation of a diagnostic reagent or kit for diagnosing gastric cancer. The invention provides application of SYK protein in preparation of a reagent or a kit for prognosis of a gastric cancer patient. The invention also provides a marker for detecting SYK as a gastric cancer specific reagent, a chip, a probe, a primer and a kit. The invention also provides an SYK-related metabolic signaling pathway as a target of a gastric cancer treatment drug.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biomedicine, and in particular to the field of gastric cancer detection systems. Background Art

[0002] Gastric cancer is one of the most common malignant tumors in the world. Early gastric cancer lacks specific signs. When most patients seek medical treatment, the lesions have progressed to the local advanced stage or have already metastasized to distant sites, and they have lost the opportunity for surgery, with an overall poor prognosis. Therefore, the treatment of pre-progressive gastric cancer has also become a hot topic and difficulty in research. In recent years, targeted drugs have gradually become an important part of clinical tumor treatment due to their high selectivity for cancer cells and their low toxicity and high efficiency. However, due to the very complex molecular mechanisms of gastric cancer occurrence and development, only a few drugs such as trastuzumab are currently effective in clinical practice. Drug resistance is prone to occur after first-line chemotherapy, which also limits the use of single targeted drugs. The rational combination of multiple mechanism chemotherapy and targeted drugs is the key to improving treatment effects and prolonging patient survival, and it is also a future research direction.

[0003] Tyrosine kinase (TK) is a transmembrane protein on the cell surface. It plays a key role in the transduction of extracellular signals into cells. Tyrosine kinase receptors play an important role in various processes of cell growth, movement, differentiation and metabolism. Therefore, tyrosine kinase inhibitors (TKIs) therapy will hopefully become a new means of tumor treatment. However, although TKIs can significantly relieve symptoms, most of them will develop drug resistance after a period of time, affecting clinical efficacy. The main reason is that they are not specific. Because most protein kinases belong to a superfamily and are structurally similar to each other, TKIs can inhibit the target protein kinase while affecting the activity of other protein kinases, which can cause unpredictable consequences. In recent years, with the successful synthesis of highly efficient and specific TKIs, the problem of low specificity has been solved to a certain extent, and protein kinase inhibitor therapy will hopefully become a new means of tumor treatment. The US FDA has approved many monoclonal antibodies that interfere with RTK activation, including cetuximab for lung cancer, panitumumab for colon cancer, and pertuzumab for breast cancer. Currently, only patients with positive HER-2 gene in the RTKs family can prolong their survival through chemotherapy combined with targeted therapy. However, not all gastric cancer patients can benefit from trastuzumab, because the HER-2 positive rate in patients with advanced gastric cancer is less than 1 / 4, and this proportion of patients in my country may be even lower. The significant effect of HER-2 inhibitors is often temporary, and most patients develop acquired drug resistance after one year of use. Therefore, it is necessary to further explore new therapeutic targets for gastric cancer.

[0004] siRNA library is a collection of multiple single-gene siRNAs, ranging from a gene family sublibrary of dozens of genes to a whole genome library of tens of thousands of genes. Most libraries are provided in the form of 96-well plates, with each well corresponding to one gene, and the number of 96-well plates obtained varies depending on the number of genes covered by the library. siRNA library is a powerful tool for high-throughput screening of functional genes / targets using RNAi technology. Its applications include screening drug targets, screening genes associated with drug effects after administration, screening functional genes related to specific cell phenotypes or disease phenotypes, and screening genes related to pathogen spread and host response. The ON-TARGET plus siRNA library purchased by the Activity Screening Platform of the Pharmaceutical Technology Center has been reported in many literatures and is mostly used to screen key signaling molecules.

[0005] The sensitivity and specificity of existing biomarkers for gastric cancer are both low; there are no mature test kit molecules for the diagnosis and efficacy assessment of gastric cancer. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the present invention provides an application of a gastric cancer marker, wherein the gastric cancer marker is spleen tyrosine kinase, namely SYK. The present invention uses SYK as a marker for specific reagents, chips, probes, primers, and kits for detecting gastric cancer.

[0007] According to one aspect of the present invention, there is provided an application of a gastric cancer marker, namely, the use of the above-mentioned SYK in the preparation of a diagnostic reagent or a kit for diagnosing gastric cancer.

[0008] According to one aspect of the present invention, a method for screening gastric cancer markers is provided. The method uses the molecular interference sequence library G-003105-01 Human siGENOME siRNA Library-Tyrosine Kinases and adopts the CellTiter-Blue assay proliferation experiment to screen out differential molecules significantly related to proliferation.

[0009] According to one aspect of the present invention, a method for screening gastric cancer markers is provided. The method uses CCK-8 proliferation assay to measure OD values ​​and calculate respective interference efficiencies after interference culture for 72 hours in four gastric cancer cell lines SGC7901, MKN-45, AGS and MGC803; and screens out molecules such as SYK that significantly affect cell proliferation.

[0010] According to one aspect of the present invention, a method for screening gastric cancer markers is provided, the method further comprising immunohistochemical analysis of the expression of the differential molecules in human gastric cancer tissue chips; construction of an in vitro transcription vector; reverse transcription and qPCR quantitative detection of the differential molecules; and bioinformatics analysis.

[0011] According to one aspect of the present invention, a gastric cancer detection system is provided. The system is any one of a specific reagent, chip, probe, primer or kit for detecting gastric cancer. The system is a system for specifically detecting SYK expression.

[0012] According to one aspect of the present invention, a gastric cancer detection system is provided, wherein the system is a SYK immunohistochemistry analysis system.

[0013] According to one aspect of the present invention, a gastric cancer detection system is provided. The above-mentioned SYK immunohistochemical analysis system uses immunohistochemical analysis of human gastric cancer tissue chips.

[0014] According to one aspect of the present invention, a gastric cancer detection system is provided, comprising a forward primer SEQ ID No. 1: 5'-GGGAGGAAGGCACACCACTA-3' and a reverse primer SEQ ID No. 2: 5'-GACCAGGCCATCAGACTCCT-3' of the target molecule SYK-qPCR.

[0015] According to one aspect of the present invention, a gastric cancer detection system is provided, the system further comprising:

[0016] Forward primer SEQ ID No.3 of target molecule SYK-siRNA#1:

[0017] 5'-GGAUCAAAGACAAAUGGAAdTdT-3';

[0018] Reverse primer SEQ ID No. 4: 5′-UUCCAUUUGUCUUUGAUCCdTdT-3′;

[0019] Forward primer SEQ ID No.5 of target molecule SYK-siRNA#2:

[0020] 5'-GGUUUGUUAAGAGUUCUUAdTdT-3';

[0021] Reverse primer SEQ ID No. 6: 5'-UAAGAACUCUUAACAAACCdTdT-3'.

[0022] According to one aspect of the present invention, there is provided a use of a SYK-related metabolic signaling pathway of a gastric cancer marker in the pharmaceutical field, wherein the SYK-related metabolic signaling pathway is used as a target for gastric cancer therapeutic drugs.

[0023] The inventors of the present invention first screened out SYK, a differential molecule significantly associated with proliferation, from a molecular interference sequence library G-003105-01Human siGENOME siRNA Library-Tyrosine Kinases containing human tyrosine kinases by using CellTiter-Blue assay proliferation experiment. The screening method for gastric cancer diagnosis of the present invention has not been reported in relevant literature or patent applications at home and abroad. The present invention provides the use of SYK as a diagnostic marker for diagnosing gastric cancer. In the technology of the present invention, the detection of SYK in tissue samples of gastric cancer patients only requires immunohistochemistry, which is simple and easy to operate, and has great feasibility for clinical application and promotion; the present invention requires that SYK can be used for gastric cancer diagnosis and rapid judgment of efficacy. The present invention provides the use of SYK in the preparation of diagnostic reagents or kits for diagnosing gastric cancer. The present invention provides the use of SYK protein in the preparation of reagents or kits for prognosis of gastric cancer patients. The present invention also provides a marker to be detected by detecting SYK as a gastric cancer-specific reagent, chip, probe, primer, and kit. The present invention also provides a SYK-related metabolic signaling pathway as a target for gastric cancer therapeutic drugs.

[0024] The Human siGENOME Tyrosine Kinase library used in the present invention, namely G-003105-01HumansiGENOME siRNA Library-Tyrosine Kinases, is a subset of molecular interference sequences containing human tyrosine kinases, containing siRNA sequences of 85 genes, the first 96-well plate is provided with siRNAs of 80 genes, the second 96-well plate is provided with siRNAs of 5 genes, the array is arranged in the second column to the 11th column, the first column and the 12th column are empty, and there are 2 96-well plates in total. The corresponding controls can be set according to the experimental needs. The inventors used CellTiter-Blue assay proliferation experiments in four gastric cancer cell lines SGC7901, MKN-45, AGS and MGC803 and a normal gastric cell line GES-1 in G-003105-01Human siGENOME siRNA Library-Tyrosine Kinases after 72 hours of interference culture, calculated the interference efficiency of each, screened out the differential molecule SYK that was significantly related to proliferation, screened new tyrosine kinase receptor sites in gastric cancer, found potential targets, and solved the problems of resistance to currently known classic tyrosine kinase inhibitors and poor effect of single targeted drugs.

[0025] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 . This is an example of the present invention using the Human siGENOME Tyrosine Kinase library, using the CCK-8 kit to perform proliferation detection on the library, and the initial screening analysis of the tyrosine receptor family molecule SYK and proliferation correlation diagram;

[0027] Figure 2 . It is a diagram of the analysis of SYK expression in gastric cancer and adjacent tissues using human gastric cancer tissue chips and immunohistochemistry kits in the embodiments of the present invention; Figure A is a diagram of two typical expression staining diagrams of 81 gastric cancer and adjacent tissues, and Figure BC is a comparative analysis diagram of SYK expression in 81 gastric cancer and adjacent tissues;

[0028] Figure 3 . is a graph showing the OS curve analysis of SYK diagnosis of gastric cancer in the embodiment of the present invention;

[0029] Figure 4 . are expression diagrams of SYK at the mRNA and protein levels in gastric cancer cells according to the embodiments of the present invention, FIG. A is a diagram showing the expression of SYK at the mRNA level in gastric cancer cell lines, FIG. B is a diagram showing the expression of SYK at the protein level in gastric cancer cell lines, FIG. C and FIG. D are diagrams showing the identification of SYK overexpression efficiency, and FIG. E is a diagram showing the identification of SYK siRNA interference efficiency; FIG. FH are diagrams showing the detection of SYK proliferation efficiency in the overexpression group and the control group, and the interference group and the control group;

[0030] Figure 5 . is a volcano diagram of differentially expressed genes after SYK sequencing in Example 1 of the present invention.

[0031] Figure 6 . is a diagram showing GO analysis after SYK sequencing in the embodiment of the present invention;

[0032] Figure 7 . is a diagram showing KEGG analysis after SYK sequencing in the embodiment of the present invention;

[0033] Figure 8 . is a GO analysis diagram based on GSEA analysis after SYK sequencing in the embodiment of the present invention;

[0034] Fig. 9 . is a KEGG analysis diagram based on GSEA analysis after SYK sequencing in the embodiment of the present invention;

[0035] Fig.10 . is a diagram showing the overall differential gene PPI after SYK sequencing in the embodiment of the present invention;

[0036] Fig.11. is a diagram showing the PPI of the top 10 genes after SYK sequencing in the embodiment of the present invention;

[0037] Fig.12 . is a diagram showing the screening of 132 differentially expressed genes by LASSO regression analysis after SYK sequencing in the embodiment of the present invention;

[0038] Fig.13 . is a diagram of the core key molecules of LASSO regression analysis after SYK sequencing in the embodiment of the present invention. DETAILED DESCRIPTION

[0039] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The above-mentioned accompanying drawings are only an overview of the technical solution of the present invention, in order to enable a clearer understanding of the technical means of the present invention, and can be implemented according to the contents of the specification. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0040] Example 1 Human siGENOME Tyrosine Kinase library was used to screen human tyrosine kinase and receptor molecules in gastric cancer cell lines.

[0041] The inventors used the Human siGENOME Tyrosine Kinase library to interfere with the culture of four gastric cancer cell lines, SGC7901, MKN-45, AGS and MGC803 for 72 hours, and then used CCK-8 proliferation assay to measure the OD value and calculate the interference efficiency of each. Based on the interference effects of the four groups of cell lines, we screened out the tyrosine kinase molecule SYK ( Figure 1 ).

[0042] The specific operation steps are: SGC7901, MKN-45, AGS, and MGC803 of the four gastric cancer cells growing in the logarithmic phase were planted in a 96-well plate, and a blank control was set up. The control group was cultured in conventional DMEM+10% FBS medium. The experimental group was added with si interfering small molecules of the siGENOME Tyrosine Kinase library. The premixed interference library was added according to the experimental instructions of the interference library company. After 48 hours and 72 hours of interference, the CCK-8 method was used to observe the proliferation of gastric cancer cells. The specific operation steps refer to the instructions of the CCK-8 kit. The preliminary experiment was repeated three times, and molecules such as SYK that significantly affected cell proliferation were screened for the next experiment.

[0043] 1.1 siRNA library presentation format:

[0044] 1) Each 96-well plate is equipped with siRNAs for 80 genes, arranged in the 2nd to 11th columns

[0045] 2) Columns 1 and 12 are empty. You can set corresponding controls according to experimental needs.

[0046] 1.2 siRNA library dissolution and dilution steps

[0047] Table 1. siRNA library dissolution and dilution method

[0048]

[0049] 1) Use a dispenser to add RNase-free water directly to the 96-well plate and dilute to the concentration of the stock solution.

[0050] 2) Divide into multiple 96-well plates according to experimental requirements to avoid repeated freezing and thawing

[0051] 3) Dosage: 0.1 nmol, stock solution dilution concentration 2uM, final concentration 25nM: 96-well plate screening (100ul / well), each screening needs 3 replicates, a total of about 4ul is required.

[0052] Example 2 Immunohistochemical analysis of SYK expression in human gastric cancer tissue microarray

[0053] Tissue chip technology, also known as tissue microarray (TMA), is a new molecular biology technology based on morphology developed in recent years. It is a high-throughput, multi-sample analysis tool. It is a high-throughput tissue section made by arranging dozens or even thousands of tiny tissue slices neatly on a glass slide to form a microarray, and hybridizes nucleic acid probes or antibody probes that mark specific genes to detect the expression of the gene in different tissues. Therefore, tissue chip is an integration of traditional nucleic acid in situ hybridization or immunohistochemistry experiments. Immunohistochemistry detects the expression of a gene in one tissue at a time, while tissue chip detects the expression of a gene in multiple tissues at a time. It has the advantages of high throughput, large sample, time-saving and fast. The present invention uses 81 pairs of gastric cancer and paracancerous tissue chips to carry out SYK immunohistochemical analysis.

[0054] 2.1 Immunohistochemical staining experimental steps:

[0055] 1) Bake the slides at 60℃ for about 60 minutes before dewaxing;

[0056] 2) Xylene dewaxing, three cylinders, 15-30 minutes per cylinder (appropriately extended in winter);

[0057] 3) Select the appropriate repair method for tissue antigens according to the requirements of each antibody;

[0058] 4) Block endogenous peroxidase with 3% H202-methanol at room temperature for 10 minutes, and wash with PBS for 5 minutes × 3 times;

[0059] 5) Add primary antibody (SYK 1:1000) and store in 4℃ refrigerator overnight;

[0060] 6) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0061] 7) Add secondary antibody (biotin-labeled goat anti-mouse / rabbit IgG) and incubate at room temperature for 10 minutes;

[0062] 8) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0063] 9) Add streptavidin-peroxidase and incubate at room temperature for 10 minutes;

[0064] 10) The DAB\AEC color development time was controlled under a microscope and the color development was terminated by washing with distilled water;

[0065] 11) Re-stain with hematoxylin, wash with water, and rinse thoroughly after differentiation. Dehydrate and make transparent in the usual way, and seal the slides with neutral gum.

[0066] Among them, step 3) repair method - antigen heat repair:

[0067] A) High temperature and high pressure thermal repair

[0068] Take a certain amount of 0.01M citrate buffer solution, pH 6.0, and heat it to boiling over high heat in a pressure cooker. Place the dewaxed and hydrated tissue chip on a high-temperature resistant plastic slice rack and put it into the boiling buffer solution. Cover the pot with a lid, close the pressure valve, and continue heating until the jet is ejected. After 1-2 minutes from the start of the jet, the pressure cooker is removed from the heat source and cooled to room temperature. Take out the slide, wash it twice with distilled water, and then wash it with PBS for 5 minutes.

[0069] B) Microwave thermal repair

[0070] Take a certain amount of 0.01M citrate buffer solution, pH 6.0, and heat it in a microwave box until it boils. Place the dewaxed and hydrated tissue chip on a high-temperature resistant plastic slice rack and put it into the boiling buffer solution. Continue to process it at high temperature for 10-15 minutes. Take it out of the microwave box and cool it to room temperature. Take out the glass slide and wash it twice with distilled water, and then wash it with PBS for 5 minutes.

[0071] 2.2 Interpretation of immunohistochemistry results

[0072] Positive staining cell counting method: Under the microscope, randomly select several non-overlapping fields of view, and count the positive staining cells manually or by machine. Gray density analysis method: Select the same area on different groups and different animal tissue sections, use image j to perform gray density analysis under the same conditions, and then perform statistical analysis. Scoring method: Under an optical microscope, the tissue sections are scored according to the degree of staining (0-3 points for negative staining, light yellow, light brown, dark brown), and the positive range (1-4 points for 0-25%, 26-50%, 51-75%, 76-100%). Finally, the scores can be added and compared. Image J software can be used to measure the average optical density value. By measuring the integrated option density (IOD) and area value of each picture, the average optical density value (mean density) is calculated, which reflects the unit area concentration of the target protein. Finally, the average value of the mean density of 5 random areas of each sample is taken as the value of this sample.

[0073] The immunohistochemistry of gastric cancer tissue chips was used to analyze the changes in gastric cancer expression and the differences in clinical pathological parameters among groups. Graphpad prism 5.0 software and student's t-test (two groups) or one-way ANOVA (three groups) statistical analysis software were used to analyze the data. The count data were summarized using frequency and percentage, and the measurement data were summarized using mean, standard deviation, median, minimum and maximum values ​​for descriptive statistics. The χ2 test or Fish's exact probability method was used to test the differences in PDC grading among people with different clinical pathological characteristics. P < 0.05 was considered statistically significant.

[0074] The results showed that SYK was found to be lower in gastric cancer than in adjacent tissues in 81 pairs of gastric cancer and adjacent tissues. Figure 2 ). Analysis of clinicopathological parameters of gastric cancer revealed that SYK was associated with TNM stage of gastric cancer, tumor visceral mucosal infiltration, and Ki-67 expression (Table 2).

[0075] Table 2 Analysis of clinical pathological parameters of SYK in the diagnosis of gastric cancer

[0076]

[0077] Kaplan-Meier analysis was used to analyze its relationship with clinical prognosis ( Figure 3 ), P<0.05 was considered statistically significant. It was found that SYK was associated with the prognosis of gastric cancer. The prognosis of gastric cancer patients with reduced SYK expression was poor, and vice versa.

[0078] The COX univariate and multivariate analysis (Table 3) revealed that SYK was lowly expressed in gastric cancer. However, SYK could not be used as an independent prognostic factor, but it could assist as an indicator of gastric cancer prognosis. The presence or absence of lymph node metastasis was an independent prognostic factor for gastric cancer.

[0079] Table 3. Univariate and multivariate analysis of SYK in the diagnosis of gastric cancer

[0080]

[0081] Example 3 Construction of in vitro transcription vector

[0082] like Figure 6 As shown: The inventors have successfully constructed a SYK stably overexpressing gastric cancer cell line (MNK45-SYK) and a control group cell line in the early stage, and found that SYK can inhibit the proliferation ability of gastric cancer cells.

[0083] Total RNA extraction

[0084] 3.1 Collect cells by centrifugation, add 1 ml TRIzol for every 5-10×106 cells, and pipette repeatedly. Do not wash cells before adding TRIzol to avoid mRNA degradation.

[0085] 3.2 Place the sample at room temperature (15-30°C) for 5 minutes to allow the nucleic acid-protein complex to completely separate.

[0086] 3.3 Add 0.2 ml of chloroform to every 1 ml of TRIzol, shake vigorously for 15 seconds, and leave at room temperature for 3 minutes.

[0087] 3.4 Centrifuge at 2-8℃, 10,000×g for 15 minutes. The sample is divided into three layers: the bottom layer is a yellow organic phase, the upper layer is a colorless aqueous phase, and an intermediate layer. RNA is mainly in the aqueous phase, and the volume of the aqueous phase is about 60% of the TRIzol reagent used.

[0088] 3.5 Transfer the aqueous phase to a new tube. If you want to separate DNA and protein, keep the organic phase. See below for further operations. Use isopropanol to precipitate RNA in the aqueous phase. Add 0.5 ml of isopropanol for every 1 ml of TRIzol used and leave at room temperature for 10 minutes.

[0089] 3.6 Centrifuge at 2-8℃, 10,000×g for 10 minutes. No RNA precipitate can be seen before centrifugation, but a gelatinous precipitate appears on the side and bottom of the tube after centrifugation. Remove the supernatant.

[0090] 3.7 Wash the RNA pellet with 75% ethanol. For every 1 ml of TRIzol used, add at least 1 ml of 75% ethanol. Centrifuge at no more than 7500 × g at 2-8°C for 5 minutes and discard the supernatant.

[0091] 3.8 Leave to dry at room temperature or vacuum dry the RNA precipitate for about 5-10 minutes. Do not vacuum centrifuge to dry, as over-drying will greatly reduce the solubility of RNA. Add 25-200μl RNase-free water or 0.5% SDS, pipette several times, and place at 55-60℃ for 10 minutes to dissolve the RNA.

[0092] Example 4 Reverse transcription and SYK qPCR quantitative detection

[0093] Using PrimeScript TM The Master Mix Kit was used to reverse transcribe RNA into cDNA. β-actin was used as an internal reference and TBGreen TM Premix Ex Taq TM II kit dye method qPCR detection of gastric cancer cell line target SYK molecule relative expression level. 2-ΔΔCt method was used to analyze the relative expression of target SYK molecule in gastric cancer cell line and normal control cell line, and it was found that it was relatively low in MNK45 and SGC7901, and relatively high in AGS ( Figure 4 A), Western blot detection of SYK protein expression level in gastric cancer cell lines ( Figure 4 B) Construction of SYK overexpressing cell line MNK45-SYK in gastric cancer cell line with relatively low SYK expression ( Figure 4 C),SGC7901-SYK( Figure 4 D), and detect its overexpression efficiency. At the same time, SYK interference cell line SYK-siRNA ( Figure 4 E), and detect its interference efficiency. CCK-8 was used to detect SYK in the overexpression and control groups, as well as between the interference and control groups and the proliferation-related factors were analyzed. The differences between the groups were statistically analyzed using Graphpad prism5.0 software student's t-test (two groups) or one-way ANOVA (three groups). It was found that SYK overexpression in cell lines MNK45 and SGC7901 can inhibit the proliferation function of gastric cancer cells ( Figure 4 F, G), Interference of SYK in gastric cancer cell line AGS can promote the proliferation of gastric cancer cells compared with the control group ( Figure 4 H).

[0094] Table 4. Primer sequences for specific detection of SYK

[0095]

[0096] Example 5 Bioinformatics Analysis

[0097] MNK45-SYK and control group cells were selected, total RNA was extracted, and sent to a sequencing company for RNAseq sequencing. A volcano map of 132 differentially expressed genes was drawn ( Figure 5 ) and conduct subsequent gene GO and KEGG pathway analysis. Figure 6 ) analysis was enriched in glucuronyl transferase activity, xenobiotic glucuronidation, flavonoid metabolism, retinoic acid binding and blood microparticles. KEGG( Figure 7 ) analysis was enriched in cofactor biosynthesis, bile secretion, interconversion of pentose and glucuronate, ascorbic acid and aldehyde salt metabolism, chemical carcinogenesis-DNA adducts, chemical carcinogenesis-receptor activation, and retinol metabolism. It was found that SYK may be involved in the above metabolic pathways.

[0098] Gene Set Enrichment Analysis (GSEA) is used to evaluate the distribution trend of genes in a predefined gene set in a gene table ranked by phenotype relevance, so as to determine their contribution to the phenotype. GSEA can also be used to find out which gene sets of specific biological significance are significantly associated with two groups of samples in terms of expression or other measurement levels, or to find out which gene sets' expression patterns or other patterns are closer to phenotype A and which are closer to phenotype B. In the present invention, MNK45-SYK and control group cells were selected for RNAseq sequencing, and the results were analyzed by GSEA. Through GSEA analysis, we found that among the top 10 of GO ( Figure 8 ) Both of them play a role in metabolic pathways such as ribosomal subunit synthesis, cytoplasmic transformation, mitochondria-protein complex, glucuronyl transferase activity, and uronic acid metabolism. The top 10 of KEGG ( Fig. 9 ) were found in the metabolic processes of ascorbic acid and ascorbic acid, porphyrin metabolism, retinol metabolism, pentose glucuronide, steroid hormone synthesis, drug metabolism, bile secretion, P450 cytochrome metabolism, etc. SYK was found to be involved in the above metabolic processes. This will help to use SYK as a targeted metabolic pathway for the treatment of gastric cancer in the future.

[0099] In order to explore the interaction relationship between genes, the present invention selected MNK45-SYK and control group cells for RNAseq sequencing, and the results were analyzed by using Cytoscape software and String database to establish a protein-protein interaction network (PPI) analysis between differentially expressed genes. Fig.10 ) and the top 10 protein interactions ( Fig.11) found that the main concentrations were UGT1A1, UGT1A3, UGT1A9, UGT1A6, UGT1A7, UGT1A4, UGT1A10, UGT1A8, CASP1 and SAMD9L 10 molecules, mainly reflected in the uridine diphosphate glucuronosyltransferase family (UGT1A). UGT1A is an important phase II metabolic enzyme in the human body, which plays an important role in metabolizing drugs and endogenous substances. It is widely distributed in various tissues and organs such as liver, kidney, respiratory tract, lung, stomach, small intestine, colon, etc. Most UGT enzymes are expressed in the liver. Analysis found that uridine diphosphate glucuronosyltransferase 1 (UGT1A1) was the most enriched protein, indicating that SYK may participate in the material metabolism process by regulating UGT1A1.

[0100] In order to explore the core factors among the numerous genes after SYK sequencing, the present invention selected MNK45-SYK and control group cells for RNAseq sequencing, and the results were subjected to LASSO regression analysis using EasyR software. 132 differential gene factors were included in the LASSO regression analysis ( Fig.12 ), and 6 molecules were found to be important predictive factors related to the SYK pathway ( Fig.13 ). These six core molecules, namely GGT1, FAM110C, STAP2, ANO2, KLRC2 and ASTN1, are significant predictors.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An application of a gastric cancer marker, characterized in that: The gastric cancer marker is spleen tyrosine kinase, namely SYK, and the SYK is used as a marker for specific reagents, chips, probes, primers, and kits for detecting gastric cancer.

2. The use of a gastric cancer marker according to claim 1, characterized in that: The use of SYK in preparing a diagnostic reagent or a kit for diagnosing gastric cancer.

3. A method for screening gastric cancer markers, characterized in that: The method uses the molecular interference sequence library G-003105-01 Human siGENOME siRNA Library-Tyrosine Kinases and uses the CellTiter-Blueassay proliferation experiment to screen out differential molecules that are significantly related to proliferation; The method is to perform interference culture in four gastric cancer cell lines, SGC7901, MKN-45, AGS and MGC803, for 72 hours, respectively, using CCK-8 proliferation assay to measure OD values, calculate respective interference efficiencies, and screen out molecules that significantly affect cell proliferation.

4. The method for screening gastric cancer markers according to claim 3, characterized in that: The method also includes immunohistochemical analysis of the expression of the differential molecules in human gastric cancer tissue chips; construction of an in vitro transcription vector; reverse transcription and qPCR quantitative detection of the differential molecules; and bioinformatics analysis.

5. A gastric cancer detection system, characterized in that: The system is any one of a specific reagent, chip, probe, primer or kit for detecting gastric cancer, and the system specifically detects SYK expression.

6. A gastric cancer detection system according to claim 5, characterized in that: The system is a SYK immunohistochemical analysis system.

7. A gastric cancer detection system according to claim 6, characterized in that: The SYK immunohistochemical analysis system uses immunohistochemical analysis of human gastric cancer tissue chips.

8. A gastric cancer detection system according to claim 5, characterized in that: The system comprises: The forward primer of the target molecule SYK-qPCR is SEQ ID No. 1: 5'-GGGAGGAAGGCACACCACT A-3'; the reverse primer is SEQ ID No. 2: 5'-GACCAGGCCATCAGACTCCT-3'.

9. A gastric cancer detection system according to claim 8, characterized in that: The system comprises: Target molecule: Forward primer SEQ ID No.3 of SYK-siRNA#1: 5'-GGAUCAAAGACAAAUG GAAdTdT-3'; Reverse primer SEQ ID No.4: 5'-UUCCAUUUGUCUUUGAUCCdTdT-3'; The forward primer of the target molecule SYK-siRNA#2 is SEQ ID No.5: 5'-GGUUUGUUAAGAGUUCU UAdTdT-3'; the reverse primer is SEQ ID No.6: 5'-UAAGAACUCUUAACAAACCdTdT-3'.

10. Use of the SYK-related metabolic signaling pathway of gastric cancer markers in the pharmaceutical field, characterized in that: The SYK-related metabolic signaling pathway is used as a target for gastric cancer therapeutic drugs.

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