Application of amino acid metabolism related genome in diagnosis of helicobacter pylori related gastric cancer

Abstract

The invention relates to the technical field of medical biological detection, provides application of an amino acid metabolism related genome in diagnosis of helicobacter pylori related gastric cancer, and provides application of the amino acid metabolism related genome in preparation of a helicobacter pylori related gastric cancer detection reagent or detection kit. The amino acid metabolism related genome is composed of ten genes, namely, SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CELA3A, DPP4 and MEP1A, and is characterized in that the amino acid metabolism related genome is composed of ten genes, namely SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11 The Hp infection increases the transport metabolism of gastric cancer cell amino acids by up-regulating the expression of amino acid metabolism related genes, thereby promoting the gastric cancer progress. Technical support and new exploration are provided for further understanding of biological functions and treatment in helicobacter pylori related gastric cancer lesions.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biological detection, and particularly to the application of amino acid metabolism-related gene sets in diagnosing Helicobacter pylori-related gastric cancer. Background Art

[0002] Helicobacter pylori (Hp) is the most common pathogen in the stomach. Among the Chinese population, the Hp infection rate in patients with non-cardia gastric cancer is 94.4%, in patients with cardiac cancer is 92.2%, and in the general population is 75.6%. After Hp infection, the risk of non-cardia gastric cancer increases by 5.94 times, and the risk of cardiac cancer increases by 3.06 times. [1] The study found that Hp infection is associated with diffuse lesions of gastric cancer, poorer differentiation, and later TNM stage, and promotes gastric cancer invasion and metastasis and induces cisplatin resistance; compared with gastric cancer patients without infection or mild infection, the 10-year progression-free survival rate and overall survival rate of patients with moderate to severe Hp infection were significantly decreased (55.6% vs. 26.3%). [2-4] .

[0003] A large amount of evidence shows that long-term infection with Hp can affect the stability of the cell genome through mechanisms such as regulating epigenetic modification, changing oncogene expression, inducing inflammatory response, and regulating metabolic pathways under the action of virulence proteins such as CagA, thereby stimulating cell proliferation, reducing epithelial cell apoptosis, inhibiting T cell development, etc., and ultimately promoting the malignant transformation of gastric epithelial cells and the malignant progression of gastric cancer. [5] However, although the role of Hp in promoting the progression of gastric cancer has been preliminarily confirmed, the molecular mechanism has not yet been fully elucidated. Therefore, exploring the mechanism of Hp in regulating the malignant process of gastric cancer and finding potential intervention targets have important biological significance and clinical social value.

[0004] The occurrence and development of tumors is the result of the long-term interaction of multiple factors. With the deepening of basic research such as genomics, the connection between carcinogenic signaling pathways and metabolic pathways has become increasingly prominent, and "disordered cellular energy metabolism" is also considered the seventh characteristic of tumors. [6] Metabolic reprogramming of tumors not only meets the increased nutritional needs of malignant cells, but also regulates many oncogenic pathways during tumor development, leading to the malignant progression of cells. [7] Studies have found that Hp infection can increase gastric cancer cell glycolysis and promote tumor cell proliferation by activating the Wnt / β-Catenin pathway, inhibiting IL-10, and upregulating pyruvate kinase PKM2 expression through the Erk pathway. [8-10]In the gastric tissue of Hp-infected mice, an increase in the abundance of 16 specific lipids, such as phosphatidylcholine and sphingomyelin, was observed; a meta-analysis of 24 independent experiments showed that after Hp radical cure, the levels of high-density lipoprotein cholesterol and triglycerides in patients increased significantly, suggesting that Hp has a regulatory effect on cellular lipid production and lipid transport. [11,12] Hp can also promote the degradation of host cell glutathione, leading to oxidative damage caused by ROS accumulation, thus causing gastric ulcers and gastric cancer.

[13] Although previous studies have found that Hp affects the host's systemic or local metabolism, the differential expression profiles of metabolites and changes in metabolic pathways have not yet been elucidated.

[0005] Therefore, it is of great significance to find the differential expression profile of metabolites and amino acid metabolism-related genes in Helicobacter pylori-related gastric cancer and further understand their biological functions in Helicobacter pylori-related gastric cancer. Currently, there is no literature report on related research. Summary of the invention

[0006] The present invention is based on the above research and aims to provide a target marker for Helicobacter pylori-related gastric cancer and also to provide the use of amino acid metabolism-related genes in the preparation of a Helicobacter pylori-related gastric cancer detection kit.

[0007] The research process of the present invention is as follows: Through non-targeted metabolomics detection and cell experiments, it was found that Hp infection regulates the reprogramming of amino acid metabolism in gastric cancer cells and promotes the progression of gastric cancer. Combined with whole transcriptome sequencing and gene re-annotation analysis, a group of amino acid metabolism-related genes regulated by Hp infection (SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CELA3A, DPP4, MEP1A) were screened, and their expression in gastric cancer was detected. According to the above research results, the differential expression spectrum of metabolites in Hp-related gastric cancer was clarified, and it was clarified that Hp infection increases the transport metabolism of amino acids in gastric cancer cells by upregulating the expression of genes related to amino acid metabolism, thereby promoting the progression of gastric cancer.

[0008] The first aspect of the present invention provides the use of an amino acid metabolism-related gene set in the preparation of a Helicobacter pylori-related gastric cancer detection reagent or a detection kit, wherein the amino acid metabolism-related gene set consists of ten genes: SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CELA3A, DPP4, and MEP1A.

[0009] This project conducted whole transcriptome sequencing and gene re-annotation analysis on Hp(+) / (-) AGS, and found 1275 mRNAs upregulated in Hp(+) samples and 1878 mRNAs downregulated in Hp(+) samples (difference fold>2.0, p<0.05) ( Figure 2 A), these differentially expressed genes are enriched in the protein digestion and absorption pathway, mainly acting on amino acid transmembrane transport ( Figure 2 B and C). Based on the above analysis results, the enriched differentially expressed genes mainly include: SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CPB1, CELA3A, DPP4, MEP1A. The expression of the above genes in Hp(+) samples increased to varying degrees. The ten genes were defined as amino acid metabolism-related genes (AAMRGs).

[0010] Preferably, the detection reagent is a reagent for detecting the expression levels of ten genes in a biological sample, and the detection kit comprises a reagent for detecting the expression levels of ten genes in a biological sample.

[0011] Further preferably, the reagent for detecting the expression levels of ten genes in a biological sample is selected from any one of the following:

[0012] (1) PCR primers with detection specificity for ten genes;

[0013] (2) Antibodies with detection specificity for the proteins corresponding to the ten genes.

[0014] In a preferred embodiment of the present invention, the primer sequences are as follows:

[0015]

[0016] The antibodies with detection specificity for the proteins corresponding to the ten genes are as follows: SLC1A5 antibody: 20350-1-AP, SLC3A1 antibody: 16343-1-AP, SLC6A19 antibody: 27575-1-AP, SLC7A9 antibody: RD81114A, SLC7A11 antibody: 26864-1-AP, SLC16A2 antibody: 20676-1-AP, SLC38A1 antibody: 28632-1-AP, CELA3A antibody: HPA028086, DPP4 antibody: 29403-1-AP, MEP1A antibody: 24640-1-AP.

[0017] Preferably, the biological sample is obtained from gastric cancer tissue of the subject, and RNA or protein is extracted from the tissue or cells for detection.

[0018] In a second aspect of the present invention, a Helicobacter pylori-related gastric cancer detection kit is provided, which comprises reagents for detecting the contents of SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CELA3A, DPP4, and MEP1A in biological samples.

[0019] Preferably, the diagnostic kit is composed of a reverse transcription system, a primer system and an amplification system, and the primer system includes the PCR primers shown in SEQ ID NOs. 1 to 22 above.

[0020] The diagnostic kit consists of an immunoblotting detection reagent and an antibody system, and the antibody system is composed of: β-actin: AF0003, SLC1A5 antibody: 20350-1-AP, SLC3A1 antibody: 16343-1-AP, SLC6A19 antibody: 27575-1-AP, SLC7A9 antibody: RD81114A, SLC7A11 antibody: 26864-1-AP, SLC16A2 antibody: 20676-1-AP, SLC38A1 antibody: 28632-1-AP, CELA3A antibody: HPA028086, DPP4 antibody: 29403-1-AP, MEP1A antibody: 24640-1-AP.

[0021] When tested, the titer of AF0003 was 1:1000, the titer of RD81114A was 1:2000, and the titers of the remaining antibodies were all 1:5000.

[0022] The third aspect of the present invention provides a method for detecting Helicobacter pylori-related gastric cancer using the above diagnostic kit, as follows:

[0023] Method 1:

[0024] A. Extract total RNA from cells or tissues;

[0025] B. Reverse transcription of the total RNA in step A to obtain cDNA;

[0026] C. qRT-PCR technology was used to quantitatively detect the copy numbers of ten genes, including SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CELA3A, DPP4, and MEP1A. The primers used in the detection process are shown in SEQ ID NOs. 1 to 22.

[0027] Method 2:

[0028] A. Extract cell proteins from cells or tissues, and further extract serum proteins;

[0029] B. The expression levels of ten proteins, SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CELA3A, DPP4, and MEP1A, were detected by immunoblotting technology. The antibodies used in the detection process are shown above.

[0030] Bioinformatics analysis showed that the mRNA and protein levels of AAMRGs increased after Hp infection, and can be used for the detection or treatment of Helicobacter pylori-related gastric cancer.

[0031] The beneficial protection and effects of the present invention are as follows:

[0032] The present invention aims at the technical problem that the differential expression spectrum of metabolites and changes in metabolic pathways in Hp-infected gastric cancer are still unclear. Combined with whole transcriptome sequencing and gene re-annotation analysis, we screened out a group of amino acid metabolism-related genes regulated by Hp infection (SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CELA3A, DPP4, MEP1A), and detected their expression in gastric cancer. Based on the above results, the differential expression spectrum of metabolites in Hp-related gastric cancer was clarified, and it was explained that Hp infection increases the transport metabolism of amino acids in gastric cancer cells by upregulating the expression of amino acid metabolism-related genes, thereby promoting the progression of gastric cancer. The present invention provides technical support and new exploration for further understanding the biological functions and treatment of Helicobacter pylori-related gastric cancer lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It shows that Hp infection regulates the amino acid metabolism of gastric cancer: A. Non-targeted metabolomics detection was performed on AGS cells before and after Hp infection, and PCA analysis was performed on the identified metabolic ions. The results showed that there were significant differences in the two groups of observed variables. B. The volcano plot shows 207 secondary metabolites with increased content and 15 secondary metabolites with decreased content identified by non-targeted metabolomics detection. C. Pathway enrichment analysis was performed on differentially expressed secondary metabolites, and the results showed that differential metabolites were enriched in the biosynthesis and metabolic pathways of amino acids. D. The normalized data of metabolites detected by positive and negative ion modes were analyzed, and the 25 metabolites with the most significant differential expression were displayed using a heat map, including essential amino acids such as phenylalanine and tyrosine.

[0034] Figure 2It shows that Hp infection regulates protein digestion and absorption and amino acid transmembrane transport: A. The whole transcriptome of AGS cells before and after Hp infection was sequenced, and heat maps were used to display genes with differential expression of more than 2 times (P<0.05). B. KEGG pathway enrichment analysis of differentially expressed genes was performed using the KOBAS database, and the results showed that differentially expressed genes were enriched in the protein digestion and absorption pathway. C. GO analysis of differentially expressed genes was performed using the KOBAS database, and the results showed that differentially expressed genes were enriched in protein hydrolysis and amino acid transport functions. D. TCGA gastric cancer data were analyzed using the GEPIA database, and the results showed that SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CELA3A, DPP4, and MEP1A were expressed at elevated levels in gastric cancer.

[0035] Figure 3 It shows that Hp infection increases AAMRGs expression and promotes gastric cancer progression: A and B. qRT-PCR and Western blot were used to detect the expression level of AAMRGs in AGS cells. The results showed that the mRNA and protein levels of AAMRGs increased after Hp infection. C. The amino acid content in AGS cells was determined by colorimetry. The results showed that the total amino acid, phenylalanine, and tyrosine content increased after Hp infection. The effect of Hp on increasing amino acid transport was inhibited after amino acid reduction culture. DF. CCK8, Transwell migration, and flow apoptosis were used to detect the proliferation, migration, and apoptosis of AGS cells, respectively. The results showed that the proliferation activity and migration ability of cells were enhanced and the apoptosis was reduced after Hp infection. DETAILED DESCRIPTION

[0036] The present invention will now be described in detail in conjunction with the embodiments and drawings, but the implementation of the present invention is not limited thereto.

[0037] The reagents and raw materials used in the present invention are all commercially available or can be prepared according to literature methods. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conventional conditions, or according to the conditions recommended by the manufacturer.

[0038] 1. Materials and Methods

[0039] (I) Cell culture, passaging, cryopreservation and recovery

[0040] 1. Cell culture

[0041] AGS gastric cancer cell line was purchased from ATCC Cell Bank (USA) and cultured in F12K medium. All cell line culture media were supplemented with 100 μg / mL streptomycin, 100 U / ml penicillin and 10% fetal bovine serum, at a temperature of 37°C and a humidity of 5% CO2.

[0042] 2. Cell passaging

[0043] Place sterilized pipettes, gun tips, centrifuge tubes and other items in the clean bench, irradiate with ultraviolet light for 30 minutes before starting the operation, discard the upper culture medium of the attached cells, wash twice with PBS, add an appropriate amount of preheated trypsin digestion solution, digest for a certain time according to the characteristics of the cells, discard the trypsin digestion solution, add an appropriate amount of preheated culture medium, gently blow the attached cells to make them fall off, collect the cell suspension, centrifuge at 1000rpm for 5 minutes, discard the upper liquid, add an appropriate amount of culture medium to resuspend, take 1 / 5-1 / 3 volume of the cell suspension and add it to the culture dish containing complete culture medium, mix the cells by cross shaking method, and culture them in the incubator.

[0044] 3. Cell cryopreservation

[0045] Cryopreservation was performed when the cells were in the logarithmic growth phase. The upper culture medium was discarded from the attached cells, and the cells were washed twice with PBS. An appropriate amount of preheated trypsin digestion solution was added. After a certain period of time, the trypsin digestion solution was discarded, and an appropriate amount of preheated culture medium was added. The attached cells were gently blown to make them fall off, and the cell suspension was collected. The cells were centrifuged at 1000rpm for 5 minutes, the upper liquid was discarded, and FBS and DMSO (volume ratio 9:1) were added. The cells were gently blown to resuspend and then transferred into the cell cryopreservation tube. The cells were frozen according to the gradient freezing method (4℃30min, -20℃2h, -80℃overnight). For long-term storage, they need to be placed in liquid nitrogen.

[0046] 4. Cell recovery

[0047] Take the frozen cells out of liquid nitrogen and quickly thaw them in a 37°C water bath. Pipette the frozen cell suspension into a centrifuge tube containing 4 times the volume of fresh culture medium, centrifuge at 1000rpm for 5 minutes, discard the upper liquid, add an appropriate amount of complete culture medium to resuspend, place the entire suspension in a small or medium dish, and culture it in a cell culture incubator.

[0048] (II) Hp strain culture and Hp strain and cell co-culture

[0049] The Hp26695 bacterial solution was inoculated on Columbia blood agar medium in a biosafety cabinet and cultured in a three-gas incubator (50mL / L O2, 100mL / L CO2, 850mL / LN2), with humidity maintained above 90%. Gastric cancer cells were cultured in a medium without antibiotics and the experiment was performed after at least 2-3 generations. After the cell confluence reached 80-90%, the cells were digested and inoculated in a 6-well plate at a density of 50-60%. Hp26695 was amplified by shaking in Bruce broth with 10% FBS one day in advance. The amplified bacterial solution was centrifuged at 7500rpm for 10 minutes, and the precipitate was resuspended in PBS. The OD600 value was detected to calculate the bacterial solution concentration. The bacterial solution was added to the cells at an MOI value of 100:1 according to the bacterial solution concentration and placed in a three-gas incubator for culture.

[0050] (III) RNA preparation and real-time fluorescence quantitative PCR (qRT-PCR)

[0051] 1. RNA extraction and reverse transcription

[0052] Total RNA was extracted from cells or tissues using TRIzol reagent. Collect cells or fresh tissues in a 1.5 mL enzyme-free centrifuge tube, add 1 mL TRIZOL and mix, place at room temperature for 5 minutes, add 200 μL chloroform, mix thoroughly, place at room temperature for 3 minutes, and centrifuge at 4°C 12000g for 15 minutes. Transfer the upper layer of liquid to a new 1.5 mL centrifuge tube, being careful not to aspirate the lower layer of liquid, add 200 μL isopropanol, mix by inversion, place at room temperature for 10 minutes, and centrifuge at 4°C 12000g for 10 minutes. Carefully discard the supernatant, retain the precipitate, add 1 mL 75% ethanol, blow off the precipitate, and centrifuge at 4°C 7500g for 5 minutes. Discard the supernatant and let it stand at room temperature for 1 hour until the RNA precipitate is dry. Add 10-40 μL DEPC water and place at 4°C for more than 1 hour until the RNA is completely dissolved, measure the RNA concentration, store at -80°C, and dilute to 0.5 μg / μl when used. According to the instructions of HiScript Q RT SuperMix for qPCR reverse transcription kit, the reverse transcription system (1μl RNA + 2μl SuperMix + 7μl ddH2O) was configured. Reverse transcription reaction: 37℃, 15 minutes. Reverse transcriptase inactivation reaction: 85℃, 5 seconds. After the reaction, the cDNA product was stored at -20℃. In the nuclear cytoplasm separation experiment, PARIS TM The kit was used to separate the nucleus and cytoplasm, and the extracted RNA was subjected to reverse transcription. When RNA was treated with RNase R, 2 μg of total RNA was digested at 37°C for 20 minutes (3 U / μg of RNase R was added), and the digested RNA was purified using the RNeasy MinElute kit.

[0053] 2. qRT-PCR

[0054] Quantitative RT-PCR was performed using the ABIPrism 7900 detection system and SYBR Green PCR Master Mix. The reaction system was configured according to the instructions of the kit. The thermal cycling conditions were set according to the instructions, with 40 cycles, GAPDH as the internal reference, and at least 3 replicates were set for each sample. -ΔΔCT The results of each sample were normalized using the results of the internal reference.

[0055] 3. Primer sequence

[0056] See Table 1 for specific sequences:

[0057] Table 1 Summary of primer sequences

[0058]

[0059]

[0060] (IV) Transwell experiment

[0061] In advance, 50 μl of diluted FN was applied to the bottom of the Transwell chamber, dried in the clean bench, placed in a 24-well plate, and placed in an incubator overnight. The cells used for the experiment were prepared into a cell suspension, and 100 ul of suspension was added to the upper layer of each chamber, with a total number of cells of 30,000 AGS. 600 ul of complete culture medium was added to the lower layer of the chamber, and 12 chambers were cultured in the incubator and taken out. The upper liquid was discarded, methanol was added for fixation, methanol was discarded after 30 minutes, PBS was gently rinsed, crystal violet staining solution was added, and staining was performed for 4 hours. The staining solution was discarded, PBA was gently rinsed 3 times, the upper cells were wiped off with a cotton swab, a drop of saline was dropped on the slide, the bottom of the chamber was immersed in saline, and the observation was taken under a microscope. A photo was taken for each of the five fields of view, and the average number of cells in the five fields of view was taken for analysis, and the measurement was repeated three times.

[0062] (V) CCK-8 cell proliferation assay

[0063] AGS (3000 cells) were inoculated in a 96-well plate and tested when the cells were attached, and 24 hours, 48 ​​hours, 72 hours, and 96 hours after attachment. The culture medium was discarded, 100 μl of diluted CCK-8 reagent was added, and the cells were incubated in a 37°C incubator for 1 hour. The absorbance value was measured at 450 nm using an ELISA reader, and at least 3 replicate wells were set for each sample.

[0064] (VI) Flow cytometry apoptosis assay

[0065] The AGS cells in the logarithmic growth phase were prepared into a cell suspension using EDTA-free trypsin and washed with PBS. Five million cells were washed and centrifuged. According to the Annexin V / PI staining flow cytometry kit manual, 1× Binding Buffer was added and the cells were adjusted to the same concentration (1×10 6 / mL), take 2×10 5 Place 100 μl of cell suspension in a flow cytometer, add Annexin V and PI labeled with fluorescent dyes, mix gently, incubate at room temperature in the dark for 15-20 min, add 300 μl PBS to resuspend the cells, and detect by flow cytometry.

[0066] (VII) Immunoblotting

[0067] 1. Protein extraction

[0068] Cell protein was extracted with RIPA lysis buffer, and serum protein was extracted with serum protein extraction kit. 80 μl lysis buffer was added to the 6-well plate, and the cells were shaken for 30 minutes at 4°C. The lysate was picked up with a cell scraper and placed in a centrifuge tube. The cells were centrifuged at 14000g for 20 minutes at 4°C, and the supernatant was transferred to a new centrifuge tube. The appropriate amount of BCA working solution was prepared according to the volume of A:B=50:1, and the standard protein was diluted to 0.5 mg / mL to prepare a standard curve. The standard protein was added to the 96-well plate at 0, 1, 2, 4, 8, 12, 16, and 20 μl, and the standard protein was added to 20 μl with saline; 1 μl of sample protein was added to the 96-well plate, and the sample was added to 20 μl with saline; 200 μl of BCA working solution was added to each well, and the plate was placed at 37°C for 30 minutes. The absorbance value was measured at 570 nm by an enzyme reader to calculate the sample protein concentration.

[0069] 2. Immunoblotting

[0070] Take 10μl of each sample protein, add 3μl 6×SDS sample buffer and 2μl β-mercaptoethanol, the total amount is 15μl, mix and boil at 100℃ for 5min. Prepare SDS-PAGE: Wash the glass plate, assemble it into the gel rack, and check for leakage. Prepare 10% separation gel solution: ddH2O 3.3mL, separation gel buffer 2mL, 30% polyacrylamide solution 2.7mL, 10% SDS 90μl, 10% APS 60μl, TEMED 6μl, mix thoroughly and add to the gap between the assembled glass plates, the filling height is about 5.5cm, then add anhydrous ethanol for sealing and pressing, let the gel polymerize at room temperature, and let it stand for about 1 hour. Prepare 5% concentrated gel solution: ddH2O 2.3mL, concentrated gel buffer 1mL, 30% polyacrylamide solution 1mL, 10% SDS 40μl, 10% APS20μl, TEMED 8μl, mix thoroughly. Discard anhydrous ethanol, add concentrated gel after it evaporates, do not generate bubbles, insert the sample comb at the top of the glass plate, fill the gap of the sample comb with concentrated gel, and let it stand at room temperature for about 30 minutes. Take out the configured glass plate, install it in the electrophoresis tank, take out the sample comb, and add 18μl of sample protein to each well. Pour the electrophoresis solution, submerge the glass plate, connect the electrodes, and perform electrophoresis at a constant voltage of 90V until the bromophenol blue indicator migrates to the downstream edge of the separation gel, then stop the electrophoresis. According to the sandwich method, place the sponge, filter paper, PVDF membrane, gel, filter paper, and sponge in order, remove all bubbles in the sandwich, fix the sieve plate, insert it into the electrophoresis tank, pour the transfer buffer, and transfer the membrane at a constant current of 220mA for 90 minutes. The PVDF membrane was blocked in 5% skim milk for 1 hour; washed 5 times with 1×TBST for 5 minutes each time. After the primary antibody was diluted, it was incubated with the PVDF membrane at 4°C overnight, washed 5 times with 1×TBST for 5 minutes each time. After the secondary antibody was diluted, it was incubated with the PVDF membrane, incubated on a shaker at 37°C for 1 hour, and washed 5 times with 1×TBST for 10 minutes each time. ECL chemiluminescent solution was evenly applied to the PVDF membrane, and photographed with a BIO-RAD chemiluminescent gel imager.

[0071] 3. Antibodies

[0072] β-actin (AF0003, 1:1000), SLC1A5 (20350-1-AP, 1:5000), SLC3A1 (16343-1-AP, 1:5 000), SLC6A19 (27575-1-AP, 1: 5000), SLC7A9 (RD81114A, 1: 2000), SLC7A11 (26864- 1-AP, 1:5000), SLC16A2 (20676-1-AP, 1:5000), SLC38A1 (28632-1-AP, 1:5000), CEL A3A (HPA028086, 1:5000), DPP4 (29403-1-AP, 1:5000), MEP1A (24640-1-AP, 1:5000)

[0073] 8. Non-targeted metabolomics analysis

[0074] AGS cells and Hp strains were co-cultured to prepare Hp(+) / (-)AGS cells. After culturing to the logarithmic growth phase, the cells were transferred to a common incubator and collected after 24 hours. The two groups of cells were frozen at 2×10 8 Eight parallel groups were prepared and Lianchuan Biotechnology Co., Ltd. was commissioned to carry out non-targeted metabolomics detection and analysis.

[0075] (IX) Amino Acid Detection

[0076] AGS (3000 cells) were inoculated in a 96-well plate. When the cells reached the logarithmic growth phase, the culture medium was discarded and a cell suspension was prepared. According to the operation manual of the amino acid detection kit (Solarbio), 5×10 6 Add the corresponding reagent to each cell and adjust the wavelength of the spectrophotometer to 570 nm for absorbance detection.

[0077] 10. Statistical analysis

[0078] Statistical results were expressed as mean ± standard deviation (X ± SD) of at least three independent experimental results. Statistical methods mainly used t-test and one-way analysis of variance to determine the statistical significance of comparisons between two or more groups. All statistical analyses were performed using SPSS software version 20.0 (SPSS Inc., USA) and GraphPad Prism software version 7.00 (GraphPad software, USA). The correlation between circSHKBP1 and miR-582 expression was established by Pearson correlation coefficient and linear regression model. The median expression level of circSHKBP1 was used as the cutoff value for survival analysis. Log-rank test was performed using GraphPad Prism software to compare Kaplan Meier survival curves. P value < 0.05 was considered statistically significant.

[0079] 2. Results and Analysis

[0080] 1. Hp infection regulates amino acid metabolism in gastric cancer

[0081] In order to study the effect of Hp infection on the metabolic profile of gastric cancer cells, Hp strains were co-cultured with human gastric cancer cell line AGS, Hp(+) / (-) AGS was constructed and non-targeted metabolomics identification was performed. The analysis report showed that there were significant differences in the metabolites between the two groups of samples ( Figure 1 A); 222 differential metabolites identified were enriched in protein absorption and amino acid metabolism pathways ( Figure 1 B and C), which is manifested by an increase in the content of essential amino acids (EAAs), mainly phenylalanine (Phe) and tyrosine (Tyr). Figure 1 D).

[0082] Studies have found that tumor cells use amino acids more economically by optimizing gene expression profiles, and reduced amino acid energy utilization costs show strong prognostic power in many cancer types

[14] EAAs such as phenylalanine and tyrosine play an important role in the occurrence and development of tumors. Adequate EAAs can inhibit the GCN2-eIF2α-ATF4 stress pathway, promote eIF4F-dependent protein translation, selectively activate the synthesis of key cancer-promoting proteins such as c-Myc, Bcl-2, and Cyclin D1, and ultimately lead to tumor progression.

[15] High levels of phenylalanine in cells can reduce the level of reactive oxygen by inhibiting PKM2 activity, protecting cells from oxidative damage, thereby reducing the killing effect on tumor cells.

[16] .

[0083] 2. Hp infection regulates protein digestion and absorption and amino acid transmembrane transport

[0084] In order to explore the causes of abnormal metabolism of EAAs, this project conducted whole transcriptome sequencing and gene re-annotation analysis on AGS of Hp(+) / (-), and found 1275 mRNAs upregulated in Hp(+) samples and 1878 mRNAs downregulated in Hp(+) samples (difference fold>2.0, p<0.05) ( Figure 2 A), these differentially expressed genes are enriched in the protein digestion and absorption pathway, mainly acting on amino acid transmembrane transport ( Figure 2B and C). Based on the above analysis results, the enriched differentially expressed genes mainly include SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CPB1, CELA3A, DPP4, and MEP1A. The expressions of the above genes in Hp(+) samples were increased to varying degrees.

[0085] SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, and SLC38A1 belong to the solute carrier (SLC) protein family, which is responsible for the absorption and transportation of substances on the cell membrane, including amino acids and nucleotides, among which 60% of SLCs have been confirmed to transport amino acids. CPB1, CELA3A, DPP4, and MEP1A are digestive enzymes in the human body, used to digest and decompose proteins. According to TCGA data analysis (http: / / gepia.cancer-pku.cn / ), except for CPB1, the expression levels of other genes in gastric cancer tissues are increased ( Figure 2 D), suggesting that these genes may play a role in promoting cancer. In this project, the above differentially expressed genes (SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CELA3A, DPP4, MEP1A) are defined as amino acid metabolism-related genes (AAMRGs).

[0086] 3. Hp infection increases AAMRGs expression and promotes gastric cancer progression

[0087] Cell experiments were performed to verify the regulation of Hp infection on AAMRGs, amino acid metabolism, and tumor development. The results showed that after Hp infection, the mRNA and protein expression levels of AAMRGs increased, and the total amino acid, phenylalanine, and tyrosine contents in AGS cells increased ( Figure 3 AC); Hp infection promoted the proliferation activity and migration ability of AGS cells and inhibited cell apoptosis, and this effect was reversed after amino acid reduction culture ( Figure 3 DF). The above results suggest that Hp infection increases the transport and metabolism of amino acids in gastric cancer cells by upregulating the expression of AAMRGs, thereby promoting the progression of gastric cancer.

[0088]

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[0103] 16.Li C,You

[0104] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of amino acid metabolism-related gene sets in the preparation of Helicobacter pylori-related gastric cancer detection reagents or detection kits, characterized in that: The amino acid metabolism-related gene set consists of ten genes: SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CELA3A, DPP4, and MEP1A.

2. The use according to claim 1, characterized in that: The detection reagent is a reagent for detecting the expression levels of ten genes in a biological sample.

3. The use according to claim 1, characterized in that: The detection kit comprises reagents for detecting the expression levels of ten genes in a biological sample.

4. The use according to claim 2 or 3, characterized in that: The reagent for detecting the expression levels of ten genes in a biological sample is selected from any one of the following: (1) PCR primers with detection specificity for ten genes; (2) Antibodies with detection specificity for the proteins corresponding to the ten genes.

5. The use according to claim 4, characterized in that: The primer sequences are shown in SEQ ID NOs. 1 to 22; The antibodies with detection specificity for the proteins corresponding to the ten genes are as follows: SLC1A5 antibody: 20350-1-AP, SLC3A1 antibody: 16343-1-AP, SLC6A19 antibody: 27575-1-AP, SLC7A9 antibody: RD81114A, SLC7A11 antibody: 26864-1-AP, SLC16A2 antibody: 20676-1-AP, SLC38A1 antibody: 28632-1-AP, CELA3A antibody: HPA028086, DPP4 antibody: 29403-1-AP, MEP1A antibody: 24640-1-AP.

6. The use according to claim 2 or 3, characterized in that: The biological sample is obtained from a gastric cancer tissue of a subject.

7. A Helicobacter pylori-related gastric cancer detection kit, comprising reagents for detecting the contents of SLC1A5, SLC3A1, SLC6A19, SLC7A9, SLC7A11, SLC16A2, SLC38A1, CELA3A, DPP4, and MEP1A in a biological sample.

8. The Helicobacter pylori-related gastric cancer detection kit according to claim 7, characterized in that: The diagnostic kit is composed of a reverse transcription system, a primer system and an amplification system, wherein the primer system includes PCR primers as shown in SEQ ID NOs. 1 to 22.

9. The Helicobacter pylori-related gastric cancer detection kit according to claim 7, characterized in that: The diagnostic kit is composed of an immunoblotting detection reagent and an antibody system, and the antibody system is composed of: β-actin: AF0003, SLC1A5 antibody: 20350-1-AP, SLC3A1 antibody: 16343-1-AP, SLC6A19 antibody: 27575-1-AP, SLC7A9 antibody: RD81114A, SLC7A11 antibody: 26864-1-AP, SLC16A2 antibody: 20676-1-AP, SLC38A1 antibody: 28632-1-AP, CELA3A antibody: HPA028086, DPP4 antibody: 29403-1-AP, MEP1A antibody: 24640-1-AP.

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