Biomarker for gastric signet ring cell carcinoma and detection kit thereof

Abstract

The invention discloses a gastric signet-ring cell carcinoma biomarker and a detection kit thereof, belongs to the technical field of biological medicines, and particularly relates to the detection kit of the gastric signet-ring cell carcinoma biomarker, which comprises a total RNA (Ribonucleic Acid) extraction reagent, an RNA purification reagent, a cDNA (Complementary Deoxyribonucleic Acid) first strand synthesis premixing reagent, a rapid fluorescent quantitative premixing reagent (SYBR Green), nuclease-free double distilled water and a specific primer. The RNA purification reagent at least comprises isopropanol, chloroform and absolute ethyl alcohol; the specific primer comprises an upstream specific primer and a downstream specific primer. The detection kit for the gastric signet-ring cell carcinoma biomarker is high in RNA purification and recovery efficiency and excellent in specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a biomarker for gastric signet ring cell carcinoma and a detection kit thereof. Background Art

[0002] Gastric cancer is one of the most common malignancies worldwide and a leading cause of cancer-related mortality. Epidemiological studies have shown that while the incidence of gastric cancer overall, including intestinal-type gastric cancer, has been declining annually, the incidence of diffuse gastric cancer, particularly gastric signet ring cell carcinoma (GSRCC), has been increasing. GSRCC develops slowly in its early stages and is generally more indolent than other gastric cancer types, only becoming invasive after infiltrating the submucosa. However, once beyond the submucosa, GSRCC rapidly disseminates and disseminates extensively, accompanied by distant metastases and pelvic implants, demonstrating a highly invasive, highly malignant, and rapidly progressive disease course. Compared with other gastric cancer types, GSRCC has a better prognosis in the early stages but a poorer prognosis in the late stages. Therefore, providing a biomarker, PTGS2, for GSRCC patients and constructing a prognostic assessment model for GSRCC patients would be valuable as a potential drug target. Summary of the Invention

[0003] The purpose of the present invention is to provide a biomarker for gastric signet ring cell carcinoma and a detection kit thereof, improve the RNA purification and recovery efficiency, and optimize the specificity of the detection kit for gastric signet ring cell carcinoma biomarker.

[0004] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are: A detection kit for gastric signet ring cell carcinoma biomarkers includes a total RNA extraction reagent, an RNA purification reagent, a cDNA first-strand synthesis premix, a rapid fluorescence quantitative premix (SYBR Green), nuclease-free double-distilled water, and specific primers. The RNA purification reagent includes at least isopropanol, chloroform, and anhydrous ethanol. The specific primers include an upstream specific primer and a downstream specific primer. The nucleotide sequence of the upstream specific primer is 5'-TTCTCGTGAAGCCCTATGA-3', and the nucleotide sequence of the downstream specific primer is 5'-GAGGCAGTGTTGATGATTTT-3'. The present invention provides a detection kit for gastric signet ring cell carcinoma biomarkers. The kit can specifically detect the expression of gastric signet ring cell carcinoma biomarkers and facilitates the construction of a prognostic assessment model for gastric signet ring cell carcinoma.

[0005] Preferably, the biomarker for gastric signet ring cell carcinoma is prostaglandin epoxide synthase 2.

[0006] Preferably, the RNA purification reagent comprises isopropanol, chloroform, anhydrous ethanol and a magnetic bead suspension.

[0007] More preferably, in the preparation of the magnetic bead suspension, oleic acid-modified ferroferric oxide magnetic particles are first reacted with ethyl silicate under the action of ammonia water, and then reacted with diethylenetriaminepropyltrimethoxysilane and 5-chloro, 4-methylvaleryl chloride. After magnetic separation, an ethanol solution is added for ultrasonic dispersion to obtain a magnetic bead suspension. The present invention uses diethylenetriaminepropyltrimethoxysilane and 5-chloro, 4-methylvaleryl chloride to modify magnetic beads, introducing a large number of active groups that facilitate the adsorption of nucleic acids, thereby efficiently purifying RNA and improving the efficiency of RNA purification and recovery. The purified RNA is reverse transcribed into cDNA, and then qRT-PCR detection is performed using the cDNA as a template, reducing the impact of impurities on qRT-PCR and helping to improve the specificity of the detection kit for gastric signet ring cell carcinoma biomarkers of the present invention.

[0008] More preferably, the usage ratio of the oleic acid modified ferroferric oxide magnetic particles and ammonia water is 1 g: 5-20 mL.

[0009] More preferably, the usage ratio of the oleic acid modified ferroferric oxide magnetic particles and ethyl silicate is 1 g: 2-10 mL.

[0010] More preferably, the usage ratio of the oleic acid modified ferroferric oxide magnetic particles and diethylenetriaminopropyltrimethoxysilane is 1 g: 2-10 mL.

[0011] More preferably, the mass ratio of the oleic acid-modified ferrosoferric oxide magnetic particles to 5-chloro, 4-methylvaleryl chloride is 1:2-10.

[0012] More preferably, the mass concentration of the ethanol solution is 75-95%.

[0013] More preferably, the ratio of the oleic acid-modified ferroferric oxide magnetic particles to the ethanol solution is 1 g: 50-150 mL.

[0014] More preferably, the preparation of the magnetic bead suspension is specifically as follows: Under a nitrogen atmosphere, oleic acid-modified ferroferric oxide magnetic particles were added to deionized water and stirred and dispersed. Ammonia water was added and stirred and dispersed. Ethyl silicate was added dropwise and stirred at 35-50°C for 1-3 hours. After the reaction was completed, it was naturally cooled to room temperature, washed with anhydrous ethanol until the pH was neutral, magnetically separated, and ultrasonically dispersed in ethanol solution to obtain a magnetic bead suspension.

[0015] More preferably, the preparation of the magnetic bead suspension is specifically as follows: Under a nitrogen atmosphere, oleic acid-modified ferroferric oxide magnetic particles were added to deionized water and stirred to disperse. After adding ammonia water, the mixture was stirred to disperse. Ethyl silicate was added dropwise, and the mixture was stirred and reacted at 35-50°C for 1-3 hours. After the reaction was completed, it was naturally cooled to room temperature. Ammonia water was added to adjust the pH to 9-10. Diethylenetriaminopropyltrimethoxysilane was added and reacted for 4-8 hours. 5-Chloro, 4-methylvaleryl chloride and triethylamine were added at 0-5°C, and the mixture was stirred and reacted at room temperature for 3-6 hours. The mixture was washed with anhydrous ethanol until the pH was neutral. After magnetic separation, an ethanol solution was added and ultrasonically dispersed to obtain a magnetic bead suspension.

[0016] More preferably, the preparation of the magnetic bead suspension is specifically as follows: Under a nitrogen atmosphere, oleic acid-modified ferroferric oxide magnetic particles are added to deionized water and stirred and dispersed, ammonia is added and stirred and dispersed, ethyl silicate is added dropwise, and the reaction is stirred at 35-50°C for 1-3 hours. After the reaction is completed, it is naturally cooled to room temperature, ammonia is added to adjust the pH to 9-10, diethylenetriaminopropyltrimethoxysilane is added and reacted for 4-8 hours, 5-chloro, 4-methylvaleryl chloride and triethylamine are added at 0-5°C, and the reaction is stirred at room temperature for 3-6 hours, then N-(tert-butyloxycarbonyl)ethanolamine, anhydrous potassium carbonate and potassium iodide are added, and the reaction is stirred at 70-80°C for 2-6 hours. After the reaction is completed, it is naturally cooled to room temperature, washed with anhydrous ethanol until the pH is neutral, subjected to magnetic separation, and ultrasonically dispersed in ethanol solution to obtain a magnetic bead suspension. The present invention further uses N-(tert-butyloxycarbonyl)ethanolamine to modify the magnetic beads, which helps to modify active groups on the surface of the magnetic beads, further improve the RNA purification and recovery efficiency, and thus further improve the specificity of the detection kit for gastric signet ring cell carcinoma biomarkers.

[0017] More preferably, the usage ratio of the oleic acid modified ferroferric oxide magnetic particles to deionized water is 1 g: 50-150 mL.

[0018] More preferably, the usage ratio of the oleic acid modified ferroferric oxide magnetic particles and ammonia water is 1 g: 5-20 mL.

[0019] More preferably, the usage ratio of the oleic acid modified ferroferric oxide magnetic particles and ethyl silicate is 1 g: 2-10 mL.

[0020] More preferably, the usage ratio of the oleic acid modified ferroferric oxide magnetic particles and diethylenetriaminopropyltrimethoxysilane is 1 g: 2-10 mL.

[0021] More preferably, the mass ratio of the oleic acid-modified ferrosoferric oxide magnetic particles to 5-chloro, 4-methylvaleryl chloride is 1:2-10.

[0022] More preferably, the usage ratio of 5-chloro, 4-methylvaleryl chloride and triethylamine is 1 g: 0.1-0.2 mL.

[0023] More preferably, the mass ratio of the oleic acid-modified ferrosoferric oxide magnetic particles to N-(tert-butyloxycarbonyl)ethanolamine is 1:5-20.

[0024] More preferably, the mass ratio of the oleic acid modified ferroferric oxide magnetic particles to anhydrous potassium carbonate is 1:1-2.

[0025] More preferably, the mass ratio of the oleic acid modified ferroferric oxide magnetic particles to potassium iodide is 1:0.1-0.5.

[0026] More preferably, the mass concentration of the ethanol solution is 75-95%, and the usage ratio of the oleic acid-modified ferroferric oxide magnetic particles to the ethanol solution is 1 g: 50-150 mL.

[0027] Preferably, the detection kit for gastric signet ring cell carcinoma biomarkers comprises: 50-100 parts total RNA extraction reagent, 20-80 parts isopropanol, 10-40 parts chloroform, 60-240 parts anhydrous ethanol, 250-1000 parts nuclease-free double-distilled water, 11-44 parts magnetic bead suspension, 0.2-0.8 parts cDNA first-strand synthesis premix, 2-10 parts SYBR Green fast fluorescence quantitative premix, 0.05-0.3 parts upstream specific primer, and 0.05-0.3 parts downstream specific primer.

[0028] The present invention also discloses a method for detecting a biomarker of gastric signet ring cell carcinoma, comprising: S1. Add total RNA extraction reagent to the tissue and homogenize it. Place it at room temperature for 3-6 minutes, centrifuge it at 10,000-12,000 rpm at 0-5°C for 5-15 minutes, collect the supernatant, add chloroform, vortex it for 10-20 seconds, place it at room temperature for 2-4 minutes, centrifuge it at 10,000-12,000 rpm at 0-5°C for 10-20 minutes, collect the aqueous phase, add isopropanol and mix well, place it at room temperature for 5-15 minutes, centrifuge it at 10,000-12,000 rpm at 0-5°C for 5-15 minutes, remove the supernatant, add ethanol solution, centrifuge it at 10,000-12,000 rpm at 0-5°C for 5-10 minutes, remove the waste liquid, dry it in the air for 2-5 minutes, add nuclease-free double-distilled water, repeatedly pipette and mix to obtain RNA solution.

[0029] S2. On a magnetic rack, mix the RNA solution and magnetic bead suspension evenly, let it stand at room temperature for 2-10 minutes, centrifuge briefly and let it stand for 2-10 minutes, add ethanol solution, pipette 2-5 times, let it stand for 1-5 minutes, remove the supernatant, repeat adding ethanol solution, pipette 2-5 times, let it stand for 1-5 minutes, remove the supernatant, then dry it at room temperature for 2-10 minutes, add nuclease-free double-distilled water and gently pipette 2-5 times, let it stand for 2-10 minutes to obtain the purified RNA solution.

[0030] S3. Dilute the purified RNA solution with nuclease-free double-distilled water to a final concentration of 10-100 ng / μL to obtain an RNA template solution. Take the RNA template solution, cDNA first-strand synthesis premix, and nuclease-free double-distilled water, react at 40-50°C for 10-20 minutes, and then at 94-97°C for 2-5 minutes to obtain a cDNA solution.

[0031] S4. Dilute the cDNA solution with nuclease-free double-distilled water to a final concentration of 20-100 ng / μL to obtain cDNA dilution solution. Take the rapid fluorescent quantitative premix reagent (SYBR Green), upstream specific primers, downstream specific primers, cDNA dilution solution and nuclease-free double-distilled water, and use a qRT-PCR instrument to detect the expression level of the target gene. The qRT-PCR reaction system is: 94-97℃ reaction for 1-3 minutes, then 94-97℃ reaction for 5-10 seconds, 50-60℃ reaction for 10-15 seconds, and 70-75℃ reaction for 10-20 seconds for 34-40 cycles.

[0032] Preferably, in step S1, the ratio of tissue to total RNA extraction reagent is 1 g: 20-40 mL.

[0033] Preferably, in step S1, the volume ratio of chloroform to the total RNA extraction reagent is 1:2-10.

[0034] Preferably, in step S1, the volume ratio of isopropanol to the total RNA extraction reagent is 1:1-2.

[0035] Preferably, in the preparation of the ethanol solution in step S1, anhydrous ethanol is diluted with nuclease-free ultrapure water to a mass concentration of 70-90%.

[0036] Preferably, in step S1, the volume ratio of the total RNA extraction reagent to the ethanol solution is 1:1-2.

[0037] Preferably, in step S1, the volume ratio of the total RNA extraction reagent to the nuclease-free double-distilled water is 1:0.1-0.2.

[0038] Preferably, the volume ratio of the RNA solution to the magnetic bead suspension in step S2 is 1:2-4.

[0039] Preferably, in the preparation of the ethanol solution in step S2, anhydrous ethanol is diluted with nuclease-free ultrapure water to a mass concentration of 70-90%.

[0040] Preferably, the volume ratio of the RNA solution to the ethanol solution in step S2 is 1:2-4.

[0041] Preferably, the volume ratio of the RNA solution to nuclease-free double-distilled water in step S2 is 1:1-2.

[0042] Preferably, the volume ratio of the RNA template solution to the cDNA first-strand synthesis premix in step S3 is 1:4-8.

[0043] Preferably, in step S3, the volume ratio of the RNA template solution to nuclease-free double-distilled water is 1:5-20.

[0044] Preferably, in step S4, the volume ratio of the cDNA dilution solution to the upstream specific primer is 1:0.2-1.

[0045] Preferably, in step S4, the volume ratio of the cDNA diluent to the downstream specific primer is 1:0.2-1.

[0046] Preferably, in step S4, the volume ratio of the cDNA diluent to the rapid fluorescence quantitative premix reagent (SYBR Green) is 1:5-20.

[0047] Preferably, in step S4, the volume ratio of the cDNA diluent to nuclease-free double-distilled water is 1:5-10.

[0048] The present invention utilizes oleic acid-modified ferroferric oxide magnetic particles that are first reacted with ethyl silicate and then with diethylenetriaminopropyltrimethoxysilane, 5-chloro, 4-methylvaleryl chloride, and N-(tert-butyloxycarbonyl)ethanolamine to produce a magnetic bead suspension. This magnetic bead suspension is then incorporated into a detection kit for gastric signet ring cell carcinoma biomarkers. This advantageous effect is achieved by: the magnetic bead suspension in the detection kit for gastric signet ring cell carcinoma biomarkers efficiently purifies RNA, with an RNA purification recovery efficiency of 85.34-98.73%. The highly pure RNA is reverse-transcribed into cDNA, which is then used as a template for qRT-PCR experiments, reducing the impact of impurities on qRT-PCR and thereby improving the specificity of the detection kit for gastric signet ring cell carcinoma biomarkers, with a specificity of 88.64-99.77%. Therefore, the present invention provides a detection kit for gastric signet ring cell carcinoma biomarkers with high RNA purification and recovery efficiency and excellent specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a TEM image of the magnetic bead suspension prepared in Example 2.

[0050] Figure 2 The mRNA expression of PTGS2.

[0051] Figure 3 The protein expression of PTGS2.

[0052] Figure 4 The migration of gastric signet ring cell cancer cells after knockdown of PTGS2.

[0053] Figure 5 The invasion of gastric signet ring cell carcinoma cells after knockdown of PTGS2.

[0054] Figure 6 The adhesion of gastric signet ring cell cancer cells after knockdown of PTGS2.

[0055] Figure 7 This is the plate cloning of gastric signet ring cell cancer cells after knocking down PTGS2.

[0056] Figure 8 The proliferation of gastric signet ring cell cancer cells after knockdown of PTGS2.

[0057] Figure 9 The soft agar colony formation ability of gastric signet ring cell carcinoma cells after knockdown of PTGS2 was investigated.

[0058] Figure 10 The migration of gastric signet ring cell carcinoma cells after celecoxib treatment.

[0059] Figure 11 The proliferation of gastric signet ring cell carcinoma cells after celecoxib treatment.

[0060] Figure 12 The migration of gastric signet ring cell carcinoma cells after overexpression of PTGS2.

[0061] Figure 13 The invasion of gastric signet ring cell carcinoma cells after overexpression of PTGS2.

[0062] Figure 14 The adhesion of gastric signet ring cell carcinoma cells after overexpression of PTGS2.

[0063] Figure 15 The proliferation of gastric signet ring cell carcinoma cells after overexpression of PTGS2.

[0064] Figure 16 The soft agar colony-forming ability of gastric signet ring cell carcinoma cells after overexpression of PTGS2.

[0065] Figure 17 The expression of adhesion molecules after overexpression of PTGS2.

[0066] Figure 18 Figure 2 shows the mRNA expression of IQGAP1 in gastric signet ring cell cancer cells after knockdown of PTGS2.

[0067] Figure 19 Figure 2 shows the mRNA expression of IQGAP1 in gastric signet ring cell carcinoma cells after overexpression of PTGS2.

[0068] Figure 20 Figure 3 shows the protein expression of IQGAP1 in gastric signet ring cell cancer cells after knockdown of PTGS2.

[0069] Figure 21 This is the protein expression of IQGAP1 in gastric signet ring cell carcinoma cells after celecoxib treatment.

[0070] Figure 22 Figure 2 shows the protein expression of IQGAP1 in gastric signet ring cell carcinoma cells after overexpression of PTGS2.

[0071] Figure 23 Figure 3 shows the protein expression of PTGS2 in gastric signet ring cell cancer cells after knockdown of IQGAP1.

[0072] Figure 24 Figure 3 shows the migration of gastric signet ring cell cancer cells after knockdown of IQGAP1.

[0073] Figure 25 The invasion of gastric signet ring cell carcinoma cells after knockdown of IQGAP1.

[0074] Figure 26 Figure 2 shows the adhesion of gastric signet ring cell cancer cells after knockdown of IQGAP1.

[0075] Figure 27 Soft agar cloning of gastric signet ring cell carcinoma cells after knockdown of IQGAP1.

[0076] Figure 28 Correlation analysis of PTGS2 and IQGAP1 mRNA expression levels.

[0077] Figure 29 Figure 2 shows the mRNA expression of ERK target genes and cell cycle molecules in gastric signet ring cell carcinoma after overexpression of PTGS2.

[0078] Figure 30 Figure 3 shows the mRNA expression of ERK target genes and cell cycle molecules in gastric signet ring cell carcinoma after knockdown of PTGS2.

[0079] Figure 31 Figure 3 is the protein expression level of MMP2 in gastric signet ring cell carcinoma cells after knockdown of PTGS2.

[0080] Figure 32Figure 3 is the protein expression level of MMP2 in gastric signet ring cell carcinoma cells after knockdown of IQGAP1.

[0081] Figure 33 Figure 3 Phosphorylation level of ERK pathway in gastric signet ring cell cancer cells after knockdown of IQGAP1.

[0082] Figure 34 Figure 2 is the phosphorylation level of ERK pathway in gastric signet ring cell carcinoma cells after overexpression of PTGS2. DETAILED DESCRIPTION

[0083] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0084] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0085] Example 1: A biomarker for gastric signet ring cell carcinoma comprising prostaglandin-endoperoxide synthase 2 (PTGS2).

[0086] Preparation of magnetic bead suspension, including, Under a nitrogen atmosphere, oleic acid-modified ferroferric oxide magnetic particles were added to deionized water and stirred to disperse. Ammonia was then added and stirred to disperse. Ethyl silicate was then added dropwise. The mixture was stirred at 40°C for 2 hours. After the reaction, the mixture was cooled to room temperature and washed with anhydrous ethanol until the pH was neutral. After magnetic separation, the mixture was ultrasonically dispersed in ethanol to obtain a magnetic bead suspension. The ratio of oleic acid-modified ferroferric oxide magnetic particles to deionized water was 1g:100mL; the ratio of oleic acid-modified ferroferric oxide magnetic particles to ammonia was 1g:10mL; and the ratio of oleic acid-modified ferroferric oxide magnetic particles to ethyl silicate was 1g:5mL. The mass concentration of the ethanol solution was 85%, and the ratio of oleic acid-modified ferroferric oxide magnetic particles to ethanol solution was 1g:100mL.

[0087] A detection kit for gastric signet ring cell carcinoma biomarkers, comprising: 100 parts total RNA extraction reagent, 40 parts isopropanol, 20 parts chloroform, 120 parts anhydrous ethanol, 500 parts nuclease-free double-distilled water, 22 parts magnetic bead suspension, 0.4 parts cDNA first-strand synthesis premix, 5 parts rapid fluorescent quantitative premix (SYBR Green), 0.15 parts upstream specific primer, and 0.15 parts downstream specific primer. Total RNA extraction reagent, nuclease-free double-distilled water, cDNA first-strand synthesis premix, and rapid fluorescent quantitative premix (SYBR Green) were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The ethanol solution was prepared by diluting anhydrous ethanol with nuclease-free double-distilled water to a final concentration of 75%. The upstream specific primer and the downstream specific primer were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The nucleotide sequence of the upstream specific primer is shown in SEQ ID No. 1, which is 5'-TTCTCGTGAAGCCCTATGA-3'; the nucleotide sequence of the downstream specific primer is shown in SEQ ID No. 2, which is 5'-GAGGCAGTGTTGATGATTTT-3'.

[0088] A method for detecting a biomarker of gastric signet ring cell carcinoma, comprising: S1. Add total RNA extraction reagent to the tissue and homogenize it. Place it at room temperature for 5 minutes, centrifuge it at 10,000 rpm at 4°C for 10 minutes, collect the supernatant, add chloroform, vortex it for 15 seconds, place it at room temperature for 3 minutes, centrifuge it at 10,000 rpm at 4°C for 15 minutes, collect the aqueous phase, add isopropanol and mix it, place it at room temperature for 10 minutes, centrifuge it at 10,000 rpm at 4°C for 10 minutes, remove the supernatant, add ethanol solution, centrifuge it at 10,000 rpm at 4°C for 5 minutes, remove the waste liquid, dry it in the air for 3 minutes, add nuclease-free double-distilled water, repeatedly pipette and mix it to obtain RNA solution. The tissue and total RNA extraction reagent usage ratio is 1 g:33 mL; the volume ratio of chloroform to total RNA extraction reagent is 1:5; the volume ratio of isopropanol to total RNA extraction reagent is 1:2; in the preparation of ethanol solution, anhydrous ethanol is diluted with nuclease-free ultrapure water to a mass concentration of 75%; the volume ratio of total RNA extraction reagent to ethanol solution is 1:1; and the volume ratio of total RNA extraction reagent to nuclease-free double-distilled water is 1:0.1.

[0089] S2. On a magnetic rack, mix the RNA solution and magnetic bead suspension evenly, let it stand at room temperature for 5 minutes, centrifuge briefly, let it stand for 5 minutes, add ethanol solution, pipette up and down five times, let it stand for 2 minutes, remove the supernatant, repeat the addition of ethanol solution, pipette up and down five times, let it stand for 2 minutes, remove the supernatant, and air dry at room temperature for 5 minutes. Add nuclease-free double-distilled water and gently pipette up and down five times, let it stand for 5 minutes to obtain the purified RNA solution. The volume ratio of RNA solution to magnetic bead suspension is 1:2; in the preparation of ethanol solution, use nuclease-free ultrapure water to dilute anhydrous ethanol to a mass concentration of 80%; the volume ratio of RNA solution to ethanol solution is 1:2; the volume ratio of RNA solution to nuclease-free double-distilled water is 1:1.

[0090] S3. Dilute the purified RNA solution with nuclease-free double-distilled water to a final concentration of 50 ng / μL to obtain an RNA template solution. Combine the RNA template solution, cDNA first-strand synthesis premix, and nuclease-free double-distilled water, and react at 45°C for 15 minutes, followed by 95°C for 3 minutes to obtain a cDNA solution. The volume ratio of RNA template solution to cDNA first-strand synthesis premix is ​​1:4; the volume ratio of RNA template solution to nuclease-free double-distilled water is 1:15.

[0091] S4. Dilute the cDNA solution to a final concentration of 50 ng / μL using nuclease-free double-distilled water to obtain a cDNA dilution buffer. Prepare 10 μL of SYBR Green rapid fluorescence quantitative premix, 0.6 μL of upstream specific primer, 0.6 μL of downstream specific primer, 1 μL of cDNA dilution buffer, and 7.8 μL of nuclease-free double-distilled water. Detect the expression of the target gene using a qRT-PCR instrument. The qRT-PCR reaction system is as follows: 95°C for 1 minute, followed by 40 cycles of 95°C for 5 seconds, 60°C for 10 seconds, and 72°C for 15 seconds. The volume ratio of cDNA dilution buffer to upstream specific primer is 1:0.6; the volume ratio of cDNA dilution buffer to downstream specific primer is 1:0.6; the volume ratio of cDNA dilution buffer to SYBR Green rapid fluorescence quantitative premix is ​​1:10; and the volume ratio of cDNA dilution buffer to nuclease-free double-distilled water is 1:7.8.

[0092] Example 2: A biomarker for gastric signet ring cell carcinoma, same as Example 1.

[0093] Preparation of magnetic bead suspension, including, Under a nitrogen atmosphere, oleic acid-modified ferroferric oxide magnetic particles were added to deionized water and stirred to disperse. After adding ammonia water, the mixture was stirred to disperse. Ethyl silicate was added dropwise and stirred to react at 40°C for 2 hours. After the reaction, it was naturally cooled to room temperature. Ammonia water was added to adjust the pH to 10. Diethylenetriaminopropyltrimethoxysilane was added and reacted for 6 hours. 5-chloro, 4-methylvaleryl chloride and triethylamine were added at 0°C and stirred to react at room temperature for 5 hours. The mixture was washed with anhydrous ethanol until the pH was neutral. After magnetic separation, ethanol solution was added and ultrasonically dispersed to obtain a magnetic bead suspension. The dosage ratio of oleic acid modified ferroferric oxide magnetic particles and deionized water is 1g:100mL; the dosage ratio of oleic acid modified ferroferric oxide magnetic particles and ammonia water is 1g:10mL; the dosage ratio of oleic acid modified ferroferric oxide magnetic particles and ethyl silicate is 1g:5mL; the dosage ratio of oleic acid modified ferroferric oxide magnetic particles and diethylenetriaminepropyltrimethoxysilane is 1g:5mL; the mass ratio of oleic acid modified ferroferric oxide magnetic particles and 5-chloro,4-methylvaleryl chloride is 1:5; the dosage ratio of 5-chloro,4-methylvaleryl chloride and triethylamine is 1g:0.15mL; the mass concentration of the ethanol solution is 85%, and the dosage ratio of oleic acid modified ferroferric oxide magnetic particles and ethanol solution is 1g:100mL.

[0094] A detection kit for gastric signet ring cell carcinoma biomarkers is provided. Compared with Example 1, except that the magnetic bead suspension is replaced with the magnetic bead suspension prepared in this example, other conditions are the same as those in Example 1.

[0095] A method for detecting a biomarker of gastric signet ring cell carcinoma is provided. Compared with Example 1, except that the magnetic bead suspension is replaced with the magnetic bead suspension prepared in this example, other conditions are the same as those in Example 1.

[0096] Example 3: A biomarker for gastric signet ring cell carcinoma, same as Example 1.

[0097] The preparation of magnetic bead suspension is the same as in Example 2.

[0098] A detection kit for gastric signet ring cell carcinoma biomarkers is provided. Compared with Example 1, except that the magnetic bead suspension is replaced by the modified magnetic bead suspension prepared in this example, other conditions are the same as those in Example 1.

[0099] A method for detecting a biomarker of gastric signet ring cell carcinoma is provided. Compared with Example 1, the magnetic bead suspension in step S2 is replaced with the modified magnetic bead suspension prepared in this example, and the volume ratio of the RNA solution to the magnetic bead suspension is changed to 1:1. Other conditions are the same as those in Example 2.

[0100] Example 4: A biomarker for gastric signet ring cell carcinoma, same as Example 1.

[0101] Preparation of magnetic bead suspension, including, Under a nitrogen atmosphere, oleic acid-modified ferroferric oxide magnetic particles were added to deionized water and stirred to disperse. After adding ammonia water, the mixture was stirred to disperse. Ethyl silicate was added dropwise, and the mixture was stirred and reacted at 40°C for 2 hours. After the reaction, it was naturally cooled to room temperature. Ammonia water was added to adjust the pH to 10. Diethylenetriaminopropyltrimethoxysilane was added and reacted for 6 hours. 5-Chloro, 4-methylvaleryl chloride and triethylamine were added at 0°C, and the mixture was stirred and reacted at room temperature for 5 hours. N-(tert-butoxycarbonyl)ethanolamine, anhydrous potassium carbonate and potassium iodide were added, and the mixture was reacted at 70°C for 5 hours. After the reaction, it was naturally cooled to room temperature, washed with anhydrous ethanol until the pH was neutral, magnetically separated, and ultrasonically dispersed in ethanol solution to obtain a magnetic bead suspension. The dosage ratio of oleic acid modified ferroferric oxide magnetic particles and deionized water is 1 g:100 mL; the dosage ratio of oleic acid modified ferroferric oxide magnetic particles and ammonia water is 1 g:10 mL; the dosage ratio of oleic acid modified ferroferric oxide magnetic particles and ethyl silicate is 1 g:5 mL; the dosage ratio of oleic acid modified ferroferric oxide magnetic particles and diethylenetriaminepropyltrimethoxysilane is 1 g:5 mL; the mass ratio of oleic acid modified ferroferric oxide magnetic particles and 5-chloro, 4-methylvaleryl chloride is 1:5; 5 -chloro, 4-methylvaleryl chloride and triethylamine are used in a ratio of 1 g:0.15 mL; the mass ratio of oleic acid modified ferroferric oxide magnetic particles and N-(tert-butyloxycarbonyl)ethanolamine is 1:10; the mass ratio of oleic acid modified ferroferric oxide magnetic particles and anhydrous potassium carbonate is 1:1; the mass ratio of oleic acid modified ferroferric oxide magnetic particles and potassium iodide is 1:0.1; the mass concentration of the ethanol solution is 85%, and the mass ratio of oleic acid modified ferroferric oxide magnetic particles and ethanol solution is 1 g:100 mL.

[0102] A detection kit for gastric signet ring cell carcinoma biomarkers is provided. Compared with Example 1, except that the magnetic bead suspension is replaced by the modified magnetic bead suspension prepared in this example, other conditions are the same as those in Example 1.

[0103] A method for detecting a biomarker of gastric signet ring cell carcinoma is provided. Compared with Example 1, except that the magnetic bead suspension in step S2 is replaced with the modified magnetic bead suspension prepared in this example, other conditions are the same as those in Example 1.

[0104] Example 5: A biomarker for gastric signet ring cell carcinoma, same as Example 1.

[0105] The preparation of magnetic bead suspension is the same as in Example 4.

[0106] A detection kit for gastric signet ring cell carcinoma biomarkers is provided. Compared with Example 1, except that the magnetic bead suspension is replaced by the modified magnetic bead suspension prepared in this example, other conditions are the same as those in Example 1.

[0107] A method for detecting a biomarker of gastric signet ring cell carcinoma is provided. Compared with Example 1, except that in step S2, the magnetic bead suspension is replaced with the modified magnetic bead suspension prepared in this example, and the volume ratio of the RNA solution to the magnetic bead suspension is changed to 1:1, other conditions are the same as in Example 1.

[0108] Comparative Example 1: A biomarker for gastric signet ring cell carcinoma, same as Example 1.

[0109] The preparation of the magnetic bead suspension was similar to that of Example 4 except that 5-chloro, 4-methylvaleryl chloride was not added. Other conditions were the same as those of Example 4.

[0110] A detection kit for gastric signet ring cell carcinoma biomarkers is provided. Compared with Example 1, except that the magnetic bead suspension is replaced by the modified magnetic bead suspension prepared in this comparative example, other conditions are the same as those in Example 1.

[0111] A method for detecting a biomarker of gastric signet ring cell carcinoma is provided. Compared with Example 1, except that the magnetic bead suspension in step S2 is replaced with the modified magnetic bead suspension prepared in this comparative example, other conditions are the same as those in Example 1.

[0112] Comparative Example 2: A biomarker for gastric signet ring cell carcinoma, same as Example 1.

[0113] The preparation of the magnetic bead suspension was similar to that of Example 4 except that diethylenetriaminopropyltrimethoxysilane and 5-chloro,4-methylvaleryl chloride were not added. Other conditions were the same as those of Example 4.

[0114] A detection kit for gastric signet ring cell carcinoma biomarkers is provided. Compared with Example 1, except that the magnetic bead suspension is replaced by the modified magnetic bead suspension prepared in this comparative example, other conditions are the same as those in Example 1.

[0115] A method for detecting a biomarker of gastric signet ring cell carcinoma is provided. Compared with Example 1, except that the magnetic bead suspension in step S2 is replaced with the modified magnetic bead suspension prepared in this comparative example, other conditions are the same as those in Example 1.

[0116] Experimental example: 1. Material characterization The magnetic bead suspension prepared in Example 2 was characterized by transmission electron microscopy (TEM).

[0117] Figure 1 This is a TEM image of the magnetic bead suspension prepared in Example 2. The magnetic bead particles in the magnetic bead suspension prepared in the present invention are regular circles, and there is accumulation and aggregation of particles.

[0118] 2. Biological experiments 2.1 Experimental Methods Three gastric signet ring cell carcinoma cell lines were established: PTGS2-overexpressing, PTGS2-knockdown, and IQGAP1-knockdown cell lines. For the PTGS2-overexpressing SCCC lines, two treatment groups were set up: a PTGS2-overexpressing control group (oe-con) and a PTGS2-overexpressing experimental group (oe-PTGS2). For the PTGS2-knockdown SCCC lines, three treatment groups were set up: a PTGS2-knockdown control group (sh-con), a PTGS2-knockdown experimental group 1 (sh1-PTGS2), and a PTGS2-knockdown experimental group 2 (sh2-PTGS2). For the IQGAP1-knockdown SCCC lines, three treatment groups were set up: an IQGAP1-knockdown control group (sh-con), an IQGAP1-knockdown experimental group 1 (sh1-IQGAP1), and an IQGAP1-knockdown experimental group 2 (sh2-IQGAP1).

[0119] Celecoxib treatment of gastric signet ring cell carcinoma cells: Celecoxib is a specific inhibitor of PTGS2. Celecoxib was diluted with sterile ultrapure water to final concentrations of 0 μmol / L, 20 μmol / L, and 40 μmol / L. Cells were treated with celecoxib at final concentrations of 0 μmol / L, 20 μmol / L, and 40 μmol / L for 24 hours.

[0120] Detection of mRNA expression level: qPT-PCR method was used to detect the mRNA expression level of target genes.

[0121] Detection of protein expression levels and pathway phosphorylation levels: Western Blot was used to detect the protein expression levels of target genes and the phosphorylation levels of pathways.

[0122] Detection of malignant phenotype of gastric signet ring cell carcinoma cells: proliferation ability was detected by CCK-8 assay and plate cloning assay; motility was detected by Transwell migration and invasion assay; adhesion and cloning ability were detected by Matrigel adhesion assay and soft agar cloning assay.

[0123] 2.2 PTGS2 expression Figure 2 is the mRNA expression of PTGS2, Figure 3 is the protein expression of PTGS2. Figure 2 and Figure 3 In the table, S1 represents the expression level of PTGS2 in paired normal tissues, and S2 represents the expression level of PTGS2 in gastric signet ring cell carcinoma tissues. Figure 2 and Figure 3 It can be seen that compared with the matched normal tissues, the mRNA expression level and protein expression level of PTGS2 in gastric signet ring cell carcinoma tissues showed an upward trend, indicating that PTGS2 is highly expressed in gastric signet ring cell carcinoma tissues.

[0124] Effect of PTGS2 on the malignant phenotype of gastric signet ring cell carcinoma cells Figure 4 To investigate the migration of gastric signet ring cell carcinoma cells after knockdown of PTGS2, Figure 5 To investigate the invasion of gastric signet ring cell carcinoma cells after knockdown of PTGS2, Figure 6 To investigate the adhesion of gastric signet ring cell carcinoma cells after knockdown of PTGS2, Figure 7 This is the plate cloning of gastric signet ring cell cancer cells after knocking down PTGS2. Figure 8 To investigate the proliferation of gastric signet ring cell carcinoma cells after knockdown of PTGS2, Figure 9 The soft agar colony formation ability of gastric signet ring cell carcinoma cells after knockdown of PTGS2. Figures 4 to 9 It can be seen that knocking down PTGS2 significantly inhibits the migration, invasion, adhesion, plate cloning, proliferation and soft agar cloning ability of gastric signet ring cell cancer cells.

[0125] Figure 10 The migration of gastric signet ring cell carcinoma cells after celecoxib treatment; Figure 11 The proliferation of gastric signet ring cell carcinoma cells after celecoxib treatment. Figure 10 and Figure 11 It can be seen that after treatment with celecoxib, the migration and proliferation abilities of gastric signet ring cell cancer cells were significantly reduced.

[0126] Figure 12 The migration of gastric signet ring cell carcinoma cells after overexpression of PTGS2. Figure 13 The invasion of gastric signet ring cell carcinoma cells after overexpression of PTGS2. Figure 14 The adhesion of gastric signet ring cell carcinoma cells after overexpression of PTGS2. Figure 15 The proliferation of gastric signet ring cell carcinoma cells after overexpression of PTGS2. Figure 16 The soft agar colony formation ability of gastric signet ring cell carcinoma cells after overexpression of PTGS2. Figures 12 to 16 It can be seen that overexpression of PTGS2 significantly promotes the migration, invasion, adhesion, proliferation and soft agar colony formation ability of gastric signet ring cell cancer cells.

[0127] Depend on Figures 4 to 16 It can be seen that PTGS2 promotes multiple malignant biological behaviors of gastric signet ring cell cancer cells, and the specific inhibitor celecoxib inhibits the malignant phenotype of gastric signet ring cell cancer cells.

[0128] 2.4 PTGS2 positively regulates IQGAP1 expression Figure 17 is the expression of adhesion molecules after overexpression of PTGS2. Figure 17 It can be seen that after overexpression of PTGS2 in gastric signet ring cell cancer cells, the expression levels of adhesion molecules changed, among which the mRNA expression level of the adhesion molecule IQGAP1 increased most significantly. Figure 18 To investigate the mRNA expression of IQGAP1 in gastric signet ring cell carcinoma cells after knockdown of PTGS2, Figure 19 Figure 2 is the mRNA expression of IQGAP1 in gastric signet ring cell carcinoma cells after overexpression of PTGS2. Figure 20 To investigate the protein expression of IQGAP1 in gastric signet ring cell carcinoma cells after knockdown of PTGS2, Figure 21 This is the protein expression of IQGAP1 in gastric signet ring cell carcinoma cells after celecoxib treatment. Figure 22 Figure 2 shows the protein expression of IQGAP1 in gastric signet ring cell carcinoma cells after overexpression of PTGS2. Figures 18 to 22 It can be seen that PTGS2 regulates the mRNA expression level and protein expression level of IQGAP1. Figure 23 The protein expression of PTGS2 in gastric signet ring cell carcinoma cells after knockdown of IQGAP1. Figure 23 It can be seen that knocking down IQGAP1 in gastric signet ring cell cancer cells did not affect the protein expression level of PTGS2, indicating that IQGAP1 is a downstream target gene of PTGS2 in gastric signet ring cell cancer cells.

[0129] 2.5 Effect of IQGAP1 on the malignant phenotype of gastric signet ring cell carcinoma cells Figure 24 To investigate the migration of gastric signet ring cell carcinoma cells after knockdown of IQGAP1, Figure 25 To investigate the invasion of gastric signet ring cell carcinoma cells after knockdown of IQGAP1, Figure 26 To investigate the adhesion of gastric signet ring cell carcinoma cells after knockdown of IQGAP1, Figure 27 The soft agar clones of gastric signet ring cell carcinoma cells after IQGAP1 knockdown. Figures 24 to 27 It can be seen that after knocking down IQGAP1, the migration, invasion, adhesion and soft agar cloning abilities of gastric signet ring cell cancer cells were weakened, which indicates that IQGAP1 promotes multiple malignant biological behaviors of gastric signet ring cell cancer cells.

[0130] Figure 28 Correlation analysis of PTGS2 and IQGAP1 mRNA expression levels showed a significant correlation between PTGS2 and IQGAP1 mRNA expression levels, with a correlation coefficient R of 0.55. This suggests that PTGS2 promotes the malignant phenotype of gastric signet ring cell carcinoma by upregulating IQGAP1 expression.

[0131] 2.6 Exploring potential downstream mechanisms by which the PTGS2-IQGAP1 axis promotes the malignant phenotype of gastric signet ring cell carcinoma Figure 29 This is the mRNA expression of ERK target genes and cell cycle molecules in gastric signet ring cell carcinoma after overexpression of PTGS2. Figure 30 Figure 2 shows the mRNA expression of ERK target genes and cell cycle molecules in gastric signet ring cell carcinoma after knockdown of PTGS2. Figure 29 and Figure 30 It can be seen that after overexpression of PTGS2, the mRNA expression levels of ERK target genes and cell cycle molecules including MMP2, c-myc, and N-cadheirn in gastric signet ring cell cancer cells were significantly increased; knockdown of PTGS2 in gastric signet ring cell cancer cells significantly inhibited the mRNA expression levels of some cell cycle molecules and ERK target genes; among them, the difference in MMP2 expression was the most significant.

[0132] Figure 31 To investigate the protein expression level of MMP2 in gastric signet ring cell carcinoma cells after knocking down PTGS2, Figure 32 The protein expression level of MMP2 in gastric signet ring cell carcinoma cells after knockdown of IQGAP1. Figure 31 and Figure 32 It can be seen that knockdown of PTGS2 and IQGAP1 both inhibited the protein expression level of MMP2 in gastric signet ring cell cancer cells. Figure 33 To investigate the phosphorylation level of the ERK pathway in gastric signet ring cell cancer cells after knocking down IQGAP1, knocking down IQGAP1 inhibited the phosphorylation level of the ERK pathway; Figure 34 Figure 3 shows the phosphorylation level of the ERK pathway in gastric signet ring cell carcinoma cells after overexpression of PTGS2. Overexpression of PTGS2 promoted the phosphorylation level of the ERK pathway, while the expression level of total ERK protein did not change significantly. This suggests that the PTGS2-IQGAP1 axis may promote the occurrence and development of gastric signet ring cell carcinoma by activating the ERK pathway and upregulating MMP2 expression.

[0133] 2.7 RNA recovery efficiency Gastric signet ring cell carcinoma tissue was collected and tested according to steps S1 and S2 of the gastric signet ring cell carcinoma biomarker detection methods described in Examples 1-5 and Comparative Example 1, respectively, to obtain RNA solutions and purified RNA solutions. The RNA concentrations in the RNA solutions and purified RNA solutions were measured using a Nanodrop microspectrophotometer. The RNA concentration in the RNA solution was designated as C1, and the volume of the RNA solution was designated as V1. The RNA concentration in the purified RNA solution was designated as C2, and the volume of the purified RNA solution was designated as V2. RNA recovery efficiency (%) = (C2 × V2) / (C1 × V1) × 100%. Table 1 shows the RNA recovery efficiency (%).

[0134] Table 1 RNA recovery efficiency (%)

[0135] As shown in Table 1, the RNA recovery efficiency of Examples 2-3 of the present invention is higher than that of Example 1. This is because the magnetic bead suspensions used in Examples 1 and 2-3 are different. In the preparation of the magnetic bead suspension, Example 1 uses oleic acid-modified ferroferric oxide magnetic particles to react with ethyl silicate to obtain a magnetic bead suspension. Example 2-3 uses oleic acid-modified ferroferric oxide magnetic particles to react with ethyl silicate first, and then with diethylenetriaminepropyltrimethoxysilane and 5-chloro, 4-methylvaleryl chloride to obtain a magnetic bead suspension. The RNA recovery efficiency of Example 2-3 is higher than that of Comparative Example 1 because 5-chloro, 4-methylvaleryl chloride is not used in Comparative Example 1 in the preparation of the magnetic bead suspension. The RNA recovery efficiency of Example 2 is higher than that of Example 3 because the amount of magnetic bead suspension used in Examples 2 and 3 is different. This shows that the magnetic bead suspension prepared by the present invention can efficiently purify RNA and improve RNA purification recovery efficiency.

[0136] The RNA recovery efficiency of Examples 4-5 of the present invention is higher than that of Example 2 because the magnetic bead suspensions used in Examples 2 and 4-5 are different. In the preparation of the magnetic bead suspension, Example 4-5 uses oleic acid-modified ferroferric oxide magnetic particles to react with ethyl silicate first, then with diethylenetriaminepropyltrimethoxysilane and 5-chloro, 4-methylvaleryl chloride, and then with N-(tert-butyloxycarbonyl)ethanolamine to obtain a magnetic bead suspension; Example 2-3 uses oleic acid-modified ferroferric oxide magnetic particles to react with ethyl silicate first, then with diethylenetriaminepropyltrimethoxysilane and 5-chloro, 4-methylvaleryl chloride to obtain a magnetic bead suspension. The RNA recovery efficiency of Example 4-5 is higher than that of Comparative Example 2 because N-(tert-butyloxycarbonyl)ethanolamine is not used in the preparation of the magnetic bead suspension. The RNA recovery efficiency of Example 4 is higher than that of Example 5 because the amount of magnetic bead suspension used in Examples 4 and 5 is different. This shows that the magnetic bead suspension prepared in Example 4 of the present invention can further improve the RNA purification and recovery efficiency.

[0137] 2.8 Specificity Test of Gastric Signet Ring Cell Carcinoma Biomarker Detection Kit Gastric signet ring cell carcinoma tissues were collected and experiments were performed according to the detection methods for gastric signet ring cell carcinoma biomarkers in Examples 1-5 and Comparative Example 1, respectively. Each experiment was performed with 4 biological replicates and 4 technical replicates. The specificity (%) = number of positive experimental groups / total number of experimental groups × 100%. Table 2 shows the specificity (%).

[0138] Table 2 Specificity (%)

[0139] As shown in Table 2, the specificity of the detection kit for gastric signet ring cell carcinoma biomarkers in Examples 2-3 of the present invention is higher than that in Example 1 and Comparative Example 1. This is because the magnetic bead suspensions used in Examples 1, Comparative Example 1, and Examples 2-3 are different. The magnetic bead suspension prepared in Examples 2-3 can efficiently purify RNA, reverse transcribe the high-purity RNA into cDNA, and then perform qRT-PCR experiments using cDNA as a template, thereby reducing the impact of impurities on qRT-PCR, thereby improving the specificity of the detection kit for gastric signet ring cell carcinoma biomarkers of the present invention; the specificity of the detection kit for gastric signet ring cell carcinoma biomarkers in Example 2 is higher than that in Example 3 because the amount of magnetic bead suspension used in Examples 2 and 3 is different. This shows that the detection kit for gastric signet ring cell carcinoma biomarkers of the present invention has excellent specificity.

[0140] The specificity of the detection kit for gastric signet ring cell carcinoma biomarkers in Examples 4-5 of the present invention is higher than that in Example 2 and Comparative Example 2. This is because the magnetic bead suspensions used in Examples 2, Comparative Example 2, and Example 4-5 are different. The magnetic bead suspensions prepared in Example 4-5 can further optimize the purification and recovery efficiency of RNA, reverse transcribe the high-purity RNA into cDNA, and then use cDNA as a template for qRT-PCR experiments, thereby reducing the impact of impurities on qRT-PCR, thereby further optimizing the specificity of the detection kit for gastric signet ring cell carcinoma biomarkers of the present invention; the specificity of the detection kit for gastric signet ring cell carcinoma biomarkers in Example 4 is higher than that in Example 5 because the amount of magnetic bead suspensions used in Examples 4 and 5 is different. This shows that the present invention uses the magnetic bead suspension prepared in Example 4 to form a detection kit for gastric signet ring cell carcinoma biomarkers, which can further optimize the specificity of the detection kit for gastric signet ring cell carcinoma biomarkers.

[0141] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0142] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any changes or modifications made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A detection kit for gastric signet ring cell carcinoma biomarkers, comprising: Total RNA extraction reagent, RNA purification reagent, cDNA first-strand synthesis premix reagent, rapid fluorescence quantitative premix reagent (SYBR Green), nuclease-free double-distilled water, and specific primers; The RNA purification reagent comprises at least isopropanol, chloroform and anhydrous ethanol; The specific primers include an upstream specific primer and a downstream specific primer. The nucleotide sequence of the upstream specific primer is 5'-TTCTCGTGAAGCCCTATGA-3', and the nucleotide sequence of the downstream specific primer is 5'-GAGGCAGTGTTGATGATTTT-3'.

2. A detection kit for gastric signet ring cell carcinoma biomarkers according to claim 1, characterized in that: The biomarker for gastric signet ring cell carcinoma is prostaglandin epoxide synthase 2.

3. A detection kit for gastric signet ring cell carcinoma biomarkers according to claim 1, characterized in that: The RNA purification reagent includes isopropanol, chloroform, anhydrous ethanol and magnetic bead suspension.

4. A detection kit for gastric signet ring cell carcinoma biomarkers according to claim 3, characterized in that: In the preparation of the magnetic bead suspension, oleic acid-modified ferroferric oxide magnetic particles are first reacted with ethyl silicate under the action of ammonia water, and then reacted with diethylenetriaminopropyltrimethoxysilane and 5-chloro, 4-methylvaleryl chloride. After magnetic separation, an ethanol solution is added and ultrasonically dispersed to obtain a magnetic bead suspension.

5. A detection kit for gastric signet ring cell carcinoma biomarkers according to claim 4, characterized in that: The usage ratio of the oleic acid modified ferroferric oxide magnetic particles and ammonia water is 1 g: 5-20 mL.

6. A detection kit for gastric signet ring cell carcinoma biomarkers according to claim 4, characterized in that: The usage ratio of the oleic acid modified ferroferric oxide magnetic particles and ethyl silicate is 1 g: 2-10 mL.

7. A detection kit for gastric signet ring cell carcinoma biomarkers according to claim 4, characterized in that: The usage ratio of the oleic acid modified ferroferric oxide magnetic particles and diethylenetriaminopropyltrimethoxysilane is 1 g: 2-10 mL.

8. A detection kit for gastric signet ring cell carcinoma biomarkers according to claim 4, characterized in that: The mass ratio of the oleic acid modified ferroferric oxide magnetic particles to 5-chloro, 4-methylvaleryl chloride is 1:2-10.

9. A detection kit for gastric signet ring cell carcinoma biomarkers according to claim 4, characterized in that: The mass concentration of the ethanol solution is 75-95%.

10. A detection kit for gastric signet ring cell carcinoma biomarkers according to claim 4, characterized in that: The usage ratio of the oleic acid modified ferroferric oxide magnetic particles and the ethanol solution is 1 g: 50-150 mL.

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