A gastric cancer biomarker and its application

Abstract

The present invention relates to a gastric cancer biomarker, specifically PISD. The inventors experimentally analyzed differentially metabolized lipids in gastric cancer tissue and adjacent normal tissue and found elevated levels of PE in gastric cancer tissue. They then identified PISD, the primary differentially metabolized enzyme that regulates PE. Subsequently, through database searches combined with experimental studies, they found that PISD is upregulated in gastric cancer. Downregulating PISD can inhibit the proliferation, migration, and invasion of gastric cancer and affect mitochondrial function. Therefore, they hypothesized that PISD could serve as a gastric cancer biomarker. By testing PISD expression in patient samples, early diagnosis of gastric cancer can be achieved, which has important implications for clinical gastric cancer diagnosis and efficacy testing and evaluation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a gastric cancer biomarker and application thereof. Background Art

[0002] Gastric cancer is a global health concern, with over 1 million new cases diagnosed and 769,000 deaths in 2020. Risk factors for the disease include Helicobacter pylori infection, age, high salt intake, and a diet low in fruits and vegetables. Gastric cancer is diagnosed histologically after endoscopic biopsy and staged using CT, endoscopic ultrasound, PET, and laparoscopy. It is a highly molecularly and phenotypic heterogeneous disease. Gastric cancer is a common malignancy with a poor prognosis, and advanced gastric cancer is generally treated with preventive and chemotherapy. Despite advances in surgical techniques, radiotherapy, chemotherapy, and neoadjuvant therapy, gastric cancer remains the third leading cause of cancer death worldwide. Although the 5-year survival rate for early-stage gastric cancer can reach >90%, the low rate of early diagnosis means that most patients present with advanced-stage disease. Therefore, prevention, early detection, and early treatment of gastric cancer remain significant challenges.

[0003] Phospholipids are the primary structural and functional components of biological membranes in most organisms, controlling their homeostasis. Phosphatidylethanolamine (PE) is one of the most abundant phospholipids in the body. PE is synthesized primarily through two pathways, one of which is decarboxylation in mitochondria by phosphatidylserine decarboxylase (PISD). PISD, located primarily in the inner mitochondrial membrane, catalyzes the decarboxylation of phosphatidylserine (PS) to PE. PISD regulates multiple aspects of mitochondrial function, playing a particularly crucial role in mitochondrial PE synthesis. Silencing PISD in mammals leads to mitochondrial defects and altered mitochondrial morphology in response to mitochondrial PE deficiency. Mitochondria are key energy-producing organelles and cellular sources of reactive species. They manage cell lifespan and death through the balance of homeostasis and structural networks. Mitochondria are essential for energy metabolism, apoptosis regulation, and cell signaling. Mitochondrial metabolism supports tumor anabolic metabolism by providing key metabolites for macromolecular synthesis and generating oncometabolites to maintain the cancer phenotype. In addition, several clinical trials are testing the efficacy of inhibiting mitochondrial metabolism as a novel cancer treatment. Mitochondria are bioenergetics and biosynthetic organelles that absorb substrates from the cytoplasm and use them to perform a range of metabolic tasks. Mitochondria in malignant cells differ structurally and functionally from those in normal cells, becoming even more important and intriguing in the cancer environment and actively participating in metabolic reprogramming. Due to altered mitochondrial metabolism and changes in membrane potential, cancer cells are more susceptible to mitochondrial-targeted therapies. Loss of functional mitochondria may lead to a halt in cancer progression or cancer cell death.

[0004] Literature has reported that breast cancer cells overexpressing PISD exhibit reduced tumor initiation potential in high-throughput microfluidic mammosphere devices and mouse xenograft models. Other studies have also shown that downregulating PISD in breast cancer cells delays autophagy. However, there are currently no reports on the role of PISD in gastric cancer. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art in the early diagnosis of gastric cancer and to provide a gastric cancer biomarker.

[0006] Technical Solution

[0007] Application of PISD (phosphatidylserine decarboxylase) as a biomarker in the preparation of diagnostic reagents for gastric cancer.

[0008] The inventors conducted lipidomics and metabolomics analysis (liposome analysis) of clinical gastric cancer tissues and adjacent tissues and found that PE was upregulated in gastric cancer tissues. They then selected a variety of gastric cancer cell lines and verified that PISD was highly expressed in gastric cancer cells compared with normal gastric epithelial cells. Through subcutaneous tumorigenesis experiments in nude mice, they found that downregulation of PISD expression could significantly inhibit the tumorigenicity of gastric cancer cells, while upregulation of PISD expression could increase the tumorigenicity of gastric cancer cells.

[0009] The gastric cancer diagnostic reagent comprises specific primers for detecting the expression level of the biomarker PISD, the sequence of which is:

[0010] Forward primer: 5'-CTTTGTACAAGTCAGTGCCAAC-3',

[0011] Back primer: 5′-CCAGATGTACAGGCTGTAGAC-3′.

[0012] Beneficial effects of the present invention:

[0013] The inventors conducted experimental analysis of differential metabolic lipids in gastric cancer tissues and adjacent normal tissues and found that PE was elevated in gastric cancer tissues, and then found the main metabolic differential enzyme PISD that regulates it. Subsequently, through database consultation combined with experimental research, they found that PISD was upregulated in gastric cancer, and downregulating PISD could inhibit the proliferation, migration and invasion of gastric cancer, and affect mitochondrial function. Therefore, it is speculated that PISD can be used as a biomarker for gastric cancer, and early diagnosis of gastric cancer can be achieved by testing the expression level of PISD in patient samples; molecular targeted agents can also be developed based on PISD as a potential target for the clinical treatment of gastric cancer; PISD can also be used as a molecular probe to explore a molecular tool for research related to mitochondrial dysfunction and gastric cancer, thereby improving the survival rate of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of lipidomics analysis in gastric cancer tissues and adjacent tissues are shown in Figure 2.

[0015] Figure 2 The test data for the expression level of PISD in gastric cancer samples and normal gastric epithelial tissues;

[0016] Figure 3 The relationship between PISD expression level and overall survival of gastric cancer patients;

[0017] Figure 4 The results of immunohistochemistry experiments on clinical gastric cancer tissue samples and gastric adjacent tissue samples;

[0018] Figure 5 Western blot test results of PISD protein expression levels in human normal gastric epithelial cells GES-1 and human gastric cancer cell lines MGC-803, SGC-7901, AGS, HGC-27, MKN28, and MKN45;

[0019] Figure 6 Western blot quantitative analysis results of PISD expression levels in AGS and MKN45 gastric cancer cell lines that stably low-express PISD;

[0020] Figure 7 The results of the proliferation activity experiment of gastric cancer cell lines with PISD knockdown are shown;

[0021] Figure 8 The results of the plate cloning experiment of the gastric cancer cell line with PISD knockdown are shown;

[0022] Figure 9 The results of in vitro transmembrane migration and invasion ability test of AGS gastric cancer cell line with PISD knockdown;

[0023] Figure 10 The results of in vitro transmembrane migration and invasion ability test of PISD-knockdown MKN45 gastric cancer cell line;

[0024] Figure 11 The results of qRT-PCR detection of PISD mRNA levels in MGC-803 cells transfected with overexpressed PISD and MGC-803 cells;

[0025] Figure 12 The results of Western Blot analysis of PISD protein levels in MGC-803 cells transfected with PISD and MGC-803 cells overexpressing PISD;

[0026] Figure 13The results of the proliferation ability experiment of gastric cancer cell lines overexpressing PISD are shown;

[0027] Figure 14 The results of the plate cloning experiment of gastric cancer cell lines overexpressing PISD;

[0028] Figure 15 The results of in vitro transmembrane migration and invasion test of gastric cancer cell lines overexpressing PISD;

[0029] Figure 16 Images obtained by monitoring tumor formation in nude mice using an in vivo real-time imaging system;

[0030] Figure 17 The tumor weight test results of the PISD overexpression group and the negative control group after 30 days of tumor growth;

[0031] Figure 18 The figures are histopathological and immunohistochemical staining of transplanted tumor specimens from nude mice with gastric cancer subcutaneous transplanted tumor models in the PISD overexpression group and the negative control group;

[0032] Figure 19 Transmission electron microscopy images of MGC-803 cells in the PISD overexpression group and the negative control group. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be further described below with reference to specific embodiments of the drawings.

[0034] Example 1 Changes of Phosphatidylethanolamine (PE) in Gastric Cancer Tissue and Para-Cancerous Tissue

[0035] Source of clinical specimens: 29 cases of clinical gastric cancer tumor tissues and 29 cases of clinical adjacent normal tissues from the First Affiliated Hospital of Anhui Medical University.

[0036] To investigate the changes in lipid metabolism in gastric cancer tissues, UPLC-MS / MS-based lipidomics analysis was performed on gastric cancer tissues and adjacent adjacent tissues in the specimens. Figure 1 The results of lipidomics analysis in gastric cancer tissues and adjacent adjacent tissues are shown. The colors are proportional to the changes in metabolites (red, up-regulated, positive correlation; blue, down-regulated, negative correlation; rows: metabolites, columns: samples). It can be seen that PE-related metabolites in gastric cancer tissues have changed significantly.

[0037] Example 2

[0038] To further explore the relevance of PISD in gastric cancer, we detected the expression levels of PISD in 415 gastric cancer samples and 34 normal gastric epithelial tissues based on the TCGA dataset, and performed statistical analysis based on these test data. The results are shown in Figure 2 .

[0039] Figure 2 Figure 2 is the test data of the expression level of PISD in gastric cancer samples and normal gastric epithelial tissues. It can be seen that the expression level of PISD in gastric cancer tissues is higher than that in normal gastric epithelial tissues.

[0040] By analyzing the Kaplan-Meier Plotter database (http: / / kmplot.com / analysis / index.php), it can be concluded that PISD expression is associated with the (5-year survival rate) and overall survival of gastric cancer patients. Figure 3 .

[0041] Example 3

[0042] To confirm whether PISD is upregulated in gastric cancer, immunohistochemical staining was performed on 29 clinical gastric cancer tissue samples and 29 adjacent normal tissues (specimens were obtained from the First Affiliated Hospital of Anhui Medical University) using the following procedures:

[0043] (1) Baking: Place the slices in a 37°C incubator and bake for 30-60 minutes;

[0044] (2) Dewaxing: Soak the slices in xylene I for 10 min → xylene II for 10 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → 90% ethanol for 5 min → 80% ethanol for 2 min → 70% ethanol for 2 min → ddH2O (twice) for 2 min;

[0045] (3) Antigen retrieval (microwave retrieval method): Prepare 1 L of sodium citrate solution in advance, place the slice rack vertically in a beaker, and make sure that the sodium citrate solution completely covers the tissue in the slice. Place it in a microwave oven on high heat for 8 minutes, stop for 2 minutes, and then on high heat for another 2 minutes. Allow to cool naturally to room temperature.

[0046] (4) Block endogenous peroxidase: add an appropriate amount of endogenous peroxidase blocker and incubate at room temperature for 10 minutes; wash with PBS three times, each time for 3 minutes;

[0047] (5) Adding primary antibody: Use an immunohistochemistry pen to draw a circle around the tissue. Depending on the size of the tissue, add 100 μL or an appropriate amount of primary antibody. Place the slice in a humidified box and refrigerator at 4°C overnight.

[0048] (6) Add enzyme-labeled goat anti-mouse / rabbit IgG polymer: After the sample is taken out of the refrigerator at 4°C overnight, it needs to be incubated at room temperature for 15 minutes to rewarm, and washed with PBS three times, each time for 3 minutes; add 100 μL or an appropriate amount of enzyme-labeled goat anti-mouse / rabbit IgG polymer, incubate at 37°C for 30 minutes; wash with PBS three times, each time for 3 minutes;

[0049] (7) Color development: Add an appropriate amount of freshly prepared DAB color development solution and observe under a microscope. The positive signal is brown or brown. The time should be controlled well to avoid excessive color development. Rinse with tap water to stop the color development.

[0050] (8) Counterstaining: Hematoxylin staining for a few seconds; hydrochloric acid differentiation, washing and blueing;

[0051] (9) Sealing: Add a drop of neutral gum next to the tissue, then cover with a cover glass, first lay one side flat, then gently put down the other side to avoid bubbles. The sealed slices are placed flat in a fume hood to dry;

[0052] (10) The dried sections can be observed under a microscope or scanned.

[0053] The immunohistochemical results of clinical gastric cancer tissue samples and gastric adjacent tissue samples are shown in Figure 4 , Figure 4 The results showed that the PISD protein level in clinical gastric cancer tissue samples was significantly higher than that in gastric adjacent cancer tissue samples from the same source.

[0054] Example 4

[0055] Western blot experiments were used to detect the protein expression levels of PISD in human normal gastric epithelial cells (GES-1) and human gastric cancer cell lines (MGC-803, SGC-7901, AGS, HGC-27, MKN28 and MKN45).

[0056] Cell sources: The cell lines used in this experiment were GES-1 (a normal human gastric epithelial cell line); AGS (a poorly differentiated human gastric adenocarcinoma cell line); MKN-45 (a poorly differentiated human gastric carcinoma cell line); MGC-803 (a poorly differentiated human gastric adenocarcinoma cell line); SGC-7901 (a moderately differentiated human gastric adenocarcinoma cell line); HGC-27 (an undifferentiated human gastric carcinoma cell line); and MKN-28 (a highly metastatic human gastric cancer cell line). GES-1, SGC-7901, MGC-803, HGC-27, MKN28, and MKN45 were purchased from the Shanghai Cell Bank; AGS was purchased from ATCC.

[0057] The Western blot test results of the protein expression levels of PISD in human normal gastric epithelial cells GES-1 and human gastric cancer cell lines MGC-803, SGC-7901, AGS, HGC-27, MKN28, and MKN45 are shown in Figure 5 , *p<0.05, **p<0.01, ***p<0.001. It can be seen that the protein expression of PISD in normal gastric epithelial cells (GES-1) was significantly lower than that in human gastric cancer cell lines (MGC-803, SGC-7901, AGS, HGC-27, MKN28 and MKN45).

[0058] Example 5

[0059] To investigate the effect of PISD on the development of gastric cancer, we used shRNA lentivirus to knock down PISD in AGS and MKN45 gastric cancer cell lines and constructed AGS and MKN45 gastric cancer cell lines that stably low-express PISD. The method is as follows:

[0060] (1) Cell transfection experiment

[0061] On the first day, 30,000 to 50,000 cells were seeded into each well of a six-well plate and incubated at 37°C to ensure that the cell confluence was approximately 20-30% when the cells were infected on the second day.

[0062] On the second day, discard the culture medium and calculate the volume of virus added based on an MOI of 10. Pre-mix complete culture medium, virus, and enhancement solution and add to each well. After 12 hours, observe the cell growth status and change the cell medium to maintain cell activity.

[0063] (2) After 48-72 hours, observe the transfection efficiency of cells under a fluorescence microscope, and a large area of ​​cells will be seen to emit green fluorescence.

[0064] (3) Add puromycin at a concentration of 2 μg / mL to screen out cells infected with the virus. Expand the cell culture, and use a culture medium containing 2 μg / mL puromycin for each culture. Observe the cell status and fluorescence intensity. After about two weeks, a polyclonal cell line stably expressing the target gene can be obtained. After the cells have stably expressed the target gene, they can be cultured in a normal complete culture medium.

[0065] (4) Transfection efficiency identification: Cell RNA was extracted and reverse transcribed, and the PISD mRNA level was identified by qRT-PCR. Western blot experiments were used to identify the PISD protein interference / overexpression efficiency.

[0066] The qRT-PCR reaction procedure was as follows: 95°C pre-denaturation for 1 minute, 95°C denaturation for 20 seconds, 60°C for 1 minute, and 35 to 45 cycles. A 20 μl reaction system consisted of: 6.5 μl of ddH2O, 3 μl of DNA template, 3 μl of 2 μM primers, and 10 μl of qPCR Super Mix Plus. The qRT-PCR reaction system is shown in Table 1, and the primers used for qRT-PCR are shown in Table 2.

[0067] Table 1 qRT-PCR premix system

[0068]

[0069] Table 2 Primer sequence information

[0070]

[0071]

[0072] Figure 6 Western blot quantitative analysis results of PISD expression levels in AGS and MKN45 gastric cancer cell lines that stably low-express PISD. Figure 6 A is the PISD expression level in the AGS gastric cancer cell line in the PISD knockdown group, Figure 6 B is the PISD expression level in the MKN45 gastric cancer cell line in the PISD knockdown group, *p<0.05, **p<0.01, ***p<0.001. The results showed that the PISD protein expression in the AGS and MKN45 gastric cancer cell lines in the PISD knockdown group was significantly reduced, and the knockdown efficiency reached more than 50%.

[0073] Example 6

[0074] 1. CCK8 activity assay was used to analyze the proliferation activity of gastric cancer cell lines with PISD knockdown:

[0075] (1) First, determine the number of cells to be seeded. Depending on the selected number of culture days, seed 2,000 to 3,000 cells in a 96-well plate and select the number of cells that will grow to approximately 95% after 48 to 96 hours.

[0076] (2) Digest and centrifuge the cells according to conventional methods, then count them using a counting plate. Take the previously determined number of cells and inoculate them evenly and quickly in a 96-well plate, following the principle that each well contains the same number of cells in 100 μL of culture medium. Try to inoculate them as evenly as possible. Set up a blank control well, that is, a zero well (no cells, only culture medium). Set up 5 to 6 replicate wells for the same well.

[0077] (3) Incubate overnight in a 37°C, 5% CO2 incubator. Incubate in the incubator for 0h, 24h, 48h, and 72h. Collect the plate after 0h, 24h, 48h, and 72h. Protect the cells from light. Add 10μL of CCK8 solution to every 100μL of culture medium. Calculate the required volume of CCK8 solution in advance, mix it with the culture medium in advance, and then add it to each well. Incubate in a 37°C incubator in the dark for 2h.

[0078] (4) Every 30 minutes, take out the 96-well plate and measure the absorbance of each well at 450 nm using a microplate reader.

[0079] The results of the proliferation activity experiment of gastric cancer cell lines with PISD knockdown are shown in Figure 7 ,in, Figure 7A is aimed at the gastric cancer AGS cell line with PISD knockdown, Figure 7 B targets the PISD-knockdown gastric cancer MKN45 cell line. It can be seen that the cell proliferation of the PISD-knockdown gastric cancer AGS and MKN45 cell lines was significantly inhibited compared with the negative control group (Vector group).

[0080] 2. Plate cloning experiments were conducted on AGS and MKN45 gastric cancer cells with PISD knockdown:

[0081] (1) Gastric cancer cells in the logarithmic growth phase were selected and digested, centrifuged, counted, and graded diluted according to conventional methods. 1000 cells were taken per well and inoculated into a six-well plate. The plates were gently shaken to evenly disperse the cells.

[0082] (2) Place the cells outside to settle for a few minutes, then place them in a 37°C, 5% CO2 incubator for culture. Do not move them in the first few days because there are very few cells. Avoid moving them so that they do not adhere to the wall. After 2 to 3 weeks of culture, change the medium as needed.

[0083] (3) After visible cell clusters appear, collect the plate, wash it twice with PBS, fix it with formalin at room temperature for 30 minutes, stain it with 0.1% crystal violet for 15 minutes, wash it three times with PBS, dry it in air at room temperature, and take photos.

[0084] The results are as follows Figure 8 As shown, Figure 8 A is aimed at the gastric cancer AGS cell line with PISD knockdown, Figure 8 Figure B shows the PISD knockdown in the MKN45 gastric cancer cell line. Compared with the negative control group (vector group), the PISD knockdown group showed poorer colony formation ability. This is consistent with the results of the CCK8 experiment, indicating that knocking down PISD expression can inhibit gastric cancer cell proliferation.

[0085] 3. Transwell migration and invasion assays were used to detect the in vitro transmembrane migration and invasion abilities of PISD-knockdown gastric cancer cell lines. The experimental methods are as follows:

[0086] 1) One day in advance, cells were starved with serum-free medium for 12 h to further eliminate the effects of serum;

[0087] 2) Take out the aliquoted Matrigel stored at -20℃ and melt it in a 4℃ refrigerator 3-4 hours in advance. Pre-cool the yellow pipette tip in a -20℃ refrigerator 1 hour in advance. Use the pre-cooled pipette tip to dilute the Matrigel with serum-free culture medium at a ratio of 1:8. The entire process should be performed on ice. Add 40μL of diluted Matrigel to the upper chamber of the bottom membrane of each Transwell chamber, covering the entire polyester membrane, and place it in a cell culture incubator for 1 to 4 hours to allow the Matrigel to solidify. Matrigel is liquid at 4℃ and will gradually solidify into a gel at 37℃, which is irreversible. Remember to operate on ice;

[0088] 3) The next day, cells were digested and centrifuged as usual, and finally digested with serum-free medium. The cells were washed twice with PBS, centrifuged again, and resuspended in serum-free medium to adjust the cell concentration to 1×10 5 / mL;

[0089] 4) Add 500 μL of complete medium containing serum to the lower chamber of a 24-well plate to create a serum concentration gradient, which can be appropriately increased. Place the Transwell chamber into the 24-well plate, ensuring that there are no bubbles between the bottom membrane of the chamber and the complete medium below.

[0090] 5) Take 200 μL of the prepared cell suspension and add it to the upper chamber of the Transwell chamber, trying to drop it in the middle;

[0091] 6) Place in a 37°C, 5% CO2 incubator and incubate for 24 to 48 hours (the specific time for membrane penetration should be determined based on your own experiments);

[0092] 7) After 24-48 hours, discard the culture medium in the 24-well plate and chambers, wash twice with PBS, and briefly air-dry the chambers.

[0093] 8) Add 500 μL of formalin solution to each well of a 24-well plate and fix for 30 minutes. Place the chamber in the 24-well plate and stain with 0.1% crystal violet for 15 minutes (either air-dry or wash three times with PBS). Recover the staining solution, gently wipe the chamber clean with cotton wool dipped in PBS, rinse the chamber gently in PBS, dry at room temperature, photograph, and count.

[0094] Figure 9 The results of the in vitro transmembrane migration and invasion test of AGS gastric cancer cell lines with PISD knockdown are shown in Figure 2. Figure 9 A is the migration ability test result, Figure 9 B is the result of invasive ability test. Figure 10 The results of in vitro transmembrane migration and invasion ability test of PISD-knockdown MKN45 gastric cancer cell line are shown in Figure 2. Figure 10 A is the migration ability test result, Figure 10 B shows the results of the invasion ability test. *p<0.05, **p<0.01, ***p<0.001 show that the average number of AGS gastric cancer cells crossing the chamber basement membrane in the PISD knockdown group was significantly reduced compared to the negative control group (Vetor group), indicating that the migration ability of gastric cancer cells was significantly inhibited by PISD knockdown. The same results were obtained in the MKN45 gastric cancer cell line. The two experimental results are consistent, demonstrating that downregulating PISD expression can inhibit the migration and invasion ability of gastric cancer cells.

[0095] Example 7

[0096] To confirm that upregulation of PISD can promote the development of gastric cancer, we used lentiviral vectors to overexpress PISD in MGC-803 gastric cancer cell lines:

[0097] (1) Cell transfection experiment

[0098] On the first day, 30,000 to 50,000 cells were seeded into each well of a six-well plate and incubated at 37°C to ensure that the cell confluence was approximately 20-30% when the cells were infected on the second day.

[0099] On the second day, discard the culture medium and calculate the volume of virus added based on an MOI of 10. Pre-mix complete culture medium, virus, and enhancement solution and add to each well. After 12 hours, observe the cell growth status and change the cell medium to maintain cell activity.

[0100] (2) After 48-72 hours, observe the transfection efficiency of cells under a fluorescence microscope, and a large area of ​​cells will be seen to emit green fluorescence.

[0101] (3) Add puromycin at a concentration of 2 μg / mL to screen out cells infected with the virus. Expand the cell culture and use a culture medium containing puromycin for each subculture. Observe the cell status and fluorescence intensity. After about two weeks, a polyclonal cell line stably expressing the target gene can be obtained. Only after the cells have stably expressed the target gene can they be cultured in a normal complete culture medium.

[0102] (4) Transfection efficiency identification: After collecting each cell and extracting total RNA, qRT-PCR was used to identify the PISD mRNA level. After extracting total protein, Western Blot was used to detect the PISD protein interference / overexpression efficiency.

[0103] The results of qRT-PCR detection of PISD mRNA levels in MGC-803 cells transfected with overexpressed PISD and MGC-803 cells are shown in Figure 2. Figure 11 As shown, compared with the negative control group (Vector group), the mRNA expression level in the gastric cancer MGC-803 cell line overexpressing PISD was significantly increased.

[0104] The results of Western Blot analysis of PISD protein levels in MGC-803 and MGC-803 cells transfected with PISD overexpression are shown in Figure 2. Figure 12 As shown, compared with the negative control group (Vector group), the protein expression of PISD in the MGC-803 gastric cancer cell line overexpressing PISD was significantly increased.

[0105] Example 8

[0106] 1. The CCK8 assay was used to detect the proliferation ability of gastric cancer cell lines overexpressing PISD.

[0107] The results of the proliferation ability experiment of gastric cancer cell lines overexpressing PISD are shown in Figure 13 Compared with the negative control group (Vector group), the cell proliferation ability of the MGC-803 gastric cancer cell line overexpressing PISD was increased.

[0108] 2. Perform plate cloning experiments on gastric cancer cell lines overexpressing PISD.

[0109] The results of the cloning plate experiment are shown in Figure 14 Compared with the negative control group (Vector group), the colony-forming ability of the MGC-803 gastric cancer cell line overexpressing PISD was enhanced. This is consistent with the results of the CCK8 experiment, indicating that overexpression of PISD can promote the proliferation of gastric cancer cells.

[0110] 3. The Transwell assay was used to detect the in vitro transmembrane migration and invasion ability of gastric cancer cell lines overexpressing PISD.

[0111] The results of the in vitro transmembrane migration and invasion ability experiments of gastric cancer cell lines overexpressing PISD are shown in Figure 15 ,in, Figure 15 A is the migration ability test result, Figure 15 B is the invasion ability test result. It can be seen that the number of transmembrane cells in the MGC-803 gastric cancer cell line overexpressing PISD is significantly increased compared with the negative control group (Vetor group), indicating that the migration and invasion ability of MGC-803 gastric cancer cells overexpressing PISD is enhanced.

[0112] Example 9

[0113] Nude mouse subcutaneous transplant tumor experiment:

[0114] Twelve BALB / c male nude mice (6 mice per group) were purchased from Jiangsu Jicui Yaokang Biological Co., Ltd., aged 6 weeks and housed under SPF conditions. They were randomly divided into two groups (PISD overexpression group and negative control group), with 6 mice per group. MGC-803 gastric cancer cells stably expressing Vector / LV-PISD in the logarithmic growth phase were collected by trypsin digestion and centrifuged at 800 rpm for 3 minutes, and the supernatant was discarded. In a sterile environment, 0.1 ml was inoculated on the back of the nude mice, with 6 × 10 cells per mouse. 6 The nude mice were returned to their cages and kept for further care, with their condition observed. It was estimated that after one week, subcutaneous nodules approximately 5 mm in size would appear in all 12 nude mice, indicating successful establishment of the gastric cancer model. Tumor size was measured and the mice were weighed every three days. Tumor volume was calculated as V = 0.5 × major diameter × minor diameter squared. One month later, the nude mice were sacrificed, and the tumors and associated organs were removed and photographed. Some were frozen at -80°C, while others were immersed in formalin for subsequent experiments.

[0115] Figure 16 This is an image obtained by monitoring tumor formation in nude mice using a live real-time imaging system. It can be seen that the fluorescence intensity at the tumor site in the PISD overexpression group was significantly stronger than that in the control group.

[0116] Figure 17 Figure 2 is the tumor weight test result of the PISD overexpression group and the negative control group after 30 days of tumor growth. It can be seen that the tumor weight of the PISD overexpression group is slightly higher than that of the control group.

[0117] Immunohistochemical staining was performed on the transplanted tumor specimens of the nude mouse subcutaneous transplanted tumor model constructed by gastric cancer cells overexpressing PISD (and the nude mouse subcutaneous transplanted tumor model constructed by gastric cancer cells with negative empty vector was set as the control group). Figure 18 Figures 2 and 3 show histopathological (H&E) and immunohistochemical (IHC) staining of nude mouse gastric cancer subcutaneous xenograft tumor specimens from the PISD overexpression group and the negative control group. As can be seen, the expression of Ki67, a cell proliferation marker, was significantly elevated in the PISD overexpression group.

[0118] Example 10

[0119] To further investigate the potential molecular mechanism by which PISD promotes tumor development, transmission electron microscopy was used to observe the ultrastructure of MGC-803 cells in the PISD-overexpressing group and the negative control group (Vector group).

[0120] Figure 19 Figure 2 is a transmission electron micrograph of MGC-803 cells in the PISD overexpression group and the negative control group (Vector group). It can be seen that the mitochondrial cristae and membrane of the PISD overexpressing MGC-803 cells are more complete and abundant.

Claims

1. Application of a reagent for detecting the expression level of the biomarker PISD in the preparation of a gastric cancer diagnostic reagent.

2. The use according to claim 1, characterized in that The reagent for detecting the expression level of the biomarker PISD comprises a detection primer, the sequence of which is: Forward primer: 5'-CTTTGTACAAGTCAGTGCCAAC-3', Back primer: 5′-CCAGATGTACAGGCTGTAGAC-3′.

Images