Marker for diagnosis, treatment and prognosis of gastric cancer peritoneal metastasis and application thereof

Abstract

The embodiment of the invention discloses a marker for diagnosis, treatment and prognosis of gastric cancer peritoneal metastasis and application of the marker. The invention discloses application of a CYS1 gene as a marker in diagnosis and prognosis of gastric cancer peritoneal metastasis. A nucleotide sequence of the CYS1 gene is shown as SEQ ID NO.1. The invention discloses a new target CYS1 for promoting gastric cancer peritoneal metastasis, the action mechanism of the target in gastric cancer peritoneal metastasis is defined, molecular diagnosis of gastric cancer is facilitated, the target CYS1 can be used as a marker for diagnosis of gastric cancer peritoneal metastasis, and gastric cancer peritoneal metastasis can be inhibited by inhibiting expression of the target CYS1. The compound has certain application value in preparation of drugs for inhibiting gastric cancer peritoneal metastasis.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biotechnology, and specifically to a marker for diagnosis, treatment and prognosis of gastric cancer peritoneal metastasis and application thereof. Background Art

[0002] Gastric cancer is the fourth most common malignant tumor in the world, and its mortality rate ranks second in the world. Peritoneal metastasis is the most classic metastasis pattern of advanced gastric cancer. It not only seriously affects the quality of life of patients, but also significantly shortens the survival time of patients. It is also the main reason for the failure of gastric cancer treatment. Although there are more and more treatments for peritoneal metastasis, the efficacy is still poor. The fundamental reason is that there are still many unknowns about the mechanism of peritoneal metastasis. Therefore, in-depth exploration of the mechanism of peritoneal metastasis of gastric cancer and finding targeted therapeutic targets are issues that need to be urgently addressed. Summary of the invention

[0003] To this end, the embodiments of the present invention provide a marker for the diagnosis, treatment and prognosis of gastric cancer peritoneal metastasis and its application. The present invention discloses a new target that promotes gastric cancer peritoneal metastasis, clarifies the mechanism of action of the target in gastric cancer peritoneal metastasis, is helpful for the molecular diagnosis of gastric cancer, can be used as a marker for the diagnosis of gastric cancer peritoneal metastasis, and inhibiting its expression can inhibit gastric cancer peritoneal metastasis, which has certain application value in the preparation of drugs for inhibiting gastric cancer peritoneal metastasis.

[0004] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0005] According to a first aspect of an embodiment of the present invention, the present invention provides use of the CYS1 gene as a marker in the diagnosis and prognosis of gastric cancer peritoneal metastasis, characterized in that the nucleotide sequence of the CYS1 gene is shown in SEQ ID NO.1.

[0006] According to a second aspect of an embodiment of the present invention, the present invention provides use of CYS1 gene as a marker in the preparation of a drug for inhibiting peritoneal metastasis of gastric cancer.

[0007] Furthermore, the drug can inhibit the invasion and migration of gastric cancer cells.

[0008] According to a third aspect of an embodiment of the present invention, the present invention provides a drug for inhibiting peritoneal metastasis of gastric cancer, wherein the drug comprises a CYS1 gene expression inhibitor or a knockout agent.

[0009] According to a fourth aspect of an embodiment of the present invention, the present invention provides a biological product for diagnosis and prognosis of peritoneal metastasis of gastric cancer, including a reagent for detecting the expression level of CYS1 gene.

[0010] Furthermore, the biological product includes a kit and a chip.

[0011] The embodiments of the present invention have the following advantages:

[0012] 1. The present invention is based on the collection of a certain amount of clinical samples for research and verification. Therefore, the research results are in line with clinical practice and have good clinical application value.

[0013] 2. The present invention has demonstrated, through a series of in vitro cell experiments and in vivo human specimen experiments, that CYS1 is highly expressed in gastric cancer tissues. Patients with high expression of CYS1 have a poor prognosis and are more susceptible to peritoneal metastasis. It can be used as a potential predictive marker for gastric cancer peritoneal metastasis and a biomarker for therapeutic drugs or gastric cancer treatment targets.

[0014] 3. The present invention discovered a new gastric cancer peritoneal metastasis protein CYS1, which can promote the peritoneal metastasis of gastric cancer by inhibiting the adhesion between gastric cancer cells, promoting the adhesion between gastric cancer cells and peritoneal mesothelial cells, and promoting the invasion and migration of gastric cancer cells. The new CYS1 marker disclosed in the present invention can be helpful for the molecular diagnosis of gastric cancer, and inhibiting its expression may be a new targeted treatment method for gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0016] Figure 1 The present invention provides an analysis of CYS1 expression and its correlation with the prognosis of gastric cancer patients; wherein, (A) the comparative results of GSE62254 data set analysis of CYS1 expression in gastric cancer and adjacent tissues; (B) the result diagram of GSE62254 data set analysis of CYS1 expression and prognosis; (C) the result diagram of TCGA data set analysis of CYS1 expression and prognosis; (D) the result diagram of immunohistochemistry of gastric cancer and adjacent tissues; (E) the quantitative analysis results of CYS1 expression in 30 gastric cancer and adjacent tissues; (F) the comparative results of GSE62254 data set analysis of CYS1 expression in patients with peritoneal metastasis (PM) and without peritoneal metastasis (NPM); (G) the result diagram of analyzing CYS1 expression and the prognosis of gastric cancer patients using 59 patients; (H) the result diagram of 59 patients verifying the correlation between CYS1 expression and peritoneal metastasis in gastric cancer patients.

[0017] Figure 2The invention provides an in vitro expression detection of CYS1; wherein, (A) qRT-PCR detection results of CYS1 expression in normal gastric epithelial cells and different human gastric cancer cells; (B) Western blot detection results of CYS1 expression in normal gastric epithelial cells and different human gastric cancer cells; (C) Western blot and qRT-PCR detection results of transient silencing efficiency of CYS1; (D) Western blot and qRT-PCR detection results of CYS1 overexpression efficiency.

[0018] Figure 3 The in vitro experiments provided by the present invention confirm that CYS1 promotes peritoneal metastasis of gastric cancer; wherein, (A) the result of the hanging drop test to detect the adhesion ability of gastric cancer cells after silencing CYS1; (B) the result of the adhesion test to detect the adhesion ability between gastric cancer cells and human peritoneal mesothelial cells (HMrSV5) after silencing CYS1; (C) the result of the Transwell test to detect the migration and invasion ability of gastric cancer cells after silencing CYS1; (D) the result of the hanging drop test to detect the adhesion ability between gastric cancer cells after overexpressing CYS1; (E) the result of the Transwell test to detect the migration and invasion ability of gastric cancer cells after overexpressing CYS1; (F) the result of the adhesion test to detect the adhesion ability between gastric cancer cells and human peritoneal mesothelial cells (HMrSV5) after overexpressing CYS1.

[0019] Figure 4 The mouse peritoneal metastasis experiment provided by the present invention verifies the relationship between CYS1 and peritoneal metastasis; wherein, (A) the mouse peritoneal metastasis model verifies the changes in mouse peritoneal nodules after silencing CYS1; (B) the quantitative analysis results of mouse peritoneal metastasis nodules; (C) the results of immunohistochemical detection of CYS1 expression in mouse peritoneal metastasis tumors. DETAILED DESCRIPTION

[0020] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1 Expression of CYS1 in gastric cancer tissue

[0022] 1.1 Human specimens: 30 paired gastric cancer and adjacent tissue samples and 59 gastric cancer specimens were selected from gastric cancer patients who underwent subtotal gastrectomy at the First Affiliated Hospital of China Medical University. This study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University and the informed consent was signed. All samples were independently diagnosed histopathologically by two gastrointestinal pathologists, and those with consistent diagnostic results were included in the study.

[0023] 1.2 Analysis methods

[0024] The expression of CYS1 in gastric cancer and adjacent tissues and its effect on the prognosis of gastric cancer patients were analyzed through online databases.

[0025] Statistical analysis was performed using SPSS 23.0 software (SPSS, Chicago, IL, USA). Graphpad Prism 7 (USA) was used for image processing. The independent sample t test was used for data comparison. The t test was used for data that met the normal distribution. The Mann-Whitney U test was used for data that did not meet the normal distribution to evaluate the expression difference between the two groups. P < 0.05 was statistically significant.

[0026] 1.3 Results

[0027] The GSE62254 dataset suggests that CYS1 is highly expressed in gastric cancer tissues ( Figure 1 A). GSE62254( Figure 1 B) and TCGA( Figure 1 C) The data set suggests that gastric cancer patients with high expression of CYS1 have a worse prognosis (P<0.05). At the tissue level, 30 pairs of paired gastric cancer tissues and adjacent controls were used to detect the expression of CYS1, confirming that CYS1 is highly expressed in gastric cancer tissues (P<0.0001) ( Figure 1 D- Figure 1 E). Analysis of the GSE62254 dataset found that the expression of CYS1 in gastric cancer tissues of patients with peritoneal metastasis (PM) was significantly increased compared with patients without peritoneal metastasis (NPM) (P<0.05) ( Figure 1 F). Using tissues from 59 patients with gastric cancer after surgery, it was confirmed that patients with high CYS1 expression had a poor prognosis and more patients had peritoneal metastasis (P = 0.0178) ( Figure 1 G- Figure 1 H).

[0028] The above content indicates that CYS1 is highly expressed in gastric cancer tissues, and gastric cancer patients with high expression of CYS1 have a poor prognosis and are more likely to have peritoneal metastasis.

[0029] Example 2 CYS1 expression silencing and overexpression

[0030] 2.1 Experimental subjects

[0031] 1) Cell lines

[0032] The present invention uses human gastric cancer cell lines: HGC27 and SGC7901, which are purchased from the Institute of Basic Research, Chinese Academy of Medical Sciences, National Experimental Cell Resource Sharing Platform, and have passed STR identification and mycoplasma detection negative identification.

[0033] 2) Experimental animals

[0034] Male BALB / C immunodeficient mice, 4-6 weeks old and weighing 14-16 g, were purchased from Beijing Weitonglihua Experimental Technology Co., Ltd. The animals were kept in an SPF animal room, and all animal experimental operations complied with the "Regulations on Laboratory Animal Management" and had been approved by the Animal Welfare Ethics Committee of China Medical University (Ethics Approval No.: CMUKT2023193).

[0035] 2.2 Cell culture

[0036] Human gastric cancer cell lines HGC27 and SGC7901 were cultured in 1640 medium containing 10% inactivated fetal bovine serum. The cells were cultured in an incubator at 37°C and 5% CO2, and fresh medium was replaced every other day.

[0037] 1) Take out the cell cryopreservation tube from the liquid nitrogen storage tank and quickly place it in a 37℃ water bath to thaw;

[0038] 2) After thawing, centrifuge the cell suspension at 1000 rpm for 5 min;

[0039] 3) Discard the supernatant, add 1 mL of complete culture medium to fully disperse the cells, then transfer the cell suspension to a T25 cell culture flask containing 3 mL of complete culture medium, mark the cell type and date, gently shake in an "8" shape to evenly distribute it, let it stand in a biosafety cabinet for 10 minutes, and then put it in a cell culture incubator;

[0040] 4) When the cells in the culture flask reached 80-90%, they were digested with 0.25% trypsin containing EDTA and then subcultured (adherent cells).

[0041] Construction of CYS1 transient knockdown cells:

[0042] HGC27 and SGC7901 cells in the logarithmic growth phase were prepared into 1*10 5 The cells were seeded at a density of 100 μg / well in a 6-well plate. After the cells grew to a confluence of 20%-30%, CYS1 silencing siRNA was added to the 6-well plate. After 48 and 72 hours of transfection, CYS1 silencing efficiency was detected by RT-qPCR and Western Blot.

[0043] Construction of CYS1 stable knockdown and overexpression gastric cancer cell lines and corresponding empty vector control cell lines:

[0044] HGC27 and SGC7901 cells in the logarithmic growth phase were prepared into 1*10 5 / well were inoculated in a 6-well plate. After the cells grew to a confluence of 20%-30%, the lentivirus was used to infect the cells according to the virus titer (MOI=30) determined in the preliminary experiment. After 12-16 hours, the virus-containing culture medium was replaced with a complete culture medium. After continuing to culture for 72 hours, the infection efficiency was observed using a fluorescence microscope. Subsequently, a complete culture medium with a puromycin concentration of 3ug / ml was prepared and added to the HGC27 and SGC7901 cell culture flasks until there was no more cell death. The infection efficiency was detected by RT-qPCR and Western Blot, and the mixed cloned stable transfection cell line was screened.

[0045] Among them, the information of siRNA used for silencing CYS1 (denoted as Si1 and Si2) and shRNA for constructing a gastric cancer cell line with stable knockdown of CYS1 (denoted as shCYS1) is as follows:

[0046] Si1: 5'-GAGCGGCAAUGUCUCUGAA-3' (SEQ ID NO. 2);

[0047] Si2: 5'-CUCCCGGAUAUUCAAAGCU-3' (SEQ ID NO.3);

[0048] shCYS1: 5'-GAGCGGCAAUGUCUCUGAA-3' (SEQ ID NO. 4).

[0049] dTdT is hanging from the 3' ends of Si1 and Si2.

[0050] 2.3 Identification of CYS1 expression

[0051] (1) RT-PCR

[0052] 1) Extraction of total RNA from cells

[0053] A. Aspirate the culture medium and wash the cells twice with pre-cooled 1× PBS;

[0054] B. Add 1 ml of Trizol reagent to the T25 culture flask, pipette the cells off, transfer them to an RNase-free EP tube, and let stand at room temperature for 10 minutes;

[0055] C. Add 200ul of chloroform to the EP tube, shake vigorously for 15s, and let it stand for 15min after it turns strawberry milk color;

[0056] D. Centrifuge at 4°C and 12,000 rpm for 15 min, then take out carefully and gently;

[0057] E. Pipette the upper colorless aqueous phase into a new RNase-free EP tube, add 500ul of isopropanol, shake and mix, let stand for 10min, and then centrifuge at 4℃, 12000rpm for 10min;

[0058] F. Carefully discard the supernatant, add 75% ethanol prepared from enzyme-free water and anhydrous ethanol, turn it upside down to make the RNA pieces bounce up, and allow it to fully contact with ethanol for 1-2 minutes, then centrifuge at 4°C and 12,000 rpm for 5 minutes;

[0059] G. Carefully discard the supernatant, dry the EP tube at room temperature for about 5 minutes, dissolve it in 30-50ul enzyme-free water and test the RNA concentration.

[0060] 2) Reverse transcription experiment

[0061] The reverse transcription reagent was purchased from TAKARA, and the reaction system is as shown in Table 1 (all operations were performed on ice):

[0062] Table 1. Reaction system

[0063]

[0064] Reaction conditions: 37°C (15 min) → 85°C (5 s) → 4°C (END); the product obtained after reverse transcription was stored in a -20°C refrigerator.

[0065] 3) Fluorescence real-time quantitative PCR

[0066] The target gene primer sequences used in this study were synthesized by BGI Biotechnology Co., Ltd. The primer information is shown in Table 2:

[0067] Table 2. PCR primer sequences

[0068]

[0069] Real-time quantitative PCR reagents were purchased from TAKARA, and the reaction system is shown in Table 3:

[0070] Table 3. PCR reaction system

[0071]

[0072] The reaction conditions are as follows:

[0073] Pre-denaturation: 95℃30s

[0074]

[0075] (2) Western Blotting

[0076] 1) Total protein extraction and quantification

[0077] A. Aspirate the culture medium and wash the cells twice with pre-cooled 1× PBS;

[0078] B. Scrape the cells with a cell scraper, blow the cells off with a pipette, and collect the cell pellet by centrifugation;

[0079] C. Add 10ul phosphatase inhibitor, 10ul protease inhibitor and 10ul PMSF to each 1ml RIPA to prepare protein lysis solution;

[0080] D. Add 250ul of protein lysis buffer to each T25 flask of cells, mix thoroughly and transfer to an EP tube. After ultrasonic disruption, place on an ice bath shaker for 30 minutes. Mix the protein mixture every 10 minutes. Centrifuge at 4℃, 12000rpm for 30 minutes after 30 minutes.

[0081] E. Pipette the supernatant into a new pre-cooled EP tube, which is the total cell protein extracted;

[0082] F. Protein quantification using BCA protein concentration detection kit;

[0083] G. Finally, add Loading Buffer and RIPA to the total protein and quantify the protein to a concentration of 2ug / ul.

[0084] 2) Gel electrophoresis

[0085] A. Prepare 8%-20% separation gel and 5% stacking gel according to the molecular weight of the protein to be tested;

[0086] B. After the gel plate is rinsed with double distilled water and leak tested, add the prepared separation gel gently from left to right into the gap of the glass plate with a pipette until it reaches 7-7.5 ml. Then add double distilled water slowly and evenly from left to right to seal the upper layer. After the gel solidifies, pour out the water on the gel and absorb it with filter paper;

[0087] C. Add concentrated gel to the separation gel, insert the comb immediately after filling, and remove the gel plate from the holder after the concentrated gel solidifies;

[0088] D. Assemble the gel plates into the electrophoresis device, add the prepared 1× electrophoresis solution into the electrophoresis tank, make the liquid levels inside and outside the tank equal, and pull out the comb at a uniform speed;

[0089] E. Add 20-50ug protein to each well and add 3-5ul marker to both lanes;

[0090] F. The electrophoresis conditions are 80V for the concentrated gel and 110V for the separated gel. The electrophoresis is terminated when the blue band reaches the lowest point of the separated gel.

[0091] 3) Transfer and ECL development

[0092] A. Prepare 1× rapid transfer buffer and activate the PVDF membrane with methanol for 10 seconds;

[0093] B. Take out the glass plate and glue block, remove the glass plate, and cut off the concentrated glue and the excess glue on both sides and below;

[0094] C. Place the sandwich plate in the order of (negative electrode) black plate-sponge-filter paper-gel-membrane-filter paper-sponge-white plate (positive electrode), avoiding bubbles and keeping the membrane moist during the process;

[0095] D. Place the sandwich into the transfer tank, add ice packs and 1× rapid transfer solution until the sandwich is covered, transfer at 400 mA for 30 minutes;

[0096] E. Block the membrane with high-efficiency Western Blot blocking solution for 20 minutes;

[0097] F. Cut the PVDF membrane according to the molecular weight and place it in the antibody incubation box. Prepare the antibody incubation solution of corresponding concentration according to the antibody instructions and incubate it with the membrane at 4°C overnight;

[0098] G. After the primary antibody is incubated overnight, the PVDF membrane is removed and washed with PBST shaker for 10 min × 4 times, and then incubated with the secondary antibody prepared in the same proportion at 4°C for 2 h;

[0099] H. After the secondary antibody incubation, the membrane was washed on a shaker with PBST for 10 min × 4 times;

[0100] I. Prepare ECL luminescent liquid in a 1:1 ratio, place the membrane on the luminescent plate of the luminometer, slowly add the luminescent liquid to evenly spread it on the membrane, and adjust the contrast to observe the stripes.

[0101] 2.4 Results

[0102] qRT-PCR and Western blot were used to detect the expression of CYS1 in normal human gastric epithelial cells and human gastric cancer cells. The results showed that the expression level of CYS1 in human gastric cancer cells was significantly higher than that in normal human gastric epithelial cells GES-1 ( Figure 1 A- Figure 1B). SGC7901 and HGC27 cells were selected for further study. The results showed that transient silencing of CYS1 expression by CYS1 siRNA was performed. qRT-PCR and Western blot showed that the relative mRNA expression of CYS1 in the Si1 and Si2 groups was significantly reduced (P < 0.001), and the brightness of the CYS1 protein band was significantly weakened (P < 0.0001) ( Figure 2 C), the results showed that knocking down the CYS1 gene can effectively reduce the expression level of the CYS1 gene at the mRNA and protein levels. CYS1 was overexpressed using lentivirus, and qRT-PCR and Western blot showed that the relative mRNA expression of CYS1 in the CYS1 group was significantly higher than that in the Vector group, and the brightness of the CYS1 protein band was significantly enhanced (P < 0.0001) ( Figure 2 D), the results showed that overexpression of CYS1 gene could effectively increase the expression level of CYS1 gene at both mRNA and protein levels.

[0103] Example 3 In vitro test to detect the effect of CYS1 knockout and overexpression on peritoneal metastasis of gastric cancer

[0104] 3.1 Hanging drop experiment

[0105] The concentration was 2.5×10 4 / 20μL of HGC27 and SGC7901 cells were suspended in serum-free and antibiotic-free culture medium and gently dripped into the lid of a 100mm culture dish, and the droplets were collected by gravity at the bottom. After culturing at 37°C and 5% CO2 for 24 hours, the density of the tumor cell spheres was observed under an inverted microscope to determine the adhesion ability. The cell suspension was evenly blown 15 times with a 20μL pipette, and the degree of cell dispersion was observed again to determine the adhesion maintenance ability of the tumor cells. After blowing the cells, three fields of view were randomly selected for observation and counting for each drop of suspension. The cells were observed under a microscope.

[0106] 3.2 Adhesion test

[0107] 10ug / mL matrix gel was spread on the bottom of a 96-well plate at 37°C overnight, and the matrix gel was blocked with PBS containing 1% bovine serum albumin to prevent nonspecific adhesion. The treated SGC7901 and HGC27 were prepared into 4.5×104 / 200μL serum-free and antibiotic-free cell suspensions, respectively, and added to the 96-well plate. Incubate at 37°C, 5% CO2 for 30 minutes and 60 minutes, respectively, and wash with PBS three times to remove non-adherent cells. Then fix with 4% paraformaldehyde for 15 minutes and air dry for 30 minutes. Use Wright stain for 1 minute, and then mix and dilute Giemsa stain for 60 minutes. After natural drying, observe under an upright microscope, randomly select 4 fields of view from each well, and count the cells.

[0108] 3.3 Cell migration and invasion assay: Transwell assay

[0109] 1) Before preparing the cell suspension, the cells were starved of serum for 12-24 hours to further remove the effect of serum;

[0110] 2) Take cells in the logarithmic growth phase, digest with trypsin to obtain a cell suspension, and adjust the cell concentration to 3-5*10 5 / ml;

[0111] 3) After Matrigel was melted in a 4°C refrigerator, it was added to serum-free culture medium to prepare a 250ug / ml working solution. 100ul of the working solution was added to each well and incubated in a cell box overnight to prevent the liquid on the membrane from evaporating. The next day, the upper liquid culture medium was removed and discarded. A thin layer of Matrigel was observed to form at the bottom of the 6-well plate.

[0112] 4) Take 100ul of cell suspension and add it to the center of the upper chamber of the Transwell chamber (add Matrigel for invasion experiment, do not add Matrigel for migration experiment), ensure the liquid level, add 600ul of culture medium containing 10% serum to the lower chamber of the Transwell chamber and remove bubbles, and incubate together for 24-48h;

[0113] 5) After the incubation, the upper chamber liquid was aspirated, washed twice with calcium-free PBS, transferred to the wells pre-added with 800ul 4% paraformaldehyde, and fixed at room temperature for 30min;

[0114] 6) Take out the chamber, dry the fixative in the upper chamber, transfer to the well pre-added with about 600ul 0.1% crystal violet staining solution, and stain at room temperature for 20 minutes;

[0115] 7) Rinse and soak the chamber several times with PBS until there is no more purple staining in the PBS solution, aspirate the upper chamber liquid, and gently wipe off the unmigrated cells in the upper layer with a wet cotton swab, while keeping the membrane intact;

[0116] 8) Under a microscope, randomly select 5-9 fields of view at 200x magnification to observe and count the cells.

[0117] 3.4 Results

[0118] The hanging drop test showed that the adhesion ability between gastric cancer cells was enhanced after silencing CYS1 (P<0.05) ( Figure 3 A), adhesion test showed that the adhesion ability between gastric cancer cells and human peritoneal mesothelial cells (HMrSV5) decreased (P < 0.05) ( Figure 3 B), Transwell assay showed that silencing CYS1 reduced the invasion and migration abilities of gastric cancer cells ( Figure 3C). Overexpression of CYS1 showed the opposite result to the above. Specifically, overexpression of CYS1 decreased the adhesion ability between gastric cancer cells (P<0.05) ( Figure 3 D), the adhesion ability to human peritoneal mesothelial cells (HMrSV5) was enhanced (P < 0.001) ( Figure 3 E), Overexpression of CYS1 enhanced the invasion and migration abilities of gastric cancer cells ( Figure 3 F).

[0119] The above content shows that the expression level of CYS1 gene or protein is closely related to peritoneal metastasis of gastric cancer. CYS1 overexpression reduces the adhesion between gastric cancer cells, causing gastric cancer cells to fall off from the primary focus of gastric cancer, increases the adhesion between gastric cancer cells and peritoneal mesothelial cells, and promotes the invasion and migration of gastric cancer cells, leading to peritoneal metastasis of gastric cancer. Inhibiting CYS1 expression can inhibit the invasion and migration of gastric cancer cells, thereby reducing the occurrence of peritoneal metastasis of gastric cancer.

[0120] Example 4 In vivo test to detect the effect of CYS1 knockout on gastric cancer peritoneal metastasis

[0121] 4.1 Determination of the metastatic ability of transplanted tumors: peritoneal dissemination experiment

[0122] 1) Take the cells of the experimental group (SGC7901-shCYS1) and the control group (SGC7901-Ctrl) in the logarithmic growth phase, digest them with trypsin to obtain a cell suspension, and adjust the cell concentration to 2*10 6 / were injected into the abdominal cavity of nude mice (5 mice in each group) at the left lower abdomen to establish the abdominal dissemination model of transplanted tumor;

[0123] 2) Monitor the condition of nude mice daily and touch the abdominal cavity and abdominal wall for tumor formation;

[0124] 3) 16 days after injection, the nude mice were killed, photographed and dissected, and the tumor nodules in the abdominal cavity and on the abdominal wall were removed. One part was fixed in 10% neutral formalin, and one part of the tumor tissue was fixed in 10% neutral formalin. Tumor tissue wax blocks were then made for observation of the tumor tissue under a HE section microscope and immunohistochemical detection; one part was directly frozen in a deep freezer for RNA and protein detection experiments.

[0125] 4.2 Pathological morphology experiment: HE staining of paraffin tissue

[0126] 1) Cut 4 μm thick sections from the wax block onto a glass slide and bake in an oven at 65°C for 15-30 min;

[0127] 2) The baked slices were placed in xylene for 15 min, 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol for 5 min each, and distilled water for 1 min;

[0128] 3) Hematoxylin staining for 3 min - rinse with running water for 5 min - 1% hydrochloric acid alcohol for 10 s - rinse with water for 1 min - 1% ammonia water for blueing for 10 s - rinse with running water for 1 min - eosin staining for 1 min - rinse with distilled water for 20 s;

[0129] 4) 80% ethanol for 1 min - 90% ethanol for 3 min - 95% ethanol for 3 min - anhydrous ethanol for 5 min - xylene for 5 min, and seal the slides with neutral gum.

[0130] 4.3 Experimental Results

[0131] The mouse peritoneal metastasis model showed that knocking down CYS1 reduced the number of gastric cancer peritoneal metastases (P < 0.01) ( Figure 4 A- Figure 4 B), Immunohistochemistry showed that CYS1 expression was downregulated in mouse peritoneal metastasis after CYS1 knockdown ( Figure 4 C).

[0132] The above content shows that inhibiting CYS1 expression can effectively reduce the number of peritoneal metastases of gastric cancer, thereby inhibiting peritoneal metastasis of gastric cancer.

[0133] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

[0134]

[0135]

[0136]

[0137]

Claims

1. The use of CYS1 gene as a marker in the diagnosis and prognosis of gastric cancer peritoneal metastasis, characterized in that: The nucleotide sequence of the CYS1 gene is shown in SEQ ID NO.

1.

2. Application of CYS1 gene as a marker in the preparation of drugs for inhibiting peritoneal metastasis of gastric cancer.

3. The use according to claim 2, characterized in that: The drug can inhibit the invasion and migration of gastric cancer cells.

4. A drug for inhibiting peritoneal metastasis of gastric cancer, characterized in that: The drugs include CYS1 gene expression inhibitors or knockout agents.

5. A biological product for diagnosis and prognosis of gastric cancer peritoneal metastasis, characterized in that: The invention comprises a reagent for detecting the expression amount of the CYS1 gene.

6. The biological product according to claim 5, characterized in that The biological products include a test kit and a chip.

Images