Application of CD18 / integrin beta2 as gastric cancer peritoneal metastasis prediction marker and treatment target

Abstract

The invention discloses application of a detection reagent for the expression level of an ITGB2 gene or an encoded protein integrinbeta2 thereof in preparation of a product for assisting in predicting gastric cancer peritoneal metastasis, belongs to the field of oncomolecular biology and the field of medicine, and particularly relates to application of CD18 / integrinbeta2 serving as a gastric cancer peritoneal metastasis prediction marker and a treatment target. The effect and molecular mechanism of CD18 / integrinbeta 2 in gastric cancer peritoneal metastasis development are defined, meanwhile, the clinical transformation value of CD18 / integrinbeta 2 is focused, a potential drug treatment target of gastric cancer peritoneal metastasis is found, a target gene participated mechanism is targeted, and an antibody is applied to clinical transformation, so that the clinical treatment effect of the gastric cancer peritoneal metastasis is improved. Theoretical basis is provided for selection of gastric cancer peritoneal metastasis treatment targets, new treatment selection is provided for gastric cancer peritoneal metastasis patients, and important clinical significance is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of tumor molecular biology and medicine, and specifically relates to the application of CD18 / integrin β2 as a predictive marker and therapeutic target for gastric cancer peritoneal metastasis. Background Art

[0002] Gastric cancer is one of the most common malignant tumors worldwide, and its incidence and cancer-related mortality rate rank among the top five among all cancer types. Gastric cancer is highly prevalent in East Asian countries. In China, gastric cancer is the third most common type of cancer and the third leading cause of cancer death in 2022. The peritoneum is one of the most common metastatic sites of gastric cancer, and approximately 30% of gastric cancer patients will experience peritoneal metastasis. The prognosis of gastric cancer peritoneal metastasis is poor, and more than 50% of patients with gastric cancer peritoneal metastasis die within one year.

[0003] Currently, the National Comprehensive Cancer Network (NCCN) and the Chinese Society of Clinical Oncology (CSCO) guidelines still recommend systemic chemotherapy for patients with advanced gastric cancer peritoneal metastasis. However, because the peritoneal vascular barrier weakens the absorption and distribution of drugs in the peritoneum, gastric cancer peritoneal metastasis responds poorly to systemic chemotherapy, which is also an important reason for the poor prognosis of gastric cancer peritoneal metastasis. In terms of local abdominal treatment, although therapeutic intraperitoneal hyperthermic chemotherapy (HIPEC) combined with cytoreductive surgery (CRS) for advanced gastric cancer peritoneal metastasis can reduce the tumor burden of patients with gastric cancer peritoneal metastasis, it has not effectively improved the short-term survival of patients with gastric cancer peritoneal metastasis due to severe postoperative complications. Overall, whether it is systemic treatment or local intraperitoneal treatment, the drug treatment of gastric cancer peritoneal metastasis is still mainly chemotherapy, and the lack of effective therapeutic targets is an important reason for the development of treatment of gastric cancer peritoneal metastasis.

[0004] The peritoneum is a characteristic metastatic site of gastric cancer. The interaction between gastric cancer cells and the peritoneal mesothelial cells on its surface, especially the adhesion of gastric cancer cells to peritoneal mesothelial cells, may become an important link in breaking through the mechanism of gastric cancer peritoneal metastasis and finding potential therapeutic targets for gastric cancer peritoneum.

[0005] The integrin family is one of the most widely studied adhesion molecule families. As transmembrane proteins, they mainly mediate cell-extracellular matrix (ECM) adhesion and extracellular-intracellular signal transduction on the cell surface. Integirnβ2 is generally believed to be expressed only on the surface of leukocytes. It adheres to endothelial cells by recognizing intracellular adhesion molecules (ICAM) on the surface of vascular endothelial cells, mediating the recruitment cascade of leukocytes under inflammatory conditions. However, there are no reports on the expression and function of integrin β2 in tumor cells. Summary of the invention

[0006] In view of the defects of the prior art, the present invention mainly provides an application of a detection reagent for the expression level of ITGB2 gene or its encoded protein integrin β2 in the preparation of a product for assisting the prediction of peritoneal metastasis of gastric cancer.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows.

[0008] The present invention provides the use of a reagent for detecting the expression level of ITGB2 gene or its encoded protein in the preparation of a product for assisting in predicting peritoneal metastasis of gastric cancer.

[0009] Furthermore, the product detects the expression level of ITGB2 gene or its encoded protein in the sample through reverse transcription PCR, real-time quantitative PCR, chip, high-throughput sequencing platform, immunofluorescence, and immunohistochemical staining.

[0010] Furthermore, the product contains specific primers for amplifying the ITGB2 gene, probes for hybridizing with the nucleotide sequence of the ITGB2 gene, or antibodies for specifically binding to the integrin β2 protein.

[0011] Furthermore, the specific primer sequences for amplifying the ITGB2 gene are shown in SEQ ID NO.1 and SEQ ID NO.2; and the antibody is a monoclonal antibody or a polyclonal antibody.

[0012] Furthermore, the product is a preparation or a kit; and the sample is a tissue or a cell.

[0013] The present invention also provides the use of an inhibitor of ITGB2 gene or its encoded protein in the preparation of a drug for treating peritoneal metastasis of gastric cancer.

[0014] The present invention also provides the use of an inhibitor of the expression level of ITGB2 gene or its encoded protein in the preparation of a drug for inhibiting the adhesion ability of gastric cancer cells.

[0015] Furthermore, the inhibitors as described above include shRNA, siRNA or CD18 / integrin β2 neutralizing antibodies specifically targeting the ITGB2 gene.

[0016] Furthermore, the nucleotide sequence of the shRNA is shown in SEQ ID NO.3; the nucleotide sequence of the siRNA is shown in SEQ ID NO.4-5.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects.

[0018] The present invention proposes for the first time the expression of adhesion molecule CD18 / integrin β2 in gastric cancer cells and for the first time explains the function of CD18 / integrin β2 in promoting the adhesion of gastric cancer cells to peritoneal mesothelial cells. CD18 / integrin β2 can be used as a potential therapeutic target for gastric cancer peritoneal metastasis. Gene knockdown or antibody blocking of cell surface CD18 / integrin β2 expression inhibits gastric cancer peritoneal metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 KEGG pathway enrichment analysis was performed on the upregulated differentially expressed genes in patients with peritoneal metastasis.

[0020] Figure 2 An in vitro detection system for evaluating gastric cancer cell adhesion to human peritoneal mesothelial cells HMrSV5 was established. A is the process of establishing gastric cancer cell adhesion to human peritoneal mesothelial cells; B is the adhesion of MKN45 and MKN45 P3 cells to HMrSV5 cells under low-power field of view, red fluorescence is DID-labeled tumor cells, original magnification 50 times, scale bar = 400μm; C is the average number of tumor cells adhered per field of view under high-power field of view (HPF), original magnification 200 times, N = 5. * P <0.05,** P <0.01.

[0021] Figure 3 The high peritoneal metastasis cell line MKN45 P3 has stronger proliferation, migration and invasion abilities. A is the MTS cell viability assay, which detects the proliferation times of MKN45 and MKN45 P3 cells at different time points compared with 0 hours, N=3; B is the Transwell assay, which detects the migration and invasion abilities of MKN45 and MKN45 P3 cells at 48 hours, the original magnification is 100 times, scale bar = 200μm; C is the average number of tumor cells that migrated per field of view, N=3; D is the average number of tumor cells that invaded per field of view, N=3. * P <0.05,** P <0.01,*** P<0.001.

[0022] Figure 4 Construction of protein interaction network based on differentially expressed genes.

[0023] Figure 5 The mRNA expression of genes encoding 18 α subunits and 8 β subunits of ITG in the high peritoneal metastasis cell line MKN45 P3 and the parental cell line MKN45. A is the mRNA expression level of integrin family genes in MKN45 and MKN45 P3 cells detected by RT-qPCR experiment, and the normalized heat map is shown; B is the relative expression fold. N=3. * P <0.05,** P <0.01,*** P <0.001, ns means no significant statistical difference.

[0024] Figure 6 RT-qPCR and Western Blot were used to detect the expression of integrin β2 at the mRNA level and protein level, respectively. A is the RT-qPCR experiment to detect the expression of CD18 / integrin β2 mRNA; B is the Western Blot experiment to detect the expression of CD18 / integrin β2 protein; C is the immunofluorescence experiment indicating the localization of CD18 / integrin β2 in cells, the original magnification is 400 times, and the scale bar = 50μm; D is the flow cytometry detection of the expression of CD18 / integrin β2 on the cell surface; E is the statistical analysis of the proportion of CD18 / integrinβ2 positive cells on the cell surface, N = 3. ** P <0.01,*** P <0.001.

[0025] Figure 7CD18 / integrin β2 promotes the adhesion of gastric cancer cells to peritoneal mesothelial cells. A is a Western Blot experiment to verify the efficiency of transient knockdown of CD18 / integrinβ2 in MKN45 P3 cells; B is a flow cytometry test of the expression of CD18 / integrin β2 on the surface of MKN45 P3 cells after transient knockdown; C is a statistical analysis of the proportion of CD18 / integrinβ2 positive cells on the cell surface, N=3; D is an in vitro tumor cell-HMrSV5 cell adhesion experiment 72 hours after CD18 / integrin β2 was knocked down in MKN45 P3 cells, the adhesion of tumor cells under low-power field of view, the red fluorescence is DID-labeled MKN45P3 cells, the original magnification is 50 times, scale bar = 400μm; E is the average number of MKN45 P3 cells adhered per field of view under high-power field of view (HPF), N=5, original magnification is 200 times. ** P <0.01,*** P <0.001, **** P <0.0001, ns means no significant statistical difference.

[0026] Figure 8 Adhesion of MKN45 P3 cells to peritoneal mesothelial cells after pretreatment with CD18 / integrin β2 neutralizing antibody (α-CD18). A shows the adhesion of tumor cells under low-power field of view after pre-incubation of MKN45 P3 cells with different concentrations of neutralizing antibody α-CD18 for 1 hour, followed by an in vitro tumor cell-HMrSV5 cell adhesion experiment. The red fluorescence is DID-labeled MKN45 P3 cells, with an original magnification of 50 times. Scale bar = 400μm; B shows the average number of MKN45 P3 cells adhered per field of view under high-power field of view (HPF), with an original magnification of 200 times, N = 5. ** P <0.01,*** P <0.001, **** P <0.0001.

[0027] Fig. 9Overexpression of CD18 / integrin β2 promotes gastric cancer cell adhesion to HMrSV5 cells. A is a Western Blot experiment to verify the efficiency of MKN45 cells overexpressing CD18 / integrin β2. B is a MKN45 blank control or stably overexpressing CD18 / integrin β2 cell. After pre-incubation with isotype control antibody or neutralizing antibody α-CD18 at 5μg / mL for 1 hour, an in vitro tumor cell-HMrSV5 cell adhesion experiment was performed. The adhesion of tumor cells under low-power field of view, red fluorescence is DID-labeled MKN45 cells, original magnification 50 times, scale = 400μm; C is the average number of MKN45 cells adhered per field of view under high-power field of view (HPF), original magnification 200 times; D is a Western Blot experiment to detect the background CD18 / integrin β2 protein expression of different gastric cancer cells; E is a Western Blot experiment to verify the overexpression of CD18 / integrin in SNU-216 cells. β2 stable transfection efficiency; F is SNU-216 blank control or stable overexpression CD18 / integrin β2 cells, after pre-incubation with isotype control antibody or neutralizing antibody α-CD18 at 5μg / mL for 1 hour, in vitro tumor cell-HMrSV5 cell adhesion experiment, tumor cell adhesion under low power field, red fluorescence is DID-labeled SNU-216 cells, original magnification 50 times, scale = 400μm; G is the average number of SNU-216 cells adhered per field of view under high power field (HPF), original magnification 200 times, N = 5. ** P <0.01,*** P <0.001, **** P <0.0001, ns means no significant statistical difference.

[0028] Fig.10 Nude mouse peritoneal metastasis model verifies that CD18 / integrin β2 promotes peritoneal metastasis of gastric cancer. A is Western Blot detection of CD18 / integrin β2 protein expression in MKN45 P3 cells with stable knockdown of CD18 / integrin β2 (shITGB2) and control (shNC) cells; B is nude mice intraperitoneally injected with 3×10 6shNC or shITGB2 MKN45 P3 cells (N=4) were added to the nude mice, and the degree of peritoneal metastasis was compared after 3 weeks. The peritoneal metastasis nodules of nude mice on day 21 (D21) are shown in the figure, and the red arrows point to the peritoneal nodules; C is the number of peritoneal nodules counted in each nude mouse on D21; D is the volume of peritoneal effusion extracted and measured in each nude mouse on D21; E is the weight of each nude mouse weighed every 5 days within 3 weeks, and the vertical axis represents the weight difference compared with D1, and a positive difference means that the weight has increased compared with D1; F is the expression of CD18 / integrin β2 in the peritoneal nodule tissue of nude mice, and HE staining and CD18 / integrin β2 were observed under the microscope. β2 staining, original magnification 200 times, scale bar = 100 μm; G is the immunohistochemical score calculated by randomly selecting 3 fields of view for each pathological section (H-score = staining intensity score × positive cell frequency score, where the staining intensity score is: negative 0 point, weak 1 point, moderate 2 points, strong positive 3 points; positive cell frequency score is: <5% is 0 point, 5%~25% is 1 point, 25%~50% is 2 points, 50%~75% is 3 points, >75% is 4 points). * P <0.05. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below by specific examples and drawings. The following examples are only used to further illustrate the present invention and should not be construed as limiting the present invention.

[0030] Example 1 Analysis of malignant phenotype of gastric cancer cells with high peritoneal metastasis.

[0031] 1. Experimental methods

[0032] 1. Data collection and processing.

[0033] The mRNA expression profiles of 300 gastric cancer tissues in the GSE62254 cohort and the clinical pathological information of the corresponding patients were downloaded from the high-throughput Gene Expression Omnibus (GEO) database (http: / / www.ncbi.nlm.nih.gov / geo / ). The R package "limma" was used to screen the differentially expressed genes (DEGs) between 54 gastric cancer samples with peritoneal metastasis and 246 gastric cancer samples without peritoneal metastasis in the GSE62254 cohort under the conditions of false discovery rate (FDR) <0.5 and fold change (FC) log value logFC>1. The R package "clusterProfiler" was used to perform Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway enrichment analysis, and finally the DEGs were selected. P The top ten pathways with a p<0.05 and the largest number of enriched genes in the pathways were considered to be pathways related to promoting the occurrence of peritoneal metastasis of gastric cancer.

[0034] 2. Cell source and cell culture.

[0035] The human gastric cancer cell line MKN45 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The gastric cancer cell line MKN45 P3 with high peritoneal metastasis was constructed by the research group in the early stage, and the construction method was referred to previous literature reports. In short, after MKN45 cells were injected into the peritoneum of BALB / c-nude mice, the cells that formed implanted nodules in the mouse peritoneum were considered to have high peritoneal metastasis potential, and tumor cells were extracted from the nodules as MKN45 P1. The above process was repeated several times, and the cells after the third generation (MKN45 P3) were verified to have stable high peritoneal metastasis ability by in vivo nude mouse peritoneal implantation experiments. MKN45 P3 has been used in other gastric cancer peritoneal metastasis-related studies of the research group. The human peritoneal mesothelial cell line HMrSV5 was provided by Professor Peng Youming of the Second Hospital of Central South University in Changsha, China and Professor Pierre Ronco of the Hospital of Tenon in France. The HMrSV5 cell line was originally established by retroviral transfection of primary human peritoneal mesothelial cells with SV40 large T antigen, and is now widely used in peritoneal-related studies.

[0036] All cell lines were cultured in RPMI-1640 medium containing 10% inactivated fetal bovine serum (FBS), 100 μg / mL streptomycin, and 100 U / mL penicillin at 37°C in an atmosphere of 95% air and 5% CO. 2All cells used in the experiment were in the logarithmic growth phase and mycoplasma tests were negative.

[0037] 3. In vitro tumor cell-human peritoneal mesothelial cell adhesion experiment.

[0038] Tumor cell lines MKN45 cells, MKN45 P3 cells and human peritoneal mesothelial cell line HMrSV5 cells were used for in vitro tumor cell-human peritoneal mesothelial cell adhesion experiments. Human peritoneal mesothelial cell line HMrSV5 cells were seeded in 12-well plates and placed at 37°C and 5% CO 2 The cells were cultured in an incubator until they were confluent as a single cell layer. After digestion and centrifugation of the tumor cells to be attached, they were washed 2 to 3 times with serum-free RPMI-1640 medium to fully remove the serum components in the cell pellet, and then DID live cell dye (Invitrogen, V22887) diluted 1:100 with serum-free RPMI-1640 medium was added to the cell suspension and incubated at 37°C, 5% CO 2 After incubation, the cells were washed 2-3 times with serum-free RPMI-1640 medium until no stain was present. 4 The cells were seeded on the HMrSV5 monolayer at a density of 10 cells / well. The cells were incubated at 37°C with 5% CO 2 After incubation under the appropriate conditions for an appropriate time, the cells were washed 2-3 times with PBS to fully remove the non-adherent tumor cells. Immediately afterwards, fluorescent photos and corresponding white light photos of at least five randomly selected fields of view were taken using an inverted fluorescence microscope, and the number of tumor cells adhering to human peritoneal mesothelial cells in each field of view under high power field (HPF) (magnification 200 times) was counted using FIJI / ImageJ software (version 1.53c) and used for subsequent statistical analysis.

[0039] 4. Cell viability determination.

[0040] After digestion and centrifugation, MKN45 and MKN45 P3 cells were fully resuspended into single cell suspensions, and 180 μL of cell suspension was inoculated into 96-well plates at a density of 3,000 cells / well. Four replicate wells were set up in each group at different time points, and 180 μL of complete medium was used as a blank control. After cell inoculation, 20 μL of MTS reagent (Promega, G3581) was added at 0 h, 24 h, 48 h, and 72 h, respectively, and the plates were placed at 37 °C and 5% CO. 2 After incubation, the 96-well plate was placed on a horizontal shaker for 20 minutes to mix thoroughly, and the absorbance was measured at 490 nm using a microplate reader.

[0041] Cell proliferation rate = (average absorbance value of different time groups - average absorbance value of blank control in the time group) / (average absorbance value of 0 hour group - average absorbance value of blank control in 0 hour group).

[0042] 5. Transwell migration and invasion assay.

[0043] MKN45 and MKN45 P3 cells were analyzed for cell migration and invasion using 8 μm Transwell chambers (Corning Life Science). After digestion and centrifugation of tumor cells, they were washed 2 to 3 times with RPMI-1640 medium without serum and double antibody to fully remove serum. For migration experiments, tumor cell suspensions were diluted to 1×10 5 The density of cells / 200 μL was placed in the upper chamber, where the culture medium in the upper chamber was RPMI-1640 medium without serum; 500 μL of RPMI-1640 medium containing 20% ​​FBS was added to the lower chamber and incubated at 37°C and 5% CO 2 Incubate for 48 hours under 4 °C conditions. For invasion experiments, Matrigel (Corning Life Sciences, 354234) was thawed overnight at 4 °C and then diluted with serum-free RPMI-1640 medium at a ratio of 1:30. 50 μL of the diluted Matrigel was added to the upper chamber on ice in advance, and then incubated at 37 °C for 1 hour to allow the Matrigel to fully solidify. Tumor cell suspension was incubated at 1.5 × 10 5 The density of cells / 200 μL was placed in the upper chamber, where the culture medium in the upper chamber was RPMI-1640 medium without serum; 500 μL of RPMI-1640 medium containing 20% ​​FBS was added to the lower chamber and incubated at 37°C and 5% CO 2 Incubate for 48 hours.

[0044] After the migration or invasion assay was completed, the culture medium in the upper chamber was gently removed with an absorbent cotton swab, and then the lower surface of the chamber was placed in 75% ethanol for at least 10 seconds to fully fix the cells on the lower surface of the chamber, and the cells retained in the upper chamber of the chamber were gently removed with an absorbent cotton swab moistened with ethanol. After the chamber was air-dried, it was stained with Wright's stain for 30 seconds, followed by 3 minutes of mixed Giemsa stain. After staining, the excess stain was gently washed off from the inside of the chamber and wiped clean with a cotton swab to dry, and at least five randomly selected fields of view were photographed using an upright fluorescence microscope. FIJI / ImageJ software (version 1.53c) was used to count the tumor cells that passed through the chamber in each field of view under high-power fields (magnification 200 times) and used for subsequent statistical analysis.

[0045] 6. Statistical analysis.

[0046] All statistical analyses were performed using GraphPad Prism software (Version 9.4.1) and R studio software (Version 1.4.1106). All experiments were repeated at least three times independently, and the experimental results are shown as mean ± standard deviation (SD). Student's t-tests were used to evaluate the differences between the two groups. P <0.05 was considered statistically significant (* P <0.05,** P <0.01,*** P <0.001, **** P <0.0001, ns means no statistically significant difference).

[0047] 2. Experimental results.

[0048] 1. First, we performed differential analysis on the expression profiles of gastric cancer patients with peritoneal metastasis (PM) and non-peritoneal metastasis (non-PM) in the GEO dataset GSE62254. KEGG pathway enrichment analysis was performed on the differentially expressed genes that were upregulated in patients with peritoneal metastasis. The focal adhesion signaling pathway ranked first, while the cell adhesion molecules and the extracellular matrix-receptor interaction pathways were also highly enriched, suggesting that the enhancement of cell adhesion ability plays an important role in promoting peritoneal metastasis of gastric cancer ( Figure 1 ).

[0049] 2. Establish an in vitro detection system to evaluate gastric cancer cell adhesion to human peritoneal mesothelial cells HMrSV5. Compared with the parental cell line MKN45, MKN45 P3 cells have stronger adhesion to human peritoneal mesothelial cells HMrSV5 at different time points ( P <0.05), the enhancement of cell adhesion ability can promote the peritoneal metastasis of gastric cancer ( Figure 2 ).

[0050] 3. Through MTS cell viability assay, it was found that the gastric cancer cell line MKN45 P3 with high peritoneal metastasis also had stronger proliferation ability ( P <0.01). Transwell assay showed that MKN45 P3 cell migration ability ( P <0.05 and more invasive ability ( Figure 3 ).

[0051] 3. Conclusion

[0052] The gastric cancer cell line MKN45 P3 with high peritoneal metastasis has stronger proliferation, metastasis and adhesion ability to human peritoneal mesothelial cells.

[0053] Example 2 Integrin β2 is an adhesion molecule that can predict high peritoneal metastasis of gastric cancer.

[0054] 1. Experimental methods

[0055] 1. Cell sources and cell culture.

[0056] The sources and culture methods of human gastric cancer cell lines MKN45, MKN45 P3 and human peritoneal mesothelial cells HMrSV5 were the same as those in Example 1. Human gastric cancer cell lines NCI-N87, HGC-27, MGC-803, BGC-823, SGC-7901, and AGS were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China), SNU-216 was purchased from the Korean KCLB Cell Bank (Seoul, Korea), and MKN74 and MKN7 were purchased from the Japanese Bioresource Research Cell Bank (Osaka, Japan). NCI-N87 cell line used high-glucose DMEM medium, AGS cell line used RPMI-1640 / F12 medium, and all other cell lines used RPMI-1640 medium. All cell lines were cultured in a medium containing 10% inactivated fetal bovine serum (FBS), 100 μg / mL streptomycin, and 100 U / mL penicillin, and placed at 37°C in 95% air and 5% CO 2 All cells used in the experiment were in the logarithmic growth phase and mycoplasma tests were negative.

[0057] 2. RNA extraction and real-time quantitative PCR (RT-qPCR).

[0058] Cellular RNA was extracted using the Eastep Super total RNA extraction kit (Promega, LS1040), and all operations were performed according to the instructions of the kit. The extracted RNA was quantified by measuring the absorbance at 260 nm using a NanoDrop ND-100 spectrophotometer. The GoScript reverse transcription kit (Promega, A2790) was used to reverse transcribe the extracted RNA into cDNA. Quantitative real-time PCR was performed using SYBR Premix Ex Taq II and run on an Applied Biosystems 7500 Real-Time PCRSystems. A total of 45 cycles were performed at 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute, followed by one cycle at 95°C for 15 seconds, 60°C for 1 minute, 95°C for 30 seconds, and 60°C for 15 seconds. The relative expression was calculated using the 2^(-∆∆Ct) method, with 18S as the internal control. The PCR primers used in this study were synthesized by Sangon Biotechnology Technology Service Co., Ltd. (Shanghai, China). The sequences of all primers are as follows: .

[0059] 3. Protein immunoblotting experiment.

[0060] After the cells were washed with cold PBS, they were lysed with an appropriate amount of lysis buffer (1% Triton X-100, 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na 3 VO 4 , 1mM PMSF, 2μg / mL aprotinin) on ice for 5 minutes. The cell lysate was further ultrasonically lysed in an ice-water mixture. The ultrasonic conditions were 150W power, 30% amplitude, 3 seconds of ultrasonication, 3 seconds of stop, and repeated 3 cycles. After the ultrasonication, the cell lysate was centrifuged at 13,000rpm at 4°C for 25 minutes, and the supernatant was aspirated to obtain the cell protein. The protein was then quantified using the Coomassie Brilliant Blue method. The volume was filled with lysis buffer, and the protein sample was fully mixed with 3× loading buffer, and then boiled in a 95°C constant temperature metal bath for 5-10 minutes to fully denature the protein.

[0061] The cooled protein samples were subjected to protein electrophoresis by SDS-PAGE. After the electrophoresis, the separation gel was placed in a transfer device and the protein was transferred to a PVDF membrane (Merke Millipore, IPVH00010) at a constant voltage of 80V for 120 minutes. After the transfer, the PVDF membrane was soaked in TBST buffer (10mM Tris-Cl pH 7.4, 150mMNaCl, 0.1% Tween-20) containing 5% skim milk and blocked with slow shaking at room temperature for 40 minutes to 1 hour. The PVDF membrane was cut according to the pre-designed method and incubated in the primary antibody diluted in TBST buffer containing 5% skim milk at an appropriate ratio, and incubated with slow shaking at room temperature for more than 6 hours or at 4°C overnight. After the incubation, it was thoroughly washed with TBST buffer (10 minutes / time, a total of 4 times). After washing, the PVDF membrane was placed in the corresponding horseradish peroxidase-labeled secondary antibody diluted 1:2000 in TBST buffer, incubated at room temperature for 40 minutes, and then washed again with TBST buffer (10 minutes / time, a total of 4 times). Finally, Western LightningPlus-ECL reagent (PerkinElmer, NEL104001EA) was used to image on an ECL luminometer using chemiluminescence. FIJI / ImageJ software (version1.53c) was used to analyze the gray value of the band for subsequent statistical analysis.

[0062] 4. Cell immunofluorescence.

[0063] Sterile coverslips were placed in 12-well plates and MKN45 and MKN45 P3 cells were plated at a density of 1 × 10 5 Cells were seeded on coverslips at a density of 100 cells / well. After the cells climbed, they were fixed with 500 μL 4% paraformaldehyde at room temperature for 15 minutes, and then permeabilized with 500 μL 0.1% TritonX-100 at room temperature for 10 minutes, and blocked with 500 μL 3% BSA solution at room temperature for 30 minutes. After blocking, the CD18 / integrin β2 primary antibody was diluted with 3% BSA solution at a ratio of 1:100, covered on the cells on the coverslip, and incubated overnight at 4°C. The next day, the corresponding species fluorescent secondary antibody was diluted with 3% BSA solution at a ratio of 1:500, and incubated at room temperature for 1 hour in the dark. After the secondary antibody incubation, DAPI stain (Thermo Scientific, 62247) was diluted with PBS solution at a ratio of 1:1000, and incubated at room temperature in the dark for 5 minutes to stain the nucleus. After counterstaining, the slides were sealed with anti-fluorescence quenching sealing solution (Beyotime, P0126) and the cell staining was observed under an upright fluorescence microscope.

[0064] 5. Cell surface flow cytometry.

[0065] MKN45 and MKN45 P3 cells were used for detection of surface antigen expression by flow cytometry. After digestion and centrifugation of the cells to be detected, they were washed 1-2 times with pre-cooled PBS solution. 2.5 μL of APC-CD18 flow antibody or isotype control antibody was diluted with 47.5 μL of flow buffer (PBS solution containing 3% FBS and 2 mM EDTA), and the cell pellet was resuspended with 50 μL of antibody diluent, and incubated at 4°C for 30 minutes in the dark. After incubation, the cells were washed 1-2 times with PBS solution and resuspended with 500 μL of flow buffer. Surface antigen expression was detected using a BD Accuri C6 flow cytometer. The results were analyzed using Flowjo software (Version 10.4).

[0066] 6. Statistical analysis.

[0067] Same as Example 1.

[0068] 2. Experimental results.

[0069] 1. To screen key molecules that promote adhesion between gastric cancer cells and peritoneal mesothelial cells, we constructed a protein interaction network using the differentially expressed genes in three pathways (focal adhesion, cell adhesion molecules, and extracellular matrix-receptor interactions) in the GSE62254 cohort through the protein-protein interaction network database STRING (http: / / string-db.org / , Version 11.5). Among them, integrin family adhesion molecules (ITGA1, ITGA8, and ITGA9) occupy a central position and are closely related to other adhesion molecules, suggesting that integrin family molecules may be an important adhesion molecule family that promotes peritoneal metastasis of gastric cancer ( Figure 4 ).

[0070] 2. For the high peritoneal metastasis cell line MKN45 P3 and the parental cell line MKN45, the mRNA expression of the genes encoding the 18 known α subunits and 8 β subunits was detected by RT-qPCR. Among them, the mRNA corresponding to ITGA11 and ITGAV was significantly overexpressed in MKN45 (P values ​​were P <0.001 and P <0.05, while ITGB2 mRNA was significantly overexpressed in MKN45 P3 ( P <0.01), and there was no significant difference in the expression of other members of the integrin family between the two cells ( Figure 5 ).

[0071] 3. RT-qPCR and Western Blot were used to detect the expression of integrin β2 at the mRNA level and protein level, respectively. Compared with the parental cells, it was significantly more highly expressed in MKN45 P3 cells. The results of cell immunofluorescence experiments showed that integrin β2 was expressed on the surface of both MKN45 and MKN45 P3 cells. Subsequently, flow cytometry surface staining results showed that the expression of integrin β2 on the surface of MKN45 P3 cells was higher than that of the parental cell line MKN45 cells ( Figure 6 ).

[0072] 3. Conclusion

[0073] CD18 / integrin β2 is an adhesion molecule highly expressed on the highly peritoneal metastatic cell line MKN45P3.

[0074] Example 3 CD18 / integrinβ2 promotes the adhesion ability of gastric cancer cells to peritoneal mesothelial cells.

[0075] 1. Experimental methods

[0076] 1. siRNA transfection.

[0077] ITGB2 small interfering RNA (siRNA) and the corresponding negative control siRNA were synthesized by Genetix Biotechnology Co., Ltd. (Wuhan, China) and Rebo Biotechnology Co., Ltd. (Guangzhou, China), and the sequences are as follows: .

[0078] siRNA was transfected using jetPRIME transfection reagent (Polyplus, 101000046), and the operation method was referred to the instructions for use of the transfection reagent. In brief, MKN45 P3 cells were transfected with 1-1.5×10 5 100 μL of siRNA was inoculated into 6-well plates. After the cells adhered to the wall, 200 μL of jetPRIME buffer, 10 μL of siRNA and 5 μL of jetPRIME transfection reagent were mixed and allowed to stand for ten minutes before adding to the cell culture system and culturing in an incubator. Cell RNA was extracted 48 hours after transfection and cell protein was extracted 72 hours after transfection to verify transfection efficiency. Other cell function experiments were performed at an appropriate time after transfection.

[0079] 2. Protein immunoblotting experiment.

[0080] Same as Example 2.

[0081] 3. Cell surface flow cytometry.

[0082] 72 hours after transfection, the cells were tested for surface antigen expression using flow cytometry, using the same method as in Example 2.

[0083] 4. Construction of a cell line stably overexpressing ITGB2.

[0084] ITGB2 overexpression (OE) and negative control lentivirus were purchased from Heyuan Biotechnology Co., Ltd. (Shanghai, China). MKN45 cells were cultured at 3 × 10 4 SNU-216 cells were seeded at a density of 2×10 4 The cells were seeded at a density of 100 / well in a 6-well plate. After the cells adhered to the wall, polybrene was added at a ratio of 1:200 to improve the transfection efficiency, and lentivirus was added (MOI = 10 for MKN45 cells and MOI = 40 for SNU-216 cells). After mixing and standing, the cells were placed in an incubator for culture. 48 hours after transfection, 5 μg / mL puromycin was used to select stably transfected cells, and the transfection efficiency was verified by Western Blot and subsequent experiments were carried out.

[0085] 5. In vitro tumor cell-human peritoneal mesothelial cell adhesion experiment.

[0086] Tumor cell lines MKN45 cells, MKN45 P3 cells, SNU-216 cells and human peritoneal mesothelial cell line HMrSV5 cells were used to conduct an in vitro tumor cell-human peritoneal mesothelial cell adhesion experiment. The specific method was the same as in Example 1.

[0087] For tumor cells transfected with siRNA, adhesion assays were performed 48-72 hours after transfection. For tumor cells treated with neutralizing antibody α-CD18, adhesion assays were performed after 1 hour of pre-incubation.

[0088] 6. Statistical analysis.

[0089] Same as Example 1.

[0090] 2. Experimental results.

[0091] 1. In the high peritoneal metastasis cell line MKN45 P3 cells that relatively highly express CD18 / integrin β2, siRNA was used to knock down CD18 / integrin β2. Western Blot confirmed that siRNA can effectively reduce CD18 / integrin β2 protein. Flow cytometry confirmed that the expression of this molecule on the surface of MKN45 P3 cells was also significantly reduced ( P <0.001), and in vitro tumor cell-HMrSV5 cell adhesion experiments found that the number of MKN45 P3 cells adhering to HMrSV5 cells was significantly reduced ( P <0.01) ( Figure 7 ).

[0092] 2. Pretreatment of MKN45 P3 cells with CD18 / integrin β2 neutralizing antibody (α-CD18) to block CD18 / integrin β2 on the cell surface significantly weakened the adhesion ability of MKN45 P3 cells to peritoneal mesothelial cells ( P <0.01, and the degree of inhibition of adhesion increased with the increase of neutralizing antibody concentration ( Figure 8 ).

[0093] 3. Compared with the blank control, the adhesion ability of MKN45 cells with high expression of CD18 / integrin β2 to peritoneal mesothelial cells was significantly enhanced ( P <0.05, while further blocking with α-CD18 significantly inhibited adhesion ( P <0.01). In addition, among gastric cancer cell lines, SNU-216 cells with low expression of CD18 / integrin β2 were selected. Compared with the blank control, the adhesion ability of SNU-216 cells with high expression of CD18 / integrin β2 to peritoneal mesothelial cells was significantly enhanced ( P <0.01, while further blocking with α-CD18 significantly inhibited the adhesion ability (P<0.01) ( Fig. 9 ).

[0094] 3. Conclusion

[0095] CD18 / integrin β2 promotes the adhesion of gastric cancer cells to peritoneal mesothelial cells.

[0096] Example 4 CD18 / integrin β2 promotes peritoneal metastasis of gastric cancer in mice.

[0097] 1. Experimental methods

[0098] 1. Construction of stable ITGB2 knockdown cell line.

[0099] ITGB2 short hairpin RNA (shRNA) and negative control lentivirus were purchased from Heyuan Biotechnology Co., Ltd. (Shanghai, China). The shITGB2 sequence was: 5′-GGAAGGACAACAACUCCAUTT-3′. MKN45 P3 cells were cultured at 3×10 4 The cells were inoculated at a density of 100 / well in a 6-well plate. After the cells adhered to the wall, polybrene was added at a ratio of 1:200 to improve the transfection efficiency, and lentivirus (MOI = 10) was added. After mixing and standing, the cells were placed in an incubator for culture. 48 hours after transfection, 5 μg / mL puromycin was used to select stably transfected cells, and the transfection efficiency was verified by RT-qPCR and Western Blot, and subsequent experiments were carried out.

[0100] 2. Protein immunoblotting experiment.

[0101] Same as Example 2.

[0102] 3. Establish a gastric cancer peritoneal metastasis model by intraperitoneal injection in nude mice.

[0103] Eight 4-5-week-old BALB / c-nude female nude mice were purchased from Jicui Yaokang Biotechnology Co., Ltd. (Jiangsu, China), housed in a special pathogen-free environment, and randomly divided into two groups. After adaptive feeding, the two groups of nude mice were injected intraperitoneally with MKN45 P3 shNC or MKN45 P3 shITGB2 cells, respectively. Each nude mouse was injected with 3×10 6 cells / 200μL PBS. The weight of each nude mouse was weighed and recorded on the day of cell injection and every 5 days after injection to observe whether ascites appeared. According to the standards of the Animal Protection Committee of our center, all nude mice were killed by cervical dislocation 3 weeks after intraperitoneal injection of cells, and the volume of peritoneal effusion of each nude mouse was measured, and the number of peritoneal nodules of each nude mouse was counted.

[0104] 4. Immunohistochemistry.

[0105] After the tissue was fixed with 10% formalin, it was embedded in paraffin and sliced ​​continuously. The slices were baked in a 70°C oven for 1 hour. After the baking was completed, the temperature dropped slightly before dewaxing and hydration began. In the order of xylene and alcohol 100%, 100%, 95%, 85%, and 75%, the slice rack was placed for 3 minutes, shaken up and down for 2-3 minutes, rinsed with tap water for 5 minutes, and soaked in distilled water. The slice box was filled with 1× sodium citrate repair solution, and antigen repair was performed using the high pressure method. After cooling to room temperature naturally, it was placed in distilled water. After rinsing with PBS twice, it was treated with solution A for 10 minutes, and after rinsing with PBS three times, it was treated with solution B for 1 hour. 200 μL of primary antibody diluted in PBS was added to each slice and incubated overnight at 4°C. The next day, after rinsing with PBS three times, solution C was added for 10 minutes. After rinsing with PBS three times, solution D was used for 20 minutes. After rinsing twice with PBS, add 200 μL DAB to each slide and color it under a microscope. When the color development is appropriate, rinse it with tap water for 10 minutes and put it in a slice box filled with distilled water. After the slide is rinsed dry, stain it with hematoxylin for 1 minute and counterstain it with nuclear staining, then rinse it with tap water for 10 minutes. In the order of alcohol 75%, 85%, 95%, 100%, 100%, and xylene 100%, 100%, put the slice rack in one at a time, shake it for 3 minutes each time, and finally leave it in xylene. Quickly drop the gum on the tissue and cover it with a coverslip. For each pathological section, three random fields were selected for immunohistochemical scoring (H-score), H-score = staining intensity score × positive cell frequency score, where the staining intensity score is: negative 0 point, weak 1 point, moderate 2 points, strong positive 3 points; positive cell frequency score is: <5% is 0 point, 5%~25% is 1 point, 25%~50% is 2 points, 50%~75% is 3 points, >75% is 4 points. This work was completed by two independent pathologists.

[0106] 5. Statistical analysis.

[0107] Same as Example 1.

[0108] 2. Experimental results.

[0109] An in vivo peritoneal metastasis model was established by intraperitoneal injection of gastric cancer cells into nude mice. MKN45 P3 cells with stable knockdown of CD18 / integrin β2 (shITGB2) and control (shNC) MKN45 P3 cells were injected into the peritoneum of nude mice. Compared with the control group, stable knockdown of CD18 / integrin β2 significantly reduced the number of tumor nodules implanted in the mouse peritoneum ( P <0.05). No malignant peritoneal effusion was observed in the peritoneal cavity of mice in the shITGB2 group, while the volume of peritoneal effusion in the peritoneal cavity of mice in the control group increased significantly and was bloody ( P<0.05). In addition, as the number of days after intraperitoneal injection of tumor cells increased, both groups of mice became emaciated and lost weight, but the weight loss of mice in the shITGB2 group was less severe than that in the control group. Immunohistochemistry results showed that compared with the shNC group, the expression of CD18 / integrin β2 in the peritoneal nodules of mice in the shITGB2 group was significantly reduced ( P <0.05) ( Fig.10 ).

[0110] 3. Conclusion

[0111] CD18 / integrin β2 promotes peritoneal metastasis of gastric cancer in mice.

[0112] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting the expression level of the ITGB2 gene or its encoded protein in the preparation of a product for assisting in the prediction of peritoneal metastasis of gastric cancer.

2. The use according to claim 1, It is characterized in that The product detects the expression level of ITGB2 gene or its encoded protein in a sample through reverse transcription PCR, real-time quantitative PCR, chip, high-throughput sequencing platform, and immunohistochemical staining.

3. The use according to claim 1, It is characterized in that The product contains specific primers for amplifying ITGB2 gene, probes hybridizing with ITGB2 gene nucleotide sequences or antibodies specifically binding to integrin β2 protein.

4. The use according to claim 3, It is characterized in that The specific primer sequences for amplifying the ITGB2 gene are shown in SEQ ID NO.1 and SEQ ID NO.2; the antibody is a monoclonal antibody or a polyclonal antibody.

5. The use according to claim 2, It is characterized in that The product is a preparation or a kit; the sample is tissue, serum or cells.

6. Use of inhibitors of ITGB2 gene or its encoded protein in the preparation of drugs for treating peritoneal metastasis of gastric cancer.

7. Use of an inhibitor of the expression level of ITGB2 gene or its encoded protein in the preparation of a drug for inhibiting the adhesion ability of gastric cancer cells.

8. The use according to any one of claims 6 to 7, It is characterized in that The inhibitor includes shRNA, siRNA or CD18 / integrin β2 neutralizing antibody specifically targeting ITGB2 gene.

9. The use according to claim 8, It is characterized in that The nucleotide sequence of the shRNA is shown in SEQ ID NO.3; the nucleotide sequence of the siRNA is shown in SEQ ID NO.4-5.

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