RANKL/RANK Pathway Promotes Peritoneal Dissemination In Gastric Cancer Cells

Objective:Tumor metastasis significantly affects the prognosis of patients with gastric cancer and is the leading cause of treatment failure.The mechanism of metastasis is complex,and the microenvironment of the tumor is rich in cytokines,growth factors,tumor secretory vesicles and other factors,which are the key factors affecting tumor metastasis.They are involved in the body’s immune and inflammatory responses and are also expressed in a variety of tumors,regulating tumor growth,invasion and metastasis,and angiogenesis.RANKL is a member of the TNF(tumor necrosis factor)family.Previous studies have confirmed that the RANKL/RANK pathway is a classical pathway for inducing osteoclast activation.In recent years,studies have confirmed that RANK is also expressed in a variety of tumor cells.It mediates the migration of various tumor cells such as prostate cancer,kidney cancer,breast cancer and lung cancer.It has been reported that gastric cancer cells are rich in RANKL in the microenvironment,and our study found that RANK is also expressed on the surface of gastric cancer cells,but whether the RANKL/RANK pathway is involved in the regulation of gastric cancer cell migration is still unclear.Lipid raft is a microdomain rich in cholesterol and sphingomyelin on the plasma membrane.It is like a protein docking platform,which is closely related to cell membrane signal transduction and protein sorting.Receptors,ligands,coupling factors,effect enzymes,and substrates involved in the same signaling pathway are usually located in the same lipid raft region,which facilitates the interaction between signal molecules.The accumulation of lipid rafts causes the signal molecules originally in the non-lipid raft or different lipid rafts to be concentrated in the same cell membrane domain,contributing to the dialogue between signal transduction and signaling pathways.Whether lipid rafts are involved in RANKL-induced gastric cancer cell migration and its specific effects are unclear.Integrins are a major family of cell surface receptors.It mediates the adhesion of cells to extracellular matrices.Its special type also induces cell-to-cell interactions during leukocyte adhesion.Integrins are widely expressed in vivo,and most cells express more than one integrin on the surface,playing a key role in a variety of life activities.Many tumor cells and extracellular matrix(ECM)interactions occur during tumor metastasis,and as a result,integrin has become more and more concerned in recent years.In this study,gastric cancer cells SGC7901,BGC823 and MGC803 were used as the research object to investigate whether RANKL can induce gastric cancer cell migration,whether lipid rafts are involved in the regulation of RANKL-induced gastric cancer cell migration,and further study the mechanism of RANKL-induced gastric cancer cell migration.The results of this study will provide a theoretical basis for clinical treatment.Methods: 1.Detection of RANK expression by flow cytometry.2.Transwell was used to measure cell migration ability.3.Immunofluorescence was used to observe the accumulation of lipid rafts,and the expression positions of Cav-1 and RANK.4.Western blot was used to detect the expression and activation of Src,Cav-1,Akt and ERK proteins.5.Transfection was performed using Lipofectamine 2000 reagent.6.Immunohistochemistry was used to detect the expression of RANK and Cav-1 in gastric cancer tissue samples.7,statistical processing: SPSS20.0 statistical software for chi-square test,Spearman rank correlation test.Each experiment was repeated 3 times and the data was expressed as ± s.Differences between the two groups were analyzed by Student’s t-test.P < 0.05 was statistically significant.Survival analysis was performed using Kaplan-Meier.The Cox model was used for single factor and multifactor survival analysis.Results:1.A variety of gastric cancer cells express RANK,and RANKL promotes gastric cancer cell migration through PI3 K and MAPK pathways.Western blot and flow surface antigen detection results indicated that RANK was expressed on the surface of MGC803,BGC823 and SGC7901 cells.RANKL(1.0 μg/ml)treatment of gastric cancer cells,MGC803,BGC823 and SGC7901 three gastric cancer cells migration capacity was significantly improved,the increase rate was 63.8%,52.3%and 56.3%.As a result of previous studies,RANKL did not promote the value-added effect on MGC803 and SGC7901 gastric cancer cells.The number of cells passing through the cell membrane compared to the control group increased the number of cells representing an increase in cell migration ability.We also detected the RANKL/RANK downstream signaling pathway,and Akt and ERK were activated after RANKL stimulation.These results suggest that the RANKL/RANK pathway is involved in gastric cancer cell migration.2.Lipid rafts participate in RANKL-induced migration of gastric cancer cellsLipid rafts are involved in many signaling pathways,and lipid rafts contain numerous signaling molecules.To test whether lipid rafts are involved in RANKL-induced gastric cancer cell migration,we treated NGC803 cells with nystatin,a lipid raft inhibitor,and RANKL for 10 minutes after nystatin addition.Immunofluorescence results suggest that RANKL can cause lipid raft aggregation,and nystatin inhibits this reaction.Detection of the downstream signals AKK and ERK of RANKL suggests that activation of Akt and ERK can be inhibited by nystatin.In the transwell experiment,it can be seen that the RANKL-induced gastric cancer cell migration rate decreased from 168.8% to 75.6%.This result indicates that lipid raft aggregation is involved in RANKL-induced gastric cancer cell migration.3.Cav-1 promotes RANKL-induced gastric cancer migrationWestern blot was used to detect the phosphorylation of Cav-1.The results indicated that Cav-1 was activated after RANKL stimulation and was time-dependent.The immunofluorescence method was used to observe Cav-1 and RANK.The results indicated that there was significant colocalization between Cav-1 and RANK.RANKL may promote the interaction between Cav-1 and RANK.Transient transfection knockout Cav-1,immunofluorescence results suggest that gastric cancer cell lipid raft aggregation is inhibited,Western blot analysis of Akt and ERK phosphorylation,the results suggest that the activation of Akt and ERK is significantly inhibited after knocking out Cav-1.Transwell method was used to detect the migration ability of MGC803.It was suggested that knocking out Cav-1 also inhibited RANKL-induced gastric cancer cell migration.These results suggest that Cav-1 promotes RANKL-induced gastric cancer cell migration4.c-Src regulates RANKL-induced gastric cancer cell migration by activating Cav-1In order to clarify the mechanism of Cav-1 activation,we examined the activation of c-Src after treatment of MGK803 cells with RANKL at different time points.The results showed that c-Src was significantly activated after RANKL treatment and reached its apex at 10 minutes.The c-Src inhibitor PP2 inhibits the activation of Cav-1,Akt and ERK.The application of immunofluorescence assay indicated that PP2 inhibited the accumulation of lipid rafts.Transwell results suggest that PP2 inhibits RANKL-induced gastric cancer cell migration.These results suggest that c-Src is involved in the regulation of RANKL-induced gastric cancer cell migration by activating Cav-1.5.Expression of Cav-1 and RANK in gastric cancer and their influence on prognosisThis study included 228 patients with gastric cancer who were willing to be treated.The clinicopathological parameters such as age,gender,tumor stage,and Lauren classification were recorded in detail and followed up regularly.Among the patients with gastric cancer,there were 162 males and 66 females with a maximum age of 80 years,a minimum age of 29 years,and a median age of 61 years.TNM staging was performed according to the seventh edition of AJCC,including 55 cases in stage I-II and 173 cases in stage III-IV.The positive expression rate of Cav-1 in 228 patients was 154/228(56.5%).The positive expression rate of RANK in 228 patients was 108/228(47.4%).Cav-1 expression was associated with histological grade and was not associated with tumor invasion depth,lymph node metastasis,stage,Lauren classification,and tumor location.Patients with positive RANK expression were significantly elevated in patients with poor histological grade and diffuse type(p<0.001).RANK expression was not associated with tumor invasion depth,lymph node metastasis,staging,and tumor location.Spearman correlation analysis showed that the expression of Cav-1 was significantly positively correlated with the expression of RANK,r=0.149,p=0.025).Univariate analysis showed that Cav-1 positive had a poor prognosis for overall survival(HR=2.284,95% CI 1.020-5.116,p=0.061),and RANK expression was also associated with a decrease in overall survival(HR=0.411,95% CI was 0.185-0.912,p=0.603).6.Effect of Cav-1 expression on prognosis and clinicopathological features in patients with RANK positive gastric cancerTo further refine the poor prognosis and to clarify the effect of Cav-1 on prognosis in different populations,we analyzed the effect of Cav-1 on prognosis in two groups with different RANK expression.The results showed that Cav-1 was not significantly associated with prognosis in patients with RANK negative,p=0.387.Among RANK-positive patients,Cav-1 positive patients had a poor prognosis,p=0.025.Univariate analysis showed that Cav-1 positive in RANK-positive patients was a poor prognostic factor for overall survival(HR=2.392,95% CI 1.082-5.289,p=0.031),and multivariate analysis showed Cav-1 positive in RANK-positive patients Patients with independent prognostic factors(HR=2.603,95% CI 1.174-5.773,p=0.019).7.RANKL can induce multiple tumor cell migration,knocking out Cav-1 can inhibit RANKL-induced tumor cell migrationIn order to verify whether the RANKL/RANK pathway is also involved in inducing migration of other tumor cells,we selected H460(lung cancer),ACHN(kidney cancer),MDA-MB-231(breast cancer)three tumor cells to detect RANK,and the results indicated that RANK was expressed in The above three tumor cell surfaces.Transfection of Cav-1 with si RNA in a timely manner significantly inhibited the migration of various tumor cells induced by RANKL.8.RANKL secreted by activated T cells can promote peritoneal metastasis of gastric cancer cellsThe peritoneal specimens of patients with gastric cancer peritoneal metastasis who had been treated in our hospital were selected.Fluorescent multicolor staining of RANKL,RANK,integrin beta2,CD8,CD48,CK and other indicators showed that NPK and integrin beta2 were expressed in peritoneal metastatic tumor tissues.T cells in the microenvironment of gastric cancer tumor tissue express RANKL.The ascites of benign patients such as ascites and cirrhosis of gastric cancer patients were collected.The detection of RANKL in ascites by ELISA showed that the RANKL in the ascites of patients with gastric cancer was higher than that in benign patients.The results of cell experiments indicated that activated T cells secrete more RANKL than unactivated T cells.The results of cell adhesion assay showed that activated T cell supernatant can promote the adhesion of gastric cancer cells to the most abundant content in the peritoneal microenvironment.Skin cells.Application of OPG can partially inhibit gastric cancer cell adhesion promoted by activated T cell supernatants.9.RANKL can enhance the adhesion of gastric cancer cells,thereby promoting peritoneal metastasis of gastric cancer We enriched the adhesion pathway by RANKL treatment of gastric cancer MGC803 cells for 48 hours.After RANKL stimulation of gastric cancer MGC803 and SGC7901 cells for 48 hours,the adhesion of gastric cancer cells increased significantly.Application of OPG can reverse the enhancement of RANKL-promoted adhesion of gastric cancer cells.10.RANKL promotes adhesion of gastric cancer cells by up-regulating integrin beta2The adhesion pathway in gastric cancer is mainly involved in the integrin family.It has been reported that integrin plays an important role in peritoneal adhesion.To further clarify the mechanism by which RANKL promotes adhesion of gastric cancer cells,we screened integrin in the geo database.Our results suggest that the expression of integrin beta2 is significantly increased in gastric cancer patients with peritoneal metastasis.The results of Westernblot showed that RANKL stimulated gastric cancer MGC803 and SGC7901 cells,and integrin beta2 was up-regulated 24 and 48 hours later.Adhesion experiments confirmed that transient transfection of integrin beta2 with si RNA inhibited RANKL-promoted gastric cancer cell adhesion.11.RANKL up-regulates integrin beta2 by down-regulating mi R335-5p,mi R1307-3p,and promotes adhesion of gastric cancer cellsTo further understand the mechanism by which RANKL up-regulates integrin beta2,we screened 52 mir RNAs that were significantly down-regulated after RANKL stimulation from the chip,and crossed with 117 mir RNAs targeting the integrin beta2 in the targetscan database to obtain five candidate mir RNAs: mir10a-5p,mir10b-5p,mir96-5p,mir335-5p,mir1307-3p.The results of rt PCR verification indicated that mir335-5p and mir1307-3p decreased significantly after 48 hours of RANKL stimulation.The results of adhesion experiments indicated that the cell adhesion ability of mimic mir10a-5p,mir10b-5p,mir96-5p,mir335-5p,mir1307-3p and mir335-5p and mir1307-3p groups was significantly decreased.Western blot results confirmed that the use of RANKL to stimulate gastric cancer MGC803 cells after mimic mir335-5p and mir1307-3p could not reverse the expression inhibition of integrin beta2.Conclusion: 1.RANKL can induce gastric cancer cell migration,and PI3 K and MAPK pathways are involved in RANKL-induced gastric cancer cell migration.2.The c-Src-Cav-1 pathway and its mediated lipid raft aggregation are the main mechanisms of action.3.RANKL induces multiple tumor cell migration by up-regulating Cav-1.4.Immunohistochemistry confirmed that Cav-1 is an independent prognostic factor for RANK-positive patients.5.RANKL secreted by activated T cells can promote peritoneal metastasis of gastric cancer cells.6.integrin beta2 is involved in RANKL-induced gastric cancer cell adhesion.7.mi R335-5p,mi R1307-3p-integrin beta2 RANKL promotes the main mechanism of adhesion of gastric cancer cells.