Mechanisms Of CYS1 And NPTX1 Promoting Metastasis In Gastric Cancer

Objective: CYS1(Cystin1)is a small molecule protein.Abnormal expression of CYS1 can inhibit the formation and function of primary cilia,leading to cilia-related diseases.In tumor-related studies,it was found that the expression of CYS1 is lower in pancreatic tumors than normal tissues and is a target gene for hepatocyte nuclear factor 6.Besides,one research has demonstrated that CYS1 was a poor predictor of gastric cancer using statistic analysis.So far,the role of CYS1 in tumors and its related biological behaviors and mechanisms has remained unclear.In this study,we aimed to investigate the ability of CYS1 in the prognosis of gastric cancer patients. Neuronal pentraxin 1(NPTX1)is a highly conserved protein involved in central nervous system diseases.It was reported low expression in tumors due to abnormal methylation.However,studies have shown that NPTX1 is associated with cell migration ability.In this study,we aimed to investigate the predictive ability of CYS1 and NPTX1 in the prognosis of gastric cancer patients and to participate in the regulation of gastric cancer metastasis,and provide a new strategy for improving the prognosis of gastric cancer patients with peritoneal metastasis.Methods: 1.The relationship between CYS1 expression and clinicopathological parameters of gastric cancer patients was analysed using TCGA(The Cancer Genome Atlas)and GEO(Gene Expression Omnibus)databases;2.The expression of mRNA and protein were detected by q-RT-PCR and Western blotting;3.SiRNA and lentivirus were used to down-regulat the target protein;4.Plasmid and lentivirus were used to upregulate the target protein;5.Hanging drop assay detected the ability of cell-cell adhesion;6.Cell-extracellular Matrix adhesion assay was used to detect the adhesion ability of cells to extracellular matrix;7.Transwell assay was used to detect the ability of migration and invasion;8.Cell-peritoneal mesothelial cell adhesion assay was used to determine the ability of adhesion between gastric cancer cells and peritoneal mesothelial cells;9.MTT was used to detect cell activity;10.Immunoprecipitation was used to detect the binding ability between proteins;11.Mass spectrometry was used to detect the proteins binding to CYS1;12.Nuclear pulp separation test was used to detect the expression of targetprotein in cytoplasm and nuclear;13.Immunofluorescence assay was used to detect the localization of protein;14.Mouse peritoneal metastasis model was used to examine the metastatic ability of CYS1 in vivo;15.Statistical analysis: each experiment was repeated for three times and the results were reported as mean ± standard deviation.Statistical analysis was performed using SPSS 17.0 statistical software,and P < 0.05 was considered statistically significant.Results: 1.High expression of CYS1 in gastric cancer is associated with poor prognosis in patients with gastric cancer.The GEO and TCGA databases showed that the expression of CYS1 in gastric cancer tissues was higher than in normal tissues,and the overall survival of patients with high expression of CYS1 was shorter compared with patients with low expression of CYS1.2.CYS1 is associated with peritoneal metastasis of gastric cancer.By analyzing the GSE62254 dataset,the proportion of peritoneal metastasis in patients with high expression of CYS1 was higher than in patients with low expression of CYS1.3.CYS1 promotes peritoneal metastasis in mice.The expression level of CYS1 in SGC7901 cells was down-regulated by lentivirus.The number of peritoneal nodules in the shCYS1 group was significantly lower than that in the control group after 16 days.4.CYS1 promotes adhesion of tumor cells to peritoneal mesothelial cells.The expression of CYS1 in SGC7901 and HGC27 was down-regulated by siRNA,and CYS1 was significantly upregulated by transfecting lentivirus.Tumor cell-peritoneal mesothelial cell adhesion assay showed that the ability of adhesion between SGC7901 and HGC27 cells and SV5 was attenuated by reducing the expression of CYS1,and the adhesion ability was increased after overexpression of CYS1.5.CYS1 inhibits cell-cell adhesion.The results of hanging drop assay indicated that the ability of cell-cell adhesion increased along with downregulation of CYS1.6.CYS1 promotes cell-extracellular matrix adhesion.Cell-extracellular matrix adhesion assay suggested that the adhesion ability of SGC7901 and HGC27 cells to the extracellular matrix is significantly reduced by decreasing the expression of CYS1,and the ability of adhesion increased after overexpression of CYS1.7.CYS1 promotes migration and invasion.Transwell assay results suggested that after downregulating CYS1,the ability of migration and invasion of SGC7901 and HGC27 cells decresed,and the ability of migration and invasion is enhanced after overexpression of CYS1.8.CYS1 inhibits adhesion junctions.The resultsof Western blotting showed that the expression of E-cadherin(E-ca)was increased after downregulating CYS1,and the expression of E-ca was down-regulated after overexpression of CYS1.There is no change in proteins involved in tight junction.9.CYS1 inhibits the formation of E-cadherin/?-catenin complex.The results of co-immunoprecipitation showed that the binding ability of E-ca to ?-catenin was increased after downregulating CYS1,and the binding ability of E-ca to ?-catenin was decreased after overexpression of CYS1.10.CYS1 promotes the competitive binding of PHB2 to E-ca with ?-catenin.Co-immunoprecipitation experiments indicated that in cells with overexpressing CYS1,PHB2 and E-ca were simultaneously precipitated by ?-catenin.Besides,the binding of ?-catenin to E-ca decreased,and the binding to PHB2 was increased.11.Overexpression of CYS1 and simultaneous silencing of PHB2 reversed the inhibition of cell-cell adhesion.The results of hanging drop assay showed that the cell-cell adhesion ability decreased after overexpression of CYS1,and the cell-cell adhesion ability was increased compared with the upregulating CYS1 group after silencing PHB2 with siRNA.12.Overexpression of CYS1 and simultaneous silencing of PHB2 reversed the down-regulation of E-ca.Western blot results showed that the expression of E-ca protein in SGC7901 and HGC27 was decreased after overexpression of CYS1,while the expression of E-ca was higher after simultaneously silencing PHB2.13.Overexpression of CYS1 and simultaneous silencing of PHB2 reversed the promotion of cell-extracelular matrix adhesion.Cell-extracellular matrix adhesion assay showed that the ability of adhesion of SGC7901 and HGC27 to cells-extracellular matrix was increased after overexpression of CYS1,and the ability after simultaneously silencing PHB2 was decreased compared with that of overexpressing CYS1.14.Overexpression of CYS1 and simultaneous silencing of PHB2 reversed the promotion of migration and invasion.Transwell assay suggested that the migration and invasion ability of SGC7901 and HGC27 cells are increased after overexpression of CYS1,which is reversed by simultaneous silencing of PHB2.15.Overexpression of CYS1 and simultaneous silencing of PHB2 could not reverse the ability of promoting tumor cell-peritoneal mesothelial cell adhesion.Tumor cell-peritoneal mesothelial cell adhesion assay showed that the adhesion ability of SGC7901 and HGC27 cells to peritoneal mesothelial cells was increased afteroverexpression of CYS1,and this phenomenon could not reversed by simultaneously silencing PHB2.16.CYS1 promotes the entry of ?-catenin into the nucleus.The results of nucleoplasmic separation assay and immunofluorescence assay showed that the expression of ?-catenin in the cytoplasm was increased and the expression of ?-catenin in the nucleus was decreased after silencing CYS1.The expression of ?-catenin in the nucleus was increased when CYS1 was overexpressed.17.CYS1 promotes the expression of ?-catenin target genes.Q-RT-PCR and Western blotting showed that the expression of target genes of ?-catenin,MMP2,MMP7,MMP9 and CD44 were down-regulated after silencing CYS1;the expression of target genes were increased after overexpression of CYS1,and the expression was reversed by simultaneous silencing of PHB2.18.The expression of NPTX1 is higher in gastric cancer and is associated with poor prognosis in patients with gastric cancer.The results of TCGA and GEO database analysis showed that the expression of NPTX1 was higher in gastric cancer tissues compared with adjacent tissues,and the overall survival of patients with high expression of NPTX1 was poorer than that of patients with low expression.19.NPTX1 promotes the abilities of migration,invasion and adhesion of gastric cancer cells.The expression level of NPTX1 in gastric cancer cell lines was detected by immunoblotting.The expression of NPTX1 in BGC823 and SNU216 was down-regulated by siRNA,and the m RNA and protein expression levels of NPTX1 in HGC27 cells were up-regulated by overexpression lentivirus.Transwell results suggest that after NPTX1 is silenced,cell migration and invasion are weakened,and cell migration and invasion are enhanced after overexpression of NPTX1.At the same time,the cell-extracellular matrix adhesion assay suggested that the adhesion of BGC823 and SNU216 cells to the extracellular matrix was significantly attenuated after silencing NPTX1,and the adhesion ability was increased after overexpression of NPTX1.20.NPTX1 is associated with ECM receptors and focal adhesion functions.Through GSEA enrichment analysis,we found that high expression of NPTX1 was significantly associated with ECM receptor and focal adhesion.21.NPTX1 is positively correlated with ITG.ITGA1 and ITGA7,which have the strongest correlation with NPTX1,were screened by VENNY.22.NPTX1 inhibited the formation of pseudopods through the ITG/FAK pathway.Western blotting showed that the ITG/FAK/Src pathway-associated protein was down-regulated after silencing NPTX1.Atthe same time,immunofluorescence assays showed that the formation of pseudopods was reduced during the invasion of BGC823 cells after silencing NPTX1.Conclusion: 1.High expression of CYS1 in gastric cancer tissues is associated with the poor prognosis of gastric cancer patients;2.CYS1 promotes peritoneal metastasis of gastric cancer;3.CYS1 competitively binds ?-catenin with E-ca via PHB2 to inhibit the formation of E-ca/?-catenin complex,inhibit gastric cancer cell-cell adhesion and promotes peritoneal metastasis in gastric cancer;4.CYS1 promotes the entry of ?-catenin into nuclear to activate its downstream target genes,promoting gastric cancer cell-extracellular matrix adhesion and the ability of migration and invasion,promotes peritoneal metastasis of gastric cancer;5.The expression of NPTX1 is higher in gastric cancer tissues,which is a poor prognosis of gastric cancer patients;6.NPTX1 promotes the formation of focal adhesion of gastric cancer cells and the formation of pseudopods of tumor cells by promoting the expression of integrin ?1 and ?7,promoting migration and invasion.