| Objective:Gastric cancer(GC)is one of the most common malignant tumors,and its morbidity and mortality are among the highest in the world.Although there are a variety of treatment strategies,such as surgery,radiotherapy,chemotherapy,targeted therapy and so on,the prognosis of patients with gastric cancer is not optimistic because gastric cancer is easy to metastasize.At present,most patients with gastric cancer are already in the advanced stage at the time of diagnosis,and most of the patients with advanced gastric cancer will occur liver,lymph node or peritoneal metastasis within 5 years after radical surgery.Among these metastases,peritoneal metastasis is the most common and fatal type of metastasis.it often brings many complications such as ascites,and due to chemotherapy resistance and rapid progression of disease,once peritoneal metastasis of gastric cancer occurs,its treatment and intervention is very difficult,which not only leads to poor prognosis,but also affects the quality of life of patients.However,there are still no valuable markers that can stably diagnose and predict peritoneal metastasis of gastric cancer.Therefore,it is urgent to clarify the mechanism of peritoneal metastasis of gastric cancer and establish an early prediction model of peritoneal metastasis.Methods: 1.The modules related to peritoneal metastasis of gastric cancer were identified by weighted gene co-expression network(Weighted Gene Co-Expression Network Analysis,WGCNA).2.The interaction network in protein level was obtained by using String database(https://string-db.org/).3.Kaplan-Meier survival curve analysis and logrank test were used to analyze the survival difference of patients with high and low expression of FSTL1.4.The receiver working characteristic curve(receiver operating characteristic,ROC)was used to evaluate the sensitivity and specificity of FSTL1 and PRIs in the diagnosis of gastric cancer peritoneal metastasis.5.Western blotting assay was used to detect the expression of protein.6.Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the expression of m RNA.7.The target gene was transiently silenced or overexpressed by si RNA or c DNA plasmid transfection technique.8.Transwell and wound healing test were used to detect the ability of cell migration and invasion.9.The adhesion test between peritoneal mesothelial cells and gastric cancer cells was used to detect the changes of adhesion ability of gastric cancer cells.10.The intraperitoneal implantation model of nude mice was used to verify whether FSTL1 could promote the peritoneum of gastric cancer in vivo.11.The differential pathways and differentially expressed genes were screened by full transcriptome sequencing.12.The signal pathways related to FSTL1 and PRIs were analyzed by using Jingdu gene and genome encyclopedia(Kyoto Encyclopedia of Genes and Genomics,KEGG)and gene enrichment analysis(Gene Set Enrichment Analysis,GSEA).13.The direct binding ability of protein to protein was detected by co-immunoprecipitation.14.Spearman correlation analysis was used to analyze the correlation between genes.15.The ss GSEA algorithm was used to estimate the score of immune cell infiltration in gastric cancer microenvironment.15.The minimum absolute contraction and selection operator algorithm(Lasso)algorithm were used to construct the prediction model of peritoneal metastasis.16.The nomogram combined with PRIs and clinical parameters were used to predict peritoneal metastasis in patients with gastric cancer.17.DCA curve and calibration curve are used to verify the prediction ability and stability of nomogram.18.Statistical analysis was carried out by Graph Pad Prism and R software.the experimental data are the average ±standard deviation(SD)of three independent repeated experiments.Student's t was used to examine and evaluate whether there is a difference between the two sets of data.P < 0.05 was considered to be statistically significant.Results: 1.Eight modules were identified by WGCNA analysis,among which turquoise module was most closely related to peritoneal metastasis.2.Combined with the results of WGCNA and string data sets,11 hub genes were identified.3.Among the 11 hub genes obtained,only FSTL1 has stable prognostic value.4.FSTL1 was highly expressed in patients with GC,and associated with poor prognosis.5.Further analysis of the GSE62254 dataset suggests that patients with peritoneal metastasis have higher levels of FSTL1 expression than patients without peritoneal metastasis.6.FSTL1 can be used as a marker for predicting peritoneal metastasis.ROC curve indicates that FSTL1 has high sensitivity and specificity in the diagnosis of gastric cancer peritoneal metastasis.7.According to the results of q RT-PCR and Western blotting experiments,HGC27 and SNU216 gastric cancer cell lines with relatively high expression of FSTL1 were selected for gene silencing,and overexpression plasmids were used to overexpress genes in MGC803 cell lines with relatively low expression of FSTL1.8.FSTL1 promotes the migration and invasion of gastric cancer cells.Adhesion and wound healing experiments showed that the adhesion and invasion ability of gastric cancer cells decreased after silencing FSTL1,while the adhesion and invasion ability of gastric cancer cells was enhanced after overexpression of FSTL1.9.FSTL1 promotes the adhesion between gastric cancer cells and peritoneal mesothelial cells(PMCs).Adhesion assay showed that after silencing FSTL1,the number of gastric cancer cells specifically adhering to PMCs decreased,on the contrary,overexpression increased.10.In vivo experiments confirmed that FSTL1 promotes peritoneal metastasis of gastric cancer.The model of peritoneal metastasis in nude mice showed that the number and weight of intraperitoneal nodules in nude mice decreased significantly after silencing FSTL1.11.FSTL1 is associated with extracellular matrixreceptor interaction(ECM-receptor interaction),adhesion plaque(focal adhesion)and PI3K-AKT signal pathways.Combined with the results of full transcriptome sequencing and bioinformatics analysis such as GSEA,KEGG,it was found that the above three pathways were significantly related to FSTL1.12.FSTL1 participates in the regulation of LAMA4 expression.The results of Western blotting showed that the level of LAMA4 protein was significantly down-regulated after silencing FSTL1,on the contrary,the overexpression was up-regulated.Spearman correlation analysis showed that the expression of FSTL1 was positively correlated with that of LAMA4.The results of full transcriptional group sequencing showed that LAMA4 was significantly down-regulated after silent FSTL1.13.FSTL1 participates in the regulation of LAMA4/integrin ? 1/FAK signal pathway.The results of Western blotting indicated that silencing and overexpressing FSTL1 changed the expression level of LAMA4,but the downstream integrin family did not change significantly.The results of co-immunoprecipitation showed that LAMA4 could directly bind to integrin ? 1,suggesting that LAMA4 played a role through direct binding to integrin ? 1,but did not affect the expression of integrin ? 1.Further Western blotting results suggested that the expression of downstream p-FAK of integrin ? 1 decreased significantly after silencing FSTL1.14.The infiltration state of immune cells in the immune microenvironment of different gastric cancer patients is different.heatmap analysis showed that the infiltration levels of 24 types of immune cells were different in different patients.Correlation analysis showed that 24 kinds of immune cells could be grouped into 5 groups,suggesting that they may participate in the same biological behavior together.15.Compared with gastric cancer patients without peritoneal metastasis,the tumor immune microenvironment of patients with peritoneal metastasis has different immune infiltration.Histogram analysis showed that among the 24 kinds of immune cells,15 species showed different infiltration states between patients with peritoneal metastasis and patients without metastasis.16.PRIs can be used as a marker for predicting peritoneal metastasis of gastric cancer.ROC curve indicates that PRIs has high sensitivity and specificity in predicting peritoneal metastasis in internal training set,internal experimental set and external verification set.17.Patients with EMT molecular subtype,pathological stage III,IV and Lauren's diffuse type have higher PRIs,suggesting that they have a higher risk of peritoneal recurrence.18.PRIs and p Stage are independent risk factors for predicting peritoneal metastasis.Univariate and multivariate analysis combined with PRIs and clinicopathological parameters suggested that only PRIs and p Stage had independent predictive value for peritoneal metastasis.19.The nomogram based on PRIs and p Stage can be used to predict peritoneal metastasis of gastric cancer.Conclusion: 1.FSTL1 is highly expressed in gastric cancer patients with peritoneal metastases,which is a poor prognostic factor in patients with gastric cancer.2.FSTL1 promotes peritoneal metastasis of gastric cancer by regulating LAMA4/ integrin ?1/FAK signal pathway which influencing biological behaviors such as invasion,migration and adhesion of gastric cancer cells.3.FSTL1 and PRIs can be used as potential markers for the diagnosis and prediction of peritoneal metastasis in patients with gastric cancer. |
PDF Full Text Request