Mechanisms Of Exosomal UBE3B And LPPR4 Promote Peritoneal Metastasis Of Gastric Cancer

Objective:Gastric cancer(GC)is one of the most common malignancies which has high incidence and mortality worldwide.Peritoneal dissemination is the main route of metastasis in advanced GC and often causes a large amount of massive ascites or intestinal obstruction.However,few reliable diagnostic or prognostic biomarkers are available for peritoneal metastasis of GC.The reason for this is that it is still unclear about the mechanism of GC peritoneal metastasis.This study aimed to investigate the mechanism of peritoneal metastasis of GC and to explore the role of key molecules in this process.Methods: 1.Exosomes from supernatants of gastric cancer cells were extracted by ultracentrifugation.2.Transmission electron microscopy(TEM)and Nano Sight measurements were used to identify exosomes.3.DID was used to label exosomes in order to track exosomes.4.The protein expression was detected by Western blotting.5.Transwell assay and wound healing assay were used to investigate the ability of cell migration and invasion.6.Tumor cell-peritoneal mesothelial cell adhesion assay was used to test the ability of tumor cells adhered to peritoneal mesothelial cells(PMCs).7.Immunofluorescence and immunohistochemistry were used to detect the expression of the marker of human peritoneal mesothelial cells(PMCs)and cancer associated fibroblasts(CAFs).8.Tumor peritoneal dissemination was investigated in mouse models in vivo.9.Metabolic level of peritoneal metastatic nodules in nude mice was determined by PET.10.Screening of differentially expressed genes was determined by the whole transcriptome sequencing.11.Bioinformatics methods were used to screen the differentially expressed genes.12.Kaplan–Meier method and log-rank test were used to analyze the survival curves.13.Receiver Operating Characteristic(ROC)was used to evaluate the sensitivity and specificity of LPPR4 in diagnosis of GC peritoneal metastasis.14.m RNA expression was detected by quantitative real time polymerase chain reaction(q RT-PCR).15.si RNA or c DNA plasmid transfection were used to transiently knock-down or over-express the target gene.16.Stable transfected cell lines were established by lentivirus transfection to knock-down or over-express the target gene.17.The Kyoto Encyclopedia of Genes and Genomics(KEGG)and Gene Set Enrichment Analysis(GSEA)were used to explore LPPR4-related signaling pathways.18.The direct binding ability of protein to protein was detected by co-immunoprecipitation.19.Spearman correlation analysis was used to analyze the correlation between genes.20.Statistical analysis: all statistical analyses were performed using Graph Pad Prism software,Social Sciences(SPSS)software version 20.0,and R software.The experimental data was displayed as means ± standard deviation(SD)of three independent experiments.Differences between two groups were assessed using the Student’s t-tests.P < 0.05 was considered to be statistically significant(*P < 0.05,**P < 0.01,***P < 0.001).Results: 1.The formation of peritoneal PMN promoted GC peritoneal metastasis.Immunohistochemistry staining of peritoneal tissues from patients with peritoneal metastasis of gastric cancer showed that the CAFs marker α-SMA was significantly overexpressed,while the PMCs marker Calretinin and WT1 were significantly underexpressed.2.A high peritoneal metastatic gastric cancer cell line MKN45 P was established and validated.The morphological changes of cells were observed under light microscope.Compared with MKN45 cells,MKN45 P cells were significantly spindle-shaped.Transwell assay showed that MKN45 P cells enhanced the ability of migration than MKN45 cells.Tumor peritoneal dissemination mouse model showed that MKN45 P could promote the formation of peritoneal metastatic nodules than MKN45 cells.3.Exosomes from supernatants of GC cells were extracted and identified.By observing the morphology of exosomes under transmission electron microscopy(TEM),it was found that exosomes had a characteristic cup shape.The particle diameter of exosomes detected by Nanosight method showed that the peak diameter of exosomes was 96.5 nm.The expression of characteristic markers of exosomes(Flotillin-1,CD63,TSG101 and CD9)was significantly higher in exosomes than that in cells assessed by Western blotting.DID labelled exosomes were effectively taken in by HMr SV5 cells assessed by internalization experiment.4.MKN45 P cells derived exosomes promoted the migration of PMCs.Compared with the PBS control group,the migration ability of HMr SV5 cells was significantly enhanced treated by MKN45 and MKN45 P cells derived exosomes,with an increasing trend detected by Transwell assay.5.MKN45 P cells derived exosomes promoted adhesion of GC cells to PMCs.Compared with the PBS control group,the number of GC cells adhering to HMr SV5 cells increased significantly treated with exosomes secreted by MKN45 and MKN45 P cells,with an increasing trend detected by tumor cell-peritoneal mesothelial cell adhesion assay.6.MKN45 P cells derived exosomes promoted the conversion of PMCs to CAFs.Compared with the PBS control group,the morphology of HMr SV5 cells treated with exosomes secreted by MKN45 and MKN45 P cells was significantly spindle-shaped by an optical microscope.Compared with the PBS control group,after treated with exosomes secreted by MKN45 and MKN45 P cells,α-SMA and vimentin were significantly up-regulated in HMr SV5 cells,while WT1 and tight junction protein(Zo-1)were dramatically down-regulated assessed by Western blotting and immunofluorescence assays to detect the markers of conversion of PMCs to CAFs.7.MKN45 P cells derived exosomes promoted GC peritoneal metastasis in nude mice.Exosomes secreted by MKN45 and MKN45 P cells or PBS were injected intraperitoneally into nude mice,and then MKN45 cells were injected intraperitoneally into nude mice.PET of animals showed that exosomes derived from MKN45 and MKN45 P cells significantly enhanced the metabolic level of peritoneal metastatic nodules compared with the PBS control group.After dissection of nude mice,it was observed that exosomes secreted by MKN45 P cells promoted the formation of GC peritoneal metastasis in nude mice to a higher degree.8.MKN45 P cells derived exosomes promoted the transformation of PMCs to CAFs in nude mice.HE staining showed that the peritoneum of the nude mice in PBS control group was mainly composed of a single continuous layer of peritoneal mesothelial cells,while the peritoneum of the nude mice in the MKN45 and MKN45 P cells derived exosomes pretreated group was significantly thickened and fibrotic.Compared with PBS control group,the expression of PMCs markers Calretinin and Mesothelin in the peritoneum of nude mice pretreated with exosomes secreted by MKN45 and MKN45 P cells was significantly decreased,while the expression of CAFs markers α-SMA was up-regulated dramatically by immunohistochemistry and immunofluorescence staining.9.Exosomal UBE3 B was a key molecule involved in the formation of peritoneal specific PMN induced by GC cell derived exosomes.By the whole transcriptome sequencing of MKN45 and MKN45 P cells,we conducted KEGG pathway enrichment analysis of differentially expressed genes.It was found that the Ubiquitin mediated proteolysis was an important differentially expressed pathway.UBE3 B might be the key molecule confirmed by Western blotting.10.GC cells derived Exosomal UBE3 B overexpression and internalization were validated.UBE3 B was overexpressed by lentivirus in MKN45 cells.Exosomal UBE3 B was effectively ingested by HMr SV5 cells,and UBE3 B was also overexpressed in exosomes derived from MKN45 cells overexpressing UBE3 B confirmed by internalization assay.11.GC cells derived Exosomal UBE3 B enhanced the migration of PMCs.The migration ability of HMr SV5 cells treated with exosomes secreted by MKN45 cells overexpressing UBE3 B significantly enhanced compared with the vector group detected by Transwell assay.12.GC cells derived Exosomal UBE3 B promoted adhesion of GC cells to PMCs.The number of GC cells adhered to HMr SV5 cells treated with exosomes secreted by MKN45 cells overexpressing UBE3 B significantly increased compared with the vector group detected by tumor cell-peritoneal mesothelial cell adhesion assay.13.GC cells derived Exosomal UBE3 B promoted the transformation of PMCs into CAFs.Compared with the vector control group,the morphology of HMr SV5 cells treated with exosomes secreted by MKN45 cells overexpressing UBE3 B was spindle-shaped by an optical microscope.Compared with the vector control group,after treated with exosomes secreted by MKN45 cells overexpressing UBE3 B,α-SMA and vimentin were significantly up-regulated in HMr SV5 cells,while WT1 and Zo-1 were dramatically down-regulated detected by Western blotting and immunofluorescence assays to detect the markers of conversion of PMCs to CAFs.14.The unfavorable prognostic and peritoneal metastasis-related genes in GC were identified by bioinformatics gene screening.In TCGA-STAD and GSE62254 datasets,differentially expressed genes,including eight genes,which had both unfavorable prognostic value and peritoneal metastasis-related effect were screened in GC.Notably,LPPR4 ranked the second highest according to fold change and might be related to tumorigenesis and progression of GC peritoneal metastasis.Therefore,LPPR4 was selected for further experimental analysis.15.LPPR4 was highly expressed in patients with GC peritoneal metastasis and was associated with poor prognosis in GC patients.A significant positive association between LPPR4 expression and peritoneal recurrence was observed in the GSE62254 cohort.Kaplan-Meier analysis was performed on the TCGA-STAD and GSE62254 cohort patients.Patients with high expression of LPPR4 in both databases were found to have significantly lower overall survival compared to patients with low expression of LPPR4.16.LPPR4 could be used as a candidate biomarker for the diagnosis and prognosis of GC peritoneal metastasis.Receiver operating characteristic(ROC)curve analysis was performed to evaluate the sensitivity and specificity of LPPR4 for the diagnosis of GC peritoneal metastasis.The area under ROC curve(AUC)of LPPR4 was 0.687,demonstrating that LPPR4 had a high sensitivity and specificity for GC peritoneal metastasis diagnosis.17.LPPR4 was up-regulated in GC cell lines.LPPR4 expression levels were significantly up-regulated in GC cell lines compared to normal gastric epithelial cell line GES-1 measured by q RT-PCR and Western blotting.According to the expression levels of LPPR4,si RNA was used for gene silencing in HGC27 and MKN74 cells with high expression of LPPR4,and overexpression plasmid was used for gene overexpression in MGC803 cells with low expression of LPPR4.18.LPPR4 promoted the migration and invasion of GC cells.The ability of GC cells to migrate and invade weakened after downregulating LPPR4,whereas the migration and invasion potential of GC cells was significantly enhanced in the LPPR4-overexpression group assessed by Transwell and wound healing assays.19.LPPR4 promoted adhesion of GC cells to PMCs.Less LPPR4-knockdown GC cells were adhered to a dense layer of HMr SV5 cells compared to the control group,whereas more LPPR4-overexpression GC cells were attached to HMr SV5 cells by tumor cell-peritoneal mesothelial cell adhesion assay.20.LPPR4 promoted peritoneal metastasis of GC cells in vivo.LPPR4 was knocked down in HGC27 cells using lentivirus.HGC27‐sh NC and HGC27‐sh LPPR4 cells were injected into the peritoneal cavity of the nude mice to evaluate the effect of LPPR4 on peritoneal implantation in vivo.HGC27‐sh LPPR4 cells developed less peritoneal metastatic nodules compared with peritoneal metastatic nodules developed by HGC27‐sh NC cells.21.LPPR4 was associated with cell adhesion molecules(CAMs)pathway.High expression of LPPR4 was found to be related to the cell adhesion molecules pathway by KEGG and GSEA enrichment analysis.22.LPPR4 was involved in regulating integrin α / FAK signaling pathway.The related protein expression in ITGA / FAK pathway was significantly down-regulated after LPPR4 silencing,whereas dramatically up-regulated after LPPR4 overexpressing in GC cells by Western blotting.Interestingly,there was no significant change in the expression of ITGB.LPPR4 was positively correlated with the expression of ITGA by Spearman correlation analysis.23.LPPR4 activated transcription of integrin α through Sp1 transcription factor.Immunoprecipitation assays failed to demonstrate a direct interaction between LPPR4 and ITGA.Sp1 was found to be a potential transcription factor of ITGA by UCSC genome website and Jaspar database.The expression of LPPR4 was positively correlated with Sp1,and the expression of Sp1 was positively correlated with ITGA by Spearman correlation analysis.The expression of Sp1 was significantly down-regulated when LPPR4 was knocked down and the expression of ITGA was significantly down-regulated when Sp1 was knocked down detected by q RT-PCR and Western blotting assays.24.LPPR4 promoted migration and invasion of GC cells by regulating the activity of Sp1.Transwell migration and matrigel invasion assays showed that Sp1 overexpression enhanced the migration and invasion abilities of GC cells.Sp1 overexpression could restore the migration and invasion ability of GC cells caused by LPPR4 gene knockdown.25.LPPR4 promoted adhesion of GC cells to PMCs by regulating the activity of Sp1.In the case of LPPR4 gene knockdown,overexpression of Sp1 was found to eliminate the reduction of GC cells adhering to HMr SV5 cells by tumor cell-peritoneal mesothelial cell adhesion assay.Conclusion: 1.Exosomes derived from gastric cancer cells promote the peritoneal metastasis of gastric cancer by promoting the formation of peritoneal specific pre-metastatic niche.2.Exosomal UBE3 B released by gastric cancer cells promotes peritoneal metastasis of gastric cancer by promoting the conversion of PMCs to CAFs to form peritoneal specific pre-metastatic niche.3.LPPR4 is highly expressed in gastric cancer peritoneal metastasis tissue,which is an unfavorable prognostic factor for patients with gastric cancer.4.LPPR4 regulates the expression of integrin α by regulating the expression of Sp1 transcription factor,via FAK,Src and AKT signal pathways,activating the target gene MMP2 to promote the migration,invasion and adhesion of gastric cancer cells,and then promotes the peritoneal metastasis of gastric cancer.5.Exosomal UBE3 B and LPPR4 can be used as potential biomarkers for the diagnosis and prognosis of peritoneal metastasis of gastric cancer.