| Part I Clinical value of salivary Fusobacterium nucleatum as a biomarker for the diagnosis of gastric cancerAim:Gastric cancer(GC)is one of the most common malignant digestive tract tumors.Since the early symptoms of GC are often not obvious,the vast majority of patients are diagnosed in the advanced stage.Fusobacterium nucleatum(Fn)colonizes mainly in the oral cavity and has been reported to be involved in the development of gastrointestinal tumors.Currently,little is known about the relationship between salivary Fn and GC.The purpose of this study was to investigate whether salivary Fn could be used as a biomarker for GC diagnosis and the effect of Fn on GC cells.Methods:1.Saliva samples were collected from 120 GC patients,31 atrophic gastritis(AG)patients,35 non-atrophic gastritis(NAG)patients,26 gastric polyp(GP)patients and 20 normal controls(NC)from Qilu Hospital of Shandong University from January 2019 to December 2020.The abundance of Fn in saliva was quantitatively determined by droplet digital polymerase chain reaction.2.Receiver operating characteristic(ROC)curve analysis was used to analyze the diagnostic value of Fn and traditional serum tumor markers CEA,carbohydrate antigen(CA)19-9 and CA72-4.3.Transwell assay and wound healing assay were used to observe the effect of Fn infection on GC cells.Western blotting assay was used to detect the expression of epithelial-mesenchymal transition(EMT)markers.Results:1.Fn level in saliva of GC patients was significantly higher than that of AG,NAG,GP patients and NC patients.Fn levels in saliva of GC patients increased with the increase of TNM stage.Fn levels in GC patients with lymph node metastasis were higher than those without lymph node metastasis.2.ROC curve analysis showed a favorable capability of Fn(73.33%sensitivity;82.14%specificity;area under the curve:0.813)in GC diagnosis,which was superior to that of CEA,CA19-9,CA72-4,ferritin,and sialic acid.3.Transwell assay and wound healing assay showed that Fn infection promoted the migration and invasion of GC cells.Western blotting analysis showed that Fn infection decreased the expression of E-cadherin and increased the expression of N-cadherin,Vimentin and Snail.Conclusion:Fn abundance in saliva can be used as a promising biomarker for the diagnosis of GC,and Fn infection can promote GC metastasis by accelerating the EMT process.Part Ⅱ Fusobacterium nucleatum induced exosomal HOTTIP promotes GC progression through miR-885-3p/EphB2Aim:Recent studies have shown that Fn is an important microorganism associated with a variety of digestive tract tumors,Fn DNA has been detected in GC tissues and it may participate in the occurrence and development of GC.However,the function and specific regulatory mechanisms of Fn infection in GC progression remain unclear.Therefore,this study aims to explore the important role and mechanism of Fn infection in the occurrence and development of GC.Methods:1.Exosomes(Fn-GCEx or GCEx)were isolated from the supernatant of MKN-28 cells treated with Fn or PBS by ultracentrifugation,and identified by transmission electron microscopy,particle size analysis and western blotting analysis.The changes of cell proliferation,migration and invasion ability were detected by EdU proliferation assay,colony formation assay,wound healing assay and transwell assay.2.The effects of Fn-GCEx on the growth and metastasis of GC tumors were evaluated by constructing mice subcutaneous tumor formation model and tumor liver metastasis model.3.RNA sequencing technology was used to screen out the up-regulated lncRNA HOTTIP in Fn-GCEx treated GC cells,and real-time fluorescence quantitative PCR(RT-qPCR)was used to verify the expression level of HOTTIP in Fn-GCEx and Fn-GCEx treated GC cells.EdU proliferation assay,colony formation assay,wound healing assay and transwell assay were used to detect the carcinogenic effect of HOTTIP in GC.4.Fluorescence in situ hybridization(FISH)was used to determine the location of HOTTIP in GC cells.The down-regulated miR-885-3p in GC cells treated by Fn-GCEx was screened by RNA sequencing,and the binding between HOTTIP and miR-885-3p was detected by RNA pull-down assay and dual luciferase reporter assay.The expression level of miR-885-3p in Fn-GCEx treated GC cells was verified by RT-qPCR analysis.EdU proliferation assay,colony formation assay,wound healing assay and transwell assay were used to detect the tumor inhibitory effect of miR-885-3p.5.The up-regulated EphB2 expression was screened in Fn-GCEx treated GC cells by RNA sequencing technology,and the combination of EphB2 and miR-885-3p was detected by dual luciferase reporter assay.RT-qPCR and western blotting experiments were used to detect the regulation of EphB2 expression by over-expression or down-expression of miR-885-3p and the expression level of EphB2 in Fn-GCEx and F-GCEx treated GC cells.6.The expression of Fn in Fn-positive GC tissues was detected by FISH assay,and the correlation between HOTTIP,miR-885-3p and EphB2 expression levels in Fn-positive GC tissues was detected by RT-qPCR analysis.RT-qPCR and western blotting analysis were used to detect the regulation of EphB2 expression by the overexpression or knockdown of HOTTIP.EdU proliferation assay,colony formation assay,wound healing assay and transwell assay were used to detect the role of EphB2 in GC progression.7.KEGG pathway enrichment analysis was used to screen out the downstream PI3K-AKT pathway,which was verified by western blotting analysis,EdU proliferation assay,colony formation assay,wound healing assay and transwell assay.Results:1.Fn-GCEx co-culture with AGS and HGC-27 cells significantly enhanced cell proliferation,migration and invasion abilities.2.In the mice subcutaneous tumor formation model,the growth rate,volume,weight and Ki-67 proliferation signal of tumor tissues in Fn-GCEx treatment groups were significantly higher than those in the control group.In the tumor liver metastasis model,the liver metastases in Fn-GCEx treatment groups were significantly more than those in the control group.3.lncRNA HOTTIP with up-regulated expression in Fn-GCEx treated GC cells was screened by RNA sequencing technology.HOTTIP expression level was increased in AGS and HGC-27 cells treated with Fn-GCEx,and also increased in Fn-GCEx.Transwell co-culture and cell experiments demonstrated that Fn infection increased the expression level of exosomal HOTTIP and transported it to the recipient GC cells,promoting the proliferation,migration and invasion of GC cells.4.HOTTIP is mainly located in the cytoplasm in GC cells.Down-regulated miR-885-3p in Fn-GCEx treated GC cells was screened by RNA sequencing technology.HOTTIP could directly bind to miR-885-3p and negatively regulate the expression of miR-885-3p,so as to participate in GC progression.5.The up-regulated expression of EphB2 in Fn-GCEx treated GC cells was screened by RNA sequencing technology,and miR-885-3p could directly bind to EphB2 and negatively regulate the expression of EphB2.miR-885-3p was negatively correlated with the expression of EphB2 in Fn-positive GC tissues.EphB2 expression was increased in AGS and HGC-27 cells treated with Fn-GCEx,but not detected in Fn-GCEx.6.HOTTIP was positively correlated with EphB2 expression in Fn positive GC tissues.HOTTIP could positively regulate the expression of EphB2 through miR-885-3p.EphB2 is the target gene of exosomal HOTTIP in GC cells,which could affect the proliferation,migration and invasion abilities of AGS and HGC-27 cells.7.EphB2 activated PI3K-AKT pathway,which promoted the proliferation,migration and invasion of GC cells.Conclusion:Fn infection induced the up-regulation of exosomal HOTTIP secreted by GC cells,and after the exosomal HOTTIP was transported to recipient GC cells,it could promote the progression of GC through miR-885-3p/EphB2/PI3K/AKT.This study revealed a potential molecular pathway for Fn to participate in GC progression and suggested that EphB2 might be a therapeutic target for GC. |
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