| Gastric cancer,which is characterized by high invasiveness and frequent distant metastasis,is the most common gastrointestinal malignant tumor in China.Nearly half of new gastric cancer cases in the world occur in China,and the mortality of gastric cancer ranks third among all malignant tumors in China.However,the specific mechanisms of gastric cancer invasion and metastasis remain poorly understood.A process known as epithelial-mesenchymal transition(EMT)can enable tumor cells to acquire a capacity for invasion,which is the beginning of tumor metastasis,yet the molecular basis of EMT has not been characterized in depthIn recent years,high-throughput sequencing technology has promoted our understanding of tumor molecular characteristics and has also opened new opportunities for EMT research.High-throughput sequencing studies of gastric cancer have revealed the presence of mesenchymal-like gastric cancer(EMT molecular subtype),which is characterized by high invasion and metastasis.To further understand the specific molecular mechanisms of gastric cancer EMT,we used a public database to study the gene expression profiles of mesenchymal-like gastric cancer,and bioinformatics analyses identified the potential key gene TNS1 in gastric cancer EMTWe propose that TNS1 promotes EMT,invasion,and metastasis in gastric cancer by activating key driver genes upstream of EMT.We support this hypothesis with cell function experiments,mechanism studies,and clinical association studies.Our work reveals the function of TNS1 in the invasion and metastasis of gastric cancer,thereby defining a therapeutic target and providing a theoretical basis for development of methods to inhibit the invasion and metastasis of gastric cancerPart 1.Molecular characteristics of mesenchymal-like gastric cancer and screening of key EMT genesBackground:According to the molecular classification of the Asian Cancer Research Group(ACRG),gastric cancer can be divided into four molecular subtypes mesenchymal-like,microsatellite instability,TP53 activation,and TP53 inactivation Among them,mesenchymal-like gastric cancer was significantly associated with elevated expression of EMT gene tags,whereas CDH1 gene expression was significantly reduced.Further study of the gene expression profile characteristics of mesenchymal-like gastric cancer should help reveal key genes affecting the occurrence and development of EMTObjective:To study the gene expression profile of mesenchymal-like gastric cancer,identify any key gene(s)affecting EMT to support further research on the specific molecular mechanism(s)of EMTMethods:mesenchymal-like and epithelial-like gastric cancer samples(including the TP53-active type and TP53-inactive type,but not the micro satellite unstable type)in the ACRG gastric cancer cohort were assessed in a comparative study of gene expression.Gene Set Enrichment Analysis(GSEA)was used to annotate the Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway and Gene Ontology(GO)annotation information for the identified differentially expressed genes.Weighted gene co-expression network analysis(WGCNA)was used to screen for genes predicted to be most closely related to mesenchymal-like gastric cancerResults:A total of 9712 significantly differentially expressed genes(p<0.05)were identified from among 46 cases of mesenchymal-like gastric cancer and 186 cases of epithelioid-like gastric cancer.GSEA showed that KEGG signaling pathways and GO entries related to EMT,"cell-matrix interactions","cell-cell interactions" and others that participate in tumor invasion and metastasis were significantly enriched in mesenchymal-like gastric cancer,highlighting the up-regulation of related genes Moreover,"cell cycle","DNA replication","DNA repair","cell division",and other KEGG signaling pathways and GO entries were significantly enriched in epithelioid gastric cancer,suggesting that related genes may be down-regulated during EMTA total of 1,230 genes were found to have logarithmic value of the expression ratio(logFC)between the mesenchymal-like and epithelial-like gastric cancer genes are greater than 1.WGCNA divided these genes into three color-coded modules.We focused on the blue-green module that positively correlates with mesenchymal-like gastric cancer:the genes in this module are highly positively correlated with mesenchymal-like gastric cancer(Spearman R=0.83,P<0.001),among which 30 were selected by scoring and ranking for predicted functional relevance.TNS1,which has not been previously associated with gastric cancer EMT,was among these top-ranking genesConclusion:Genes related to tumor invasion and metastasis are significantly enriched in mesenchymal-like gastric cancer,among which TNS1 is a potential key EMT genePart 2.Verification of a key candidate gene TNS1 for gastric cancer EMTBackground:The EMT process contributes to the ability of malignant tumor cells to acquire invasion and metastasis capacity,but its specific molecular mechanism(s)are not clear.TNS1 is a member of the angiotensin adhesion family,and is known to function in biological processes including cell adhesion,movement,and signal transduction.It has been reported that TNS1 is involved in the invasion and metastasis of malignant tumors,but its specific role is thought to depend on specific tumor type For example,TNS1 inhibits invasion and metastasis in breast cancer and prostate cancer cells,while TNS1 promotes invasion in bowel cancer,bladder cancer,and lung cancer cells.The relationship between TNS1 and invasion and metastasis of gastric cancer has not been reported,so any mechanisms through which TNS1 may affect tumor invasion and metastasis remain unknown.Based on the potential association between TNS1 and EMT that we found in Part 1,we hypothesized that TNS1 may improve invasion and metastasis of gastric cancer by activating EMT;this portion of the research project examined this hypothesisObjective:To functionally validate that TNS 1 promotes the invasion and metastasis of gastric cancer by promoting EMT using cell function experiments,mechanistic research,and clinical verificationMethods:We used siRNA to construct the TNS1 knockdown gastric cancer cell lines SGC-7901 and HGC-27.Cell scratches and Transwell cell migration and invasion experiments were used to study the effect(s)of TNS 1 on the migration and invasion of gastric cancer cells.Based on protein and mRNA expression data of gastric cancer from public databases,Parametric GSEA(PGSEA)was used to perform EMT scoring by adopting several well-established EMT signatures.The correlations between TNS1 expression and the EMT scores were analyzed.qPCR and western blot were used to study the effect of TNS1 knockdown on the expression of the key driver molecule snail that is known to function upstream of EMT and the mesenchymal cell marker vimentin.Immunohistochemical staining of TNS1 protein was performed in the Affiliated Hospital of Jiangsu University(AHJU)gastric cancer cohort to analyze any relationships between the TNS1 protein expression level and distant metastasis(M1 staging).Transcriptome data of gastric cancer from the cohorts ACRG and GEO 15459 were utilized to analyze any relationships between the TNS1 mRNA expression level and the M1 status.Results:TNS1 knockdown drastically inhibited the migration and invasion of gastric cancer cells(p<0.05).TNS1 expression at both the mRNA and the protein levels were positively correlated with the EMT scores,and TVS1 knockdown significantly reduced the mRNA and protein expression of snail and vimentin(p<0.05),suggesting that TNS1 promotes EMT by upregulating snail.In the AHJU cohort,the proportion of M1 was 9.6%,and the proportion of M1 with high TNS1 protein expression was significantly higher than that with low TNS1 protein expression(12%vs 3.2%,p=0.045).In the ACRG and GE015459 cohorts,the proportion of M1 was 25.7%and 31.3%,respectively.The proportion of M1 with high TNS1 mRNA expression was also significantly higher than that with low TNS1 mRNA expression(ACRG:36.3%vs 19.3%,p=0.001;GE015459:36.7%vs 22.2%,p=0.037).Conclusion:TNS1 promotes the invasion and metastasis of gastric cancer by upregulating snail to activate EMT.Part 3.Relationships between TNS1 and clinicopathological characteristics and prognosis of gastric cancerBackground:Gastric cancer is highly heterogeneous,and patients in the same stage may have very different prognoses,suggesting the existence of molecular heterogeneity.Identifying molecular markers related to the prognosis of gastric cancer should be helpful for the early identification of patients at high risk of recurrence and metastasis after surgery,and should also be helpful for the optimization of personalized treatment strategies.Our earlier studies functionally confirmed a role for TNS1 in gastric cancer EMT,invasion,and metastasis;however,the relationships between TNS1 and clinicopathological characteristics and the prognosis of gastric cancer remains to be studied.Objective:To reveal the clinical significance of TNS1 by analyzing the relationship between TNS1 and clinicopathological characteristics and prognosis of gastric cancer.Methods:The relationship between TNS1 protein/mRNA expression level and clinicopathological characteristics were analyzed for three independent gastric cancer cohorts,including the AHJU,ACRG,and GE015459.Kaplan-Meier survival analysis combined with the Log-rank test was used to study the effects of TNS1 high expression on overall survival(OS),while univariate and multivariate Cox proportional hazard models were used to analyze factors affecting OS.Results:In at least two cohorts,the high expression rate of TNS1 was significantly higher in patients with positive nerve invasion than in those with negative nerve invasion(p<0.05),in ?/?-stage than in ?B/?-stage gastric cancer(p<0.05),in diffuse than in intestinal type gastric cancer(p<0.05).In the AHJU cohort,patients with high TNS1 protein expression had significantly shorter OS than those with low TNS1 protein expression(p<0.001).Similarly,in the ACRG and GE015459 cohorts,patients with high TNS1 mRNA expression also had significantly shorter OS than those with low TNS1 mRNA expression(p<0.001 and p=0.008).After univariate selection for suitable variables,the multivariate Cox model showed that TNS1 expression levels were independent OS predictors in all three of the examined gastric cancer cohorts.Conclusion:The high expression of TNS1 indicates a poor prognosis and can be used as a prognostic biomarker in gastric cancer. |
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