Investigation Of Clinical Significance,Biological Functions And Regulation Mechanism Of B7H4 In Cancer Associated Fibroblasts Of Gastric Carcinoma

Part 1:Clinicopathological features and prognostic analysis on the expression of B7H4 in cancer-associated fibroblasts and ACOT4 in gastric cancer cellsObjective:To investigate the clinical significance of the combined expression of B7H4 in cancer-associated fibroblasts and ACOT4 in gastric cancer cells as well as its relationship with the prognosis of gastric cancer patients.Methods:In this study,308 gastric adenocarcinoma patients who underwent radical gastrectomy for gastric cancer were selected in the Affiliated Hospital of Jiangnan University from July 2006 to November 2011.The postoperative pathological tissues of patients were collected to construct tissue chips.In this study,immunohistochemical methods were used to detect the expression of B7H4 in cancer-associated fibroblasts(CAFs)and the expression of ACOT4 in tumor tissues.At the same time,the relationship of the expression of ACOT4 in gastric adenocarcinoma cells and B7H4 in CAFs with the survival rate as well as the various clinical baseline characteristics and pathology was analyzed.The survival analysis was performed using the multiplicative limit method(Kaplan-Meier),and survival curves were drawn.Finally,Cox proportional hazards regression models were employed to perform the univariate and multivariate analyses of overall survival in patients with gastric adenocarcinoma.Results:1.The immunohistochemical results of gastric cancer patients showed that B7H4 was highly expressed in CAFs,and ACOT4 was mainly expressed in gastric cancer cells.The positive rates of ACOT4 and B7H4 in 308 tissues detected by immunohistochemistry were 49.0%(151/308)and 56.5%(174/308),respectively;2.The expression of ACOT4 in GC cells was significantly correlated with that of B7H4 in CAFs(P<0.05).3.In this study,median survival time of patients with gastric cancer was 768.07 days.According to the results of univariate and multivariate analysis on the overall survival rate,the expression of ACOT4 in tumor cells(P=0.027)and the expression of B7H4 in CAFs(P=0.045)were independent prognostic factors,respectively.The mortality risk of patients with positive ACOT4 expression was higher than that in patients with negative ACOT4 expression,and the mortality risk of patients with positive B7H4 expression was higher than that in patients with negative B7H4 expression;4.The survival prognosis of patients with negative ACOT4 expression in gastric cancer cells was significantly better than that with positive ACOT4 expression in cancer tissues(P=0.037),and the survival prognosis of patients with negative B7H4 expression in CAFs was significantly better than that with positive B7H4 expression in CAFs(P=0.024);The co-expression of B7H4 in CAFs and ACOT4 in gastric cancer cells was significantly correlated with the prognosis of gastric cancer patients(P<0.05).Conclusion:The expression of B7H4 in CAFs and ACOT4 in gastric cancer cells are significantly correlated with the prognosis of gastric cancer patients.The expression of ACOT4 in gastric cancer cells and that of B7H4 in CAFs may be independent prognostic factors for gastric cancer patients,and gastric cancer patients with high expression of ACOT4 and B7H4 in CAFs showed poor prognosis.Part 2:The significance of the expression of ACOT4 and B7H4 in gastric cancer tissues and the role of fibroblasts in the development of gastric cancerObjective:To investigate the significance of the expression of B7H4 in CAFs of gastric cancer tissue and ACOT4 in gastric cancer cells based on single-cell sequencing analysis,and to analyze the role of fibroblasts in the development and progression of gastric cancer cells based on differential genes expression analysis.Methods:Gastric cancer single-cell transcriptome datasets(GSE122796 and GSE134520)of gastric cancer patients were extracted from NCBI-GEO,which were clustered according to the expression patterns of marker genes in gastric epithelial cells using UMAP,and the expression profiles of the pseudo-differentiation trajectory map of human gastric mucosa in carcinogenesis in precancerous lesions and at early stages were drawn.The transcriptome sequencing data(GSE122796)of gastric cancer patients were extracted from NCBI-GEO to obtain single-cell genomic data of cancer tissues and paracancerous tissues.In this study,deconvolution was used to perform algorithm performs deconvolution analysis on the infiltrating cells in the microenvironment of gastric cancer tissue and adjacent tissue,and GSEA gene enrichment analysis was carried out to screen the pathways of differential gene expression between cancer tissues and adjacent tissues.Results:1.Fibroblasts play a significant role in intestinal metaplasia.Gastric epithelial cells are always accompanied by the presence of fibroblasts in the process of gastrointestinal epithelial metaplasia and redevelopment into gastric cancer cells;2.During the intestinal metaplasia process of gastric epithelial cells,the fibroblasts,aggregated,thereby affecting the malignant progression of gastric cancer cells;3.The expression profile of pseudo-differentiation trajectory map of gastric epithelial cells’ showed that ACOT4 was mainly expressed on epithelial cells,while B7H4 was mainly enriched in fibroblasts.During the malignant process of gastric epithelial cells,B7H4 and ACOT4 could interact with each other and may promote the malignant progression.4.GO and KEGG analysis demonstrated that two types of cells showed different expression pathways,and the genes with higher expression in gastric cancer tissue than those in adjacent tissue were more concentrated on these pathways,such as Focal adhesion kinase,ECM-receptor pathway,cAMP signaling pathway,Cytokine interaction pathway,Actin cytoskeleton regulation,Hippo signaling pathway,which may be related to tumor progression;5.For the deconvolution analysis on microenvironment infiltrating cells in cancer tissue and paracancerous tissue,only fibroblasts were higher in cancer tissue than in paracancerous tissue(P<0.05),and the proportion of B cells was higher in paracancerous tissue(P<0.01).The results of Wilcox T test showed that,by comparing with the adjacent tissue,the propotion of cancer-associated fibroblasts(P<0.05),neutrophils(P<0.05),CD4+TH1(P<0.01))and endothelial cells(P<0.05)was significantly higher in the cancer tissues,while the propotion of B cells(P<0.01),CD8+ central memory cells(P<0.01),CT4+effector memory cells(P<0.05),and class-switching memory cells B cells(P<0.05)was lower in cancerous tissues,with statistically significant difference;6.GSEA gene enrichment analysis screened out three signaling pathways for differential genes between cancerous tissue and paracancerous tissue,including WNT/β-catenin,TGF and EMT pathways.Gene sets under pathways were statistical significant and thereinto,WNT/β-catenin exhibited the greatest significance(LogFC>2,Adjust p-val<0.05,FDR q-val<0.25).Conclusion:During the intestinal metaplasia of gastric epithelial cells,fibroblasts aggregated and affected the malignant progression of gastric cancer cells.ACOT4 was mainly expressed on epithelial cells,whileB7H4 is mainly enriched on fibroblasts.B7H4 and ACOT4 may interact with each other and promote malignant progression in the process of malignant transformation.The propotion of fibroblasts in gastric cancer tissue was significantly higher than that in adjacent tissue,and the promoting effect of cancer-associated fibroblasts on gastric cancer cells may be mainly through the WNT/β-catenin pathway.Part 3:CAFs activates β-Catenin signaling to promote the occurrence and development of gastric cancer through the action of B7H4 on ACOT4 in gastric cancer cellsObjective:To further explore the regulatory mechanism and role of the expression of B7H4 in gastric cancer tissue CAFs and ACOT4 in gastric cancer cells during the malignant progression of gastric cancerMethods:Co-immunoprecipitation was firstly conducted to verify the correlation and interaction between B7H4 and ACOT4.The combination of B7H4 with ACOT4 was detected by co-immunoprecipitation and ubiquitin detection on B7H4 could stablize ACOT4 and reduce its degradation.In this study,gastric cancer cell lines with highly-and low-expressed ACOT4 and fibroblast cell lines with highly-and low-expressed B7H4 were constructed,and MGC-803 cells were selected to construct transient cell lines with highly-and low expressed ACOT4,which were co-incubated with serum of fibroblasts with highly-and low-expressed B7H4,respectively(MGC-803-siNC,MGC-803-siACOT4,MRC5-siNC+MGC-803,MRC5-siB7H4+MGC-803).The protein expression of ACOT4,B7H4,GSK3-β,pGSK3-β and β-Catenin was detected by Western Blot,with GAPDH as an internal reference.In order to explore the effect of B7H4 in CAFs on tumor cell progression,Transwell assay was used to detect the differences in migration and invasion abilities of co-cultured gastric cancer cells after down-regulation of B7H4 expression in fibroblasts.Finally,a lentiviral vector with down-regulated expression of B7H4 was utilized to transfect human gastric cancer cell MGC803,so as to construct a stable transcription cell line,which was subcutaneously injected into the axilla of BalB/c nude mice to form tumors.Then,a tumor-bearing model of nude mice was constructed to detect the effect of down-regulated B7H4 on the development of gastric cancer cells.Results:1.The results of co-immunoprecipitation showed that HEK293 cells were transformed into B7H4 plasmid.The level of ACOT4 protein bound by B7H4 was detected.B7H4 could be pulled down to ACOT4,and the two molecules could be combined with each other;siRNA was selected to down-regulate the expression of B7H4 in MGC-803 gastric cancer cells,and ubiquitin plasmid was adopted to transfect wild-type and B7H4 knockdown gastric cancer MGC 803 cells,respectively.Western blotting indicated that ACOT4 polyubiquitination appeared in the B7H4 knockdown MGC 803 cell group,and B7H4 could bind with ACOT4 and prevent ACOT4 ubiquitination and degradation;2.Western Blot results showed that:① After the researchers transfected small interfering RNA into MGC-803 gastric cancer cells to down-regulate ACOT4,along with the down-regulation of ACOT4 protein expression,the expression of B7H4 in the nucleus of MGC-803 gastric cancer cells transfected with siRNA present no change(P>0.05),while the expression of pGSK3-β and β-catenin decreased with statistically significant difference(P<0.05,P<0.05);②The researchers down-regulated B7H4 through transfecting small interfering RNA into MRC5 fibroblasts,to co-culture with MGC-803 gastric cancer cells.Compared with MRC5-siNC+MGC-803,the protein expression level of ACOT4 in MRC5-siB7H4+MGC-803 cells was significantly decreased(P<0.05).The protein expression levels of B7H4,pGSK3-β and β-catenin were decreased,and the difference was statistically significant(P<0.05).The protein expression of B7H4 in CAFs of gastric cancer tissue was positively correlated with that of ACOT4 in gastric adenocarcinoma cells.B7H4 in CAFs of gastric cancer could regulate β-catenin by affecting the expression of ACOT4 and activate WNT/β-catenin Pathway;3.In control cells(MRC5-siNC+MGC-803)and cells with B7H4 downregulated by siRNA(MRC5-siB7H4+MGC-803),Transwell assay confirmed the effect of B7H4 in CAFs of gastric cancer on the invasion and migration ability of gastric cell(P<0.01);4.Compared with sh-NC group,the tumor formation ability of sh-B7H4 group(B7H4 expression stably down-regulated group)was significantly weakened,and the tumor diameter and growth rate were significantly lower than those of the negative control group,with statistical significance(P<0.01).Conclusion:The protein expression of B7H4 in CAFs of gastric cancer tissue is positively correlated with that of ACOT4 in gastric cancer cells;B7H4 can bind to ACOT4 to prevent ACOT4 ubiquitination degradation and stabilize the expression of ACOT4.Low expression of B7H4 can reduce the subcutaneous tumorigenicity of gastric cancer cells;B7H4 in CAFs may regulate tumor metabolism by binding to ACOT4,and activate the WNT/β-catenin pathway through β-catenin,thereby affecting the malignant progression of gastric cancer.