Study On Biological Function And Mechanism Of Transcription Factor FOXS1 In Gastric Cancer

Gastric cancer is the third most common malignant tumor disease in the world.At present,the molec?lar mechanism of the occurrence and development of gastric cancer is still unclear.The transcription factor FOXS1 is one of the genes that are significantly differentially expressed between gastric cancer tissues and normal tissues based on TCGA sequencing dataset analysis and is located at chr20q11.21.Both TCGA gastric cancer data and GEO database analysis showed that FOXS1 gene was highly expressed in gastric cancer;and RT-PCR and IHC demonstrated that FOXS1 was highly expressed in gastric cancer.These res?lts suggest that FOXS1 gene can promote gastric cancer development.The Fox transcription factor family is named for its conserved fork-headed DNA-binding domain,and its members play a very important role in tumorigenesis.The Fox family has become a hot topic in cancer research.However,FOXS1 is currently a new member of the Fox family's latest discovery.Its biological function and molec?lar mechanism in tumors have not been reported so far.Therefore,this paper focuses on the relationship between transcription factor FOXS1 and clinical significance of gastric cancer and investigates its effect on cell proliferation,cell cycle,invasion and migration,and elucidates the molec?lar mechanisms underlying their biological functions.Objective: To investigate the expression and clinical value of the transcription factor FOXS1 in gastric cancer.To investigate the effects of FOXS1 gene on the cell growth,cell cycle and colony formation ability of gastric cancer cells in vitro.In vivo,detection of the effect of FOXS1 gene on the tumorigenic ability of gastric cancer cells in nude mice.To examine the effect of FOXS1 gene on invasion,migration and EMT of gastric cancer cells.To clarify the upstream promoter region and upstream reg?latory mechanisms of the FOXS1 gene.To investigate the signaling pathways FOXS1 gene involved in gastric cancer.To investigate FOXS1-related miRNAs and study their reg?latory mechanisms.Methods: The gastric cancer dataset was collected from the Cancer Genome Atlas(TCGA)database,and the FOXS1 gene expression profile data and clinical information were downloaded.The expression of FOXS1 in gastric cancer tissues and normal tissues was analyzed by SPSS,and its relationship with clinicopathological parameters and prognosis was summarized.Kaplan-Meier m?ltiplication method and Cox ratio were used.The risk regression model analyzes its impact on the patient's clinical outcome.The GSE19826,GSE13911 and GSE51575 data sets in the GEO database are used as validation datasets.The online analysis tool GEPIA obtained different expression level of the FOXS1 gene in various tumors.The expression of FOXS1 was detected by RT-PCR and WB in various gastric cancer cells and normal gastric epithelial cells GES-1,respectively.RT-PCR,WB,and IHC further validated their expression at mRNA and protein levels respectively in gastric cancer and paracancerous tissues collected clinically.IHC detects the expression of FOXS1 in gastric cancer tissue chips.FOXS1 gene were knocked down by siRNA silencing method.FOXS1 gene were overexpressed by LV5-FOXS1 lentivirus infected methods.The effects of FOXS1 gene on cell growth were measured by cell proliferation assay and cell cycle were detected by flow cytometry.Plate cloning experiments were performed to examine the effect of FOXS1 gene on cell clone formation.The effect of overexpression of FOXS1 gene on the tumorigenic ability of gastric cancer cells was detected in nude mice.Cell scratch assay and Transwell assay were used to detect the effect of overexpression or knockdown of FOXS1 gene on the migration and invasion of gastric cancer cells.IF,WB and IHC were used to detect the expression of EMT markers in gastric cancer cells after overexpression or knockdown of FOXS1 gene.The luciferase reporter gene recombinant plasmid with different lengths in the promoter region of FOXS1 gene was constructed.The dual luciferase reporter gene was used to analyze the position of the promoter region of FOXS1 gene.Bioinformatics predicts transcription factors that bind to the FOXS1 promoter region and is initially validated using a dual luciferase reporter assay.Western Blot,RT-PCR further verified the reg?lation of transcription factors on FOXS1 gene expression.Bioinformatics predicts the binding motif sequence of the transcription factor to the FOXS1 promoter region,and performs site-directed mutagenesis of the sequence.Dual luciferase reporter assay and ChIP assay to verify whether it binds directly to the sequence of FOXS1 promoters.According to the expression level of FOXS1 gene in the RNA-seq data of patients in the GC database from TCGA,the data were divided into FOXS1 high expression group and FOXS1 low expression group.The two groups of genes were analyzed by GSEA enrichment and statistical analysis.Bioinformatics methods predict miRNAs associated with FOXS1 expression in gastric cancer.The effects of miRNAs on FOXS1 gene expression were verified by RT-PCR and WB experiments.The wild type or mutant binding site fragment of the FOXS1 3'UTR was inserted into the downstream of the firefly luciferase gene in the GP-miRGLO vector to construct a vector.The dual luciferase gene reporter assay was used to detect the binding of miRNAs to FOXS1 3'-UTR.Results: Analysis of the TCGA database revealed that the expression of FOXS1 was significantly up-reg?lated in gastric cancer tissues compared to normal gastric tissues.The high expression of FOXS1 was significantly associated with age and T stage of gastric cancer patients.KM plotter res?lts showed that high expression of FOXS1 was significantly associated with poor prognosis in patients with gastric cancer.Cox proportional hazard regression models indicated that high FOXS1 expression was an independent risk factor for poor prognosis in patients with gastric cancer.Both the GEPIA and GEO databases showed a significant high expression of FOXS1 gene in gastric cancer.In five gastric cancer cells,the expression of FOXS1 at mRNA and protein levels was significantly higher than that of GES-1.RT-PCR confirmed FOXS1 was highly expressed in 35 pairs of gastric cancer and adjacent tissues collected clinically.Western Blot and immunohistochemistry in the first 8 pairs of clinically collected tissues showed that the FOXS1 gene was also highly expressed at the protein level in gastric cancer.IHC res?lts of gastric cancer tissue microarray(10 normal liver tissues,15 early gastric cancer tissues,55 advanced gastric cancer tissues)showed that 7 cases of FOXS1 protein were highly expressed in early gastric cancer tissues compared with normal gastric tissues,and 45 cases in advanced gastric cancer tissues,suggesting that FOXS1 can better indicate advanced gastric cancer.FOXS1 knockdown significantly inhibited the growth of gastric cancer cells,the activation of the G1/S phase detection pathway and the clonality of gastric cancer cells.The overexpression of FOXS1 gene produced the opposite effect.In addition,FOXS1 overexpression promotes the tumorigenic ability of gastric cancer cells in vivo.Overexpression of FOXS1 gene promoted the migration and invasion of gastric cancer cells and decreased the expression of E-cad,and increased the expression of N-cad,while FOXS1 knockdown had the opposite effect.The res?lts of the dual luciferase reporter gene assay showed that the FOXS1 gene promoter region was located at-660 ~ 0.NFKB1 is mainly expressed in the-1100 ~-660 region.11 potential SP1 binding sites,4 potential STAT3 binding sites and 7 potential E2F1 binding sites are located in the FOXS1 promoter-380 ~ + 1 region.The dual luciferase reporter gene assay showed that NFKB1 significantly inhibited the activity of the FOXS1 promoter from-1100 to-660,while SP1,E2F1 and STAT3 significantly promoted the promoter activity of the FOXS1 promoter-380 ~ +1 region.RT-PCR and WB experiments confirmed that NFKB1 overexpression significantly inhibited the expression of FOXS1 gene.After mutation of the NFKB1 binding site in the FOXS1 promoter region,the NFKB1 transcription factor no longer has a reg?latory effect.ChIP experiments demonstrated that NFKB1 cannot bind directly to the FOXS1 promoter region.These res?lts suggest that NFKB1 can indirectly bind to the FOXS1 promoter region to transcriptionally inhibit its expression.GSEA data analysis showed that the FOXS1 gene was most significantly enriched in the Hh signaling pathway.Correlation analysis showed that GLI1 and FOXS1 genes have a very high correlation in gastric cancer.Overexpression of GLI1 significantly inhibited the expression of the FOXS1 gene at the mRNA and protein levels,whereas GLI1 knockdown had the opposite effect.In addition,online software predictive analysis,FOXS1 gene promoter-660 ~ +1 region has a transcription factor GLI1 binding site.Dual luciferase reporter assays showed that GLI1 overexpression significantly inhibited the activity of the FOXS1 gene promoter,whereas GLI1 knockdown had the opposite effect.After the GLI1 binding site important base mutation,the GLI1 knockdown has a reversal effect on the reg?lation of the activity of the FOXS1 promoter.ChIP res?lts showed that the GLI1 gene can directly bind to the promoter region of the FOXS1 gene.Bioinformatics methods initially screened two miRNAs(miR-328-3p and miR-125a-5p).RT-PCR res?lts showed that both miRNAs did not affect the expression of FOXS1 at the mRNA level.Western Blot res?lts showed that only overexpression of miR-125a-5p significantly inhibited the expression level of FOXS1 protein,while the use of miR-125a-5p inhibitor had opposite res?lts.The dual luciferase reporter assay showed that the luciferase activity of the FOXS1 3'-UTR region with the miR-125a-5p binding site mutated was no longer up-reg?lated after miR-125a-5p inhibition.Conclusion: In conclusion,FOXS1 can be used as one of the prognostic indicators of gastric cancer.The expression of FOXS1 was associated with the malignant phenotype of gastric cancer,and has a good indication for advanced gastric cancer.Mechanismly,GLI1 can directly bind to the FOXS1 promoter region and inhibit the high expression of FOXS1 gene,while miR-125a-5p can directly bind to the FOXS1 3'-UTR region to inhibit the translation of FOXS1 protein,providing new ideas for treating gastric cancer by targeting FOXS1.