Exploring The Effect And The Mechanism Of GFPT2 In Control Of EMT Process In Laryngocarcinoma

Background and Objectives:Cancer is the leading cause of death worldwide.Laryngeal cancer is the second most common malignancy of upper respiratory digestive tract after lung cancer.Most are squamous cell carcinomas.Despite advances in treatment,the five-year overall survival rate for this malignancy is still close to 50 percent,and many patients already have metastases at the time of diagnosis.Epithelial-mesenchymal transition(EMT)is a process in which epithelial cells transform into mesenchymal cells and acquire the ability of invasion and migration,which is closely related to the invasion and metastasis of tumor cells.transforming growth factor-β(TGF-β)signaling pathway controls cell cycle by regulating gene transcription and affects cell proliferation,differentiation,adhesion,metastasis and apoptosis.Its function is mainly realized by transforming target gene expression in specific cells.Glutamino-fructose-6-phosphate aminotransferase 2(GFPT2),a rate-limiting enzyme in the hexosamine biosynthesis pathway(HBP),is responsible for glycosylation.However,the relationship between GFPT2 and laryngeal squamous cell carcinoma(LSCC)has not been clarified.In this study,the expression of GFPT2 in patients with LSCC and its relationship with patients’ survival and prognosis were investigated,and the role and function of GFPT2 in the pathogenesis of LSCC were further discussed.This will provide a new experimental basis for the clinical diagnosis and treatment of LSCC and a full understanding of the pathogenesis and development of laryngeal cancer.Methods:(1)The gene expression profile data of The Cancer Genome Atlas(TCGA)were downloaded to analyze the expression of GFPT2 and its relationship with the prognosis of patients with head and neck squamous cell carcinoma(HNSCC).(2)Western blot and Real-time PCR were used to detect the expression levels of GFPT2 in laryngeal cancer tissues and clinical tissue samples adjacent to cancer,and the differences between the two were analyzed.(3)The expression changes of GFPT2 gene in laryngeal cancer cell lines with SMAD3 and SMAD4 knocked out after TGF-β stimulation were analyzed.(4)The association between GFPT2 and stromal cell infiltration was analyzed using public databases.Gene set enrichment analysis(GSEA)was used to study the potential mechanism of GFPT2.(5)The influence of GFPT2 on EMT molecular protein markers in laryngeal cancer cells was explored by overexpression/knockdown assay,and its influence on migration and invasion was explored by cell scratch and Transwell assay.Results:(1)In HNSCC patients,mRNA expression level of GFPT2 increased and was closely associated with poor prognosis.(2)Compared with the adjacent tissues,the expression level of GFPT2 in laryngeal cancer tissues was significantly increased.(3)GSEA analysis showed that TGF-β signaling pathway was significantly positively correlated with the expression of GFPT2,and cytokine TGF-β could activate the expression of GFPT2.Knockout SMAD3 and SMAD4 significantly inhibited TGF-β1-induced GFPT2 gene expression level.(4)GFPT2 was expressed in both immune and stromal cell single-cell subsets.Cancer-associated fibroblasts(CAFs)are the main cells that express GFPT2.The GSEA gene of GFPT2 showed that GFPT2 was significantly enriched in EMT.(5)Cell experiments confirmed that overexpression of GFPT2 promoted the migration and invasion of laryngeal cancer cells,while knockdown of GFPT2 significantly inhibited the migration and invasion of laryngeal cancer cells.Conclusion:Our analysis suggests that GFPT2 may be a novel prognostic biomarker for HNSCC.It was confirmed that the expression of GFPT2 was significantly increased in laryngeal carcinoma tissues and laryngeal carcinoma cells,and could promote the migration and invasion of laryngeal carcinoma cells.TGF-β1 activates the transcription activity of SMAD3/SMAD4 complex,which activates the expression of GFPT2.