Role Of PFKFB3 In Gastric Cancer With Hyperglycemia And Its Molecular Mechanism

Background Gastric cancer（GC）is one of the most deadly malignancies with high morbidity worldwide.Cancer cells exhibited higher level of glucose catabolism than normal cells to meet the needs of rapid growth,and emerging evidences indicate that hyperglycemia has positive effects on the progression of tumor.As a vital regulator of glycolysis,PFKFB3 can up-regulate the glycolysis level of cells and it was confirmed to have a higher expression level in tumor tissue and correlated with the prognosis of GC patients.It is widely known that cancer patients complicated with hyperglycemia often have a worse prognosis than that with euglycemia.Although PFKFB3 has been proven to be overexpressed in a variety of tumor tissues,including gastric cancer tissues,but the role of PFKFB3 in GC patients with hyperglycemia remains unclear.MethodThe data from the cancer genome atlas（TCGA）,gene expression omnibus（GEO）and Kaplan-Meier Plotter database were utilized to analyze the expression level of PFKFB3 and conducted survival analysis of GC patients.Western blot assay was used to detect gene expression at the protein level.Small interfering RNA（si RNA）transfection assay was conducted to down-regulated the expression of PFKFB3,western blot was used to detect the expression of epithelial-mesenchymal transition-related markers in transfected cells.Cell function assays,including cell total number assay,MTT,cell colony formation,migration assay and wound-healing assay,were carried out to reflect the ability of cell proliferation and migration.The Target Scan online analysis tool and gene set enrichment analysis（GSEA）were used to explore how hyperglycemia induced PFKFB3 overexpression and promoted malignant phenotype in GC.Finally,we also designed a series of rescue experiments to verify the above experimental results.ResultsPFKFB3 was significantly upregulated and its overexpression was associated with poor prognosis of GC patients.Besides,hyperglycemia stimulated the higher expression of PFKFB3 along with the enhanced proliferation,migration and epithelial-mesenchymal transition（EMT）in GC cells.PFKFB3 knockdown effectively reversed the effects of high glucose concentration on GC malignant phenotype.We screened out the most likely upstream regulatory factor of PFKFB3 by Target Scan--miR-26-5p,and inhibition of miR-26-5p enhanced the expression of PFKFB3 and also promoted the malignant phenotype of gastric cancer.Finally,the result of gene set enrichment analysis revealed that the potential downstream pathway of PFKFB3 was the transforming growth factorβ（TGF-β）signaling pathway,which was also confirmed by western blot.Therefore,PFKFB3 silencing also inhibited the activation of TGF-β signaling pathway.In conclusion,the expression of PFKFB3 was enhanced by down-regulation of miR-26-5p in high glucose environment,and promoted the proliferation,migration,colony formation and epithelial-mesenchymal transition of gastric cancer cells through activating TGF-β signaling pathway.ConclusionOur results indicated that the down-regulation of miR-26-5p induced by high glucose environment enhanced the expression of PFKFB3 and promoted malignant phenotype of gastric cancer cells in GC patients with hyperglycemia,including the proliferation,migration,colony formationand,epithelial-mesenchymal transition of gastric cancer cells.