The Study On The Mechanism Of PLCE1 Regulates ENO1 Through The E3 Ubiquitin Ligase FBXW7 To Promote Glycolysis In Esophageal Squamous Cell Carcinoma

Objective: Previous studies have verified that PLCE1 can promote glycolysis through ENO1 in esophageal squamous cell carcinoma.This study aims to further verify this conclusion and preliminically explore the molecular mechanism of PLCE1 regulating ENO1 and promoting glycolysis through E3 ubiquitin ligase FBXW7 in esophageal squamous cell carcinoma.Methods:(1)Serum ENO1 levels were detected by enzyme-linked immunosorbent assay.(2)The effect of PLCE1 on the extracellular acidification rate of esophageal squamous cell carcinoma cells through ENO1 was detected by the Seahorse cell energy metabolism analyzer.(3)The effect of PLCE1 on tumor growth and glycolysis of esophageal squamous cell carcinoma through ENO1 was detected by tumor-forming experiment in nude mice.(4)Immunoprecipitation protein spectrum was used to screen ubiquitin enzymes interacting with PLCE1 in esophageal squamous cell carcinoma cells.(5)The protein expression of FBXW7 after PLCE1 silencing was detected by Western Blot;FBXW7 expression level was detected in normal esophageal epithelial cells and esophageal squamous cell carcinoma cells.(6)The interaction between FBXW7 and ENO1 in esophageal squamous cell carcinoma cells was detected by immunoprecipitation.(7)The protein expression of FBXW7 in esophageal squamous cell carcinoma tissues and normal esophageal epithelial tissues was detected by immunohistochemistry.(8)Mutation of FBXW7 in esophageal squamous cell carcinoma tissues and esophageal squamous cell carcinoma cells was detected by PCR resequencing.(9)ENO1 protein expression in esophageal squamous cell carcinoma cells after overexpression of wild-type FBXW7 and mutant FBXW7 and ENO1 protein stability after addition of actinomycin was detected by Western Blot.(10)The interaction between wild type and mutant FBXW7 and ENO1 in 293 T cells was detected by immunoprecipitation.The ubiquitination of ENO1 in 293 T cells with lysine sites mutation was detected by immunoprecipitation.(11)CCK8,plate cloning,cell scratch,Transwell migration and invasion assay was conducted to detect proliferation,migration and invasion of esophageal squamous cell carcinoma cells after overexpression of FBXW7.(12)Statistical analysis was performed by SPSS 25.0 software.Results:(1)Serum ENO1 expression was elevated in patients with esophageal squamous cell carcinoma.(2)PLCE1 could improve the extracellular acidification rate of esophageal squamous cell carcinoma by ENO1.(3)PLCE1 increased body weight,tumor volume,tumor weight,glucose uptake,pyruvate production,lactic acid production and ATP production by ENO1.(4)PLCE1 interacts with FBXW7 in esophageal squamous cell carcinoma cells and FBXW7 expression is reduced after silencing PLCE1.(5)ENO1 interacts with FBXW7 in esophageal squamous cell carcinoma cells.(6)FBXW7 expression level was lower in esophageal squamous cell carcinoma cells and tissues,and correlated with the differentiation degree and prognosis.(7)Overexpression of wild-type FBXW7 in esophageal squamous carcinoma cells resulted in down-regulation of ENO1 protein expression and decreased protein stability.After overexpression of mutant FBXW7(except V341 A mutation),ENO1 protein expression was not significantly changed and protein stability was unchanged.(8)Reduced binding ability of mutant FBXW7(except V341 A mutation)to ENO1 in 293 T cells.(9)ENO1 ubiquitination was decreased in 293 T cells after mutation of ENO1 at K406,K420,K422 and K434 sites.(10)Overexpression of FBXW7 inhibited the proliferation,migration and invasion of esophageal squamous cell carcinoma cells.Conclusion:(1)PLCE1 can promote the growth and glycolysis of esophageal squamous cell carcinoma through ENO1.(2)PLCE1 regulates ENO1 protein levels through FBXW7-mediated ENO1 ubiquitination.(3)FBXW7 mutations reduce ENO1 binding and thus inhibit ENO1 ubiquitination.(4)Reduced FBXW7 expression suggests poor patient prognosis,and overexpression of FBXW7 inhibits proliferation,migration and invasion of esophageal squamous carcinoma cells.