Expression Of TRAIP In Human Tongue Squamous Cell Carcinoma And Its Effects On Cells Proliferation,Invasion,and Migration

Objective To explore the expression of tumor necrosis factor receptor-associated factor interaction protein(TRAIP)in tongue squamous cell carcinoma(TSCC)tissues and cells,and study the relationship between TRAIP and the clinicopathological data of patients with TSCC.To explore the effects of TRAIP and DEx D-Box Helicase 39A(DDX39A)on the proliferation and metastasis of TSCC and the related molecular mechanism.Methods TRAIP expression in head and neck squamous cell carcinoma(HNSC)was analyzed using TCGA database.Western blot tested the expression of TRAIP in9 pairs of fresh TSCC tissues and adjacent normal tissues.Immunohistochemistry detected the expression of TRAIP in 65 pairs of TSCC tissues and adjacent normal tissues.Bioinformatics,mass spectrum and immunoprecipitation analyzed the relationship between TRAIP and DDX39 A.Tongue squamous cell carcinoma cell lines were transfected with TRAIP and DDX39 A overexpression or knockdown lentivirus.CCK-8,colony formation,scratch assay,Transwell and flow cytometry detected the effects of TRAIP and DDX39 A on the proliferation and migration abilities of TSCC cells.Western blot detected the effects of TRAIP and DDX39 A on the relative proteins expression of epithelial mesenchymal transition(EMT)and Wnt/ β-catenin signaling pathway.CAL 27 cells with TRAIP knockdown were injected into the subcutaneous or tail vein of nude mice and the effect of TRAIP on tumor proliferation and metastatic ability were tested.Results TCGA database showed that TRAIP expression of was significantly higher in HNSC tissues than that in adjacent normal tissues(P <0.001),and Western blot results showed that TRAIP was overexpressed in TSCC tissues compared with adjacent normal tissues(P <0.01).The immunohistochemistry results showed that TRAIP was mainly located in cell membrane and cytoplasm.The expression of TRAIP in TSCC tissues was higher than that in adjacent tissues,the immuno-staining is stronger positive in lymph node metastasis than that in the primary tumor tissues.The TRAIP expression level was higher in the primary tumors with lymph node metastasis compared with those without lymph node metastasis(P <0.01),and was significantly associated with patient clinical stages,differentiation grade and recurrence(P <0.05).The results of bioinfermatics,mass spectrometry and immunoprecipitation indicated a possible interaction between TRAIP and DDX39 A.CCK-8 and colony formation assays showed that cell proliferation capacity was significantly reduced after silencing TRAIP or DDX39 A,while overexpression of TRAIP or DDX39 A significantly enhanced the proliferation capacity of TSCC cells.Flow cytometry cycle experiments showed more cells in G1 phase and fewer cells in S phase after TRAIP or DDX39 A knockdown,while the results were opposite after TRAIP or DDX39 A overexpression.The scratch assay and Transwell results showed that cell migration and invasion were significantly decreased after TRAIP or DDX39 A knockdown,while the cell migration and invasion was enhanced after overexpressing TRAIP or DDX39 A,and DDX39 A knockdown could reverse the enhanced effect of TRAIP overexpression.Results of subcutaneous tumorigenesis experiment and lung metastasis model in nude mice showed reduced cell proliferation and metastasis ability after silencing TRAIP gene.The expression of EMT-related proteins N-cadherin,Vimentin,Slug,Snail,MMP 2 and MMP 9 were decreased after TRAIP or DDX39 A knockdown,while the E-cadherin expression showed no significant change.But the proteins expression except E-cadherin were increased after TRAIP or DDX39 A overexpression,the E-cadherin expression still showed no significant change.DDX39 A knockdown could reverse the activation effect of TRAIP overexpression on EMT.The expression of p-β-catenin,c-Myc,and cyclin D1 decreased after silencing TRAIP or DDX39 A,but no significant change in β-catenin expression.While the expressions of p-β-catenin,c-Myc,and cyclin D1 were increased after overexpressing TRAIP or DDX39 A,and no significant change in β-catenin expression.Conclusion TRAIP is upregulated in tongue squamous cell carcinoma,which may activate the EMT and Wnt / β-catenin signaling pathway by interacting with DDX39 A,thus promoting the proliferation and metastasis of tongue squamous cell carcinoma cells.