TRAIP enhances progression of tongue squamous cell carcinoma through EMT and Wnt/β-catenin signaling by interacting with DDX39A

Abstract

Background: Tongue squamous cell carcinoma (TSCC) is one of the most common malignant tumors with high mortality and poor prognosis. Its incidence rate is increasing gradually. Tumor necrosis factor receptor-associated factor interacting protein (TRAIP), as a factor related to several tumors, reveals that its gene expression is different between normal tissue and primary tumor of head and neck squamous cell carcinoma using bioinformatics analysis.

Method: In our study, TCGA database, immunohistochemistry, proliferation assay, colony formation, wound healing assay, Transwell, cell cycle analysis and tumor xenografts model were used to determine the expression and functions of TRAIP in TSCC.

Result: We found that TRAIP may promote the proliferation, migration and invasion of TSCC. Furthermore, the results of bioinformatics analysis, mass spectrometry and co-immunoprecipitation suggested that DDX39A may be a TRAIP interacting protein. DDX39A has been proven to be an oncogene in several tumors, which may have an important effect on cell proliferation and metastasis in multiple tumors. In addition, the high expression of DDX39A implies the poor prognosis of patients. Our study demonstrated that TRAIP probably interact with DDX39A to regulate cell progression through epithelial-mesenchymal transition and Wnt/β-catenin pathway. In addition, we show that the necessary domain of DDX39A for the interaction between DDX39A and TRAIP region.

Conclusion: These results indicate that TRAIP is important in occurrence and development of TSCC and is expected to become the new promising therapeutic target.

Keywords: DDX39A; EMT; Progression; TRAIP; Tongue squamous cell carcinoma; Wnt/β-catenin signaling.

Fig. 1

The expression level of TRAIP is upregulated in TSCC. A The level of TRAIP expression in different human normal tissues. B TRAIP levels in unpaired HNSC tissues and normal tissues in TCGA. C TRAIP levels in paired HNSC tissues and normal tissues in TCGA. D TRAIP levels in paired HNSC tissues and normal tissues in GSE. E ROC for TRAIP to diagnose HNSC. F TRAIP protein expression levels in TSCC were higher than that in normal tissues. T: tumor, N: normal tissue. G Representative IHC images of TRAIP expression. IHC findings showed that the expression level of TRAIP in TSCC tissues(b, IHC score: 6) was higher than that in adjacent tissues(a, IHC score: 4). The TRAIP expression level was higher in the primary tumors with lymph node metastasis(d, IHC score: 7) than that without lymph node metastasis(c, IHC score: 5) .The immuno-staining is strongly positive in lymph node metastasis(f, IHC score: 8) than in primary tumor tissues(e, IHC score: 6). a:100X ; b-f : 200X *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

TRAIP promotes TSCC cell proliferation, migration and invasion in vitro. Cell viability was tested using proliferation assay (A). Cell proliferation ability was detected using colony formation assay (B). Wound healing assay (C) and transwell assay (D) were used to detect the migration and invasion abilities of TSCC cell. E The distribution of cell phase in TSCC cell lines after TRAIP was silenced or overexpressed. Data are shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

TRAIP knockdown inhibited TSCC cell growth and metastasis in vivo. A The weight of mice. B Dissected tumors generated by TRAIP knockdown or negative control CAL27 cell (n = 5). C, D The growth curve (C) and weights of xenograft tumors (D). E The metastatic nodules of nude mice lungs after tail vein injection of TRAIP knockdown or negative control CAL27 cell. F Representative HE images of lung tissues after tail vein injection (left : 200X ; right : 400X ; upper: shTRAIP#1 group; lower: shNC group). *P < 0.05

Fig. 4

TRAIP interacts with DDX39A in TSCC cells. A, B Whole-cell lysates of CAL27, SCC15 and SCC9 cells were used in Co-IP with IgG (control) and anti-TRAIP or anti-DDX39A antibodies. C-F Western blot analysis was used to measure the expression of TRAIP and DDX39A in these two factors silenced or overexpressed cells. G Schematic of DDX39A domains. H Co-immunoprecipitation analysis of the interaction of Flag-TRAIP and WT or truncated MYC-DDX39A

Fig. 5

DDX39A promotes TSCC cell proliferation and migration in vitro. Cell growth ability was determined using proliferation assay (A) and colony formation assay (B), (C-D) The distribution of cell phase after DDX39A was silenced or overexpressed. The migration abilities were measured using wound healing assay (E-F). DDX39A promotes TSCC cell migration and invasion in vitro. The migration and invasion abilities were measured using Transwell assay (G-L) *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

TRAIP promotes TSCC cell migration and invasion by EMT. Western blot analysis was used to determine the protein levels of E-cadherin, N-cadherin, Vimentin, Slug, Snail, MMP2, MMP9 after TRAIP or DDX39A was silenced (A-F). TRAIP promotes TSCC cell migration and invasion by EMT. Western blot analysis was used to determine the protein levels of E-cadherin, N-cadherin, Vimentin, Slug, Snail, MMP2, MMP9 after TRAIP or DDX39A was overexpressed (G-J). CAL27 and SCC9 cells were transfected with lentiviruses. Cells were used to detect the EMT relative proteins expression (K-N)

Fig. 7

TRAIP and DDX39A activate Wnt/β-catenin pathway in TSCC cells. Western blot analysis of Wnt/β-catenin pathway relative proteins after TRAIP or DDX39A knockdown (A-F). TRAIP and DDX39A activate Wnt/β-catenin pathway in TSCC cells. Western blot analysis of Wnt/β-catenin pathway relative proteins after TRAIP or DDX39A overexpressed (G-J). CAL27 and SCC9 cells were transfected with lentiviruses. Cells were used to detect the Wnt/β-catenin pathway relative proteins expression (K-N)

Fig. 8

The schematic diagram of TRAIP regulates TSCC cells proliferation, migration and invasion by interacting with DDX39A via Wnt/β-catenin pathway. By BioRender