YPEL3 suppresses epithelial-mesenchymal transition and metastasis of nasopharyngeal carcinoma cells through the Wnt/β-catenin signaling pathway

Abstract

Background: Metastasis remains the major cause of death in nasopharyngeal carcinoma (NPC). Yippee-like 3 (YPEL3) plays an important role in tumorigenesis. However, its function and mechanism in NPC has not been systematically explored.

Methods: We evaluated YPEL3 expression in NPC cell lines and tissues using real-time PCR and western blotting. Then, we established NPC cell lines that stably overexpressed YPEL3 and knocked down YPEL3 expression to explore its function in NPC in vitro and in vivo. Additionally, we investigated the potential mechanism of YPEL3 action by identifying the Wnt/β-catenin signaling pathway downstream genes using western blotting.

Results: YPEL3 was downregulated in NPC cell lines and tissue samples. Ectopic expression of YPEL3 inhibited NPC cell migration and invasion in vitro; while silencing of YPEL3 promoted NPC cell migration and invasion. Further study indicated that overexpression of YPEL3 inhibited NPC cell epithelial-mesenchymal transition (EMT) and that silencing it enhanced EMT. Overexpression of YPEL3 suppressed NPC cell lung metastasis in vivo. The mechanism study determined that YPEL3 suppressed the expression levels of Wnt/β-catenin signaling pathway downstream genes and the nuclear translocation of β-catenin.

Conclusions: YPEL3 suppresses NPC EMT and metastasis by suppressing the Wnt/β-catenin signaling pathway, which would help better understanding the molecular mechanisms of NPC metastasis and provide novel therapeutic targets for NPC treatment.

Keywords: Epithelial–mesenchymal transition; Metastasis; Nasopharyngeal carcinoma; Wnt/β-catenin; YPEL3.

Fig. 1

YPEL3 mRNA and protein expression levels in NPC cell lines and tissues. a-b Quantitative RT-PCR (a) and western blotting analysis (b) of YPEL3 expression levels in NPC cell lines. c Quantitative RT-PCR analysis of YPEL3 mRNA expression levels in NPC (n = 12) and normal nasopharyngeal epithelial tissues (n = 12); d western blotting analysis of YPEL3 protein levels in NPC (T, n = 4) and normal nasopharyngeal epithelial tissues (N, n = 4). All of the experiments were performed at least three times. Data presented are the mean ± SD; the P-value was calculated using the Student t-test

Fig. 2

Effects of YPEL3 overexpression on NPC cell migration and invasion in vitro. a Representative western blotting analysis of YPEL3 overexpression in CNE-2 and SUNE-1 cells. GAPDH served as the loading control. b-d Representative images and quantification of the effects of YPEL3 overexpression on the migratory and invasive abilities of CNE-2 and SUNE-1 cells as determined by wound healing (b), Transwell migration (c), and invasion (d) assays. All of the experiments were performed at least three times. Data presented are the mean ± SD; **P < 0.01 compared with control using Student t-test

Fig. 3

Effects of YPEL3 silencing on NPC cell migration and invasion in vitro. a Representative western blotting analysis of YPEL3 silencing in CNE-2 and SUNE-1 cells. GAPDH served as the loading control. b-d Representative images and quantification of the effects of YPEL3 silencing on the migratory and invasive abilities of CNE-2 and SUNE-1 cells as determined by wound healing (b), Transwell migration (c), and invasion assays (d). All of the experiments were performed at least three times. Data presented are the mean ± SD; **P < 0.01 compared with control using Student t-test

Fig. 4

YPEL3 overexpression inhibited EMT and its correlation with EMT markers. a Immunofluorescence staining and quantification analysis of E-cadherin and vimentin expression levels (×100). b, c Western blot analysis of EMT marker expression levels in NPC cells in which YPEL3 was overexpressed (b) or silenced (c). d Correlations between YPEL3 expression and E-cadherin and vimentin expression in NPC tissue samples (n = 10). All of the experiments were performed at least three times. Data presented are the mean ± SD; **P < 0.01, *P < 0.05 compared with control using Student t-test

Fig. 5

YPEL3 suppressed lung metastasis in vivo. Nude BALB/C mice were intravenously injected via the tail vein with SUNE-1 cells stably overexpression YPEL3 or vector (n = 9 in each group). a Representative images of macroscopic lung metastases, arrowheads indicate the metastatic nodes; b Quantification of the average number of macroscopic metastatic nodes formed on the lung surface; c Representative images of HE staining (×100); d Quantification of the average number of microscopic metastatic nodes formed in the lungs based on pathological analysis of HE-stained sections. Data presented are the mean ± SD; **P < 0.01 compared with control using Student t-test

Fig. 6

YPEL3 inhibited the Wnt/β-catenin signaling pathway. a Representative western blotting and quantification analysis of GSK-3β, β-catenin, c-MYC, and cyclin D1 expression levels after YPEL3 overexpression. b Representative western blotting and quantification analysis of GSK-3β, β-catenin, c-MYC, and cyclin D1 expression levels after YPEL3 silencing. c YPEL3 inhibited the nuclear (Nu) translocation of β-catenin. Cyto, cytoplasmic. All of the experiments were performed at least three times. Data presented are the mean ± SD; *P < 0.05 and **P < 0.01 compared with control using Student t-test