EBV-miR-BART8-3p induces epithelial-mesenchymal transition and promotes metastasis of nasopharyngeal carcinoma cells through activating NF-κB and Erk1/2 pathways

Abstract

Background: Epstein-Barr virus (EBV) is ubiquitously associated with nasopharyngeal carcinoma (NPC). EBV encodes two groups of microRNAs (miRNAs) which are divided into BamHI fragment H rightward open reading frame 1 (BHRF1) and BamHI-A rightward transcripts (BART) microRNAs. EBV miR-BART has been found to be involved in the development and progression of NPC. However, so far the role of EBV-miR-BART8-3p in NPC progression remains unknown. This study aimed to investigate the role of EBV-miR-BART8-3p in NPC and explore the underlying mechanisms.

Methods: miRNA expression was profiled in NPC and normal nasopharyngeal mucosal specimens using miRNA sequencing. EBV-miR-BART8-3p and RNF38 expression was quantified with qPCR assay. The migration, invasion and metastasis of NPC cells were evaluated using CCK-8, colony-forming, wound-healing, and migration and invasion assays. The expression levels of epithelial-mesenchymal transition (EMT)-related markers,metastasis-related markers and NF-κB and Erk1/2 signaling proteins were determined using Western blotting. Tumorigenic assay was performed to evaluate the pulmonary metastatic ability of NPC cells in vivo.

Results: EBV BART miRNAs were highly over-expressed and co-expressed in NPC and might be associated with deactivated immune response in NPC according to the sequencing analysis. EBV-miR-BART8-3p expression was significantly higher in human NPC specimens than in normal nasopharyngeal mucosal specimens. EBV-miR-BART8-3p was found to promote NPC migration, invasion and metastasis, drove an EMT process and upregulated expression of metastasis-related proteins expression in NPC cells. Our data showed EBV-miR-BART8-3p directly targeted RNF38 in NPC cells.

Conclusion: The present study demonstrates that EBV-miR-BART8-3p plays a significant role in inducing EMT and promoting metastasis through directly targeting RNF38 in NPC cells via the activation of NF-κB and Erk1/2 signaling pathways. Our findings suggest that EBV-miR-BART8-3p is a potential therapeutic target for NPC.

Keywords: EBV-miR-BART8-3p; Epithelial-mesenchymal transition; Epstein-Barr virus; Erk1/2 signaling; Metastasis; NF-κB signaling; Nasopharyngeal carcinoma.

Fig. 1

EBV-miR-BART8-3p expression is upregulated and co-expressed in NPC. a Heat map of 86 miRNAs differentially expressed between NPC specimens and normal nasopharyngeal mucosal specimens (CTRL). b Topological overlap matrix (TOM) plot of the microRNA co-expression network. The x- and y-axes represent 1315 microRNAs and the color intensity indicates interaction strength between microRNAs with red for the strongest interaction and white for no interaction. The color bars along the x- and y-axes denote the module membership. c The 20 most highly upregulated EBV BART miRNAs identified between NPC specimens and normal nasopharyngeal mucosal specimens

Fig. 2

EBV-miR-BART8-3p promotes NPC cell migration and invasion in vitro and lung metastasis in vivo. a qPCR assay reveals higher EBV-miR-BART8-3pexpression in NPC specimens than in normal nasopharyngeal mucosal specimens (CTRL). b qPCR assay reveals higher EBV-miR-BART8-3p expression in NPC cells transfected withEBV-miR-BART8-3p precursors than in those transfected with control vectors. miRNA levels are normalized to U6 snRNA expression. c Representative images and quantification of the wound-healing assay in CNE-1 andSUNE-1cells. d Representative images and quantification of migration and invasion assays in CNE-1 andSUNE-1. e Representative images and quantification of the wound-healing assay in C666–1 cells. f Representative images and quantification of migration and invasion assays in C666–1 cells. ND, not detectable. Data is presented as the mean ± SD. ** P < 0.01; *** P < 0.001

Fig. 3

Overexpression of EBV-miR-BART8-3p promotes lung metastasis of NPC in vivo. a Nude mice were intravenously injected with SUNE-1-BART8-3p cells or control vector-transfected SUNE-1 cells via the tail veins, and were sacrificed 6 weeks post-injection. Representative images in vivo were obtained by the whole-body imaging system. b Representative images of metastatic nodules in mouse lungs. c Number of metastatic nodules in mouse lungs. d Weight of mouse lungs. *P < 0.05

Fig. 4

EBV-miR-BART8-3pregulatesEMT and increases metastasis-related markers expression in NPC cells. a Western blotting reveals that upregulation of EBV-miR-BART8-3p results in a reduction in the E-cadherin expression and an increase in the Snail, N-cadherin, Vimentin expression. GAPDH serves as an internal control. b Morphology changes are observed using phase contrast microscopy (magnification, × 200). c Western blotting reveals that upregulation of EBV-miR-BART8-3presults in an increase in the MMP2 and MMP9 expression

Fig. 5

EBV-miR-BART8-3p regulates NF-κB and Erk1/2 signaling pathways. a Upregulation of EBV-miR-BART8-3p activates NF-κB and Erk1/2 signaling pathways in CNE-1 and SUNE-1 cells. b Downregulation of EBV-miR-BART8-3p inhibits NF-κB and Erk1/2 signaling pathways in C666–1 cells

Fig. 6

RNF38 is a direct target of EBV-miR-BART8-3p. a EBV-miR-BART8-3p and its putative binding sequence in 3’UTR of RNF38 mRNA, and mutations are generated as indicated. b Quantification of RNF38protein expression by Western blotting in CNE-1, SUNE-1 and C666–1 cells. c The relative luciferase activity in HEK293 cells after co-transfection with wild-type (WT) or mutant (MT) RNF38 3’UTR reporter genes and EBV-miR-BART8-3p or control. d Relative RNF38 expression is quantified in 10 normal nasopharyngeal specimens and 19 NPC specimens by qPCR, and GAPDH serves as an internal control. e Spearman’s correlation analysis reveals a negative correlation between the EBV-miR-BART8-3p and RNF38 expression in NPC specimens (n = 19)

Fig. 7

Restored RNF38 rescues the phenotypes of NPC cells. a Reconstitution of RNF38 reduces the migration and invasion of CNE-1-BART8-3p and SUNE-1-BART8-3p cells. b Reconstitution of RNF38 reverses the expression of EMT-associated markers and metastasis-related proteins. c The proposed model shows the role of EBV-miR-BART8-3p in regulation of NPC metastasis. **P < 0.01; *** P < 0.001