Neuropilin-1 Promotes Epithelial-to-mesenchymal Transition In Oral Squamous Cell Carcinoma

Background:Oral squamous cell carcinoma(OSCC) is the most common malignant oral cancer.OSCC is characterized by severe progression, partnered with a high potential for both nodal metastasis and locoregional invasion. Accumulating lines of evidence have shown that the epithelial-to-mesenchymal transition(EMT) contributes to tumor metastasis and invasion. Neuropilin-1(NRP1) is a multifunctional type I transmembrane glycoprotein that plays an important role in cancer progression and is associated with poor prognosis. It also may play an essential role both in the induction and the maintenance of EMT process in several cancer cells. NRP1-induced EMT process may be an important mechanism for promoting invasion and metastasis of OSCC.Objective1. To test the expression level of NRP1 of oral squamous cell carcinoma samples,analyse the relationship between the level of expression of NRP1 and metastasis as well as prognosis of OSCC.2. To investigate whether NRP1 overexpression would promoted EMT and alters the biological behavior of OSCC cells;3. To explore the molecular biological mechanism of NRP1-induced EMT in OSCC cell lines;Methods1. Immunohistochemical staining was used to investigate the expression of NRP1 in 60 OSCC and 30 normal tissue samples. The correlation of the NRP1 level with patients’ clinicopathological parameters and prognosis was analyzed using the Chi-square analysis.2. The purified pc DNA3.1 and pc DNA3.1-NRP1 plasmids were transfected into CAL27,HN4 and HN6 cells to obtain experiment group cells which stably express NRP1 and control group cells with low NRP1 expression.The variations in gene and protein expression of the epithelial and mesenchymal markers of OSCC cell lines were evaluated by Real-time RT-PCR and western blotting. Wound healing, transwell invasion, colony-formation and drug resistance assay were performed to measure the biological properties of the NRP1 overexpression cells.3. Western blotting and RT-PCR were conducted to assess the activation of the components of the NF-κB pathway in NRP1- overexpressing OSCC cells and control.We then addressed whether selective inhibition of NF-κB suppresses NRP1-mediated EMT.Results1. Immunohistochemical staining results showed that NRP1 was positive stained for NRP1 in all of the tumor specimens, while only 6 cases were positive for NRP1 expression among the 30 normal oral epithelial tissues evaluated. NRP1 was significantly highly expressed in tumor tissues(p<0.001). There was also a significant correlation between higher NRP-1 expression levels and lymph node metastasis(p<0.005) as well as Poor Prognosis(p<0.005).2. NRP1 overexpression promoted EMT in OSCC cell lines with morphological changes and variations in gene and protein expression of the epithelial and mesenchymal markers. The mesenchymal cells present enhanced invasive and metastatic properties and elevated cell proliferation and drug resistance ability of OSCC cells. The induction of EMT promoted the acquisition of some cancer stem cell(CSC)-like characteristics in OSCC cells.3. NRP1-mediated EMT occurs through activation of the NF-κB signaling pathway.Selective inhibition of NF-κB suppresses NRP1-mediated EMT process of NRP1 overexpressing cells. These cells displayed significantly lower migration and invasion abilities than the corresponding parental cell lines.Conclusions Our results indicate that Higher NRP1 expression levels are associated with lymph node metastasis and poor prognosis in OSCC patients. NRP1 may regulate the EMT process in OSCC cell lines through NF-κB activation. The induction of EMT promoted the acquisition of some cancer stem cell(CSC)-like characteristics in OSCC cells. Selective inhibition of NF-κB suppresses NRP1-mediated EMT process and diminishes the migration and invasion abilities of NRP1 overexpressing cells.