Neuropilin-1 regulated by miR-320 contributes to the growth and metastasis of cholangiocarcinoma cells

Background Neuropilin-1 (NRP-1) activates signaling pathways as multifunctional co-receptors in cancer cells. However, its role and how it is regulated by miRNAs in cholangiocarcinoma (CCA) have not yet been investigated. Methods The expression of NRP-1, miR-320 and key molecules involved in cell proliferation, migration and related signaling pathways were detected by immunohistochemistry, immunoblotting and qRT-PCR. Stable transfectants depleted of NRP-1 were generated. The regulatory effect of miR-320 on NRP-1 was evaluated by luciferase reporter assays. Cell proliferation, cell cycle distribution and migration were examined. Xenograft tumor models were established to assess tumorigenesis, tumor growth and lung metastasis. Results CCA tissues expressed higher levels of NRP-1 than adjacent normal biliary tissues, and its expression negatively correlated with miR-320. NRP-1 depletion inhibited cell proliferation and induced cell cycle arrest in the G1/S phase by upregulating p27, and downregulating cyclin E and cyclin-dependent kinase 2; and reduced cell migration by inhibiting the phosphorylation of focal adhesion kinase. NRP-1 depletion suppressed tumorigenesis, tumor growth and lung metastasis by inhibiting cell proliferation and tumor angiogenesis in experimental animals. Depletion of NRP-1 inhibited the activation of VEGF/VEGFR2, EGF/EGFR and HGF/c-Met pathways stimulated by respective ligands. MiR-320 negatively regulated the expression of NRP-1 by binding to the 3’-UTR of NRP-1 promoter, and miR-320 mimics inhibited cell proliferation and migration, and the growth of established tumors in animals by downregulating NRP-1. Conclusions The present results indicate that NRP-1 is negatively regulated by miR-320, and both of them may be potentially therapeutic targets for CCA. This article is protected by copyright. All rights reserved.