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Abstract
Neuropilin-1 regulated by miR-320a participates in the progression of cholangiocarcinoma by serving as a co-receptor that activates multiple signaling pathways. The present study sought to investigate upstream lncRNAs that control the expression of miR-320a/neuropilin-1 axis and dissect some of the underlying mechanisms. Here we report lncRNA TTN-AS1 (titin-antisense RNA1) acts as a sponging ceRNA to downregulate miR-320a and is highly expressed in human cholangiocarcinoma tissues and cells. The expression of the above three molecules is correlated with the clinicopathologic parameters of cholangiocarcinoma patients. In this study, multiple bioinformatics tools and databases were employed to seek potential lncRNAs that have binding sites with miR-320a and TTN-AS1 was identified because it exhibited the largest folds of alteration between cholangiocarcinoma and normal bile duct epithelial cells. The regulatory role of TTN-AS1 on miR-320a was further evaluated by luciferase reporter and RNA pulldown assays, coupled with in situ hybridization and RNA immunoprecipitation analyses, which showed that TTN-AS1 bound to miR-320a through an argonaute2-dependent RNA interference pathway in the cytoplasm of cholangiocarcinoma cells. Knockdown and overexpression assays showed that the regulatory effect between TTN-AS1 and miR-320 was in a one-way manner. TTN-AS1 promoted the proliferation and migration of cholangiocarcinoma cells via the miR-320a/ neuropilin-1 axis. The function of TTN-AS1 on tumor growth and its interaction with miR-320a were confirmed in animal models. Further mechanistic studies revealed that TTA-AS1, through downregulating miR-320a, promoted cell cycle progression, epithelial–mesenchymal transition, and tumor angiogenesis by upregulating neuropilin-1, which co-interacted with the hepatocyte growth factor/c-Met and transforming growth factor (TGF)-β/TGF-β receptor I pathways. In conclusion, the present results demonstrate that lncRNA TTA-AS1 is a sponging ceRNA for miR-320a, which in turn downregulates neuropilin-1 in cholangiocarcinoma cells, indicating these three molecules represent potential biomarkers and therapeutic targets in the management of cholangiocarcinoma.
Highlights
Cholangiocarcinoma (CCA) arises from the epithelial cells facing the lumen of the biliary trees and is the second most frequent primary hepatic tumor after hepatocellularDespite the latest progress in the development of molecular targeted therapies, the prognosis for this devastating cancer remains grim[3]
We have recently demonstrated that human CCA tissues expressed higher levels of NRP-1, which co-activates the vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF)-mediated pathways involved in the progression of CCA14
LncRNAs that have binding sites with miR-320a were screened by using multiple bioinformatics tools and databases, and 10 potential candidates were selected based on the criteria of free energy ≤10 kcal/mol and score >140 (Supplementary Table S1)
Summary
Cholangiocarcinoma (CCA) arises from the epithelial cells facing the lumen of the biliary trees and is the second most frequent primary hepatic tumor after hepatocellularDespite the latest progress in the development of molecular targeted therapies, the prognosis for this devastating cancer remains grim[3]. There is great urgency in further elucidating the molecular mechanisms and pathways underpinning this disease so that the clinical outcome of CCA patients could be improved. Neuropilin-1 (NRP-1) is a non-tyrosine kinase transmembrane molecule overexpressed in gastrointestinal cancers[8,9], and serves as a co-receptor for several cellular signaling pathways involved in cancer progression[10,11,12,13]. We have recently demonstrated that human CCA tissues expressed higher levels of NRP-1, which co-activates the vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF)-mediated pathways involved in the progression of CCA14. In exploring the miRNA-mediated mechanisms that lead to the overexpression of NRP-1, we have shown that miR-320a negatively regulates NRP-1 by binding to the 3′-UTR of its promoter and is expressed at low levels in CAA tissues and cells[14].
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