Clonorchis sinensis legumain promotes migration and invasion of cholangiocarcinoma cells via regulating tumor-related molecules

Clonorchis sinensis infection causes serious pathological changes in the bile duct and is highly correlated with cholangiocarcinoma. The excretory-secretory products (ESP) of C. sinensis play a critical role in the oncogenesis and progression of cholangiocarcinoma, while the components and precise mechanism remain unclear. Here, we evaluated the function of C. sinensis legumain (Cslegumain) in promoting the invasion and migration of cholangiocarcinoma cells and the mechanism involved.The structural and molecular characteristics of Cslegumain were predicted and analyzed using the online program Phyre2. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining were performed to test the transcriptional level of Cslegumain and its localization in the adult. Native Cslegumain was detected by western blotting assay. The effects of Cslegumain on the proliferation, invasion and migration of cholangiocarcinoma cells were checked using CCK-8 assay, Matrigel transwell assay and scratch wound healing assay. Expression levels of tumor-related molecules regulated by Cslegumain were evaluated by qRT-PCR and western blotting assay.Cslegumain showed high similarity with human legumain in the secondary and tertiary structures and displayed higher transcriptional levels in the adult worm than in the metacercariae. Native Cslegumain was detected in a catalytic form and was localized mainly in the intestine of the C. sinensis adult and epithelial cells of the intrahepatic bile duct. After transfection into RBE cells, Cslegumain showed high ability in promoting the invasion and migration but not the proliferation of cholangiocarcinoma RBE cells. Furthermore, the expression levels of some molecules including E-cadherin and N-cadherin were downregulated, while the levels of α-actinin 4, β-catenin and inducible nitric oxide synthase (iNOS) were upregulated.Our findings indicated that Cslegumain showed very similar structures as those of human legumain and could promote the invasion and migration of cholangiocarcinoma cells by regulating some tumor-related molecules.