Clonorchis sinensis granulin: identification, immunolocalization, and function in promoting the metastasis of cholangiocarcinoma and hepatocellular carcinoma

Background Long-term infections by Clonorchis sinensis are associated with cholangitis, cholecystitis, liver fibrosis, cirrhosis, and even liver cancer. Molecules from the worm play vital roles in disease progress. In the present study, we identified and explored molecular characterization of C. sinensis granulin ( Cs GRN), a growth factor-like protein from C. sinensis excretory/secretory products ( Cs ESPs). Methods The encoding sequence and conserved domains of Cs GRN were identified and analysed by bioinformatics tools. Recombinant Cs GRN (r Cs GRN) protein was expressed in Escherichia coli BL21 (DE3). The localisation of Cs GRN in adult worms and Balb / c mice infected with C. sinensis was investigated by immunofluorescence and immunohistochemistry, respectively. Stable Cs GRN-overexpressed cell lines of hepatoma cells (PLC-GRN cells) and cholangiocarcinoma cells (RBE-GRN cells) were constructed by transfection of eukaryotic expression plasmid of pEGFP-C1- Cs GRN. The effects on cell migration and invasion of Cs GRN were assessed through the wound-healing assay and transwell assay. The levels of matrix metalloproteinase 2 and 9 (MMP2 and MMP9) in PLC-GRN or RBE-GRN cells were detected by real-time PCR (qRT-PCR). The levels of E-cadherin, vimentin, N-cadherin, zona occludens proteins (ZO-1), β-catenin, phosphorylated ERK (p-ERK) and phosphorylated AKT (p-AKT) were analysed by Western blotting. Results Cs GRN, including the conserved GRN domains, was confirmed to be a member of the granulin family. Cs GRN was identified as an ingredient of Cs ESPs. Cs GRN was localised in the tegument and testes of the adult worm. Furthermore, it appeared in the cytoplasm of hepatocytes and biliary epithelium cells from infected Balb / c mouse. The enhancement of cell migration and invasion of PLC-GRN and RBE-GRN cells were observed. In addition, Cs GRN upregulated the levels of vimentin, N-cadherin, β-catenin, MMP2 and MMP9, while it downregulated the level of ZO-1 in PLC-GRN/RBE-GRN cells. In total proteins of liver tissue from r Cs GRN immunised Balb / c mice, vimentin level decreased, while E-cadherin level increased when compared with the control groups. Meanwhile, the levels of p-ERK reached a peak at 4 weeks post immunisation and the level of p-AKT did at 2 weeks after immunisation. Conclusions The encoding sequence and molecular characteristics of Cs GRN were identified. As a member of granulin superfamily, Cs GRN induced mesenchymal characteristics of PLC and RBE cells and was found to regulate the activities of the downstream molecules of the ERK and PI3K/AKT signalling pathways, which could contribute to the enhancement of cell migration and invasion.