Clonorchis sinensis granulin promotes malignant transformation of human intrahepatic biliary epithelial cells through interaction with M2 macrophages via regulation of STAT3 phosphorylation and the MEK/ERK pathway

Background Clonorchis sinensis granulin ( Cs GRN), a component of the excretory/secretory products of this species, is a multifunctional growth factor that can promote the metastasis of cholangiocarcinoma cells. However, the effect of Cs GRN on human intrahepatic biliary epithelial cells (HIBECs) is unclear. Here, we investigated the effect of Cs GRN on the malignant transformation of HIBECs and its possible underlying mechanism. Methods The malignant transformation phenotypes of HIBECs after Cs GRN treatment were estimated by EdU-488 incorporation assay, colony formation assay, wound-healing assay, Transwell assay and western blot. The biliary damage of Cs GRN-treated mice was detected by western blot, immunohistochemical staining and hematoxylin and eosin staining. The phenotypes of the macrophages [human monocytic leukemia cell line (THP-1)] were analyzed by flow cytometry, immunofluorescence and immunohistochemistry, both in vitro and in vivo. A co-culture system was developed to explore the interaction between THP-1 and HIBECs in Cs GRN-containing medium. Enzyme-linked immunosorbent assay and western blot were used to detected the activation of interleukin 6 (IL-6), phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. An inhibitor of the MEK/ERK pathway, PD98059, was used to determine whether this pathway is involved in Cs GRN-mediated cell interactions as well as in STAT3 phosphorylation and malignant transformation of HIBECs. Results Excessive hyperplasia and abnormal proliferation of HIBECs, enhanced secretion of hepatic pro-inflammatory cytokines and chemokines, as well as biliary damage, were observed in vitro and in vivo after treatment with Cs GRN. The expression of the markers of M2 macrophages significantly increased in Cs GRN-treated THP-1 cells and biliary duct tissues compared with the controls. Moreover, following treatment with Cs GRN, the HIBECs underwent malignant transformation in the THP-1-HIBECs co-culture group. In addition, high expression of IL-6 was observed in the Cs GRN-treated co-culture media, which activated the phosphorylation of STAT3, JAK2, MEK and ERK. However, treatment with an inhibitor of the MEK/ERK pathway, PD98059, decreased expression of p-STAT3 in Cs GRN-treated HIBECs and further repressed the malignant transformation of HIBECs. Conclusions Our results demonstrated that, by inducing the M2-type polarization of macrophages and activating the IL-6/JAK2/STAT3 and MEK/ERK pathways in HIBECs, Cs GRN promoted the malignant transformation of the latter. Graphical abstract