Clonorchis sinensis Granulin Promotes Malignant Transformation of Hepatocyte Through EGFR-Mediated RAS/MAPK/ERK and PI3K/Akt Signaling Pathways

Abstract

The biological functions of growth factor, such as granulins, have been explored in parasites, and we elucidated that <i>Clonorchis sinensis</i> granulin (<i>Cs</i>GRN) promoted the metastasis of hepatocellular carcinoma in our previous study. However, it is still unclear for the malignant transformation role of <i>Cs</i>GRN in normal human hepatocytes. In this study, by transfecting pEGFP-C1-<i>Cs</i>GRN eukaryotic expression plasmid, a cell line with stable overexpression of <i>Cs</i>GRN in normal hepatocyte (LO2-GRN cells) was constructed. The effects on cell proliferation were detected by carrying out cell counting kit-8 (CCK8) assay and colony formation assay. Additionally, we conducted flow cytometry analysis to determine whether the proliferation of <i>Cs</i>GRN was due to cell cycle arrest. Subsequently, the migration ability and the invasion ability of LO2-GRN cells were evaluated through wound-healing assay and transwell assay. Meanwhile, the levels of the markers of RAS/MAPK/ERK and PI3K/Akt signaling pathways activation in LO2-GRN cells were assessed by quantitative RT-PCR and Western blot. Our results indicated that <i>Cs</i>GRN promoted the proliferation of LO2 cells by regulating the expression of cell-cycle-related genes. Moreover, the overexpression of <i>Cs</i>GRN regulates malignant metastasis of liver cells by inducing the upregulation of epithelial-mesenchymal transition (EMT) marker proteins. Furthermore, both mRNA and protein expression levels of p-EGFR, RAS, p-ERK, p-AKT, p-PI3K, and p-braf have been enhanced by <i>Cs</i>GRN. These results showed that <i>Cs</i>GRN promoted the malignant transformation of hepatocytes by regulating epidermal growth factor receptor (EGFR)-mediated RAS/MAPK/ERK and PI3K/Akt signaling pathways, which suggested that <i>Cs</i>GRN could serve as a novel oncoprotein during <i>Clonorchis sinensis</i>-associated malignant transformation of hepatocytes.