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

The biological functions of growth factor, such as granulins, have been explored in parasites, and we elucidated that granulin (GRN) promoted the metastasis of hepatocellular carcinoma in our previous study. However, it is still unclear for the malignant transformation role of GRN in normal human hepatocytes. In this study, by transfecting pEGFP-C1-GRN eukaryotic expression plasmid, a cell line with stable overexpression of 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 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 GRN promoted the proliferation of LO2 cells by regulating the expression of cell-cycle-related genes. Moreover, the overexpression of 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 GRN. These results showed that 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 GRN could serve as a novel oncoprotein during -associated malignant transformation of hepatocytes.