Mechanism Exploration On Malignant Transformation Of Heterogeneous Oval Cell Subset Based On GPC3-dependent Wnt/β-catenin Pathway

Objective To explore a method of long-term cultivation of hepatic oval cells in vitro and elucidate whether GPC3could promote the malignant transformation of heterogeneous oval cell subsets depending on Wnt/β-catenin pathway, which could provide a promising target for further anti-HCC-therapy.Methods1. Hepatic oval cells(HOCs) were isolated from the established2-AAF/PH SD rat model and cultured in DMEM/F12(1:1) containing10%FBS、20ng/ml EGF、10ng/ml LIF, and then EGF and LIF were removed after four months. The characteristic of HOCs was determined by morphological observation and detection of molecular markers ALB、CK-19and AFP.2. Expressions of key elements such as β-catenin and target genes such as c-myc and cyclinD1in Wnt/β-catenin signaling pathway were detected by western blotting to clarify whether the signaling pathway was activated or not after recombinated plasmids pcDNA3.1(+)/GPC3were transfected into HepG2cells. The growth rates of HepG2of both groups were assessed by using the MTT assay; 3. Expressions of key elements such as βcatenin and target genes such as c-myc and cyclinD1in Wnt/β-catenin signaling pathway were detected by western blotting to clarify whether the signaling pathway was activated or not after recombinated plasmids pcDNA3.1(+) containing GPC3-EGFP chimeric gene were transfected into HepG2cells.Results1. Hepatic oval cells isolated from2-AAF/PH SD rat model and cultured in DMEM/F12(1:1) containing10%FBSΟ20ng/ml EGFΟlOng/ml LIF still expressed both hepatocyte marker ALB and biliary epithelial cell marker CK-19after four months’ cultivation, proving their potential of bilateral-differentiation, while in the absence of EGF and LIF, the expression of fetal liver marker AFP, which is also one marker of oval cell, decreased quickly;2.There were significant differences in the expressions of GPC3and key elements such as P-catenin and target genes such as c-myc and cyclinDl in Wnt/β-catenin pathway between the GPC3-transfected HepG2cells and the untransfected cells, so was the growth rates of cells (P<0.05);3.There were no significant differences in the expressions of GPC3and key elements such as β-catenin and target genes such as c-myc and cyclinD1in Wnt/β-catenin pathway between the GPC3-transfected hepatic oval cells and the untransfected cells (P>0.05). Conclusion1. Hepatic oval cells can proliferate in DMEM/F12(1:1) containing10%FBS、20ng/ml EGF、10ng/ml LIF and maintain stem/progenitor cell activities for several months;2. GPC3could probably promote the growth of HepG2cells by activating Wnt/β-catenin pathway;3. Whether GPC3promotes malignant transformation of heterogeneous oval cells depending on Wnt/β-catenin signaling pathway still needs to be confirmed.