No abstract

On the basis of their characteristics, we presume that developmental stage-specific hepatocytes should have the ability to induce maturation of hepatoma cells. A regulatory circuit formed by hepatocyte nuclear factor (HNF)-4α, HNF-1α, HNF-6 and the upstream stimulatory factor (USF-1) play a key role in the maturation of embryonic hepatocytes; however, it is unclear whether the regulatory circuit mediates the embryonic induction of hepatoma cell maturation. In this study, 12.5-d to 15.5-d mouse embryonic hepatocytes or their medium were used to coculture or treat HepG2 cells, and the induced maturation was evaluated in vitro and in vivo. In the induced HepG2 cells, the components of the regulatory circuit were detected, their cross-regulation was evaluated and HNF-4α RNA interference was performed. We found that 13.5-d to 14.5-d embryonic hepatocytes could induce HepG2 cell maturation, demonstrated by morphological changes, increased maturation markers and decreased c-Myc and α-fetoprotein (AFP) in vitro. The majority of HepG2 tumors were eliminated by 13.5-d embryonic induction in vivo. All components of the regulatory circuit were upregulated and the binding of HNF-4α, HNF-1α, HNF-6 and USF-1 to their target sites was promoted to rebuild the regulatory circuit in the induced HepG2 cells. Moreover, RNA interference targeting HNF-4α, which is the core of the regulatory circuit, attenuated the induced maturation of HepG2 cells with downregulation of the regulatory circuit. These results revealed that developmental stage-specific hepatocytes could induce the maturation of HepG2 cells by rebuilding the regulatory circuit. is believed to be a controller and marker of mature hepatocytes (27,28). As a downstream target of HNF-4α, HNF-1α is also essential for hepatocyte maturation (29,30). USF-1, which competes with the oncogene c-Myc for the E-box regulatory element, is another downstream target of HNF-4α that plays crucial roles in the maturation and formation of functional hepatocytes (31,32). USF-1 can activate HNF-6, which is another important transcription factor for hepatocyte maturation that has been found to regulate 33). Therefore, HNF-4α, HNF-1α, HNF-6 and USF-1 form a positive feedback loop that drives hepatocyte maturation in liver development ( Figure 1A). Because the transcription factor binding sites are generally believed to scatter within 10 kb upstream of the transcriptional start site (34), the 10-kb gene promoters were all analyzed. On the basis of previous reports (25), the predicted binding sites of HNF-4α HNF-1α, HNF-6 and USF-1 were identified, respectively ( Figure 1B). It remains unknown whether developmental stage-specific hepatocytes rebuild the regulatory circuit to induce the maturation of HepG2 cells.In this study, HepG2 cells were cocultured with mouse embryonic hepatocytes at gestation of 12.5-15.5 d, because the mouse hepatocytes differentiated at 12.5 d, and the liver structure became firmly established between 12 and 15 d of gestation (35)(36)(37). The induced maturation of HepG2 cel...