In the present study, potential molecular biomarkers of NASH hepatocarcinogenesis were investigated using the STAM mice NASH model, characterized by impaired insulin secretion and development of insulin resistance. In this model, 2-days-old C57BL/6N mice were subjected to a single subcutaneous (s.c.) injection of 200 μg streptozotocin (STZ) to induce diabetes mellitus (DM). Four weeks later, mice were administered high-fat diet (HFD) HFD-60 for 14 weeks (STAM group), or fed control diet (STZ group). Eighteen-week-old mice were euthanized to allow macroscopic, microscopic, histopathological, immunohistochemical and proteome analyses. The administration of HFD to STZ-treated mice induced significant fat accumulation and fibrosis development in the liver, which progressed to NASH, and rise of hepatocellular adenomas (HCAs) and carcinomas (HCCs). In 18-week-old animals, a significant increase in the incidence and multiplicity of HCAs and HCCs was found. On the basis of results of proteome analysis of STAM mice HCCs, a novel highly elevated protein in HCCs, cache domain-containing 1 (CACHD1), was chosen as a potential NASH-HCC biomarker candidate. Immunohistochemical assessment demonstrated that STAM mice liver basophilic, eosinophilic and mixed-type altered foci, HCAs and HCCs were strongly positive for CACHD1. The number and area of CACHD1-positive foci, and cell proliferation index in the area of foci in mice of the STAM group were significantly increased compared to that of STZ group. In vitro siRNA knockdown of CACHD1 in human Huh7 and HepG2 liver cancer cell lines resulted in significant inhibition of cell survival and proliferation. Analysis of the proteome of knockdown cells indicated that apoptosis and autophagy processes could be activated. From these results, CACHD1 is an early NASH-associated biomarker of liver preneoplastic and neoplastic lesions, and a potential target protein in DM/NASH-associated hepatocarcinogenesis.
Keywords: CACHD1; HCC; NASH; STAM mice; hepatocarcinogenesis.