Animals, Experimental Design, and Treatments

In the present study, we used liver tissue samples from male mice subjected to NASH-associated hepatocarcinogenesis. This is the first mouse model that mimics the development of HCC in diabetes-associated NASH patients (Fujii et al., 2013; Takakura et al., 2014). The complete experimental design and pathomorphological description of the STAM NASH-related hepatocarcinogenesis model have been described previously by Fujii et al. (2013). Briefly, 2-day-old male C57BL/6J mice were injected with streptozotocin (200 μg/mouse) and were continuously fed a high-fat diet (CLEA Japan, Tokyo, Japan) starting from 4 weeks of age throughout the duration of the study. Control male mice were not injected with streptozotocin and were maintained on standard animal chow for the duration of the study. Liver tissue samples of STAM mice at non-alcoholic fatty liver (NAFL; 6 weeks), NASH-fibrotic (12 weeks), and full-fledged HCC (20 weeks) stages of hepatocarcinogenesis and liver samples from age-matched control mice were purchased from the SMC Laboratories., Inc. (Tokyo, Japan). All experimental procedures were performed according to the Japanese Pharmacological Society Guidelines and experimental protocols were approved by the SMC Laboratories, Inc. Research Animal Care and Use Committee.