Nonalcoholic fatty liver disease (NAFLD) is one of the major causes of hepatocellular carcinoma (HCC). Although the intracellular cholesterol accumulation has been demonstrated to regulate the gene expression responsible for steatohepatitis, the role played by cholesterol in the development of NAFLD-associated HCC has not been fully elucidated. In this study, using microarray analysis, we investigated the molecular mechanisms governing cholesterol-mediated progression of NAFLD. To ensure hepatic cholesterol accumulation, either a high-fat and high-cholesterol (HFHC) diet or a high-fat and high-cholesterol with cholic acid (HFHCCA) diet was fed to diethylnitrosamine (DEN)-injected C57BL/6J mice for 10 weeks. While an HFHC diet increased hepatic triglyceride levels, an HFHCCA diet induced hepatic cholesterol accumulation by reducing bile acid biosynthesis in DEN-injected mice. Livers from both HFHC and HFHCCA groups exhibited increases in steatosis and necrosis; however, histological features of HCC were not observed in any of the experimental groups. Hepatic gene expression profile of the HFHCCA group was different from those of other groups. Functional analysis showed that cholic acid supplementation upregulated differentially expressed genes (DEGs) associated with inflammation, proliferation, apoptosis, chemical drug response, and cancer signaling pathway. Downregulated DEGs were associated with steroid metabolism, mitochondrial function, and oxidative phosphorylation pathway. Furthermore, hepatic cholesterol accumulation lowered the expression of DEGs associated with macronutrients and energy metabolism, especially amino acid metabolism. Taken together, feeding the HFHCCA diet to DEN-injected mice accelerated the progression of NAFLD to the procarcinogenic state based on global gene expression profile, demonstrating the possible role played by hepatic accumulation of cholesterol.

非酒精性脂肪性肝病（NAFLD）是肝细胞癌（HCC）的主要原因之一。尽管已证明细胞内胆固醇积累可调节导致脂肪性肝炎的基因表达，但胆固醇在 NAFLD 相关 HCC 发展中所起的作用尚未完全阐明。在这项研究中，我们使用微阵列分析，研究了控制胆固醇介导的 NAFLD 进展的分子机制。为了确保肝脏胆固醇积累，将高脂肪和高胆固醇 (HFHC) 饮食或高脂肪和高胆固醇加胆酸 (HFHCCA) 饮食喂给注射了二乙基亚硝胺 (DEN) 的 C57BL/6J 小鼠 10周。虽然 HFHC 饮食会增加肝脏甘油三酯水平，HFHCCA 饮食通过减少 DEN 注射小鼠的胆汁酸生物合成来诱导肝脏胆固醇积累。HFHC 和 HFHCCA 组的肝脏都表现出脂肪变性和坏死的增加；然而，在任何实验组中均未观察到 HCC 的组织学特征。HFHCCA 组的肝基因表达谱与其他组不同。功能分析表明，补充胆酸上调了与炎症、增殖、凋亡、化学药物反应和癌症信号通路相关的差异表达基因 (DEG)。下调的 DEG 与类固醇代谢、线粒体功能和氧化磷酸化途径有关。此外，肝脏胆固醇积累降低了与宏量营养素和能量代谢相关的 DEGs 的表达，尤其是氨基酸代谢。总之，根据全局基因表达谱，向注射了 DEN 的小鼠喂食 HFHCCA 饮食加速了 NAFLD 向致癌状态的进展，证明了胆固醇在肝脏中积累可能发挥的作用。