Abstract

Background: Hepatic cholesterol accumulation and hypercholesterolemia are considered as the risk factors of hepatocellular carcinoma (HCC). However, the therapeutic effects of cholesterol lowering drugs for the treatment of HCC are controversial, indicating that the relationship between cholesterol metabolism and HCC is more complex than anticipated.

Methods: FVB/NJ mice were hydrodynamically injected with c-Myc and Sleeping Beauty to induce HCC. UPLC-MS/MS was used to analyze metabolites of glycolysis and the pentose phosphate pathway (PPP). ¹⁴C-acetate sodium and ³H-thymidine were used to measure cholesterol synthesis and DNA synthesis, respectively. A dual-AAV8-based CRISPR/Cas9 was used to ablate the binding site of miR-206 within the 3’UTR (untranslated region) of its target genes.

Results: Activation of cholesterol synthesis, glycolysis and the pentose phosphate pathway (PPP) was observed in HCC patients and c-Myc HCC mouse model. A positive feedback between cholesterol synthesis and the PPP was formed in tumors of c-Myc mice. Blocking the PPP prevented cholesterol synthesis and thereby development of HCC in c-Myc mice, while ablation of glycolysis did not affect cholesterol synthesis and failed to prevent c-Myc-induced HCC. Unexpectedly, HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) and G6PD (glucose-6-phosphate dehydrogenase), the rate-limiting enzymes of cholesterol synthesis and the PPP, were identified as direct targets of microRNA-206. By targeting Hmgcr and G6pd, microRNA-206 disrupted the positive feedback between cholesterol synthesis and the PPP, thereby inhibiting DNA synthesis and hepatocyte proliferation. Hydrodynamic injection (HDI) of microRNA-206 fully prevented HCC in c-Myc mice, while 100% of control mice dies of HCC. Disrupting the interaction of microRNA-206 with Hmgcr and G6pd restored cholesterol synthesis, the PPP and HCC growth in microRNA-206-treated c-Myc mice.

Conclusion: This study identified a previously undescribed positive feedback loop between cholesterol synthesis and the PPP, which drives HCC development by providing substrate of DNA synthesis and energy for hepatocyte proliferation, while microRNA-206 prevents HCC by disrupting this loop. Cholesterol synthesis as a process rather than cholesterol itself is the major contributor of HCC, which at least in part explains the controversial findings of cholesterol-lowering drugs for the treatment of HCC.