Simple Summary: For hepatocellular carcinoma (HCC), the second most common cause of cancerrelated death, effective therapeutic approaches are lacking. As aberrant gene methylation is a major contributor to the development of HCC, demethylating drugs such as 5-azacytidine (5-Aza) have been proposed. However, despite the potential efficacy of 5-Aza in HCC, most of its mechanisms of action are still unknown. Here, we investigate the phenotypic/molecular effects of 5-Aza with a focus on miR-139-5p. Using multiple in vitro and in vivo models of HCC, we show for the first time that 5-Aza can impair HCC development via upregulation of miR-139-5p, which in turn downregulates the ROCK2/cyclin D1/E2F1/cyclin B1 pro-proliferative pathway and the ROCK2/MMP-2 pro-migratory pathway. These observations elucidate the mechanisms of action of 5-Aza in HCC, strengthen its therapeutic potential, and provide novel information about the crosstalk among ROCK2/cyclin D1/E2F1/cyclin B1/MMP-2 in HCC.

Abstract: Background: For hepatocellular carcinoma (HCC), effective therapeutic approaches are lacking. As aberrant gene methylation is a major contributor to HCC development, demethylating drugs such as 5-azacytidine (5-Aza) have been proposed. As most 5-Aza mechanisms of action are unknown, we investigated its phenotypic/molecular effects. Methods: 5-Aza effects were examined in the human HCC cell lines JHH-6/HuH-7 and in the rat cell-line N1-S1. We also employed a xenograft mouse model (HuH-7), a zebrafish model (JHH-6), and an orthotopic syngeneic rat model (N1-S1) of HCC. Results: 5-Aza downregulated cell viability/growth/migration/adhesion by upregulating miR-139-5p, which in turn downregulated ROCK2/cyclin D1/E2F1 and increased p27kip1, resulting in G1/G0 cell accumulation. Moreover, a decrease in cyclin B1 and an increase in p27kip1 led to G2/M accumulation. Finally, we observed a decrease in MMP-2 levels, a stimulator of HCC cell migration. Aza effects were confirmed in the mouse model; in the zebrafish model, we also demonstrated the downregulation of tumor neo-angiogenesis, and in the orthotopic rat model, we observed impaired N1-S1 grafting in a healthy liver. Conclusion: We demonstrate for the first time that 5-Aza can impair HCC development via upregulation of miR-139-5p, which in turn impairs the ROCK2/cyclin D1/E2F1/cyclin B1 pro-proliferative pathway and the ROCK2/MMP-2 pro-migratory pathway. Thus, we provide novel information about 5-Aza mechanisms of action and deepen the knowledge about the crosstalk among ROCK2/cyclin D1/E2F1/cyclin B1/p27kip1/MMP-2 in HCC.