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.

CEINGE Advanced Biotechnologies via Gaetano Salvatore 486 1 80145 Napoli Italy

Clinical and Experimental Onco Haematology Unit Centro di Riferimento Oncologico Istituto di Ricovero a Cura a Carattere Scientifico IRCCS 33081 Aviano Italy

Department of Chemical Materials and Industrial Production Engineering University of Naples Federico 2 Piazzale 5 Tecchio 80 1 80125 Naples Italy

Department of Chemistry and Biology 10 University MaRS Discovery District West Tower 661 University Avenue Toronto ON M5G 1M1 Canada

Department of Clinic Surgical Sciences Laboratory of Experimental Surgery and Animal Facility University of Pavia Via Ferrata 9 1 27100 Pavia Italy

Department of Engineering and Architecture University of Trieste Via Valerio 6 A 1 34127 Trieste Italy

Department of Experimental Oncology Institute of Oncology Ljubljana Zaloska 2 SI 1000 Ljubljana Slovenia

Department of General Surgery Maggiore Hospital Largo Donatori del Sangue 1 1 26900 Lodi Italy

Department of Life Sciences Cattinara University Hospital Trieste University Strada di Fiume 447 1 34149 Trieste Italy

Department of Medical Surgical and Health Sciences University of Trieste Cattinara Hospital Strada di Fiume 447 1 34149 Trieste Italy

Department of Paediatrics University of Cambridge Addenbrooke's Hospital Hills Road Cambridge CB2 0QQ UK

Faculty of Health Sciences University of Primorska Polje 42 SI 6310 Izola Slovenia

Industrial Engineering Department University of Padova Via Francesco Marzolo 9 1 35131 Padova Italy

International Clinical Research Center of St Anne's University Hospital CZ 65691 Brno Czech Republic

Nealys SRL Via Flavia 23 1 1 34148 Trieste Italy

Stem Cell Research and Application Laboratory VNUHCM University of Science Ho Chi Minh City 72711 Vietnam

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