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5-Azacytidine Downregulates the Proliferation and Migration of Hepatocellular Carcinoma Cells In Vitro and In Vivo by Targeting miR-139-5p/ROCK2 Pathway

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Department of Life Sciences, Cattinara University Hospital, Trieste University, Strada di Fiume 447, I-34149 Trieste, Italy
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Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia
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Faculty of Health Sciences, University of Primorska, Polje 42, SI-6310 Izola, Slovenia
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Department of Medical, Surgical and Health Sciences, University of Trieste, Cattinara Hospital, Strada di Fiume 447, I-34149 Trieste, Italy
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Department of Chemical, Materials and Industrial Production Engineering, University of Naples “Federico II”, Piazzale V. Tecchio 80, I-80125 Naples, Italy
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CEINGE Advanced Biotechnologies, via Gaetano Salvatore, 486, I-80145 Napoli, Italy
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Department of Engineering and Architecture, University of Trieste, Via Valerio 6/A, I-34127 Trieste, Italy
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Department of Clinic-Surgical Sciences, Laboratory of Experimental Surgery and Animal Facility, University of Pavia, Via Ferrata 9, I-27100 Pavia, Italy
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Department of General Surgery, Maggiore Hospital, Largo Donatori del Sangue 1, I-26900 Lodi, Italy
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International Clinical Research Center (ICRC) of St Anne’s University Hospital, CZ-65691 Brno, Czech Republic
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Department of Paediatrics, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0QQ, UK
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Clinical and Experimental Onco-Haematology Unit, Centro di Riferimento Oncologico, Istituto di Ricovero a Cura a Carattere Scientifico IRCCS, 33081 Aviano, Italy
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Industrial Engineering Department, University of Padova, Via Francesco Marzolo, 9, I-35131 Padova, Italy
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Nealys SRL, Via Flavia 23/1, I-34148 Trieste, Italy
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Stem Cell Research and Application Laboratory, VNUHCM, University of Science, Ho Chi Minh City 72711, Vietnam
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Department of Chemistry and Biology, X University, MaRS Discovery District, West Tower 661 University Avenue, Toronto, ON M5G 1M1, Canada
*
Authors to whom correspondence should be addressed.
Academic Editor: Anne Corlu
Cancers 2022, 14(7), 1630; https://doi.org/10.3390/cancers14071630
Received: 30 December 2021 / Revised: 14 February 2022 / Accepted: 18 March 2022 / Published: 23 March 2022
(This article belongs to the Section Cancer Therapy)
For hepatocellular carcinoma (HCC), the second most common cause of cancer-related 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.
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. View Full-Text
Keywords: hepatocellular carcinoma; 5-azacytidine; cell cycle; migration; miR-139-5p; ROCK2; E2F1; MMP-2 hepatocellular carcinoma; 5-azacytidine; cell cycle; migration; miR-139-5p; ROCK2; E2F1; MMP-2
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MDPI and ACS Style

Tonon, F.; Cemazar, M.; Kamensek, U.; Zennaro, C.; Pozzato, G.; Caserta, S.; Ascione, F.; Grassi, M.; Guido, S.; Ferrari, C.; Cansolino, L.; Trotta, F.; Kuzmanov, B.G.; Forte, G.; Martino, F.; Perrone, F.; Bomben, R.; Gattei, V.; Elvassore, N.; Murano, E.; Truong, N.H.; Olson, M.; Farra, R.; Grassi, G.; Dapas, B. 5-Azacytidine Downregulates the Proliferation and Migration of Hepatocellular Carcinoma Cells In Vitro and In Vivo by Targeting miR-139-5p/ROCK2 Pathway. Cancers 2022, 14, 1630. https://doi.org/10.3390/cancers14071630

AMA Style

Tonon F, Cemazar M, Kamensek U, Zennaro C, Pozzato G, Caserta S, Ascione F, Grassi M, Guido S, Ferrari C, Cansolino L, Trotta F, Kuzmanov BG, Forte G, Martino F, Perrone F, Bomben R, Gattei V, Elvassore N, Murano E, Truong NH, Olson M, Farra R, Grassi G, Dapas B. 5-Azacytidine Downregulates the Proliferation and Migration of Hepatocellular Carcinoma Cells In Vitro and In Vivo by Targeting miR-139-5p/ROCK2 Pathway. Cancers. 2022; 14(7):1630. https://doi.org/10.3390/cancers14071630

Chicago/Turabian Style

Tonon, Federica, Maja Cemazar, Urska Kamensek, Cristina Zennaro, Gabriele Pozzato, Sergio Caserta, Flora Ascione, Mario Grassi, Stefano Guido, Cinzia Ferrari, Laura Cansolino, Francesco Trotta, Biljana G. Kuzmanov, Giancarlo Forte, Fabiana Martino, Francesca Perrone, Riccardo Bomben, Valter Gattei, Nicola Elvassore, Erminio Murano, Nhung H. Truong, Michael Olson, Rossella Farra, Gabriele Grassi, and Barbara Dapas. 2022. "5-Azacytidine Downregulates the Proliferation and Migration of Hepatocellular Carcinoma Cells In Vitro and In Vivo by Targeting miR-139-5p/ROCK2 Pathway" Cancers 14, no. 7: 1630. https://doi.org/10.3390/cancers14071630

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