Human Cancer Biology MicroRNA-221 Targets Bmf in Hepatocellular Carcinoma and Correlates with Tumor Multifocality

Deregulated cell proliferation and apoptosis play a major role in hepatocellular carcinoma (HCC). MicroRNAs participate in the modulation of key molecules linked to hepatocarcinogenesis. Purpose: This study aims to investigate the role of miR-221 in the modulation of Bmf, a proapoptotic BH3-only protein, and to characterize miR-221 contribution to hepatocarcinogenesis through modulation of apoptosis. Experimental Design: Transfection of miR-221 and anti-miR-221 in HCC-derived cell lines and luciferase reporter assay were used to assess Bmf as a target of miR-221. Modulation of miR-221 and Bmf expression contributed to characterize their role in anoikis. Primary HCC tissues were analyzed to assess the clinical relevance of in vitro findings. Results: Enforced miR-221 expression caused Bmf down-regulation, whereas anti-miR221 induced its up-regulation. A luciferase reporter assay confirmed Bmf as a target of miR-221. Following matrix detachment, miR-221 silencing led to increased apoptotic cell death. The analysis of HCC tissues revealed an inverse correlation between miR221 and Bmf expression and a direct correlation between Bmf and activated caspase-3, as a marker of apoptosis. High miR-221 levels were associated with tumor multifocality and reduced time to recurrence after surgery. Conclusions: Our results indicate that miR-221, by targeting Bmf, inhibits apoptosis. Moreover, in HCC, miR-221 overexpression is associated with a more aggressive phenotype. These findings, together with the previously reported modulation of CDKN1B/ p27 and CDKN1C/p57, show thatmiR-221 simultaneously affectsmultiple pro-oncogenic pathways and suggest miR-221 as a potential target for nonconventional treatment against HCC. (Clin Cancer Res 2009;15(16):5073–81) Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, with an increasing trend in incidence (1). HCC results from the deregulation of multiple signaling pathways. Initial steps involve the disruption of a set of interdependent pathways controlling cell growth and apoptosis. At later stages, cells may acquire angiogenic, invasive, and metastatic properties in a process that involves the interactions of neoplastic cells with the surrounding microenvironment. Among oncogenic factors in HCC, microRNAs (miRNA) participate in several carcinogenic mechanisms (2). We and other groups have reported previously the altered expression of miRNAs in human HCC (3–14). miRNAs are short (19-25 nucleotides) RNA sequences able to modulate the expression of a wide range of target genes by pairing homologous sequences within 3′-untranslated region (3′-UTR) of mRNAs, thus preventing or impairing their translation or promoting RNA degradation. Authors' Affiliations: Dipartimento di Medicina Interna e Gastroenterologia e Centro di Ricerca Biomedica Applicata and Dipartimento di Scienze Chirurgiche e dei Trapianti, Università di Bologna, Bologna, Italy; Dipartimento di Medicina Sperimentale e Diagnostica e Centro Interdipartimentale per la Ricerca sul Cancro, Università di Ferrara, Ferrara, Italy; Department of Experimental Therapeutics, University of Texas M.D. Anderson Cancer Center, Houston, Texas;andComprehensiveCancerCenter,OhioStateUniversity,Columbus,Ohio Received 1/14/09; revised 3/26/09; accepted 3/31/09; publishedOnlineFirst 8/11/09. Grant support:Associazione Italiana per la Ricerca sul Cancro (Regional), Italian Ministero dell'Università e della Ricerca Scientifica, and Italian Ministero della Salute (M. Negrini); Associazione Italiana per la Ricerca sul Cancro (Regional) and Fondazione CARISBO (L. Bolondi); National Cancer Institute Program Project Grants (C.M. Croce); Kimmel Foundation scholar award (G.A. Calin); Associazione Italiana per la Ricerca sul Cancro fellowship (F. Fornari); and Fondazione Italiana per la Ricerca sul Cancro fellowship (M. Ferracin). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Laura Gramantieri, Dipartimento di Medicina Interna e Gastroenterologia, Università di Bologna, Via Albertoni, 15, 40138 Bologna, Italy. Phone: 39-51-6362579; Fax: 39-51-6362725; E-mail: laura. [email protected] or Massimo Negrini, Dipartimento di Medicina Sperimentale e Diagnostica, Università di Ferrara, Via Luigi Borsari, 46, 44100 Ferrara, Italy. Phone: 39-53-2455399; Fax: 39-53-2247618; E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-09-0092 5073 Clin Cancer Res 2009;15(16) August 15, 2009 www.aacrjournals.org Research. on April 12, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Published OnlineFirst August 11, 2009; DOI: 10.1158/1078-0432.CCR-09-0092