HepatomiRNoma: The proposal of a new network of targets for diagnosis, prognosis and therapy in hepatocellular carcinoma

G Model

ARTICLE IN PRESS

ONCH-2049; No. of Pages 10

Critical Reviews in Oncology/Hematology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Critical Reviews in Oncology/Hematology journal homepage: www.elsevier.com/locate/critrevonc

Review

HepatomiRNoma: The proposal of a new network of targets for diagnosis, prognosis and therapy in hepatocellular carcinoma Fabrizio Bronte a,1 , Giuseppe Bronte b,1 , Daniele Fanale b , Stefano Caruso b , Enrico Bronte b , Maria Grazia Bavetta a , Eugenio Fiorentino b , Christian Rolfo c , Viviana Bazan b , Vito Di Marco a , Antonio Russo b,∗ a

Section of Gastroenterology, DiBiMIS, University of Palermo, Italy Department of Surgical, Oncological and Oral Sciences, University of Palermo, Palermo, Italy c Department of Oncology, University Hospital of Antwerp, Edegem, Belgium b

Contents 1. 2. 3. 4.

5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 MiRNA biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 MicroRNAs and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 HEPATOmiRNOMA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.1. miR-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2. miR-9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.3. miR-15b and miR-130b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.4. miR-16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.5. miR-18a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.6. miR-21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.7. miR-34a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.8. miR-92a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.9. miR-101 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.10. miR-122 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.11. miR-129-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.12. miR-132 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.13. miR-195 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.14. miR-199a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.15. miR-221 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.16. miR-222 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.17. miR-223 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.18. miR-224 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.19. miR-375 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.19. miR-500 and miR-885-5p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.20. miR-675 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.21. miR-1274a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Current issues and clinical implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author at: Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology, University of Palermo, Via del Vespro 129, 90127 Palermo, Italy. Fax: +39 091 6554529. E-mail address: [email protected] (A. Russo). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.critrevonc.2015.09.007 1040-8428/© 2015 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Bronte, F., et al., HepatomiRNoma: The proposal of a new network of targets for diagnosis, prognosis and therapy in hepatocellular carcinoma. Crit Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.09.007

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a r t i c l e

i n f o

Article history: Received 14 May 2015 Received in revised form 6 August 2015 Accepted 29 September 2015 Keywords: Hepatocellular carcinoma MicroRNA Diagnosis Targeted therapy Serum Prognosis

a b s t r a c t The diagnosis and treatment of hepatocellular carcinoma (HCC) underwent a huge advancement in the last years. Recently, microRNAs (miRNAs) have been also studied to provide a new tool for early diagnosis of high risk patients, for prognostic classification to identify those patients who benefit cancer treatment and for predictive definition to select the right targeted drug. In this review we revised all the available data obtained to explore the role of miRNAs in HCC. This analysis led to identification of miRNAs which could gain a diagnostic, prognostic or predictive role. The results of studies on miRNAs involved in HCC are initial and far from providing scientific evidences to translate into clinical practice. We propose a classification of these miRNAs, that we could name HepatomiRNoma as a whole. Anyway prospective studies have to be designed to clarify the real clinical impact of this new tool. © 2015 Published by Elsevier Ireland Ltd.

1. Introduction

2. MiRNA biogenesis

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death, among both men and women (Siegel et al., 2014). Although diagnosis and treatment of HCC underwent a huge advancement in the last years, the identification of new biomarkers for the early diagnosis of the tumor, to predict prognosis, or to identify new therapeutic targets is needed. Micro-ribonucleic acids (miRNAs) are a class of naturallyoccurring, evolutionarily conserved, small non-coding RNA molecules, about 21–24 nucleotides in length (Lagos-Quintana et al., 2001). MiRNAs negatively regulate gene expression via translational repression or mRNA degradation, by binding the 3 untranslated region (UTR) of their target mRNA (Bartel, 2004). Since the discovery of the first miRNA, lin-4, in 1993 through studies on Caenorhabditis elegans, a large number of miRNAs has been recently identified (Lee et al., 1993). To date, approximately 1000 miRNAs have been detected in humans and each of these ones has been classified with an identification number (Griffiths-Jones et al., 2008). Because they regulate about 30% of genes, a crucial role is exerted in different biological processes, such as proliferation, differentiation, apoptosis and metabolism. In addition, recent evidences showed altered expression of miRNAs in various diseases, including cancer (Corsini et al., 2012). MiRNA online databases (e.g. TarBase, miRBase, miRò) describe their nomenclature, targets, functions and implications in different diseases (Griffiths-Jones et al., 2008). Interestingly, a single miRNA can regulate a great number of mRNAs, while a single mRNA can be target of different miRNAs (Caruso et al., 2012). In this review we considered both preclinical findings and clinical data as regards the role of each miRNA in hepatocellular carcinogenesis and the perspectives of their use as diagnostic or prognostic biomarkers or therapeutic tool in HCC patients. The changes in miRNA expression pattern may represent a new opportunity for HBV-related HCC diagnosis and therapy. Some studies focused on miRNA polymorphisms responsible for HCC susceptibility, others have identified several miRNAs altered in HBV-related HCC tissue and cells (Petrini et al., 2015). A literature review was performed on these aspects of miRNAs in HCC. We searched on PubMed by these keywords (“microRNAs”[MeSH Terms] OR “microRNAs”[All Fields] OR “miRs”[All Fields]) AND (“carcinoma; hepatocellular”[MeSH Terms] OR “liver neoplasms”[MeSH Terms] OR “hepatocellular carcinoma”[All fields] OR “HCC”[All fields] OR (“liver”[All fields] AND “cancer”[All fields])). Among all the articles found by this search we selected those about the role of miRNAs in liver carcinogenesis; diagnosis; prognosis and therapy of HCC. Further articles not found by this search were searched manually among references of relevant articles and reviews.

MiRNA biogenesis is a multistep process that begins in the nucleus where the RNA polymerase II or RNA polymerase III, after binding to the promoter, transcribes a long primary transcript called pri-miRNA, an inactive form about 1 Kb in length (Lee et al., 2004; Borchert et al., 2006). The pri-miRNA is then processed by the Microprocessor complex consisting of Drosha, an RNase III endonuclease, its partner DGCR8 and associated proteins, into approximately 70-nucleotide incomplete, hairpin-like miRNA precursor (pre-miRNA) (Gregory et al., 2004). Pre-miRNA, originated by cleavage of pri-miRNA, is transported from nucleus into the cytoplasm through the Esportin-5, which recognizes and binds to the precursor. In the cytoplasm, the pre-miRNA is cleaved by Dicer, an RNase III endonuclease, into a 20–24 base long RNA duplex, which contains both the mature miRNA strand and its complementary strand. RISC (RNA-induced silencing complex) incorporates only the mature miRNA strand, while the complementary strand is degraded. Therefore, the mature microRNA carried by the RISC complex can negatively regulate one or more target mRNAs by binding to specific sequences at the 3 UTR (Kim, 2005). In particular, there are two mechanisms. When miRNA binds its target mRNA with a perfect complementarity is able to degrade the mRNA and thereby inhibits its expression. In contrast, when miRNA-mRNA binding occurs with an imperfect complementarity, mRNA translation is stopped (Bartel, 2004; Kim, 2005). 3. MicroRNAs and cancer Although miRNAs are able to negatively regulate the expression of target mRNAs, their expression is also regulated by different mechanisms such as specific translational regulation, methylation and histone deacetylation, DNA copy alteration and gene mutations affecting proteins involved in processing and maturation (Li et al., 2009a; Cummins and Velculescu, 2006). Furthermore, it has been reported that more than 50% of microRNA genes are often located in the fragile sites of chromosomes and in genomic regions which are prone to deletion, amplification and mutations (Corsini et al., 2012). It is now recognized that the aberrant expression of various microRNAs is closely related to the etiology and clinical outcome of several human cancers, including hepatocellular carcinoma (HCC). MiRNAs involved in carcinogenesis can have two different roles. In fact, they can be microRNA oncogenes, called oncomirs, if they promote tumor development through the negative regulation of tumor suppressor genes. Alternatively, tumor suppressor microRNA, called anti-oncomiRs, are able to inhibit tumor growth by targeting oncogenes (Corsini et al., 2012; Visone and Croce, 2009). For these reasons, oncomiRs are up-

Please cite this article in press as: Bronte, F., et al., HepatomiRNoma: The proposal of a new network of targets for diagnosis, prognosis and therapy in hepatocellular carcinoma. Crit Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.09.007

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Table 1 HEPATOmiRNOMA. Hepato-antioncomir inhibits tumor growth by targeting oncogenes

Hepato-oncomir promotes tumor development through the negative regulation of tumor suppressor genes miRNA

T-cell/tissue

T-serum

miRNA

T-cell/tissue

T-serum

miR-9 miR-15b miR-130b miR-16 miR-18a miR-21 miR-92a miR-221 miR-222 miR-224 miR-500 miR-885-5p miR-675

Up Down – Up Up Up Up Up Up – – Up

– Up Down Up Up Down/up Up Up Up Up Up –

miR-1 miR-34a miR-101 miR-122 miR-129-2 miR-132 miR-195 miR-199a miR-223 miR-375

Down Down Down Down Down Down Down Down Down Down

– – Up/down Up – – Down Down Up Down

regulated in cancer cells while anti-oncomiRs are down-regulated. It has been reported that tumors may have both high and low miRNA expression levels, depending on tissue and cancer characteristics (Corsini et al., 2012; Li et al., 2009a). For this reason, currently have been identified 23 miRNAs (miR-1, miR-9, miR-15b, miR-130b, miR-16, miR-18a, miR-21, miR-34, miR-92a, miR-101, miR-122, miR-129-2, miR-132, miR-195, miR-199a, miR-221, miR222, miR-223, miR-224, miR-375, miR-500, miR-675, miR-885-5p) detectable in peripheral blood and tissues, which are involved in hepatocellular carcinoma development and progression and we can name them HepatomiRNoma as a whole. Similarly, we can differentiate two types of HEPATOmiRNAs: HEPATOoncomirs, if they promote tumor development through the negative regulation of tumor suppressor genes; HEPATOanti-oncomiRs, if they are able to inhibit tumor growth by targeting oncogenes. In this review we want to focus on and explain the role of these serum and tissue miRNAs in hepatocellular carcinoma (Table 1). 4. HEPATOmiRNOMA 4.1. miR-1 miR-1 is implicated in mitogenesis, proliferation, angiogenesis, cell invasion and metastatic process. In vitro studies on HepG2 cells have shown that inserting of miR-1 in those cells lacking it inhibits proliferation. The mechanisms by which miR-1 acts are various. Among the various mechanisms of carcinogenesis the hypermethylation of related genes results in a reduction of the miR-1 levels. It results in the overexpression of certain oncogenes, FoxP1 and MET, as potential targets, thus promoting cell proliferation (Datta et al., 2008). Two other mechanisms of miR-1 action are given by (Wei et al., 2012; Li et al., 2012a). They showed that low levels of miR-1 induce the overexpression of endothelin-1 (ET-1) gene responsible for the degradation of extracellular matrix. Thus the migration and metastasis development of HCC are favored. Besides the vascular tone and vascular growth are stimulated through IGF-1 and 2, PDGF, and EGF. These properties have allowed us to identify serum miR-1 as potential prognostic factor in HCC as suggested by the study of Koberle et al. (2013), since it is independent of the stage Barcelona Clinic Liver Cancer (BCLC), Cancer of the Liver Italian Program (CLIP) and treatment of tumors. 4.2. miR-9 miR-9 has also a role in the metastatic process. In fact, in 2013, Sun et al. (2013) showed that miR-9 is overexpressed in Hep12 cell lines and, once transfected those cell lines with low metastatic potential such as HepG2 and SMMC7721, they acquire a high inva-

siveness. Probably, the molecular target of this miRNA is the KLF17 gene, a negative regulator of epithelial-mesenchymal transition (EMT) and metastasis. The levels of KLF17 expression are reduced when miR-9 is overexpressed. The role in the aggressiveness of this miRNA in the tumor process was confirmed by Tan et al. (2010). These authors have shown that miR-9 inhibits the production of E-cadherin promoting tumor migration. Moreover, the miR-9 upregulation in HCC cancer tissues is significantly correlated with aggressive clinicopathological features and higher tumor staging and risk, and may be considered as an independent prognostic factor (Cai and Cai, 2014). 4.3. miR-15b and miR-130b The miR-15b, which is associated with the miR-130b, exerts its carcinogenetic role by suppressing the antiapoptotic protein Bcl-w. For this reason, these two miRNAs were identified as responsible for the growth and recurrence processes of HCC. Furthermore, it was shown that the tissue levels of these miRNAs (Chung et al., 2010) are reduced in contrast to serum levels that are increased in HCC patients. This characteristic could be exploited for diagnostic purposes with the area under the ROC curve of 0.981 (Liu et al., 2012). The pathogenic role of these miRNAs is even more supported by Wu et al. (2014), who showed that the HBx protein, a viral protein of HBV, induces inhibition of miR-15b. This would explain the high carcinogenetic power of HBV during viral suppression. This mechanism would lead to the overexpression of fucosyltransferase 1 and 2 (FUT1 and 2) and of Globo H, two proteins involved in cell proliferation and migration (Wu et al., 2014). Recently, it was reported that miR-130b promotes cell aggressiveness by inhibiting peroxisome proliferator-activated receptor gamma (PPAR-␥) in human HCC. The down-regulation of this miRNA restores the PPAR-␥ expression and subsequently inhibits the epithelial-mesenchymal transition in HCC cells (Tu et al., 2014). 4.4. miR-16 The miR-16 is another miRNA related to HBx protein. It belongs to a class of miRNAs, including miR-15a, miR-15b and miR-16, whose genes are located on chromosome 13q and 3 and cotranscribed from DLEU2 and SMC4, respectively, a gene family that regulates the cell cycle by activating the G1/S phase. They target CCND1, CCND3, CCNE1 and CDK6. This class of genes represses several oncogenes such as c-Myb, Bmi-1, BCL-2, Wnt3a, and Wip1. In particular, miR-16 target CCDN1 and c-myc inhibiting apoptosis and promoting the entry of cells in the G1 phase of the cell cycle (Wu et al., 2011). This role was further confirmed by Tsang and Kwok (2010), who showed that the introduction of epigallocathechinagallate (EGCG) increases tissue levels of miR-16 favoring

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the suppression of Bcl-2. Similarly, it has been reported that serum levels of miR-16 are significantly lower in HCC than in chronic liver disease (CLD) patients or control subjects (Ge et al., 2014). In addition, these authors confirmed how this test may be used in association with other markers reaching a good diagnostic performance with high sensitivity and specificity, in particular, if the analysis is restricted to tumors more than 3 cm as diameter (Qu et al., 2011). 4.5. miR-18a miR-18a is involved in cell proliferation through inhibition of ER-alpha. Serum and tissue levels of this miRNA are high in HCC. The miRNA serum levels are able to differentiate patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma with a good diagnostic performance: area under ROC curve is 0.775 with a sensitivity of 77.2% and a specificity of 70.0% (Liu et al., 2009a; Li et al., 2012b). Recently, Li et al. (2015) showed that increased p53 levels induce the processing of miR18a reducing the ER-alpha levels in female HCC. 4.6. miR-21

While Yang et al. (2012a) explained how the HBV chronic infection increases the expression of TGF-␤, which reduces the miR-34a expression. This event increases the expression of the chemokine CCL22 that stimulates T regulatory cells and favours the immune escape. As a consequence the colonization and dissemination of HCC are also favored. Serum levels of this miRNA (Cermelli et al., 2011) are elevated in patients with chronic hepatitis C infection than in control individuals and these are related to the degree of fibrosis. Recently, Xu et al. (2015) found that miR-34a induces cellular senescence through the negative modulation of telomere pathway in human HCC by targeting c-Myc and FoxM1 involved in the activation of telomerase reverse transcriptase (hTERT) transcription. 4.8. miR-92a Little is known about the miR-92a. The only study conducted by Shigoka et al. (2010) showed that it is highly expressed in HCC tissue and is reduced in the plasma of HCC patients. However, after surgical resection, it tends to decrease in the tissue and to increase in the plasma, thus indicating a prognostic role. Conversely, other studies demonstrated that miR-92a is up-regulated in serum of patients with HBV-positive HCC compared to control groups (Li et al., 2010a; Giray et al., 2014).

mir-21, one of the most studied in HCC, shows proliferative actions and promotes cell migration. In vitro studies have shown that its tissue levels are increased in HCC tumors and cell lines. This condition leads to the failure of some tumor suppressor, for example phosphatase and tensin homolog (PTEN) (Meng et al., 2007), RHOB (Connolly et al., 2010) and programmed cell death 4 (PDCD4) (Zhu et al., 2012), while stimulates the production of phospho-cJun, matrix metalloproteinases (MMP-2 and MMP-9) (Zhu et al., 2012). These mechanisms lead to the stimulation of cell proliferation promoting migration and invasion. Tomimaru et al. (2010) have demonstrated that miR-21 has an effect in the modulation of the response to IFN-␣/5-FU favoring the clinical response and survival. This miRNA is the first to be studied as a therapeutic tool and other two studies (Connolly et al., 2008; Zhu et al., 2011) have shown that the transfection of tumor cells with antisense oligonucleotides specific for mir-21 leads to a reduction of the cell growth and survival. Likewise studies by Xu et al. (2011a), Bihrer et al. (2011), Marquez et al. (2010) have shown that serum levels of miR-21 are increased with a progressive course from chronic hepatitis to cirrhosis and hepatocellular carcinoma. Multivariate analyses demonstrate that the only parameters to be associated with this miRNA are histological necroinflammation and viral load. Furthermore, Wang et al. (2014) have recently demonstrated that the miR-21 levels are significantly higher in exosomes than in exosome-depleted supernatants or the whole serum, suggesting for exosomal miR-21 a potential role of biomarker for HCC diagnosis.

miR-101 expression was significantly downregulated in most of HCC tissues and all cell lines. This miRNA inhibits cell proliferation through the inhibition of three proteins EZH2, c-myc and MCL-1 involved in apoptotic process. Once their control is lost, these induce cell proliferation and migration (Xu et al., 2014). Interestingly, serum miR-101 levels were found to have an inverse correlation with tissue levels. The expression of serum miR-101 in patients with HBV-related HCC (HBV–HCC) was significantly increased than in the healthy controls, and this increase was related to hepatitis B surface antigen positivity, HBV DNA levels and tumor size (Fu et al., 2013). Conversely, Xie et al. (2014) reported that the serum miR-101 levels were significantly downregulated in HBV–HCC patients compared with healthy controls, HBV-associated liver cirrhosis patients (HBV–LC) and patients with chronic hepatitis B. These results suggest that serum miR-101 can serve as a potential non-invasive biomarker to differentiate HBV–HCC from HBV–LC. Recently, it was shown that enforced overexpression of miR-101 by lentivirus-mediated systemic delivery strongly inhibits HCC in vivo by repressing multiple molecular targets such as EZH2, STMN1, COX2 and ROCK2 and blocking angiogenesis and epithelial-mesenchymal transition of HCC cells (Zheng et al., 2015a).

4.7. miR-34a

4.10. miR-122

miR-34a inhibits tumor proliferation by different mechanisms. Indeed, it reduces the expression of c-MET reducing phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) which inhibit cell proliferation (Li et al., 2009b). Another mechanism implicated in the inhibition of cell proliferation is shown by Cheng et al. (2010). These authors have demonstrated that this miRNA reduces the expression of MACF1 and TPM4, linker protein of mitotic process, inhibiting the G1 phase of cell cycle, with subsequent blockade of lamin A/C, a microtubule actin cross-linking factor, a-tubulin chain 1B and Glial fibrillary acidic protein. However, it seems also implicated in the Wnt mechanism of action. It also seems to reduce the levels of CDK6 and CyclinD1 cell cycle regulators (Guo et al., 2011). Lou et al. (2013) showed that this miRNA reduces the expression of Bcl-2 and SIRT1, a regulator of apoptosis.

mir-122 is involved in migration, cell invasion and progression processes through various biomolecular mechanisms and perhaps it is the most important among the miRNAs involved in the process of hepatocarcinogenesis. Its tissue levels are reduced in the course of HCC, but those in serum are increased (Liu et al., 2009b). One of the most important mechanisms is represented by the inhibition of bcl-w, an antiapoptotic protein of bcl-2 family. High levels of mir122 down-regulate bcl-w promoting apoptosis (Lin et al., 2008; Young et al., 2010; Xu et al., 2011b). Another important role was demonstrated by Fornari et al. (2009). These authors showed that the underexpression of miR-122 up-regulates the levels of cyclin G1 and down-regulate p53. The increase of cyclin G1 favors cell growth. This mechanism seems to be implicated in tumor growth and subsequently in the tumor recurrence process. Other minor

4.9. miR-101

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processes in which miR-122 is implicated seem to include the migration-related processes. Indeed low levels of miR-122 promote overexpression of disintegrin and metalloproteinase as ADAM17 (Tsai et al., 2009) and ADAM10 (Bai et al., 2009) that promote migration. This action seems to be correlated with chronic viral infection. Besides in the study by Bai et al. (2009) cell growth and survival of HCC cells expressing miR-122 were significantly reduced upon treatment with sorafenib, a multi-kinase inhibitor, which is actually approved for HCC patients. Li et al. (2013), Fan et al. (2011) showed that low miR-122 levels stimulated HBV replication and overexpression of viral antigen with subsequent cellular damage, overgrowth and hepatocellular fibrosis. Indeed, low levels of miR-122 stimulated Klf6, a fibrosis activator (Tsai et al., 2012). Circulating levels of miR-122 are elevated with good diagnostic performance. With a cut-off 0.474 we have an Area Under ROC curve of 0.869 with high sensitivity and specificity, 81,6% and 83,3%, respectively (Wang et al., 2012). This performance improves with progression disease (Xu et al., 2011a). For this reason, it has been demonstrated that miR-122 is negatively related to MELD score and necroinflammation (Koberle et al., 2013). The role in viral replication supported the hypothesis that the use of the vector plasmid pLMP-miR-122 on HepG2 cells promotes the inhibition of HBV replication (Huang and Liu, 2009). Therefore, miR-122 shows an important role as a potential biomarker in diagnosis, prognosis and therapy of HCC (Thakral and Ghoshal, 2015). 4.11. miR-129-2 Low levels of miR-129-2 arising by DNA hypermethylation induce overexpression of SOX4, a proto-oncogene that promotes cell proliferation of HCC cell lines. For this reason, its potential utility as an early diagnostic marker for HCC was studied (Chen et al., 2013; Lu et al., 2013). 4.12. miR-132 As well as the miR-122 is involved in the viral replication of HBV also miR-132 interacts with HBV. It seems that HBx, an HBV protein, suppresses this miRNA through the hypermethylation of its gene, with subsequent AKT hyperphosphorylation and cyclin D1 overexpression which favour cell growth (Wei et al., 2013; Wen et al., 2015). 4.13. miR-195 miR-195 appears to be reduced both at tissue and serum level. To a certain extent, Xu et al. (2009) demonstrated that tissue levels of this miRNA are decreased in hepatocarcinoma tissue and cell lines, explaining the mechanism of the loss of suppression of cyclin D1, CDK6, and E2F3 resulting in pRB phosphorylation. These events include an increase in E2F levels with an higher number of cells in G1/S phase of cell cycle. Another mechanism indicated by Yang et al. (2012b) is given by the loss of the Bcl-w expression reducing the number of cells in apoptosis. Qu et al. (2011) suggest that serum levels of this miRNA could be useful for differential diagnosis between viral hepatitis and HCC. Recent studies reported that miR-195 acts as a tumor suppressor by directly targeting CBX4 and Wnt3a in HCC cells (Zheng et al., 2015b; Yang et al., 2014a). 4.14. miR-199a miR-199a is perhaps the most widely recognized miRNA as a molecular target for new targeted therapies. Fornari et al. (2010) showed that miR-199a negatively regulates the mTOR cascade and c-met. Elevating levels of miR-199a reduce the amount of cells in G1 phase of the cell cycle with subsequent reduction of invasive

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potential, and increase the susceptibility to hypoxia and the sensitivity of doxorubicin-induced apoptosis. Probably, the action of this miRNA is on Discoidin domain receptor-1 (DDR1) tyrosine kinase, a protein that increases the cell invasiveness (Shen et al., 2010) and perhaps also on CD44, increasing the sensitivity of neoplastic cells to doxorubicin (Henry et al., 2010), and PAK4, a protein that inhibits the PAK4/Raf/MEK/ERK cascade by reducing cell proliferation (Hou et al., 2011). Furthermore, Song and colleagues showed that miR-199a modulates the survival and cell proliferation by targeting Frizzled type 7 receptor (FZD7), the most important Wnt receptor involved in cancer development and progression (Song et al., 2014). Recently, Shen et al. (2015) showed that the expression of miR-199a and miR-125b is greatly modulated by DNA hypermethylation that plays a key role in hepatocarcinogenesis. In conclusion, serum miR-199a levels are reduced so that using a cut-off of 10 it can be used as a diagnostic test for the differentiation between chronic hepatitis and hepatocellular carcinoma (Qu et al., 2011; Yin et al., 2015). 4.15. miR-221 miR-221 is one of the most studied miRNAs in the field of hepatology. In fact, it is been identified as a major part of hepatocellular proto-oncogenes. The mechanisms by which miR-221 is implicated in hepatocarcinogenesis are different. Its relationship to cancer stage has been shown. In fact, the levels are higher in stages III-IV. This finding suggests a prognostic role as demonstrated by Karakatsanis et al. (2013) and Rong et al. (2013). Its prognostic value is reported in the work of Li et al. (2011a), in which it was shown that serum levels of miR-221 correlate with the stage of cirrhosis and cancer. These many ways by which miR-221 exerts these effects indicate that miR-221 targets CDKN1B/p27 of cyclin-dependent kinase inhibitor (CDKI) CDKN1C/p57 (Fornari et al., 2008). It is shown in this work that themiR-221 upregulation leads to a downregulation of CDKN1B/p27 and CDKN1C/p57. The interaction occurs in particular with 3 UTR of CDKN1C/p57 mRNA. This process leads to an increase of the hepatocellular cell growth by increasing the number of cells in the S phase of the cell cycle. Gramantieri et al. (2009) demonstrated how overexpression of miR-221 leads to a reduction of Bmf, an inducer of caspase-3. A reduction of Bmf thus reduces the process of apoptosis. The authors concluded that this miRNA is responsible for the prognosis of HCC and reduced time to recurrence. Pineau et al. (2010) indicate miR-221 as a tumor growth inducer of murine liver progenitor cells through the stimulation of DNA damage-inducible transcript 4 (DDIT4), a modulator of mTOR. The overexpression of miR-221 also leads to a decreased expression of p27 (Kip1), a cell cycle inhibitor both being associated with the tumor stage and the presence of metastases (Fu et al., 2011). In 2010, it has been showed how the suppression of miR-221 protects against apoptosis induced by endoplasmic reticulum stress through the activation of the cell cycle mediated by p27 (Kip1) and MEK/ERK (Dai et al., 2010). Another way by which the overexpression of miR-221 exerts its action is explained by Santhekadur et al. (2012). Indeed, they show how a Staphylococcal nuclease domain-containing 1 (SND1) protein stimulates NF-kB and miR221 resulting in the overexpression of angiogenic factors such as angiogenin and CXCL16. Additional mechanisms are explained by Yuan et al. (2013). miR-221 leads to an increase of cell proliferation through the induction of the S phase of the cell cycle having as molecular targets p27 and p57. miR-221 also seems to be a good molecular target. In fact, it has been reported that the 2 -Omethylphosphorothioate-modified oligonucleotide anti-miR-221 reduces cell proliferation and increases the markers of apoptosis and cell cycle arrest, increases the doubling time of the tumor and growth thereby increasing the survival in mice (Park et al., 2011). So also Callegari et al. (2012) showed that in transgenic mice in

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which cirrhosis and cancer are induced by diethylnitrosamine, the overexpression of miR-221 determines an inhibition of its target: CDKI1b/p27 and CDKI1c/p57. Recently, novel genetically modified adenoviral vectors were developed to drive the expression of multiple binding sites for miR-221, the “miR-221 sponge”, which was designed to sequester miR-221 molecules (Moshiri et al., 2014). 4.16. miR-222 miR-222, overexpressed at the tissue level and in the serum (Li et al., 2011a), seems to exert its action through the phosphorylation of AKT. This effect confers a metastatic potential in cancer cells as demonstrated by Wong et al. (2010). Yang et al. (2014b) suggested that MiR-222 overexpression promotes proliferation of human HCC cells by downregulating the p27 expression at post-transcriptional level. 4.17. miR-223 Little is known about the miR-223, whose expression is reduced in tissue (Zhao et al., 2011), while it is increased in the serum of HCC patients (Xu et al., 2011a). Probably its repression leads to an increase of the c-myc expression. The re-expression of miR-223 in HBV-, HCV-, and non-HBV- non-HCV-related HCC cell lines revealed a consistent inhibitory effect on cell viability (Wong et al., 2008). Furthermore, Yang et al. (2013) demonstrated that miR-223 overexpression plays an important role in the regulation of multidrug resistance (MDR) through downregulation of the ABCB1 expression by increasing the HCC cell sensitivity to anticancer drugs. Therefore, miR-223 may act as a potential therapeutic biomarker for HCC patients who show MDR resistancecaused by increased ABCB1 expression. 4.18. miR-224 miR-224 is implicated in cell survival and the processes of invasiveness. Indeed, during hepatocellular carcinogenesis it inhibits apoptosis by blocking apoptosis inhibitor-5 (API-5) (Wang et al., 2008). But it also stimulates some targets such as PAK4 and MMP9 increasing migration and tumor progression in HepG2 cells (Li et al., 2010b). Serum levels seem to be higher in patients with hepatocellular carcinoma (Li et al., 2011a). Also, miR-224 promotes cell proliferation by targeting SMAD family member 4 (SMAD4), and this event is significantly associated with lower patient survival (Wang et al., 2013). In a recent work, Zhuang and Meng (2015) reported that HCC patients with high miR-224 serum level showed poor survival compared to that with low serum miR-224 level. Moreover, serum miR-224 concentration reflects the tumor stage and liver damage. Finally, the combined high expression of miR-224 and pAKT may be a potential indicator for predicting unfavorable prognosis in HCC patients (Yu et al., 2014). 4.19. miR-375 miR-375 inhibits autophagy through the regulation of ATG7, a gene responsible for auto-phagocytosis (Chang et al., 2012). Liu et al. (2010) showed that the expression of this miRNA is reduced in tissue of patients with hepatocellular carcinoma. They also demonstrated that miR-375 is an important regulator of yes-associated protein (YAP) oncogene, implicating a potential therapeutic role in HCC treatment. It also seems that the reduction of this miRNA involves an increase of alpha-fetoprotein as demonstrated by Ho et al. (2011). However, its serum levels seem high. Also, miR-375 seems to be the only one which allows to specifically differentiate HBV-related HCC patients from HCC patients without HBV. It has

Table 2 Potential role of miRNAs in HCC diagnosis, prognosis and therapy. miRNA miR-1 miR-9 miR-15b miR-130B miR-16 miR-18a miR-21 miR-34a miR-92a miR-101 miR-122 miR-129-2 miR-132 miR-195 miR-199a miR-221 miR-222 miR-223 miR-224 miR-375 miR-500 miR-885-5p miR-675 miR-1274a

Diagnosis

+ + + + + + + + + + + + + + + + + +

Prognosis + + + + + +

Therapeutic target

+ +

+ + +

+ +

+ + + + +

+ + + + +

+ +

been reported an AUC of ROC curve equal to 0.96 and a sensitivity and specificity of 96% and 100%, respectively (Li et al., 2011b). Recent data suggested that circulating miR-375 could be a novel potential predictive biomarker in early-stage HCC (Yin et al., 2015). 4.19. miR-500 and miR-885-5p There are no studies that demonstrate the mechanisms by which these miRNAs act in hepatocellular carcinoma. However, Yamamoto et al. (2009) and Gui et al. (2011) showed that serum levels of both miRNAs have a good diagnostic performance in differentiating healthy subjects from patients with liver disease. 4.20. miR-675 The expression of miR-675 in HCC is associated with a dramatic increase of proliferative and growth capacity with inhibition of motility in HCC cells. Pathway analyses of the effect of miR-675 overexpression in HCC cells revealed a predominant induction of cell adhesion and cell cycle initiation pathways. It has been reported that miR-675 causes increases in proliferation and an accumulation of cells with tetraploid DNA content associated with a repression of Rb. In addition, the overexpression of miR-675 alters cellular morphology, reduces invasive potential, and increases anchorageindependent growth capacity. These findings are consistent with a mesenchymal-to-epithelial transition associated with a reduction in the expression of the key EMT mediator, Twist1 (Hernandez et al., 2013). 4.21. miR-1274a miR-1274a was showed to be able to greatly reduce ADAM9 expression in HCC cells. This observation could be a consequence of its direct binding to ADAM9 3 UTR. miRNA expression profile was evaluated in HepG2 cells treated with sorafenib versus vehicle as control. Fourteen miRNAs have shown an altered expression after treatment with sorafenib. MiR-1274a was up-regulated by sorafenib treatment. This effect could be mediated by a significant repression of ADAM9, a protease that is involved in the effect of sorafenib on HCC cells (Zhou et al., 2011). In a recent work,

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the authors suggested a potential role of miR-1274a in paclitaxelinduced apoptosis of HCC cells (Yan et al., 2013). 5. Current issues and clinical implications The miRNAs analyzed in this review represent those which were studied for at least the relationship with cancer-related biological processes with an involvement in hepatocellular carcinogenesis and HCC progression. Several pathways seem to be related to these miRNAs, i.e. the apoptosis/survival pathways by Bcl, c-myc, CDKs and RB, the cell invasion and metastasis pathways by MMPs and chemokines, and the signaling transduction pathways by EGF, IGF1, PDGF, MET. Indeed, miRNAs can differentially regulate tumor suppressor genes and oncogenes through the multiple steps of cancer development and progression (Russo et al., 2013). Some of these miRNAs were also studied for a clinical perspective. On the basis of these findings we argue to identify new diagnostic, prognostic and predictive biomarkers for HCC patients. It is even more important to detect these biomarkers in serum because we can gather relevant information about HCC development and aggressiveness in each patient in a minimally invasive manner. Circulating miRNAs for HCC diagnosis would allow to screen the high risk patients affected by liver diseases or even the larger population of healthy subjects. miRNAs in serum could also help to define prognosis, mainly if we compare their circulating levels to tissue expression. During treatment, miRNAs from peripheral blood could also be used to monitor drug efficacy and could help to identify the development of resistance mechanisms. Namely in this review we can focus on some of the reported miRNAs according to their clinical potential. In most of analyzed studies, serum miRNAs were extracted from peripheral blood samples, while tissue miRNAs were isolated from formalin-fixed paraffin embedded (FFPE) or fresh tissues. Tissue and serum miRNA expression levels were evaluated in patients and healthy controls using mostly microarray and RNA-Seq technologies. Selected candidate miRNAs from both technologies were then verified by quantitative Real Time polymerase chain reaction (qRT-PCR) from independent sample sets. As regards the use of serum miRNAs for HCC diagnosis we could suppose a diagnostic role for miR-16, miR-18a, miR-101, miR-122, miR-195, miR-199a, miR-224, miR-375, miR-500 and miR-885-5p. Among these miRNAs miR-375 achieves the highest sensitivity, specificity and Area Under ROC Curve. These diagnostic serum miRNAs could be also useful to differentiate healthy subjects from HCC patients (miR-101, miR-122, miR-224, miR-500, mir-885-5p), patients with benign liver disease from HCC patients (miR-16, miR18a, miR-195, miR-199a), and HBV-related HCC patients from those with HCC without HBV. A prognostic role can be recognized for miR-1, miR-92a and miR-101. Indeed, miR-1 and miR-101 seem to be related to tumor stage.An increase of miR-92 expression was observed after surgery and it could help to identify those patients who are relapse-free after curative resection. Serum miRNAs could represent an alternative to tissue prognostic and predictive biomarkers which have been identified in various solid malignancies until now (Russo et al., 2009; Bronte et al., 2010; Rizzo et al., 2010; Bronte et al., 2011; Caraglia et al., 2011). On the basis of some findings reported in this review we argue that some miRNAs could have a role to monitor the effect of sorafenib. This function can be attributed to miR-122, which is reduced by sorafenib, and miR-1274a which is upregulated under treatment with sorafenib in HCC cells. Since miRNAs are involved in various cancer-related processes, such as proliferation, angiogenesis, cell invasion and metastasis, if we are able to identify what miRNAs are exerting their function in a specific malignancy, we could also identify a targeted anti-miRNA to use as treatment

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(Amodeo et al., 2013). In HCC patients just one targeted agent has been approved yet, sorafenib. No molecular biomarkers are actually available for the selection of patients suitable for this treatment. They are only selected by clinical features. We should try to search for new biomarker miRNAs in serum. These biomarkers could be also be useful for the other targeted agents under clinical development for HCC patients (Bronte et al., 2014). For example, in some malignancies like non-small-cell lung cancer, circulating miRNAs were identified as predictive factors to chemotherapy-induced antitumor activity (Franchina et al., 2014). And also for other malignancies the need for predictive biomarkers is much more relevant because the number of new targeted agents in non-haematological tumors is rapidly arising (Di et al., 2011; Bronte et al., 2013). We propose here the term HepatomiRNoma to summarize all those miRNAs which are related to HCC biology, diagnosis and prognosis. Indeed miRNAs do not work individually within a cancer cell, but they represent a network of interactions with various genes involved in cancer development and progression. However, these miRNAs taken together should be evaluated concomitantly in specific studies. This approach would be more expensive than single analyses, but it could provide a more useful tool to manage the screening for HCC in high risk patients with liver diseases and to help to define prognostic classification of HCC patients. 6. Conclusions Many evidences have been collected about miRNA in HCC as regards both their biological, diagnostic and prognostic role (Table 2). However little is known about how these small molecules interact each other and with other pathogens such as HBV and HCV, the major inducers of liver carcinogenesis. Both preclinical and clinical research on miRNAs in HCC proceeds through the analysis of individual miRNAs in cell lines, tissue or serum. Nowadays, biologists, pathologists, oncologists and gastroenterologists could improve their knowledge about HCC-related tumor biology, diagnosis and prognosis by the complete mapping of these miRNAs. For this reason, we reviewed all the available evidences about miRNAs in HCC to provide a tool, which could help researchers to address their work on HCC research. Conflict of interest The authors declare no conflicts of interest. References Amodeo, V., Bazan, V., Fanale, D., et al., 2013. Effects of anti-miR-182 on TSP-1 expression in human colon cancer cells: there is a sense in antisense? Expert Opin. Ther. Targets 17 (11), 1249–1261. Bai, S.M., Nasser, M.W., Wang, B., et al., 2009. MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to sorafenib. J. Biol. Chem. 284 (46), 32015–32027. Bartel, D.P., 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116 (2), 281–297. Bihrer, V., Waidmann, O., Friedrich-Rust, M., et al., 2011. Serum MicroRNA-21 as marker for necroinflammation in hepatitis C patients with and without hepatocellular carcinoma. PLoS One 6 (10). Borchert, G.M., Lanier, W., Davidson, B.L., 2006. RNA polymerase III transcribes human microRNAs. Nat. Struct. Mol. Biol. 13 (12), 1097–1101. Bronte, G., Rizzo, S., La Paglia, P., et al., 2010. Driver mutations and differential sensitivity to targeted therapies: a new approach to the treatment of lung adenocarcinoma. Cancer Treat. Rev. 36 (Suppl. 3), S21–S29. Bronte, G., Terrasi, M., Rizzo, S., et al., 2011. EGFR genomic alterations in cancer: prognostic and predictive values. Front. Biosci. (Elite Ed.) 3, 879–887. Bronte, G., Cicero, G., Cusenza, S., et al., 2013. Monoclonal antibodies in gastrointestinal cancers. Expert Opin. Biol. Ther. 13 (6), 889–900. Bronte, F., Bronte, G., Cusenza, S., et al., 2014. Targeted therapies in hepatocellular carcinoma. Curr. Med. Chem. 21 (8), 966–974. Cai, L., Cai, X., 2014. Up-regulation of miR-9 expression predicate advanced clinicopathological features and poor prognosis in patients with hepatocellular carcinoma. Diagn. Pathol. 9 (1), 1000.

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Please cite this article in press as: Bronte, F., et al., HepatomiRNoma: The proposal of a new network of targets for diagnosis, prognosis and therapy in hepatocellular carcinoma. Crit Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.09.007

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ARTICLE IN PRESS F. Bronte et al. / Critical Reviews in Oncology/Hematology xxx (2015) xxx–xxx

Biographies Fabrizio Bronte obtained his MD degree from University of Palermo in 2006. He has been specialised in Gastroenterology since 2012. Actually he is responsible of long-term monitoring of chronic liver diasease HBV-related and working in the Gastroenterology Unit at the University Hospital of Palermo. He has participated as Investigator in several Clinical Trials with Good Clinical Practice criteria about antiviral therapy in patients with chronic HBV and HCV infection. His main researches include: viral hepatitis, antiviral therapy, epidemiology of liver disease, natural history of chronic hepatitis, hepatocellular carcinoma, viral hepatitis in patients with hemoglobinopathy. He has published many full papers in top-rated international journals. Giuseppe Bronte received his MD degree from University Medical School of Palermo (Italy) in 2004. His post-graduate specialty was in Medical Oncology in 2008. He received his PhD degree in Experimental and Clinical Oncology from the same University in 2012. Co-investigator and Data manager in Multicenter Clinical Trials, managed by different Clinical Research Cooperative Groups, according to Good Clinical Practice. He is a member of scientific societies and he is actively involved in the teaching and research of oncology fellows and students. He is author more than 40 publications in top-rated international cancer journals. Daniele Fanale PhD, received his doctorate degree in “Molecular and Cellular Oncopathology” from the University of Palermo (Italy) in 2010. In the recent years, he acquired experience and expertise in the field of the microarray technologies, in particular, in gene and miRNA expression analysis and gene Copy number Variation analysis. In these years, he has been involved in translational oncology research aimed at identifying new potential biomarkers in cancer for diagnostic, prognostic and predictive purposes. In this context, he has been concerned with the molecular genetics of sporadic, hereditary and familial tumors. He has authored or co-authored over 20 peer-reviewed publications in top-rated international journals. Stefano Caruso PhD, received his doctorate degree in “Clinical and Molecular Oncology” from the University of Palermo (Italy) in 2015. In the recent years, he acquired experience and expertise in the field of MicroRNAs and Noncoding RNAs, focusing on the role of these molecules as new potential biomarkers in cancer. He has authored or co-authored over 10 peer-reviewed publications in top-rated international journals. Enrico Bronte received his MD degree from University Medical School of Palermo (Italy) in 2012. He is co-investigator and data manager in Multicenter Clinical Trials, managed by different Clinical Research Cooperative Groups, according to Good Clinical Practice. His research is focused on Cardio-Oncology and Targeted Therapy in Cancer Treatment. He is author of more than 10 peer-reviewed publications in top-rated international journals. Maria Grazia Bavetta received her MD degree from University Medical School of Palermo (Italy) in 2010. She works in the Gastroenterology Unit at the University Hospital of Palermo. She is Co-Investigator in Clinical Trials according to Good Clinical Practice about antiviral therapy in patients with chronic HCV infection. Her main researches include: viral hepatitis, antiviral therapy, epidemiology of liver disease, natural history of chronic hepatitis, hepatocellular carcinoma and especially HCV cirrhosis. Eugenio Fiorentino MD is Full Professor of Surgery at University School of Medicine of Palermo, Department of Surgical and Oncological Sciences. He is leading expert in gastroesophageal reflux disease and clinical director of the Esophageal Diseases Clinical Program at University Hospital Policlinico in Palermo. He has authored over 100 scientific publications mainly on gastroesophageal reflux disease and edited one book on acid reflux. Current area of research interest include gastroesophageal reflux, Barrett’s esophagus, and esophageal cancer.

at the Antwerp University Hospital, Belgium. He is board certified in oncology at the National Cancer Institute, University of Milan, Italy, where he worked for more than 6 years with Luca Gianni in clinical trials. In 2004, he moved to Spain, working in clinical research in thoracic tumors with Rafael Rosell at the Spanish Lung Cancer Group. He received his PhD in experimental and clinical research in oncology from the University of Palermo, Italy. His research focus is target therapies, drug development and resistance. Viviana Bazan PhD, is Professor of Technical Sciences of Medicine of Laboratory at University Medical School of Palermo, Department of Surgical and Oncological Sciences (Italy). From July 2008 to July 2011, she has been an Adjunct Assistant Professor and since August 2011 is Adjunct Associate Professor at Temple University’s College of Science and Technology, Philadelphia (USA). She has been Co-Editor of Annals of Oncology (Volume 17, 2006 and Volume 18, 2007). Over the last few years, she has been implicated in clinical oncology research aimed at identifying biomolecular prognostic features and treatment response. In this context she has been concerned with the molecular genetics of sporadic, hereditary and familial tumors. She is the author of more than 130 publications in top-rated cancer journals. Vito Di Marco obtained his medical degree (1982) and specialization in Gastroenterology (1987) in the University of Palermo. Research Fellow from 1996 to 2000 and Senior Lecturer of Gastroenterology from 2000 to 2010 at University of Palermo. Actually, he is Associate Professor of Gastroenterology at the University of Palermo and the chief of the program for the diagnosis and the therapy of chronic viral hepatitis and cirrhosis in the Section of Gastroenterology and Hepatology in the Department of Internal Medicine. He has partecipated as principal investigator or subinvestigator at more than 50 clinical trials and observational studies on liver diseases (Chronic hepatitis B, Chronic hepatitis C, Cirrhosis, Hepatocellular carcinoma). He has published more than 150 full papers in international journals and has made many presentations at both national and international meetings. His main research interests include: epidemiology of acute and chronic liver disease; non-invasive methods for the diagnosis of chronic liver disease; diagnosis, natural history and therapy of chronic hepatitis C; diagnosis, natural history and therapy of chronic hepatitis B; therapy of HBV and HCV cirrhosis; autoimmune hepatitis, stress and NAFLD, diagnosis and therapy of hepatocellular carcinoma; virus infection, siderosis and liver damage in Thalassemia patients. Antonio Russo MD, is Professor of Medical Oncology at University Medical School of Palermo, Department of Surgical, Oncological and Stomatological Sciences (Italy). From 2004 to July 2011 he has been an Adjunct Associate Professor and since August 2011 Adjunct Full Professor at Temple University’s College of Science and Technology, Philadelphia (USA). Since February 2012 is Director of Medical Oncology Unit and Director of Regional Reference Center for Prevention, Diagnosis and Treatment of Rare Tumors and Heredo-familial Solid Tumors in Adults , AOUP “P. Giaccone”, Palermo (Italy). Since April 2012 is Director of the Specialization School in Medical Oncology, University of Palermo, School of Medicine, Palermo, Italy. Since November 2013 Medical Oncology Unit directed by Prof A Russo has been recognized as a 2013 ESMO Designated Centres of Integrated Oncology and Palliative Care. Since 2001 he has been a coordinator with Prof D. Kerr (University of Oxford, UK) and Prof B. Iacopetta (Western Australia University) of the “CRCP53 International Collaborative Study”. Since 2003 he has been an expert member of INSERM (Institut National de la Santè et de la Recherche Mèdical, France), since 2007 of Scientific Committee INCA (Institut National du Cancer, France) and of NWCRF (North West Cancer Research Fund, UK). He is member of Editorial board of Journal of Carcinogenesis & Mutagenesis (since 2011) and World Journal of Gastrointestinal Oncology and World Journal of Clinical Oncology (since 2012). Since 2013 is Associate Editor of Journal of Solid Tumors. Since 2008 he has been Guest Editor of Annals of Oncology (2006, 2007). The central theme of his studies is translational research, meaning the application of molecular genetics in cancer management. He is PI in several national and international clinical trials. He is the author of more than 300 peer-reviewed publications listed on Medline-PubMed.

Christian Rolfo is Associate Professor of the University of Antwerp, Belgium, and is also Coordinator of the Phase I Early Clinical Trials Unit, Department of Oncology

Please cite this article in press as: Bronte, F., et al., HepatomiRNoma: The proposal of a new network of targets for diagnosis, prognosis and therapy in hepatocellular carcinoma. Crit Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.09.007