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Diagnostic and therapeutic potential of miRNA signatures in patients with hepatocellular carcinoma

  • Florie Borel
    Affiliations
    Department of Research & Development, Amsterdam Molecular Therapeutics, Meibergdreef 61, 1105 BA Amsterdam, The Netherlands

    Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
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  • Pavlina Konstantinova
    Affiliations
    Department of Research & Development, Amsterdam Molecular Therapeutics, Meibergdreef 61, 1105 BA Amsterdam, The Netherlands
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  • Peter L.M. Jansen
    Correspondence
    Corresponding author. Tel.: +31 205668981; fax: +31 206917033.
    Affiliations
    Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
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Open AccessPublished:February 06, 2012DOI:https://doi.org/10.1016/j.jhep.2011.11.026

      Summary

      MicroRNAs (miRNAs) are evolutionary conserved small non-coding RNAs that regulate gene expression by mediating post-transcriptional silencing of target genes. Since miRNAs are involved in fine-tuning of physiological responses, they have become of interest for diagnosis and therapy of a number of diseases. Moreover, the role of dysregulated miRNAs in maintaining the malignant phenotype has profound implications for cancer therapy. We will review the best defined cellular miRNAs and changes in their expression profile in hepatocellular carcinoma (HCC). Cellular miRNAs can also be released into the circulation, and these miRNAs are detected in most body fluids. Circulating miRNAs are associated with HCC and are possible biomarkers. Finally, by affecting several clinically relevant targets, artificially increasing or decreasing the expression level of a given miRNA offers fascinating therapeutic perspectives. We will therefore highlight recent developments in miRNA-based gene therapy with a focus on their therapeutic potential for HCC.

      Abbreviations:

      miRNA (microRNA), RNAi (RNA interference), HCC (hepatocellular carcinoma), UTR (untranslated region), AHL (adjacent healthy liver), HL (healthy liver), CLD (chronic liver disease), HCV (hepatitis C virus), HBV (hepatitis B virus), TACE (transarterial chemoembolization), SNP (single nucleotide polymorphism), AFP (α-fetoprotein), AFP-L3 (Lens culinaris agglutinin-reactive AFP), DCP (des-γ-carboxyprothrombin)

      Keywords

      miRNA expression profiles associated with HCC

      miRNA biogenesis and mechanism of action

      miRNAs are endogenous ∼22-nt long single stranded RNAs. There are currently 1492 human miRNA sequences registered in the miRBase database (http://www.mirbase.org). miRNAs are non-coding but are implicated in post-transcriptional regulation of genes involved in fundamental cell processes and in diseases [
      • Bushati N.
      • Cohen S.M.
      MicroRNA functions.
      ]. The miRNA gene is usually transcribed by RNA polymerase II in the nucleus into a primary transcript called pri-miRNA (Fig. 1A) of approximately 1–4 kb [
      • Saini H.K.
      • Griffiths-Jones S.
      • Enright A.J.
      Genomic analysis of human microRNA transcripts.
      ]. These transcripts can be either monocistronic – a single miRNA gene behind a promoter – or polycistronic – expressed from one transcript as a cluster containing several miRNA gene products e.g. the miR-17∼92 miRNA polycistron [
      • Lee Y.
      • Jeon K.
      • Lee J.T.
      • Kim S.
      • Kim V.N.
      MicroRNA maturation: stepwise processing and subcellular localization.
      ]. Depending on their genome position, globally about 50% of all miRNA genes are intragenic, the so-called mirtrons – likely to be regulated through their host gene – but they can also be located in intergenic regions i.e. likely to be independent transcriptional units [
      • Griffiths-Jones S.
      Annotating noncoding RNA genes.
      ]. The pri-miRNA is then cleaved by the microprocessor complex which consists of the nuclease Drosha and the double-stranded RNA-binding protein DiGeorge syndrome critical region gene 8 (DGCR8) into a precursor miRNA (pre-miRNA) (Fig. 1B). This ∼70-nt long pre-miRNA is exported to the cytoplasm via Exportin-5 (Fig. 1C) where it will be cleaved by another nuclease, Dicer, into an imperfect miRNA–miRNA duplex (Fig. 1D) of ∼18–25 nucleotides [
      • Hammond S.M.
      • Bernstein E.
      • Beach D.
      • Hannon G.J.
      An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.
      ]. While the passenger strand (miRNA, in black in Fig. 1) is commonly degraded, the mature miRNA guide strand (in red in Fig. 1) is loaded into the RNA-induced silencing complex (RISC; Fig. 1E) where further regulations will be undertaken, depending on the level of complementarity between the miRNA and its target in the 3′ untranslated region (3′ UTR) of the messenger RNA (mRNA). In case of perfect complementarity, the mRNA will be cleaved by RISC and degraded; in case of imperfect complementarity, translation will be repressed [
      • Bartel D.P.
      MicroRNAs: target recognition and regulatory functions.
      ]. In mammals, decreased mRNA levels were shown to be preceding protein decrease in 84% cases [
      • Guo H.
      • Ingolia N.T.
      • Weissman J.S.
      • Bartel D.P.
      Mammalian microRNAs predominantly act to decrease target mRNA levels.
      ]. Functional target sites within the mRNA usually consist of a 6–7-nt long sequence which is complementary to the miRNA sequence, followed by an adenosine, the so-called miRNA “seed” sequence. Target mRNAs end up in cytoplasmic processing-bodies (P-bodies) where they are degraded [
      • Liu J.
      • Valencia-Sanchez M.A.
      • Hannon G.J.
      • Parker R.
      MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies.
      ]. Interestingly, other recently discovered classes of non-coding RNAs can also participate in regulation of gene expression and/or have been associated with HCC. Ender et al. showed that the small nucleolar RNA (snoRNA) ACA45 is processed by Dicer into RNAs of miRNA-like length (20- to 25-nt long) that will bind to Argonaute proteins (Ago), moreover they demonstrated the miRNA-like function of ACA45 by luciferase reporter assays [
      • Ender C.
      • Krek A.
      • Friedlander M.R.
      • Beitzinger M.
      • Weinmann L.
      • Chen W.
      • et al.
      A human snoRNA with microRNA-like functions.
      ]. Yang et al. identified a long non-coding RNA (lncRNA) named High Expression in Hepatocellular Carcinoma (lncRNA-HEIH) that is differentially expressed in HCC and whose expression level is positively associated with tumor recurrence and negatively correlated with survival. In addition, they showed that shRNA-mediated downregulation of lncRNA-HEIH significantly inhibited the growth of tumors in a xenograft mouse model [
      • Yang F.
      • Zhang L.
      • Huo X.S.
      • Yuan J.H.
      • Xu D.
      • Yuan S.X.
      • et al.
      Long noncoding RNA high expression in hepatocellular carcinoma facilitates tumor growth through enhancer of zeste homolog 2 in humans.
      ].
      Figure thumbnail gr1
      Fig. 1miRNA biogenesis. miRNA biogenesis involves multiple steps requiring (A) RNA Pol II for transcription of the 1–4 kb primary transcript called pri-miRNA, (B) nuclease Drosha-DGCR8 for cropping of the single-stranded sequences flanking double-stranded stem–loop structure of the pre-miRNA precursor of ∼70-nt long, (C) export of the pre-miRNA via Exportin-5 from the nucleus to the cytoplasm and (D) nuclease Dicer cleavage of the loop to generate the mature ∼22-nt long miRNA that will be incorporated into the RISC (E). miRNA guide strand is represented in red, and passenger strand is represented in black.

      Regulation of miRNA processing in association with HCC

      Figure thumbnail fx8

      miRNA polymorphisms

      Besides the possible alterations in the miRNA processing, miRNA polymorphisms can also be associated with an increased risk of HCC. A miRNA polymorphism consists of a single nucleotide polymorphism (SNP) in the miRNA gene. Although rare, a SNP in a miRNA can affect its transcription, processing, or target recognition. Since binding of a miRNA to its mRNA target is limited only to the seed sequence, even one nucleotide change would result in a different group of genes that would be regulated. Recently, two groups of investigators have described that a variant of miR-196a-2 is positively associated with HCC susceptibility, in two populations of distinctive ethnical background [
      • Akkiz H.
      • Bayram S.
      • Bekar A.
      • Akgollu E.
      • Ulger Y.
      A functional polymorphism in pre-microRNA-196a-2 contributes to the susceptibility of hepatocellular carcinoma in a Turkish population: a case–control study.
      ,
      • Qi P.
      • Dou T.H.
      • Geng L.
      • Zhou F.G.
      • Gu X.
      • Wang H.
      • et al.
      Association of a variant in MIR 196A2 with susceptibility to hepatocellular carcinoma in male Chinese patients with chronic hepatitis B virus infection.
      ]. Yet, the field of HCC-associated miRNA polymorphisms and their relevance to disease progression as a result of regulation of different pools of genes is only starting to develop.

      Detection of miRNAs

      Since miRNAs are involved in fine-tuning of physiological responses, they have become of interest for diagnosis and therapy of a number of diseases; nevertheless, reliable miRNA detection is a key requirement. Currently, the three most commonly used detection methods are microarray, RT-qPCR and next-generation sequencing (NGS). Much less common is the use of Northern blot, in situ hybridization and bead-based flow cytometry. Microarray is based on annealing of DNA oligonucleotides to the homologous sequences, on a microchip. Main advantages are the relatively low price and the high throughput, but the method has a low sensitivity and specificity, i.e. miRNAs with similar sequences (miRNA families) can hybridize with the same probe. The use of DNA locked nucleic acid (LNA) oligonucleotides in microarrays ensures a greater specificity by increasing the melting temperature. In addition, the sensitivity has also been increased [
      • Castoldi M.
      • Schmidt S.
      • Benes V.
      • Noerholm M.
      • Kulozik A.E.
      • Hentze M.W.
      • et al.
      A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA).
      ]. miRNA RT-qPCR is based on a stem–loop primer binding to the mature miRNA during the reverse transcription, making it a highly specific technique that can distinguish 1-nt differences between related miRNAs [
      • Chen C.
      • Ridzon D.A.
      • Broomer A.J.
      • Zhou Z.
      • Lee D.H.
      • Nguyen J.T.
      • et al.
      Real-time quantification of microRNAs by stem–loop RT-PCR.
      ]. Although preamplification step sometimes required before the RT-qPCR can induce some bias and underestimate the concentration of lowly expressed miRNAs, this method is more sensitive than microarray. Despite its higher cost, it is currently the method of choice for validation of miRNA signatures. NGS is a high-throughput technology that provides global information on all miRNAs in a certain sample. Costs are much higher and data analysis is more laborious, but NGS provides quantitative data, allows miRNA discovery and provides data on miRNA polymorphisms and differential processing. Finally, the nCounter developed by Nanostring Technologies (Seattle, WA) is based on annealing of a fluorescent barcode probe followed by single molecule imaging, without preamplification step, offering high sensitivity and specificity. This technology offers high-throughput when using up to 800 multiplexed targets.

      Key miRNAs dysregulated in HCC

      Dysregulation of miRNAs in cancer has been repeatedly described, e.g. deregulated miRNAs in prostate, bladder, and kidney cancer [
      • Catto J.W.
      • Alcaraz A.
      • Bjartell A.S.
      • De Vere W.R.
      • Evans C.P.
      • Fussel S.
      • et al.
      MicroRNA in prostate, bladder, and kidney cancer: a systematic review.
      ], breast cancer [
      • Corcoran C.
      • Friel A.M.
      • Duffy M.J.
      • Crown J.
      • O’Driscoll L.
      Intracellular and extracellular microRNAs in breast cancer.
      ], colon cancer [
      • Song B.
      • Ju J.
      Impact of miRNAs in gastrointestinal cancer diagnosis and prognosis.
      ]. Importantly, miRNAs are predominantly downregulated in tumor tissue [
      • Lu J.
      • Getz G.
      • Miska E.A.
      • Alvarez-Saavedra E.
      • Lamb J.
      • Peck D.
      • et al.
      MicroRNA expression profiles classify human cancers.
      ]. Hepatocellular carcinoma is no exception and various HCC-specific miRNA signatures have been described (Table 1). Screens of clinical samples are qualitatively heterogenic, firstly because of the variability in the technical procedure, from method of sampling (surgery or biopsy), time to and procedure of freezing, RNA isolation, to method of detection. Most miRNA screens are done using miRNA RT-qPCR, but some publications report microarray and NGS as described in Table 1. Secondly, the disease etiology is a significant factor of variation. This should be taken into account when pooling data, as the patient group can have a single etiology (alcohol or viral) or mixed etiologies (alcohol plus viral). Thirdly, the stage of the disease should also be considered, although miRNA dysregulations occur from an early stage [
      • Gao P.
      • Wong C.C.
      • Tung E.K.
      • Lee J.M.
      • Wong C.M.
      • Ng I.O.
      Deregulation of microRNA expression occurs early and accumulates in early stages of HBV-associated multistep hepatocarcinogenesis.
      ], it is not clear how miRNA expression changes during disease progression. Finally, the control tissue used for normalization is also of importance, as it can be the healthy liver from patients with a different pathology or no known pathology, or non-tumoral liver tissue from the same patient, i.e. with the same underlying liver disease (e.g. cirrhosis, viral infection), the latter allowing to look only at intra-individual changes [
      • Borel F.
      • Han R.
      • Visser A.
      • Petry H.
      • van Deventer S.J.
      • Jansen P.L.
      • et al.
      Adenosine triphosphate-binding cassette transporter genes up-regulation in untreated hepatocellular carcinoma is mediated by cellular microRNAs.
      ]. Nevertheless, dysregulation of several key miRNAs appears to be common to different screens, as described in Table 1.
      Table 1Key cellular miRNAs dysregulated in HCC (studies based on patient material) compared to the healthy liver (HL).
      NI, no information; RT-qPCR, reverse-template quantitative PCR; NGS, next-generation sequencing, HCV, hepatitis C virus; HBV, hepatitis B virus.

      Use of miRNAs in molecular classification of HCC and in prognosis

      Key miRNAs are affected in HCC, and different dysregulation patterns can be used to discriminate tumors based on molecular characteristics. For instance, downregulation of miR-375 has been associated with β-catenin mutation, and downregulation of miR-107 with hepatocyte nuclear factor 1α (HNF1α) [
      • Ladeiro Y.
      • Couchy G.
      • Balabaud C.
      • Bioulac-Sage P.
      • Pelletier L.
      • Rebouissou S.
      • et al.
      MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations.
      ]. Toffanin et al. recently proposed a miRNA-based classification of HCC in three subclasses: the wingless-type MMTV integration site, interferon-related, and proliferation subclasses [
      • Toffanin S.
      • Hoshida Y.
      • Lachenmayer A.
      • Villanueva A.
      • Cabellos L.
      • Minguez B.
      • et al.
      MicroRNA-based classification of hepatocellular carcinoma and oncogenic role of miR-517a.
      ]. Such miRNA-based determination of molecular subclasses of HCC could allow subtype-specific treatment. miRNA signatures can also be used to determine disease prognosis, e.g. Budhu et al. identified a 20-miRNA signature as a predictor of survival and recurrence [
      • Budhu A.
      • Jia H.L.
      • Forgues M.
      • Liu C.G.
      • Goldstein D.
      • Lam A.
      • et al.
      Identification of metastasis-related microRNAs in hepatocellular carcinoma.
      ]. In addition, low tumoral miR-26 expression has been associated with high interleukin-6 (IL6) expression, and shorter survival [
      • Ji J.
      • Shi J.
      • Budhu A.
      • Yu Z.
      • Forgues M.
      • Roessler S.
      • et al.
      MicroRNA expression, survival, and response to interferon in liver cancer.
      ]; Ji et al. showed a better response of these tumors to interferon therapy compared to tumors with high miR-26 levels [
      • Ji J.
      • Shi J.
      • Budhu A.
      • Yu Z.
      • Forgues M.
      • Roessler S.
      • et al.
      MicroRNA expression, survival, and response to interferon in liver cancer.
      ]. It hence appears that miRNA profiling may play a crucial role in the clinic, not only for HCC classification and subtype-specific treatment allocation, but also for prognosis.

      Gene targets of miRNAs and their association with HCC

      Oncogenic and tumor-suppressive miRNAs

      Prior to inhibition of gene expression, mature miRNAs are loaded into RISC which will mediate recognition of the target mRNAs and lead to either mRNA degradation or translational repression. This negative regulation of gene expression by miRNAs leads to a balance between miRNA and gene expression level. In the context of HCC, miRNAs can act either as oncogenes, by inducing progression of a cell to cancer, or as tumor-suppressors, by preventing cell progression to cancer miRNAs (Fig. 2, in red) repressing the expression of oncogenic targets; when downregulated in HCC, a higher expression of their targets is allowed, hence promoting the malignant phenotype. Alternatively, upregulation of oncogenic miRNAs (Fig. 2, in green) in HCC will cause downregulation of their gene targets, again promoting the malignant phenotype.
      Figure thumbnail gr2
      Fig. 2miRNAs and their oncogenic and tumor-suppressing targets associated with HCC. Tumor suppressing miRNAs which are downregulated in HCC are indicated in red and oncogenic miRNAs which are upregulated in HCC are indicated in green. miRNAs post-transcriptionally repress the expression of genes involved in cell cycle regulation, (blue); cell proliferation, (grey); apoptosis, (yellow); cell migration and invasion, (white); and of proto-oncogenes, (purple). Genes having a positive effect on the cell process are marked in the dark shade, while genes having a negative effect on the cell process are marked in the light shade. For example, CCND1 is marked in dark blue, because it causes cell cycle progression, and has been linked to the development and progression of cancer. Vice-versa, PTEN is marked in light blue as it causes cell cycle arrest. Additionally, one gene can be targeted by several miRNAs, for example tumor-suppressor PTEN was shown to be simultaneously repressed by oncogenic miR-21 and miR-221. Bar-headed lines indicate post-transcriptional repression of gene expression. Data presented in this figure is non-exhaustive and based on literature.

      Targets of tumor-suppressing miRNAs downregulated in HCC

      Being downregulated in HCC, tumor-suppressor miRNAs cause upregulation of oncogenic target genes, stimulate and/or increase cellular mechanisms such as cell proliferation, cell cycle regulation, cell migration and invasion, apoptosis, and hence participate in the establishment and maintenance of the cancer phenotype, as described in Fig. 2. Cyclin G1 (CCNG1) is one of the most well-characterized targets of miR-122 [
      • Gramantieri L.
      • Ferracin M.
      • Fornari F.
      • Veronese A.
      • Sabbioni S.
      • Liu C.G.
      • et al.
      Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma.
      ]. However, miR-122 also targets the anti-apoptotic BCL-W [
      • Lin C.J.
      • Gong H.Y.
      • Tseng H.C.
      • Wang W.L.
      • Wu J.L.
      MiR-122 targets an anti-apoptotic gene, Bcl-w, in human hepatocellular carcinoma cell lines.
      ] and ADAM17 (a disintegrin and metalloprotease family 17) involved in metastasis [
      • Tsai W.C.
      • Hsu P.W.
      • Lai T.C.
      • Chau G.Y.
      • Lin C.W.
      • Chen C.M.
      • et al.
      MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma.
      ]. Additional validated miRNA targets are described in Fig. 2.

      Targets of oncogenic miRNAs upregulated in HCC

      Oncogenic miRNAs are upregulated in HCC, thus causing downregulation of target genes and decrease of cell mechanisms such as apoptosis, which eventually leads to onset and progression of the disease, as described in Fig. 2. Overall, less miRNAs are upregulated than downregulated in cancer [
      • Lu J.
      • Getz G.
      • Miska E.A.
      • Alvarez-Saavedra E.
      • Lamb J.
      • Peck D.
      • et al.
      MicroRNA expression profiles classify human cancers.
      ]. miR-221 targets the cyclin-dependent kinase inhibitors CDKN1B/p27 [
      • Galardi S.
      • Mercatelli N.
      • Giorda E.
      • Massalini S.
      • Frajese G.V.
      • Ciafre S.A.
      • et al.
      MiR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27Kip1.
      ] and CDKN1C/p57 [
      • Fornari F.
      • Gramantieri L.
      • Ferracin M.
      • Veronese A.
      • Sabbioni S.
      • Calin G.A.
      • et al.
      MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma.
      ], which results in an increase in the G1 to S phase shift and induces cell growth. Another target of miR-221 is the pro-apoptotic BMF of the BCL-2 family [
      • Gramantieri L.
      • Fornari F.
      • Ferracin M.
      • Veronese A.
      • Sabbioni S.
      • Calin G.A.
      • et al.
      MicroRNA-221 targets Bmf in hepatocellular carcinoma and correlates with tumor multifocality.
      ], therefore in HCC, BMF downregulation inhibits apoptosis. Enforced miR-221 expression also induces downregulation of DNA damage-inducible transcript 4 (DDIT4), leading to modulation of the mTOR pathway [
      • Pineau P.
      • Volinia S.
      • McJunkin K.
      • Marchio A.
      • Battiston C.
      • Terris B.
      • et al.
      MiR-221 overexpression contributes to liver tumorigenesis.
      ] and of the tumor suppressors thymidylate synthase (TIMP3) and PTEN [
      • Garofalo M.
      • Di L.G.
      • Romano G.
      • Nuovo G.
      • Suh S.S.
      • Ngankeu A.
      • et al.
      MiR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation.
      ], which results in enhanced cell migration.

      Circulating miRNAs

      Origin and clinical relevance of circulating miRNAs

      Figure thumbnail fx9

      Circulating miRNAs associated with HCC

      As described above, many miRNAs are dysregulated in HCC. Therefore, it is anticipated that circulating miRNAs are also affected during HCC progression. A few studies reported altered levels of circulating miRNAs in association with HCC (Table 2). For instance, the serum level of miR-221 was shown to be 4.8-fold elevated in HCC patients [
      • Li J.
      • Wang Y.
      • Yu W.
      • Chen J.
      • Luo J.
      Expression of serum miR-221 in human hepatocellular carcinoma and its prognostic significance.
      ]. Additionally, high level of miR-221 positively correlated with cirrhosis, tumor size and tumor stage, and negatively correlated with overall survival. These promising results should be validated in a larger patient cohort; nevertheless, miR-221 serum level monitoring could be of clinical relevance as a potential diagnosis tool and biomarker of treatment efficacy. Indeed, no optimal blood tumor marker has been developed so far, the performance of α-fetoprotein (AFP), Lens culinaris agglutinin-reactive AFP (AFP-L3) and des-γ-carboxyprothrombin (DCP) [
      • Marrero J.A.
      • Feng Z.
      • Wang Y.
      • Nguyen M.H.
      • Befeler A.S.
      • Roberts L.R.
      • et al.
      Alpha-fetoprotein, des-gamma carboxyprothrombin, and lectin-bound alpha-fetoprotein in early hepatocellular carcinoma.
      ] is limited in a surveillance mode and for early HCC detection. In addition, the American Association for the Study of Liver Diseases (AASLD) Practice Guidelines (July 2010) discarded AFP for surveillance and diagnosis. Therefore, there is a need for novel markers that would combine the less invasiveness of a blood test and serve as a reliable early detection method. miRNAs definitely have this potential because not only they can be detected in plasma, but their sensitivity and stability are suitable for a clinical setting. Depending on the method, as little as one copy can be detected (see paragraph on detection). Nevertheless, appropriate controls should be used, since HCC is most often accompanied by viral infection, cirrhosis, or other underlying liver conditions. Therefore, in order to assess the HCC-specificity of a miRNA, it is critical to ensure not only an age- and gender-matched control group but they should also be matched for etiology and severity of underlying liver disease. For instance, the miRNA profile of three patient groups was compared: 105 patients with HCC (19.1% HBV, 62.9% HCV, 17.1% other etiology), 107 with chronic liver disease (CLD; 7.5% HBV, 55.1% HCV, 37.4% other etiology) and 71 normal controls [
      • Qu K.Z.
      • Zhang K.
      • Li H.
      • Afdhal N.H.
      • Albitar M.
      Circulating microRNAs as biomarkers for hepatocellular carcinoma.
      ]. In another study, miR-16 and miR-199a levels were decreased in serum and significantly associated with HCC [
      • Qu K.Z.
      • Zhang K.
      • Li H.
      • Afdhal N.H.
      • Albitar M.
      Circulating microRNAs as biomarkers for hepatocellular carcinoma.
      ]. miR-16 was more sensitive as HCC detection marker than AFP, DCP, and AFP-L3. Combination of miR-16 with AFP, DCP, and AFP-L3 allowed detection of 92.4% HCC cases with a high specificity (78.5%), and interestingly, it could detect tumors ⩽3 cm with the same sensitivity (92.4%). This research demonstrates the feasibility of plasma markers for diagnosis of HCC. Circulating miRNAs could therefore be used as a first-line testing in HCC patients if they would outperform the currently used tumor markers. The discovery of circulating miRNAs offers interesting clinical perspectives but this field of research is quite recent and more work has to be done. It remains to be established which miRNA can sensitively and reliably be correlated with the presence of HCC at early stages of disease development and prognosis.
      Table 2Circulating miRNAs candidate biomarkers for HCC.
      HL, healthy liver; HCC, hepatocellular carcinoma; CH, chronic hepatitis; LC, liver cirrhosis, CLD, chronic liver diseases.

      Cellular miRNAs as therapeutic targets in HCC

      Potential of miRNA-based gene therapy

      RNAi was identified in Caenorhabditis elegans in 1998 by Fire and Mello [
      • Fire A.
      • Xu S.
      • Montgomery M.K.
      • Kostas S.A.
      • Driver S.E.
      • Mello C.C.
      Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
      ] and in mammalian cells in 2001 by Tuschl [
      • Elbashir S.M.
      • Harborth J.
      • Lendeckel W.
      • Yalcin A.
      • Weber K.
      • Tuschl T.
      Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
      ]. Since then, RNAi has generated increasing interest and publications in diverse research areas. The combination of RNAi with the latest developments in the field of gene therapy which rendered it safer, and the delivery more efficient, opens the door to novel therapeutic perspectives. Among the many possibilities currently investigated, the use of cellular miRNAs as therapeutic agents is one of the most promising from a clinical point of view. Many miRNAs are downregulating genes that are highly relevant to HCC and therefore contribute to disease progression. Because a single miRNA could potentially affect several clinically relevant targets, artificially increasing or decreasing the expression level of a given miRNA offers interesting therapeutic perspectives. Such therapy could even be combined with local chemotherapy via the transarterial route (transarterial chemoembolization, TACE) to increase the treatment effectiveness. Nevertheless, because a miRNA can affect the expression of several downstream targets, modulating the expression of a miRNA of interest could also lead to undesirable off-target effects.

      miRNA-based gene therapy for HCC

      The main question raised by RNAi-based gene therapy is the delivery of the effector molecule, which should preferably be controllable, sustained and tissue-specific. Several groups have chosen for non-viral delivery of synthetic miRNA molecules. miRNA mimics or miRNA antagomirs can be repeatedly delivered locally or systemically and that would cause transient suppression of target gene expression. To prevent rapid degradation of naked molecules, miRNAs are modified or conjugated to improve stability or target delivery to a specific tissue. They can be incorporated into stable nucleic acid lipid particles (SNALPs), a lipid bilayer coated by polyethylene glycol (PEG) which will protect them from degradation, prevent immunostimulation and facilitate their uptake in endosomes [
      • Morrissey D.V.
      • Lockridge J.A.
      • Shaw L.
      • Blanchard K.
      • Jensen K.
      • Breen W.
      • et al.
      Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.
      ]. Similarly, 2′O-methyl modifications increase the stability of synthetic molecules, additionally preventing off-targeting [
      • Fedorov Y.
      • Anderson E.M.
      • Birmingham A.
      • Reynolds A.
      • Karpilow J.
      • Robinson K.
      • et al.
      Off-target effects by siRNA can induce toxic phenotype.
      ]. Mimicking the external viral protein structure, virus-like particles (VLP) can also be used for synthetic miRNA delivery, yet they are not suitable for all applications since they stimulate the immune response [
      • Takamura S.
      • Niikura M.
      • Li T.C.
      • Takeda N.
      • Kusagawa S.
      • Takebe Y.
      • et al.
      DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration.
      ]. However, VLP vehicles take advantage of the natural virus tropism, e.g. HBV in the liver, and can efficiently mediate hepatic gene transfer [
      • Brandenburg B.
      • Stockl L.
      • Gutzeit C.
      • Roos M.
      • Lupberger J.
      • Schwartlander R.
      • et al.
      A novel system for efficient gene transfer into primary human hepatocytes via cell-permeable hepatitis B virus-like particle.
      ]. Finally, miRNA conjugation to HDL, LDL or cholesterol will also lead to hepatic uptake [
      • Wolfrum C.
      • Shi S.
      • Jayaprakash K.N.
      • Jayaraman M.
      • Wang G.
      • Pandey R.K.
      • et al.
      Mechanisms and optimization of in vivo delivery of lipophilic siRNAs.
      ]. However, even with the described improvements, miRNAs would need to be delivered monthly or bimonthly. Landford et al. inhibited miR-122 expression in 4 chimpanzees using SPC3649 LNA-modified oligonucleotides. Because miR-122 stimulates HCV RNA accumulation, miR-122 inhibition leads to an efficient suppression of HCV replication and stable reduction of viremia in chimpanzees [
      • Lanford R.E.
      • Hildebrandt-Eriksen E.S.
      • Petri A.
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      Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.
      ]. A phase I trial for SPC3649 (Miravirsen from Santaris Pharma) showed that SPC3649 was well-tolerated and the drug is now in phase II trial. This approach holds promise for HCV patients and has the advantage that it should not allow the development of viral escape variants. Nevertheless Li et al. reported that mutations in miR-122 binding site in HCV 5′ UTR reduced SPC3649 treatment efficacy [
      • Li Y.P.
      • Gottwein J.M.
      • Scheel T.K.
      • Jensen T.B.
      • Bukh J.
      MicroRNA-122 antagonism against hepatitis C virus genotypes 1-6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR.
      ]. This indicates that viral escape could still be possible.
      Alternatively, gene therapy using virally-delivered miRNAs is desirable when chronic and genetic diseases need to be treated. Viral delivery indeed can offer sustained expression after single dosing, however, in clinical perspective, it raises several questions concerning safety. Pri-miRNA can be delivered as an expression cassette using different types of viral vectors. The advantages and disadvantages of the viral vectors used in gene therapy clinical trials are summarized in Table 3. Briefly, the main disadvantage of lentivirus and retrovirus is their integration into the host genome, which raises safety issues. On the contrary, adeno-associated virus (AAV) genome remains episomal, which gives it an advantageous safety profile. However, the episomal presence of AAV questions its relevance for cancer therapy. Up to now, adenovirus has been widely used in HCC gene therapy clinical trials [
      • Habib N.
      • Salama H.
      • Abd El Latif Abu Median A.
      • Isac Anis I.
      • Abd Al Aziz R.A.
      • et al.
      Clinical trial of E1B-deleted adenovirus (dl1520) gene therapy for hepatocellular carcinoma.
      ,
      • Li N.
      • Zhou J.
      • Weng D.
      • Zhang C.
      • Li L.
      • Wang B.
      • et al.
      Adjuvant adenovirus-mediated delivery of herpes simplex virus thymidine kinase administration improves outcome of liver transplantation in patients with advanced hepatocellular carcinoma.
      ,
      • Makower D.
      • Rozenblit A.
      • Kaufman H.
      • Edelman M.
      • Lane M.E.
      • Zwiebel J.
      • et al.
      Phase II clinical trial of intralesional administration of the oncolytic adenovirus ONYX-015 in patients with hepatobiliary tumors with correlative p53 studies.
      ,
      • Mazzolini G.
      • Alfaro C.
      • Sangro B.
      • Feijoo E.
      • Ruiz J.
      • Benito A.
      • et al.
      Intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas.
      ,
      • Palmer D.H.
      • Mautner V.
      • Mirza D.
      • Oliff S.
      • Gerritsen W.
      • van der Sijp J.R.
      • et al.
      Virus-directed enzyme prodrug therapy: intratumoral administration of a replication-deficient adenovirus encoding nitroreductase to patients with resectable liver cancer.
      ,
      • Sangro B.
      • Mazzolini G.
      • Ruiz J.
      • Herraiz M.
      • Quiroga J.
      • Herrero I.
      • et al.
      Phase I trial of intratumoral injection of an adenovirus encoding interleukin-12 for advanced digestive tumors.
      ,
      • Tian G.
      • Liu J.
      • Zhou J.S.
      • Chen W.
      Multiple hepatic arterial injections of recombinant adenovirus p53 and 5-fluorouracil after transcatheter arterial chemoembolization for unresectable hepatocellular carcinoma: a pilot phase II trial.
      ], as well as Vaccinia virus [
      • Park B.H.
      • Hwang T.
      • Liu T.C.
      • Sze D.Y.
      • Kim J.S.
      • Kwon H.C.
      • et al.
      Use of a targeted oncolytic poxvirus, JX-594, in patients with refractory primary or metastatic liver cancer: a phase I trial.
      ]. Despite the fact that many virus-delivered “classical” gene therapy products have been developed for HCC and are currently progressing through clinical trial phases, no virus-delivered miRNA-based gene therapy has been tested in clinical trials yet. Indeed, more research still needs to be done to carefully evaluate potential risks of this approach. Kota et al. showed that self-complementary AAV serotype 8 (scAAV8)-delivery of miR-26a in tumor-bearing tet-o-myc; LAP-tTA mice restored miR-26a expression [
      • Kota J.
      • Chivukula R.R.
      • O’Donnell K.A.
      • Wentzel E.A.
      • Montgomery C.L.
      • Hwang H.W.
      • et al.
      Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model.
      ]. Re-expression of miR-26a specifically reduced cancer cell proliferation, induced tumor-specific apoptosis, and suppressed tumorogenesis. At 3 weeks post-transduction, most liver tissue in the control group was replaced with tumor while in 8 out of 10 mice of the treated group no or small tumors only were found [
      • Kota J.
      • Chivukula R.R.
      • O’Donnell K.A.
      • Wentzel E.A.
      • Montgomery C.L.
      • Hwang H.W.
      • et al.
      Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model.
      ]. This research demonstrated for the first time the therapeutic potential of restoring the expression of a dysregulated miRNA in the liver. Additional advantages of this approach are that the miRNA is well-tolerated, given that it is only downregulated in tumor cells, and therefore only tumor cells are affected. However, its significance in patients still needs to be determined.
      Table 3Gene therapy vectors used in clinical trials.
      Kb, kilobases.
      (1)Source: gene therapy clinical trials worldwide database, The Journal of Gene Medicine, 2011.
      With the first successes of RNA agents in clinical trials, it becomes clear that miRNAs and their inhibitors hold a great potential as therapeutics for different cancers including HCC.

      Conclusions

      It is now well-established that miRNAs are key players in many various biological processes, including development, cellular proliferation, apoptosis, and oncogenesis [
      • Bushati N.
      • Cohen S.M.
      MicroRNA functions.
      ]. In HCC, miRNAs have aberrant processing and expression profiles, in addition, the profile of circulating miRNAs is also affected, which renders them potential biomarkers, with possible applications in diagnosis, especially for early, pre-symptomatic disease, and prognosis of HCC. One miRNA may target several genes that are involved in the development and maintenance of the HCC phenotype. Therefore, miRNA-based gene therapy offers promising perspectives compared to classical gene therapy for HCC. An additional advantage of miRNAs is that since they encode no protein, they are generally not immunogenic. However, activation of Toll-like receptors (TLRs), involved in initiation of inflammatory responses to pathogens, can occur, as reviewed by O’Neill et al. [
      • O’Neill L.A.
      • Sheedy F.J.
      • McCoy C.E.
      MicroRNAs: the fine-tuners of Toll-like receptor signalling.
      ], indicating that unanticipated off-target effects can occur in a clinical setting. For instance, the let-7 family regulates the expression of TLR4 and this can create off-target effects [
      • Androulidaki A.
      • Iliopoulos D.
      • Arranz A.
      • Doxaki C.
      • Schworer S.
      • Zacharioudaki V.
      • et al.
      The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs.
      ]. A positive aspect of gene therapy for HCC is that the delivery route seems not to be questioned, up to now direct imaging-guided intratumoral injection has been the most used strategy in clinical trials, but tumor-selective intra-arterial administration could be a good alternative [
      • Tian G.
      • Liu J.
      • Zhou J.S.
      • Chen W.
      Multiple hepatic arterial injections of recombinant adenovirus p53 and 5-fluorouracil after transcatheter arterial chemoembolization for unresectable hepatocellular carcinoma: a pilot phase II trial.
      ]. In HCC, combination of classical and miRNA-based therapies appears a desirable goal. First, chemo- or radiation therapy can improve gene transfer efficiency and transgene expression [
      • Egami T.
      • Ohuchida K.
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      • Onimaru M.
      • Toma H.
      • et al.
      Chemotherapeutic agents potentiate adenoviral gene therapy for pancreatic cancer.
      ,
      • Zhang M.
      • Li S.
      • Li J.
      • Ensminger W.D.
      • Lawrence T.S.
      Ionizing radiation increases adenovirus uptake and improves transgene expression in intrahepatic colon cancer xenografts.
      ,
      • Connolly E.
      • Melegari M.
      • Landgraf P.
      • Tchaikovskaya T.
      • Tennant B.C.
      • Slagle B.L.
      • et al.
      Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype.
      ,
      • Huang X.H.
      • Wang Q.
      • Chen J.S.
      • Fu X.H.
      • Chen X.L.
      • Chen L.Z.
      • et al.
      Bead-based microarray analysis of microRNA expression in hepatocellular carcinoma: miR-338 is downregulated.
      ,
      • Huang Y.S.
      • Dai Y.
      • Yu X.F.
      • Bao S.Y.
      • Yin Y.B.
      • Tang M.
      • et al.
      Microarray analysis of microRNA expression in hepatocellular carcinoma and non-tumorous tissues without viral hepatitis.
      ,
      • Jiang J.
      • Gusev Y.
      • Aderca I.
      • Mettler T.A.
      • Nagorney D.M.
      • Brackett D.J.
      • et al.
      Association of MicroRNA expression in hepatocellular carcinomas with hepatitis infection, cirrhosis, and patient survival.
      ,
      • Meng F.
      • Henson R.
      • Wehbe-Janek H.
      • Ghoshal K.
      • Jacob S.T.
      • Patel T.
      MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer.
      ,
      • Murakami Y.
      • Yasuda T.
      • Saigo K.
      • Urashima T.
      • Toyoda H.
      • Okanoue T.
      • et al.
      Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues.
      ,
      • Su H.
      • Yang J.R.
      • Xu T.
      • Huang J.
      • Xu L.
      • Yuan Y.
      • et al.
      MicroRNA-101, down-regulated in hepatocellular carcinoma, promotes apoptosis and suppresses tumorigenicity.
      ,
      • Varnholt H.
      • Drebber U.
      • Schulze F.
      • Wedemeyer I.
      • Schirmacher P.
      • Dienes H.P.
      • et al.
      MicroRNA gene expression profile of hepatitis C virus-associated hepatocellular carcinoma.
      ,
      • Wang Y.
      • Lee A.T.
      • Ma J.Z.
      • Wang J.
      • Ren J.
      • Yang Y.
      • et al.
      Profiling microRNA expression in hepatocellular carcinoma reveals microRNA-224 up-regulation and apoptosis inhibitor-5 as a microRNA-224-specific target.
      ,
      • Wong Q.W.
      • Lung R.W.
      • Law P.T.
      • Lai P.B.
      • Chan K.Y.
      • To K.F.
      • et al.
      MicroRNA-223 is commonly repressed in hepatocellular carcinoma and potentiates expression of Stathmin1.
      ,
      • Zhang R.
      • Wang L.
      • Yang A.G.
      Is microRNA-143 really a turncoat of tumor suppressor microRNA in hepatitis B virus-related hepatocellular carcinoma?.
      ,
      • Gui J.
      • Tian Y.
      • Wen X.
      • Zhang W.
      • Zhang P.
      • Gao J.
      • et al.
      Serum microRNA characterization identifies miR-885-5p as a potential marker for detecting liver pathologies.
      ,
      • Shigoka M.
      • Tsuchida A.
      • Matsudo T.
      • Nagakawa Y.
      • Saito H.
      • Suzuki Y.
      • et al.
      Deregulation of miR-92a expression is implicated in hepatocellular carcinoma development.
      ,
      • Xu J.
      • Wu C.
      • Che X.
      • Wang L.
      • Yu D.
      • Zhang T.
      • et al.
      Circulating MicroRNAs, miR-21, miR-122, and miR-223, in patients with hepatocellular carcinoma or chronic hepatitis.
      ,
      • Yamamoto Y.
      • Kosaka N.
      • Tanaka M.
      • Koizumi F.
      • Kanai Y.
      • Mizutani T.
      • et al.
      MicroRNA-500 as a potential diagnostic marker for hepatocellular carcinoma.
      ]. Second, in the case of combined chemo- and gene- therapies, a direct co-injection of both via the intravenous route used for the TACE procedure, could be suitable, offering a gene therapy delivery route already clinically approved and in practice. The discovery of miRNA-mediated gene regulation as a fundamental post-transcriptional mechanism increases the complexity of cancer genetics. However, understanding the molecular mechanisms by which miRNAs regulate development and tumorogenesis may lead to novel concepts in the diagnosis and treatment of cancer. Besides the fact that miRNAs have shown promising results in pre-clinical studies, miRNA-based gene therapy is not yet suitable for routine clinical practice.

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
      Amsterdam Molecular Therapeutics declared no commercial interest in the conclusions.

      Financial support

      By Amsterdam Molecular Therapeutics.

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