MicroRNAs in Hepatocellular Carcinoma: Regulation, Function, and Clinical Implications

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the third cause of cancer-related death. Poor understanding of the mechanisms underlying the pathogenesis of HCC makes it difficult to be diagnosed and treated at early stage. MicroRNAs (miRNAs), a class of noncoding single-stranded RNAs of ~22 nucleotides in length, posttranscriptionally regulate gene expression by base pairing with the 3′ untranslated regions (3′UTRs) of target messenger RNAs (mRNAs). Aberrant expression of miRNAs is found in many if not all cancers, and many deregulated miRNAs have been proved to play crucial roles in the initiation and progression of cancers by regulating the expression of various oncogenes or tumor suppressor genes. In this Paper, we will summarize the regulations and functions of miRNAs aberrantly expressed in HCC and discuss the potential application of miRNAs as diagnostic and prognostic biomarkers of HCC and their potential roles in the intervention of HCC.


Introduction
Being one of the most frequently diagnosed cancer worldwide, liver cancer is the second leading cause of cancerdeath in men and the sixth leading cause of cancer-related death in women [1]. Hepatocellular carcinoma (HCC), which account for 70% to 85% of the primary liver cancer cases, is rarely detected at its early stage, resulting in a short survival of few months [2]. About 90% of HCC cases arise from cirrhosis, which can be attributed to a wide range of factors including chronic viral hepatitis B or C (HBV or HCV) infections, alcohol abuse, nonalcoholic steatohepatitis (NASH), autoimmune hepatitis, primary biliary cirrhosis (PBC), and carcinogens exposure [3]. Considerable progresses on unraveling molecular mechanisms of HCC have been achieved recently, paving the way to the early detection and treatment of HCC.
MicroRNAs (miRNAs), a class of noncoding RNAs of ∼22 nucleotides in length found both in plants and animals, have emerged as key posttranscriptional regulators of gene expression. It has been reported that many miRNAs are involved in human cancers, such as lung, breast, brain, liver, colorectal cancer, and leukemia. By targeting different genes in tumor development, miRNAs function as oncogenes or tumor suppressor genes. In this paper, we will summarize the process and the regulation of miRNA biogenesis, as well as our current knowledge about the biological relevance of miRNAs to HCC. Then, we will discuss the potential application of miRNAs as predictive, diagnostic, and prognostic biomarkers of HCC and their potential roles in cancer treatment.
Since the first discovered miRNA lin-4 by Victor Ambros and his colleagues in Caenorhabditis elegans, more than 20,000 miRNAs have been identified in 193 species (Sanger miRBase release 19; http://www.mirbase.org/) [4]. miRNAs downregulate the expression of specific genes predominantly by base pairing with the 3 untranslated regions (3 UTRs) of target messenger RNAs (mRNAs), leading to translational inhibition, transcript destabilization, or both [5]. However, recently, findings indicate that miRNAs can also target the 5 UTRs and coding regions of mRNAs [6,7].

The Biogenesis and Maturation of miRNAs
1In the nucleus, miRNAs genes are transcribed mainly by RNA polymerase II to generate primary miRNA transcripts (pri-miRNAs) that consist of one or more hairpin structures and finally produce one or more functional miRNAs [8]. 2 The Scientific World Journal Like protein-coding mRNAs, pri-miRNAs are usually capped at the 5 end and polyadenylated at the 3 end [9]. Pri-miRNAs are then cleaved into ∼70 nt hairpin-structured precursors (pre-miRNAs) with a 5 phosphate and a 3 2nt overhang by a multiprotein complex called microprocessor which consists of Drosha, an RNase III enzyme, and DGCR8/Pasha, a double-stranded RNA-binding domain protein (dsRBD) [10]. The 3 2nt overhang is recognized by exportin-5 which transports pre-miRNAs to the cytoplasm via an Ran-GTP-dependent mechanism [11]. In the cytoplasm, pre-miRNAs are further processed to ∼22 nt duplex by Dicer, a second RNase III endonuclease, and the dsRBD proteins TRBP/PACT [12]. Finally, the two miRNA strands are unwound, and one of the strands associates with an argonaute (AGO) protein within the RNA-induced silencing complex (RISC) where they regulate gene expression by mRNA degradation or translational repression, while another miRNA strand is quickly degraded [13] (Figure 1).
However, some miRNAs are not generated as described earlier. Mirtrons, for example, are produced from spliced introns as debranched introns that mimic the structural features of pre-miRNAs and enter to miRNA-processing pathway without Drosha-mediated cleavage [14]. In addition, some small nucleolar RNAs (snoRNAs) [15], transfer RNAs (tRNAs) [16], and endogenous short hairpin RNAs (shRNAs) [17] can also be processed into miRNA-like molecules in a microprocessor-independent manner.

Transcriptional Regulation of miRNAs.
Transcription is an important step for miRNA expression regulation. Many characteristics of protein-coding genes, such as CpG islands, TATA box, TFIIB recognition, initiator elements, and histone modifications, also present in the promoters of miRNA genes [18], suggesting that the transcription regulators of miRNA like transcription factors (TFs), enhancers, and silencing elements may be similar to protein-coding genes. For instance, myogenin and myoblast determination 1 (MyoD1) can bind to the upstream of miR-1 and miR-133 loci and induce their transcription during myogenesis [19]. The proto-oncogene c-Myc which regulates ∼10-15% of human genes, binds to E-boxes and activates the transcription of miR-17-92 cluster [20], whereas the tumor suppressor p53 transactivates miR-34, which consequently suppresses the transcriptional activity of -catenin [21].
Furthermore, miRNAs can autoregulate their own transcription by targeting some transcription factors to establish negative or positive feedback loops. For example, miR-133b can regulate the maturation and function of midbrain dopaminergic neurons within a negative feedback circuit that includes the paired-like homeodomain transcription factor PITX3, the regulator and direct target of miR-133b [22]. Zincfinger E-box-binding protein ZEB1/SIP1 and the miRNA-200 family represent another example of a complex doublenegative feedback regulation. miRNAs in miR-200 family play major roles in maintaining the epithelial phenotype by preventing the expression of ZEB1/SIP1, which can bind to the transcription start site of miR-200 family genes and repress their expression in mesenchymal cells [23]. Similar to protein-coding genes, the expression of miR-NAs is also regulated by epigenetic mechanisms including DNA methylation and specific histone modifications. MiR-127, the first reported epigenetically regulated miRNA, is upregulated after treatment with demethylation agents 5aza in several cancer cell lines [24]. Moreover, inhibition of histone deacetylases (HDACs) results in transcriptional changes of ∼40% miRNAs genes [25]. For example, Sampath and his colleagues demonstrated that HDACs mediated the silencing of miR-15a, miR-16, and miR-29b in chronic lymphocytic leukemia [26].

Posttranscriptional Regulation of miRNAs.
The expression of miRNAs is also controlled by the posttranscriptional maturation. For example, many pri-miRNAs are expressed during early mouse development but are not efficiently processed into mature miRNAs [27]. Moreover, the expression of individual miRNAs in a genomic cluster and processed from the same pri-miRNA is sometimes different at the mature form level [28].
The first step of miRNA processing is catalyzed in the nucleus by Drosha which is associated with DGCR8 and other proteins to form the microprocessor complex. Downregulation of the expression level of either Drosha or DGCR8 by RNAi leads to the reduction of both pre-miRNAs and mature miRNAs [10], and defects in the Drosha processing step contribute to widespread downregulation of miRNAs in primary tumors [27]. Some transcription factors, such as p53, receptor-regulated SMADs (R-Smads), and estrogen receptor (ER ), can also participate in the rapid regulation of miRNA expression in response to extracellular stimuli by interacting with the DEAD-box RNA helicases p68 (DDX5) and/or p72 (DDX17). Both of them are components of the Drosha microprocessor complex [29][30][31].
The expression of let-7 miRNA controlled by Lin-28 shows a complicated model of posttranscriptional regulation of miRNA expression. The RNA-binding protein Lin28 selectively represses the let-7 family miRNAs biogenesis by binding to the terminal loop of pre-and pri-let-7 miRNAs [32]. During cell differentiation, the increase of mature let-7 results from the decrease of Lin28 [33]. Interestingly, Lin28 is downregulated by let-7 miRNAs, presenting a doublenegative feedback loop in cell differentiation [34].

miRNAs and HCC-Associated Virus Infection.
Chronic infections with either HBV or HCV increase the relative risk of liver cancer greatly. These chronic viral infections are present in more than 70% of HCC cases, and iatrogenic interventions against these viruses significantly reduce the risk of HCC development [35]. Cellular miRNAs have shown able to regulate HBV infection at the transcription level either by targeting cellular transcription factors required for HBV gene expression or by a directly binding to HBV transcripts [36]. For instance, miR-155 can downregulate HBV transcription by inhibiting the expression of CCAAT/enhancer-binding protein (C/EBP-), which binds to the Enhancer II, core promoter and S-promoter of HBV cccDNA and activates the transcription of HBV cccDNA [37]. ER -targeting miR-18a is overexpressed in female HCC tissues, thus blocking ERmediated suppression of HBV transcription [38]. This maybe explain why hepatocellular carcinoma predominantly affects men, with an incidence typically more than twofold higher in males than in females [1]. miRNAs can target important players in DNA methylation and histone modification that play crucial roles in HBV cccDNA transcription. For example, miR-152 and miR-148a target DNA (cytosine-5)-methyltransferase 1 (DNMT-1) can methylate viral DNA and inhibit HBV replication [39]. Similarly, miR-1 regulates the expression of HDAC4 that can inhibit the replication of HBV [40]. On the other hand, HBVencoded proteins can influence cellular miRNA expression. Wang et al. compared the expression of 286 cellular miRNAs before and after the expression of HBx protein in HepG2 cells [41]. HBx was found to significantly upregulate the expression of 7 miRNAs but downregulate the expression of 11 cellular miRNAs such as the let-7 family miRNAs [42,43]. miRNAs in the let-7 family are commonly downregulated in various cancers including HCC and target and downregulate a number of proteins playing important roles in tumorigenesis and metastasis, such an Ras [44], high-mobility group AThook 2 (HMGA2) [45], myc [46], and signal transducer and activator of transcription 3 (STAT3) [41].

miRNAs and Other Factors Related with HCC Development.
Cirrhosis caused by chronic alcohol consumption is another risk factor of HCC especially in western countries. MiR-217 could promote ethanol-induced fat accumulation in hepatocytes by downregulating SIRT1 [48]. In a miRNA profiling study, Ladeiro et al. found that miR-126 * was decreased in alcohol-related HCC [49]. Hepatic specimens from mice fed with an ethanol-containing diet indicated a decreased expression of miR-199 and miR-200, which are commonly downregulated in HCC [50].
miRNAs are also involved in the pathogenesis of NASH, an increasingly important risk factor for HCC in recent years. Unsaturated fatty acids have been shown to increase miR-21 expression which downregulates the expression of tumor suppressor phosphatase and tensin homolog (PTEN) [51]. MiR-155 which targets another tumor suppressor C/EBP- [52] is consistently upregulated in choline-deficient and amino acid-defined (CDAA) fed mice [37].
Carcinogen exposure induces malignant transformation often accompanied with miRNA deregulation. For instance, 4 The Scientific World Journal after continuously being exposed to a low concentration of microcystin-LR, a hepatocarcinogen, the human hepatic cell line WRL-68 showed aberrant miRAN expression including the up-regulation of oncogenic miRNA, miR-21, and miR-221 [53]. Deregulation of miRNA expression also was found in the liver of mice treated with microcystin-LR and such deregulated miRNAs including miR-125b, miR-34a, and miR-21 which play crucial roles in liver tumorigenesis [54]. Furthermore, miR-191 that was known to regulate a variety of oncogenic pathways was found to be upregulated by dioxin, a known liver carcinogen [55]. These miRNA alterations may be used to develop methods monitoring environmental carcinogens.

miRNAs Deregulated in HCC.
Many reports had shown miRNA deregulation in HCCs and a list of aberrantly expressed miRNAs in HCC, has been summarized in Table 1. Like transcription factors, miRNAs play key roles in regulating diverse cellular pathways. Moreover, aberrant miRNA expression can be used as the signature to detect or characterize different type of HCC.

Biological Relevance of Deregulated miRNAs in HCC
miRNAs have been proved to exert their functions either as oncogenes or tumor suppressor genes in human cancers. Deregulated miRNAs in cancer cells have been found to contribute to most if not all hallmarks of malignant transformation including sustained proliferative signaling, resistant to cell death, immortality, angiogenesis, invasion, and metastasis [56].

Regulation of Cell Proliferation and Survival by Deregulated MicroRNAs in HCC.
Under normal physiological conditions, cell proliferation and death are finely balanced by many regulators involved in cell cycle, growth, and apoptosis. However, these regulators are targeted by miRNAs deregulated in HCC. Several miRNAs have been reported to be implicated in cell cycle regulation. For example, miR-26a and miR-195, which were found to be frequently downregulated in HCC, cooperate to overcome the G1/S cell cycle blockade through the repression of E2F expression [57,58]. In contrast, E2F1-targeting miR-106b and miR-93 promote the pathogenesis of HCC by inhibiting E2F1-induced apoptosis [59]. As a downstream target of tumor suppressor p53, miR-34a functions as a link between p53 signaling and the cell cycle regulation by targeting cyclin D1, cyclin-dependent kinase 4 (CDK4) and CDK2 in HCC [60]. MiR-221 and miR-222 have been reported to target CDKN1B/p27/Kip1 and CDKN1C/p57/Kip2, while miR-223 participates in regulating the G2/M transition mediated by stathmin-1 [61]. In addition, miR-193b and miR-250b can suppress colony forming ability in vitro and tumorigenesis in vivo by inducing cyclin D1mediated G1 phase arrest [62,63].

miRNAs Related Genetic Variations and HCC Risk Prediction.
Single nucleotide polymorphisms (SNPs) in some miR-NAs or their targets are associated with risk of HCC (Table 2). Since the binding of a miRNA to its target mRNA is largely attributed to the seed sequence, even one nucleotide variation in the seed sequence would result in dramatic changes in the efficiency of miRNA-gene interaction. For instance, the rs11614913 (C → T) SNP in miR-196a-2 is positively associated with HCC susceptibility in Chinese [163,164] and Turkish [165]. The "TTCA" insertion (rs3783553) disrupts the binding site in the 3 -UTR of IL-1alpha for miR-122 and miR-378, leading to the up-regulation of IL-1alpha expression and the promotion of HCC development [168]. However, conflicted results were achieved from three studies on the association between polymorphism of miR-499a and HCC risk in three populations with different ethnical backgrounds [159][160][161]. Therefore, polymorphisms of miRNAs or the 3 -UTR of their targets may be useful in HCC risk prediction.

miRNAs as Diagnostic and Prognostic Markers of HCC.
The differential expression of miRNAs in hepatocellular carcinoma cells compared with their expression in normal hepatocytes indicates potential values of miRNA detection in HCC diagnosis and prognosis predication. For example, HCCs can be divided into three main clusters based on miRNA profiling [175]. Using a human miRNA microarray, Murakami and colleagues identified three significantly upregulated miRNAs and five downregulated ones in 25 HCC tissues compared with the nontumorous samples. The algorithm based on the detection of these deregulated miRNAs showed an overall prediction accuracy of 97.8% for HCC diagnosis. In addition, the expression levels of miR692, miR620, and miR618 were inversely correlated with the degree of HCC differentiation [176]. Downregulation of miR-139 was associated significantly with poor prognosis of patients and features of metastatic tumors including venous invasion, microsatellite formation, absence of tumor encapsulation, and reduced differentiation [107]. Inversely, high levels of miR-222 and C19MC miRNA were correlated with poor clinicopathological features such as increased risk of tumor recurrence and shorter overall survival [177,178]. More studies on the clinical value of miRNAs detection in HCC are summarized in Table 3. Extracellular miRNAs in the circulation are stable, suggesting that miRNAs may serve as novel diagnostic markers [191]. Yamamoto et al. first reported an increased amount of miR-500 in the sera of HCC patients and its levels dropped to normal after the surgical treatment [192]. Interestingly, Shigoka and coworkers found that the relative amount of miR-92a in the plasmas from HCC patients, was decreased 8 The Scientific World Journal compared with that from the healthy donors [193]. In addition, serum level of miR-25, miR-375, and let-7f can clearly separate HCC cases from non-HCC controls, and miR-375 level alone displays the potential for the detection of HCC [194]. So far, more than 20 circulating miRNAs have been identified as diagnostic markers of HCC.

Potential
Roles of miRNAs in HCC Therapy. As miRNAs have confirmed to function as oncogenes or tumor suppressors and exogenous expression of tumor suppressor miRNAs or inhibition of onco-miRs resulted in alterations in malignance phenotypes of HCC cells in vitro, it might be possible to use artificial miRNAs to repress cancer development in vivo. Indeed, systemic administration of miR-26a, a miRNA expressed at high levels in normal tissues but downregulated in HCC, resulted in the inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity in a mouse model of HCC. These findings suggest that delivery of miRNAs that are highly expressed in normal tissues but lost in disease cells may provide a general strategy for miRNA replacement therapies without significant toxicity [58]. Two independent studies had reported that decreasing miR-221 level led to prolonged survival or a reduction of the number and size of tumor nodules in mice HCC models by using anti-miR-221 oligonucleotides or cholesterol-modified isoform of anti-miR-221 [195,196]. On the other hand, restoration of miR-122 [88], miR-143 [132], and miR-124 [102] individually significantly inhibited tumorigenesis and metastasis in vivo.
Moreover, miRNAs have also been shown to influence sensitivity of tumors to anticancer drugs. HCC cells transfected with pre-miR-21 were resistant to the cytotoxicity induced by IFN-/5-FU, and miR-21 expression in clinical HCC specimens was associated with the poor clinical response to the IFN-/5-FU combination therapy [136]. In addition, miR-181b can enhance resistance of HCC cells to doxorubicin [134]. Therefore, antagomirs targeting miR-21 or miR-181b might be useful in increasing drug efficacy. In contrast, restoration of miR-122 in HCC cells renders them more sensitive to adriamycin and vincristine through downregulating the expression of multidrug resistance (MDR) proteins [197].

Conclusion and Perspectives
Deregulation of miRNAs significantly contributes to the development of HCC. miRNAs mainly functions to downregulate the expression of targeted genes. However, they may have other yet unknown functions including the activation of gene transcription. The discovery of new types or novel functions of miRNAs provides us with more and deeper insights into the molecular mechanism underlying the pathogenesis of HCC. On the other hand, the miRNA expression profiles altered in HCC paves the way to early detection and treatment of HCC. With the advantage of target multiple genes simultaneously, miRNAs as therapeutic targets would be more efficient than other single-gene targeted therapeutics such as RNAi-based therapy, thus representing a new avenue for the development of anti-HCC treatments.