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Department of Gastroenterology and Neurology, Kagawa University School of Medicine, Kagawa, Japan
Correspondence: Profesor Tsutomu Masaki, Department of Gastroenterology and Neurology, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki-cho, Kida-gun, Kagawa 761-0793, Japan. Email: firstname.lastname@example.org
Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. Despite improvements in HCC therapy, the prognosis for HCC patients remains poor due to a high incidence of recurrence. An improved understanding of the pathogenesis of HCC development would facilitate the development of more effective outcomes for the diagnosis and treatment of HCC at earlier stages. miRNA are small, endogenous, non-coding, ssRNA that are 21–30 nucleotides in length and modulate the expression of various target genes at the post-transcriptional and translational levels. Aberrant expression of miRNA is common in various human malignancies and modulates cancer-associated genomic regions or fragile sites. As for the relationship between miRNA and HCC, several studies have demonstrated that the aberrant expression of specific miRNA can be detected in HCC cells and tissues. However, little is known about the mechanisms of miRNA-related cell proliferation and development. In this review, we summarize the central and potential roles of miRNA in the pathogenesis of HCC and elucidate new possibilities that may be useful as diagnostic and prognostic markers, as well as novel therapeutic targets in HCC.
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miRNA are small, interfering, non-coding RNA that are 21–30 nucleotides in length, and it has been predicted that there are approximately 1000 of these sequences in the human genome. Each miRNA negatively regulates its target genes by binding to multiple mRNA. Binding between miRNA and mRNA triggers the RNA-mediated RNAi pathway, in which the mRNA transcripts are cleaved by an miRNA-associated RNA-induced silencing complex (miRISC). In most animals, single-stranded miRNA act by binding to imperfectly complementary sites within the 3′-untranslated regions of their target mRNA, inhibiting translation or initiating degradation via the miRISC. Recruitment of the miRISC can modulate the expression of targeted protein-coding genes.[4, 5] Surprisingly, each miRNA can promote the targeting and modulation of more than 200 mRNA.[6, 7] In humans, a total of 2000 miRNA have been discovered.
Although their importance is recognized in regulating protein-coding gene expression, the precise functions of miRNA remain elusive. It is now apparent that miRNA play an important role in human carcinogenesis.[6, 9-12] In addition, the expression of miRNA is generally downregulated in tumor tissues compared with normal tissues, indicating that a subset of miRNA act as tumor suppressors. Therefore, the discovery of miRNA has expanded our knowledge of post-transcriptional gene regulation during cancer development. Interestingly, more than half of all genes that encode miRNA are located at fragile sites or in cancer-associated regions of the genome, suggesting that miRNA may serve as diagnostic markers or therapeutic targets. The first report of altered miRNA expression in cancer involved a frequent chromosomal deletion and two miRNA, miRNA-15 (miR-15) and miRNA-16 (miR-16), thought to target B-cell lymphoma 2 (BCL-2), which is the anti-apoptotic factor in chronic lymphocytic leukemia. Recent reports have shown that miRNA are associated with the pathogenesis of various types of cancers,[9-12] and these findings have increased our understanding of epigenetic modification during oncogenesis.
Several studies have recently reported a relationship between miRNA and hepatocellular carcinoma (HCC).[12, 15-18] Among several miRNA implicated in HCC, including miR-21, miR-221 and miR-222, the aberrant levels of miRNA expressions were upregulated.[12, 16, 17] However, other miRNA, including miR-122a, miR-145, miR-199a and miR-223, were decreased in HCC compared to normal tissues.[15, 19] Surprisingly, Huang et al. have shown that, among aberrant miRNA in HCC, miR-338 affects several clinical features, such as tumor size, tumor–node–metastasis classification, vascular invasion and intrahepatic metastasis. These studies suggest that the modulation of miRNA may play an important role in the progression of HCC.
Biogenesis and Regulation of miRNA
Several steps are involved in the biogenesis of miRNA: transcription, cleavage, export, further cleavage, strand selection and interaction with mRNA (Fig. 1). The genes encoding miRNA are primarily transcribed by RNA polymerase II into initial transcripts that are then processed through the canonical pathway or the “mirtron” pathway to form primary miRNA transcripts (pri-miRNA) that include one or more hairpin structures. These pri-miRNA are capped at the 5′-end and polyadenylated at the 3′-end and then cleaved into approximately 70-nucleotide (nt) hairpin-structured precursors (pre-miRNA) with a 5′-phosphate and a 3′-2-nt overhang by a multiprotein complex that includes an RNase III enzyme named Drosha and a dsRNA-binding domain protein (dsRBD) named DGCR8/Pasha. Subsequently, exportin-5 translocates pre-miRNA from the nucleus to the cytoplasm through a Ran-guanine-triphosphate (GTP)-dependent mechanism. These translocated pre-miRNA are cleaved by a second RNase III endonuclease named Dicer and the dsRBD proteins TRBP/PACT. Finally, one strand of the pre-miRNA interacts with the Argonaute (AGO) protein and is degraded in the RNA-induced silencing complex that modulates mRNA degradation and translational repression.
In addition to the canonical miRNA pathway, some miRNA are processed into miRNA-like molecules in a Microprocessor-independent manner, including certain snoRNA, tRNA and endogenous shRNA. Furthermore, more than 1 million Drosophila small-RNA sequences were recently generated using 454 pyrosequencing, and these sequences identified 14 short introns with predicted hairpin structures. These short introns were named “mirtrons” after analyzing their characteristics. Primary mirtron precursors include mirtronic introns and flanking exonic sequences that typically lack the lower stem (∼11 bp) that is found in miRNA, which mediates their recognition and cleavage via the Pasha (DGCR8)/Drosha complex. The “AG” splice acceptor of a typical mirtron adopts a 2-nt 3′-overhang for these hairpins, allowing mirtronsto enter the miRNA-processing pathway without Drosha-mediated cleavage.
miRNA regulate various biological processes by modulating specific mRNA, and their expression is tightly regulated in normal cells.[29, 33] Each miRNA can potentially be controlled independently at the transcriptional level by various regulators or at the epigenetic level via DNA methylation.[33-35] Interestingly, the regulatory elements that control protein-coding genes, such as CpG islands, TATA boxes, transcription factor II B recognition sites, initiator elements and histone modifications, are also found in the promoters of miRNA genes, indicating that the transcription factors are similar between protein-coding genes and miRNA. Therefore, the expression of various processing components is simultaneously regulated to modulate the activity of mature miRNA.
In addition, miRNA can also autoregulate their own transcription by controlling various transcription factors in positive or negative feedback loops. The miR-200 family of miRNA plays an important role by inhibiting the expression of zinc finger E-box-binding homeobox 1 (ZEB1) and survival of motor neuron protein-interacting protein 1 (SIP1), which act as transcriptional repressors in epithelial cells, to regulate the epithelial to mesenchymal transition; however, ZEB1 and SIP1 also suppress the miR-200 family, including miR-200a and miR-200b, by binding their promoter regions. This result indicates that miRNA and their target molecules are tightly regulated by each other at the transcription level.
Moreover, miRNA are also regulated by epigenetic processes, such as DNA methylation and specific histone deacetylation. Seventeen of the 313 human miRNA were upregulated more than threefold after treatment with the chromatin-modifying drugs 5-aza-2′-deoxycytidine and 4-phenylbutyric acid. Among these miRNA, miR-127 was highly upregulated after treatment. In addition, inhibiting histone deacetylases rapidly downregulated 22 miRNA and upregulated five miRNA.
Furthermore, miRNA expression is also controlled at the post-transcriptional level. A large fraction of miRNA genes are post-transcriptionally regulated, including those of the Let-7 family. Individual miRNA expression processed from the same pri-miRNA is occasionally different at the mature miRNA level. Initially during post-transcriptional regulation, miRNA are processed by Microprocessor, which consists of Drosha and the dsRNA-binding protein DGCR8 in the nucleus. A loss of Drosha or DGCR8 function leads to a decrease in pre-miRNA and mature miRNA. In the cytoplasm, pre-miRNA that are exported from the nucleus in a Ran/GTP/Exportin-5-mediated event are further regulated by the RNase III enzyme Dicer to become mature RNA.
miRNA Expression and HCC Development
miRNA have been reported to influence the critical functions of cellular differentiation, proliferation, apoptosis, invasion and metastasis. The miRNA expression profiles in tumors are different from those in normal tissues and also vary according to the type of tumor. Interestingly, the direct targets of miRNA are also protein-coding genes of the cell cycle, apoptosis and metastasis in HCC. Recently, microarray analysis has revealed that a subset of miRNA are up- and downregulated during the development of HCC. Reductions in the expression of miRNA are frequently observed in HCC, and the targets of these downregulated miRNA may be putative oncogenes. Conversely, some of the upregulated miRNA act as oncogenic miRNA in HCC and may be targets of tumor suppressor genes.
miRNA are associated with various chronic liver diseases, such as alcoholic liver disease, non-alcoholic steatohepatitis (NASH)[46-48] and viral hepatitis.[49-51] In addition to these chronic diseases, miRNA are also involved in the development of HCC. In alcoholic liver disease, miR-217 induces ethanol-induced fat accumulation in hepatocytes by inhibiting the expression of SIRT1. The miRNA miR-126, miR-27b, miR-182, miR-183, miR-199, miR-200a, miR-214 and miR-322 were downregulated in alcohol-related HCC.[51, 52] The observed reduction in miR-27 in alcoholic steatohepatitis was the result of epigenetic events that occurred in response to alcohol.
The development and exacerbation of NASH, both of which are also associated with miRNA, increase the risk of HCC. A recent study indicated that miRNA play a critical role in activating hepatic stellate cells (HSC) during the development of NASH. Free cholesterol was observed to accumulate due to an enhancement of both sterol regulatory element-binding protein-2 (SREBP2) and miR-33a signaling via the inhibition of peroxisome proliferator-activated receptor-γ signaling together with HSC activation and disruption of the SREBP2-mediated cholesterol-feedback system in HSC. The upregulation of miR-21, which downregulates the expression of tumor suppressor phosphatase and tensin homolog (PTEN), is induced by unsaturated fatty acids in hepatocytes. In addition, miR-155 suppresses another tumor suppressor gene, CCAAT-enhancer-binding protein-β, and has been also shown to be upregulated in mice fed a choline-deficient amino acid-defined diet.[47, 48]
Viral hepatitis, such as that caused by the hepatitis B virus (HBV) and hepatitis C virus (HCV), is the most frequent cause of HCC and cirrhosis. The 5-year cumulative risk of hepatocarcinogenesis with liver cirrhosis ranges 5–30%. Among HBV cases, only two miRNA, miR-210 and miR-199-3p, were observed to affect HBV gene expression and replication in an experimental model. In contrast, many cellular miRNA indirectly regulate the HBV life cycle by affecting virus-relevant cellular proteins. Ura et al. examined the expression of 188 miRNA in HCC and adjacent normal tissues obtained from 12 HBV positive and 14 HCV positive patients. In these groups, the expression of six miRNA was decreased in HBV patients, and that of 13 miRNA was reduced in HCV patients. These data suggest that there are distinct patterns in the miRNA profiles between HBV and HCV infection. Several reports demonstrated that miR-96 or miR-26 were significantly upregulated in HBV-related HCC tissues.[52, 58] Takazawa et al. reported comprehensive miRNA profiles using sequencing methods that provided more than 314 000 reliable reads from HCC tissue and more than 268 000 reliable reads from adjacent normal liver. Bioinformatic-based analysis revealed several miRNA altered in HCC, including miR-122, miR-21 and miR-34a. Therefore, further studies that characterize the miRNA associated with HBV-related HCC may yield a novel therapeutic tool for HCC patients with HBV infection.
In addition, miR-196 plays an important role through the inhibition of Bach1 (a basic leucine zipper mammalian transcriptional repressor) and upregulation of hemeoxygenase 1 in HCV-related HCC. Diaz et al. also demonstrated 18 miRNA among 2226 human miRNA that were exclusively expressed in HCV-related HCC. One of these 18 miRNA has been associated with networks that include p53, PTEN and retinoic acid and is involved in the pathogenesis of HCC.[62, 63] These data suggest that miRNA pathways are essential to the development of HCC during HCV infection.
Clinical Significance of miRNA in HCC
Single nucleotide polymorphisms of miRNA and the risk of HCC
Single nucleotide polymorphisms in miRNA and their targets have been associated with an increased risk of HCC. Because miRNA must closely recognize binding sites in their target genes, a variation in even 1 nt may produce dramatic changes in the post-transcriptional regulation of target genes. Polymorphisms in miR-101-1/rs7536540, miR-101-2/rs12375841, miR-34b/c/rs4938723 and miR-106b-25-cluster/rs999885 are positively associated with an increased risk of HCC. In contrast, miR-371–373/rs3859501 and miR-149c/rs2292832 are negatively involved in HCC risk. However, conflicting results were obtained from several studies, such as with miR-499a/rs3746444[68-70] and miR-196a-2/rs11614913.[68, 71-73]
miRNA as biomarkers for HCC
miRNA can be characterized as prognostic or diagnostic markers. Downregulation of the miRNA miR-let-7g, miR-22, miR-26, miR-29, miR-99a, miR-122, miR-124, miR-139, miR-145 and miR-199b has been implicated in cell proliferation, apoptosis, angiogenesis, recurrence, shorter disease-free survival and poor prognosis (Table 1). In contrast, upregulation of miR-10b, miR-17-5p, miR-21, miR-135a, miR-155, miR-182, miR-221 and miR-222[118, 156] has been associated with metastasis, angiogenesis and poor prognosis (Table 2). In addition, miRNA profiling classified HCC into three main clusters. These results indicate the potential value of miRNA detection for the prediction of survival in HCC. Furthermore, Yamamoto et al. reported that miR-500 was increased in the sera of HCC patients and decreased after surgical treatment. In addition, other miRNA, such as miR-25, miR-375 and let-7f, can be used to distinguish HCC from normal control tissue. Interestingly, extracellular miRNA are stable in circulation, indicating that miRNA may serve as useful diagnostic markers for HCC.
Table 1. Downregulated miRNA in hepatocellular carcinoma
Because miRNA function as oncogenes or tumor suppressors, alterations in these miRNA influence the malignant phenotypes of HCC cells. In tumor tissues, miRNA associated with tumor suppressors are downregulated during tumorigenesis, tumor development and metastasis. These miRNA may be potential therapeutic targets, and strategies for miRNA replacement therapies have been developed using miR-26a, miR-122 and miR-124 in a HCC mouse model. In contrast, inhibition of miR-221 lengthened survival, reduced the nodule number and retarded tumor development.[177, 199] Furthermore, no toxicity was observed when miRNA-targeted therapy was used to treat HCC in a mice model. miRNA have been also shown to affect the sensitivity of tumors to anticancer drugs. Several reports have indicated that miRNA profiles are dramatically altered following metformin treatment for various cancers: gastric cancer, esophageal cancer and HCC. Interestingly, overexpression of miR-21 and miR-181b induced resistance to interferon–5-fluorouracil combination therapy and doxorubicin treatment in HCC. In contrast, Bai et al. demonstrated that restoring expression of the tumor suppressive miR-122 makes HCC cells more sensitive to sorafenib via the downregulation of multidrug resistance genes.
miRNA are being considered as new biomarkers and potential therapeutic targets for HCC. To date, many miRNA have been identified as regulators of various target genes during HCC development. In addition, although miRNA-based therapy is not currently used in the clinic, its innovative applications are growing in various fields. However, the details regarding targetable miRNA and the mechanisms of miRNA-induced anticancer effects remain unclear. Further analyses and new technology for miRNA research will elucidate novel concepts in the pathogenesis of HCC. Consequently, analyzing miRNA profiles and their signaling pathways offers deeper insights into the treatment options for HCC.