miRNAs (microRNAs) are important regulators of gene expression. In higher eukaryotes, the tightly controlled expression of different miRNAs, each of which regulates multiple target mRNAs, is crucial for the maintenance of tissue type and the control of differentiation. miR-122 is a highly liver-specific miRNA that is important in hepatitis C virus infection, cholesterol metabolism and hepatocellular carcinoma. In the present review, we discuss the effects of miR-122 on liver physiology and pathology. Recent evidence of pathways involved in the regulation of miR-122 expression is also considered.
- circadian rhythm
- hepatitis C virus
- microRNA (miRNA)
miRNAs (microRNAs) were first identified in Caenorhabditis elegans when a gene involved in the heterochronic pathway, lin-4, was shown to express a short non-coding RNA product instead of a protein . Subsequently, many different miRNA molecules have been found in a broad range of eukaryotes . miRNAs are encoded in the genome and are transcribed, usually by RNA polymerase II, within long pri-miRNA (primary miRNA transcript) hairpins (Figure 1). miRNAs may be encoded individually or as part of a cluster, often with related targets, and pri-miRNAs may be unique transcripts or introns of coding mRNAs . Pri-miRNAs undergo successive nuclear and cytoplasmic processing by the RNAse III enzymes Drosha and Dicer respectively (Figure 1). The resulting mature 21–23-nt single-stranded miRNA molecule is incorporated into an active protein complex, usually known as the miRISC (miRNA-induced silencing complex) (Figure 1), as it shows much similarity to the RISC (RNA-induced silencing complex) that mediates siRNA (short interfering RNA) activity . In animals, miRNAs generally regulate the expression of specific genes by binding with imperfect complementarity to sites in the 3′-UTR (untranslated region) of target mRNAs. Each miRNA has multiple target mRNAs, and an individual mRNA may be targeted by multiple miRNAs. miRNA binding results in repression of protein synthesis and in mRNA degradation, with both effects observed to different extents in different experimental systems. The mechanism behind this is not yet fully understood .
Many miRNAs show specific expression patterns that are governed by both tissue type and developmental stage. A particularly striking example of this regulation is miR-122, which was first identified when miRNAs were isolated from several different mouse tissues. miR-122 accounted for 72% of all the miRNAs cloned from the liver, and was not detected in any of the other tissues analysed . miR-122 levels in the liver increase during embryogenesis, reaching approx. 66000 copies per cell in adult mouse liver, and 135000 copies per cell in primary human hepatocytes . The sequence of miR-122 is conserved from humans to zebrafish, and in situ hybridization in zebrafish indicated that the liver-specific expression pattern of miR-122 is also highly conserved . miR-122 is expressed as a unique miRNA within a single transcript, which is designated hcr .
The high-level expression of miR-122 in liver makes it an attractive candidate for study, as it is likely to have important roles in liver physiology. Identification of miR-122 target genes demonstrated that it regulates significant liver processes, particularly cholesterol metabolism [8,9]. miR-122 is also associated with disease and has a major positive role in HCV (hepatitis C virus) infection , and a negative role in HCC (hepatocellular carcinoma) . Despite the extensive effects of miR-122 on gene expression in the liver, inactivation of this miRNA does not appear to be detrimental to liver function in animals [8,9,12].
In the present review, we discuss the targets of miR-122 and its role in normal and diseased liver function, and the pathways governing its expression.
Regulation of HCV by miR-122
HCV is a positive-sense single-stranded RNA virus that infects approx. 3% of the global population . Chronic HCV infection can lead to liver cirrhosis and eventually to HCC. Development of an effective treatment has become a major international endeavour .
miR-122 is required for HCV replication in cultured Huh7 human hepatocytes [9,15]. miR-122 is recruited directly to two adjacent binding sites in the 5′-UTR of HCV RNA, and this binding is responsible for enhanced viral RNA synthesis . Initial observations suggested that miR-122 does not affect HCV protein synthesis, implying that the miRNA has a stimulatory effect on viral RNA synthesis . Subsequent analysis has shown that miR-122 does, in fact, have a positive effect on viral translation [16,17]. This translational stimulation is not sufficient to explain the effect of miR-122 on viral replication, which led to the conclusion that miR-122 regulates both HCV translation and a second stage in the replication cycle . This second stage remains unidentified, but miR-122 does not affect HCV RNA synthesis either in cells or in isolated replication complexes [18,19], raising the possibility that RNA stability may be regulated.
The lack of a protective vaccine against HCV and poor patient tolerance of and response to the established therapy of PEGylated IFN (interferon) α combined with ribavirin mean that there is an urgent need for better treatments to be developed . The positive effect of miR-122 on the HCV life cycle implies that miR-122 inhibition could be used as a novel approach in the treatment of HCV-infected individuals. However, quantification of miR-122 and HCV RNA in infected human liver suggests that the relationship between the miRNA and the virus is more complex in tissue than in cultured cells. miR-122 levels did not correlate with viral load, and individuals with lower pre-treatment miR-122 levels tended to respond poorly to IFN therapy . In Huh7 cells, miR-122 was found to be down-regulated in response to IFNβ . This was thought to be one of the antiviral effects of IFN treatment, but it was shown subsequently that PEGylated IFNα in humans and IFNα in mice do not affect miR-122 levels . Despite the lack of correlation between miR-122 and HCV load in infected patients, a recent animal study has provided exciting data to support miR-122 inhibition as an anti-HCV therapy. miR-122 sequestration and inactivation using a complementary LNA (locked nucleic acid) molecule had a potent antiviral effect in HCV-infected chimpanzees . This validates the principle of miR-122-targeted HCV treatment, but clinical trials in humans will be required before it can be established whether this approach will bear fruit.
The endogenous targets of miR-122 and its role in cholesterol metabolism
The first target of miR-122 to be identified was CAT-1 (cationic amino acid transporter 1) in the liver . It was shown subsequently that miR-122 repression of CAT-1 synthesis is relieved under conditions of amino acid starvation by the protein HuR binding to the CAT-1 3′-UTR . It is not known whether any other miR-122 targets show similar complexity of regulation. Microarray analysis following miR-122 inhibition in mice indicated that miR-122 regulates hepatic fatty acid and cholesterol synthesis, and controls plasma cholesterol levels [8,9]. It was possible to identify direct miR-122 targets by looking for mRNAs that show increased expression when miR-122 is sequestered and contain predicted miR-122 target sites in the 3′-UTR . Several such mRNAs were validated as targets by testing the effects of miR-122 on luciferase–3′-UTR reporters, and included mRNAs encoding proteins that are usually repressed in hepatocytes, such as aldo-A (aldolase A) and Ndrg3 (N-myc downstream regulated gene 3) . This suggests that miR-122 plays a role in maintaining the adult liver phenotype. The effects on cholesterol metabolism are likely to be indirect, as mRNAs involved in cholesterol biosynthesis tended to be down-regulated when miR-122 was sequestered and to lack miR-122 seed matches [8,9].
A decline in plasma cholesterol levels alongside enhanced fatty acid oxidation was observed in normal mice after treatment with an antisense oligonucleotide against miR-122 . Additionally, miR-122 inhibition led to a decline in plasma cholesterol levels and a significant improvement in liver steatosis in a mouse obesity model . No significant side effects of miR-122 sequestration were observed in mice or chimpanzees, supporting the potential of miR-122 inhibition as a therapeutic target for metabolic disease [8,9,12]. A specific reduction in damaging LDL (low-density lipoprotein) cholesterol levels in chimpanzees treated with miR-122 inhibitors is encouraging when considering this approach as a treatment for hypercholesterolaemia .
miR-122 and cancer
It is well established that specific miRNAs play a significant role in cancer . A number of miRNAs are located in fragile sites within the genome, and the dysregulation of miRNA expression is a key factor in tumorigenesis . Analysis of the levels of specific miRNAs has also proved to be an effective predictor of stage and treatment outcome in various tumours .
A comparison of miR-122 levels in hepatocytes and primary human HCC cells revealed that miR-122 is down-regulated in HCC cells . It was also shown that reintroduction of miR-122 into HCC cells can reverse the tumorigenic properties of these cells, whereas inhibition of miR-122 in Huh-7 human hepatoma cells stimulates the appearance of such properties [11,25]. This suggests that miR-122 acts as a tumour suppressor in the liver. Some miR-122 targets that may be involved in tumorigenesis have been identified. Cyclin G1 is expressed at high levels in a number of tumours, and miR-122 inhibits cyclin G1, leading to improved sensitivity of HCC cells to the common chemotherapeutic compound doxyrubicin . Additionally, it was shown that miR-122 acts as a tumour suppressor in the liver by down-regulating expression of a protein commonly associated with tumour metastasis, ADAM17 (a disintegrin and metalloproteinase 17) .
These results suggest that miR-122-based therapeutic strategies may be useful in the treatment of HCC. These observations are also important when considering miR-122 inhibition as a therapy for HCV or hypercholesterolaemia. No adverse effects were observed over the course of the animal trials carried out to date [8,9,12,28], but a link between low miR-122 levels and HCC implies that long-term inactivation of the miRNA might be inadvisable, and that it will be very important to follow up treated animals for a long period after the treatment has ended. An increased understanding of the association between miR-122 expression, HCV infection and HCC will be important for clinical development of miR-122-based drugs.
miR-122 and the circadian rhythm
Circadian rhythms are mechanisms by which an organism anticipates environmental rhythms and changes its physiological or behavioural state accordingly . The maintenance of such rhythms, particularly in highly metabolic organs such as the liver, is essential for normal function . Specific miRNAs have been identified as key players in regulation and maintenance of circadian gene expression .
miR-122 expression and function have been shown to be important in the circadian output of the liver . Transcription of the miR-122 gene is circadian (Figure 1), with pri- and pre-miR-122 levels fluctuating in a circadian rhythm in mouse liver . Mature miR-122 levels appear to remain constant throughout the day, owing to the long half-life and large steady-state pool of this miRNA. Despite this, miR-122 depletion was linked to circadian changes in expression of many mRNAs, some of which were shown to be direct miR-122 targets . The circadian transcription of pri-miR-122 was found to be governed by the orphan nuclear receptor REV-ERBα , but the question of how a constant level of miR-122 can regulate fluctuating target levels remains to be answered. It is possible there may be a specific role for newly synthesized miR-122 in regulation of circadian targets, or that functionally distinct subpopulations of miR-122 may exist.
miRNA expression can be regulated by changes in transcription and processing, but modification of the mature miRNA sequence may also affect its level or function. There are a few specific examples of modification of animal miRNAs, such as 3′-uridylation of certain miRNAs leading to destabilization and loss of function . miR-122 was shown recently to be subject to post-synthesis modification in the form of 3′-adenylation. This is catalysed by the cytoplasmic GLD-2 poly(A) polymerase, which adds a single adenine to the 3′-end of miR-122 (Figure 1). This results in increased stability of miR-122, presumably by impeding a 3′-end-dependent degradation process . Non-templated 3′-adenines have also been detected on various other miRNAs, but the functional consequences for these miRNAs remain unclear .
The high expression of miR-122 in the liver appears to correlate with a central role in various functions of normal and diseased livers. It provides a very attractive target for treatment of HCV and hypercholesterolaemia, and can be effectively sequestered in vivo using antisense oligonucleotides. Rather surprisingly, given the high intracellular levels and numerous targets of miR-122, inactivation of the miRNA does not have any apparent adverse effects on liver physiology. However, reduced miR-122 expression does show an association with hepatocellular carcinoma, and further work will be necessary before any anti-miR-122-based therapies can be administered to human patients.
miR-122 has several unusual functions that make it a useful miRNA to study that is likely to provide insight into miRNA mechanisms in general. The positive regulation that miR-122 exerts by binding to HCV RNA is only partially understood, but shows that animal miRNAs can modulate processes other than post-transcriptional repression of gene expression by binding to 3′-UTR targets. The relief of miR-122-imposed repression of CAT-1 by HuR binding is also an important example of regulated miRNA activity. The regulation of miR-122 activity by circadian transcription, despite maintenance of miR-122 levels, suggests that it may be wrong to assume that miRNA quantification provides an accurate indication of the functional pool. A more detailed insight into all of these processes will be important for our understanding of liver biology and the regulation and mechanism of miRNAs.
Our research is supported by a Biotechnology and Biological Sciences Research Council David Phillips Fellowship to C.L.J. [grant number BB/F02360X/1].
We thank Ashley Roberts for a critical reading of the paper.
Post-Transcriptional Control: mRNA Translation, Localization and Turnover: A Biochemical Society Focused Meeting held at University of Edinburgh, U.K., 8–10 June 2010. Organized and Edited by Matthew Brook (Edinburgh, U.K.), Mark Coldwell (Southampton, U.K.), Simon Morley (Sussex, U.K.) and Nicola Gray (Edinburgh, U.K.).
Abbreviations: CAT-1, cationic amino acid transporter 1; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; IFN, interferon; miRNA, microRNA; pri-miRNA, primary miRNA transcript; UTR, untranslated region
- © The Authors Journal compilation © 2010 Biochemical Society