Liver-specific microRNA miR-122 Research Articles

Enhanced Virus Translation Enables miR-122-Independent Hepatitis C Virus Propagation.

The 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome forms RNA structures that regulate virus replication and translation. The region contains an internal ribosomal entry site (IRES) and a 5'-terminal region. Binding of the liver-specific microRNA (miRNA) miR-122 to two binding sites in the 5'-terminal region regulates viral replication, translation, and genome stability and is essential for efficient virus replication, but its precise mechanism of action is still unresolved. A current hypothesis is that miR-122 binding stimulates viral translation by facilitating the viral 5' UTR to form the translationally active HCV IRES RNA structure. While miR-122 is essential for detectable replication of wild-type HCV genomes in cell culture, several viral variants with 5' UTR mutations exhibit low-level replication in the absence of miR-122. We show that HCV mutants capable of replicating independently of miR-122 display an enhanced translation phenotype that correlates with their ability to replicate independently of miR-122. Further, we provide evidence that translation regulation is the major role for miR-122 and show that miR-122-independent HCV replication can be rescued to miR-122-dependent levels by the combined impacts of 5' UTR mutations that stimulate translation and by stabilizing the viral genome by knockdown of host exonucleases and phosphatases that degrade the genome. Finally, we show that HCV mutants capable of replicating independently of miR-122 also replicate independently of other microRNAs generated by the canonical miRNA synthesis pathway. Thus, we provide a model suggesting that translation stimulation and genome stabilization are the primary roles for miR-122 in promoting HCV. IMPORTANCE The unusual and essential role of miR-122 in promoting HCV propagation is incompletely understood. To better understand its role, we have analyzed HCV mutants capable of replicating independently of miR-122. Our data show that the ability of viruses to replicate independently of miR-122 correlates with enhanced virus translation but that genome stabilization is required to restore efficient HCV replication. This suggests that viruses must gain both abilities to escape the need for miR-122 and impacts the possibility that HCV can evolve to replicate outside the liver.

In Vivo Assessment of Drug-Induced Hepatotoxicity Using Xenopus Embryos.

Failure to predict drug-induced toxicity reactions is a major problem contributing to a high attrition rate and tremendous cost in drug development. Drug screening in X. laevis embryos is high-throughput relative to screening in rodents, potentially making them ideal for this use. Xenopus embryos have been used as a toxicity model in the frog embryo teratogenesis assay on Xenopus (FETAX) for the early stages of drug safety evaluation. We previously developed compound-screening methods using Xenopus embryos and believe they could be used for in vitro drug-induced toxicity safety assessment before expensive preclinical trials in mammals. Specifically, Xenopus embryos could help predict drug-induced hepatotoxicity and consequently aid lead candidate prioritization. Here we present methods, which we have modified for use on Xenopus embryos, to help measure the potential for a drug to induce liver toxicity. One such method examines the release of the liver-specific microRNA (miRNA) miR-122 from the liver into the vasculature as a result of hepatocellular damage, which could be due to drug-induced acute liver injury. Paracetamol, a known hepatotoxin at high doses, can be used as a positive control. We previously showed that some of the phenotypes of mammalian paracetamol overdose are reflected in Xenopus embryos. Consequently, we have also included here a method that measures the concentration of free glutathione (GSH), which is an indicator of paracetamol-induced liver injury. These methods can be used as part of a panel of protocols to help predict the hepatoxicity of a drug at an early stage in drug development.

Impact of a patient-derived hepatitis C viral RNA genome with a mutated microRNA binding site.

Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5’ end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester miR-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5’ genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5’ end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5’ end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood.

Precursor microRNA-122 inhibits synthesis of Insig1 isoform mRNA by modulating polyadenylation site usage.

The insulin-induced gene 1 protein (Insig1) inhibits the cholesterol biosynthesis pathway by retaining transcription factor SREBP in the endoplasmic reticulum, and by causing the degradation of HMGCR, the rate-limiting enzyme in cholesterol biosynthesis. Liver-specific microRNA miR-122, on the other hand, enhances cholesterol biosynthesis by an unknown mechanism. We have found that Insig1 mRNAs are generated by alternative cleavage and polyadenylation, resulting in specific isoform mRNA species. During high cholesterol abundance, the short 1.4-kb Insig1 mRNA was found to be preferentially translated to yield Insig1 protein. Precursor molecules of miR-122 down-regulated the translation of the 1.4-kb Insig1 isoform mRNA by interfering with the usage of the promoter-proximal cleavage–polyadenylation site that gives rise to the 1.4-kb Insig1 mRNA. These findings argue that precursor miR-122 molecules modulate polyadenylation site usage in Insig1 mRNAs, resulting in down-regulation of Insig1 protein abundance. Thus, precursor microRNAs may have hitherto undetected novel functions in nuclear gene expression.

Hepatitis C Virus Core Protein Promotes miR-122 Destabilization by Inhibiting GLD-2.

The liver-specific microRNA miR-122, which has essential roles in liver development and metabolism, is a key proviral factor for hepatitis C virus (HCV). Despite its crucial role in the liver and HCV life cycle, little is known about the molecular mechanism of miR-122 expression regulation by HCV infection. Here, we show that the HCV core protein downregulates the abundance of miR-122 by promoting its destabilization via the inhibition of GLD-2, a non-canonical cytoplasmic poly(A) polymerase. The decrease in miR-122 expression resulted in the dysregulation of the known functions of miR-122, including its proviral activity for HCV. By high-throughput sequencing of small RNAs from human liver biopsies, we found that the 22-nucleotide (nt) prototype miR-122 is modified at its 3′ end by 3′-terminal non-templated and templated nucleotide additions. Remarkably, the proportion of miR-122 isomers bearing a single nucleotide tail of any ribonucleotide decreased in liver specimens from patients with HCV. We found that these single-nucleotide-tailed miR-122 isomers display increased miRNA activity and stability over the 22-nt prototype miR-122 and that the 3′-terminal extension is catalyzed by the unique terminal nucleotidyl transferase activity of GLD-2, which is capable of adding any single ribonucleotide without preference of adenylate to the miR-122 3′ end. The HCV core protein specifically inhibited GLD-2, and its interaction with GLD-2 in the cytoplasm was found to be responsible for miR-122 downregulation. Collectively, our results provide new insights into the regulatory role of the HCV core protein in controlling viral RNA abundance and miR-122 functions through miR-122 stability modulation.

Human MicroRNA-602 Inhibits Hepatitis C Virus Genotype 1b Infection and Promotes Tumor Suppressor Gene Expression in a Hepatoma Cell Line

Objective: Hepatitis C virus (HCV) infects 200 million people worldwide, inducing cirrhosis and hepatocellular carcinoma. Liverspecific microRNA (miR) miR-122 facilitates HCV replication, and Locked Nucleic Acid (LNA)-anti-miR-122 was used successfully as a mono-therapy in HCV infected patients. Studies show that anti-miR-122 may negatively impact hepatocyte metabolism. Previously, we showed that human (hsa) miR-602 relative intracellular expression was up-regulated in microarray profiling when transfected with HCV genotype 1b, and hsa-miR-602 lowered HCV accumulation in infected Huh-7.5 cells. This study compares the effectiveness of hsa-miR-602 and LNA-anti-miR-122 on inhibiting HCV replication and regulating the expression of tumor suppressor genes p53, p73, and RASSF1A. Methods: Huh-7.5 cells were transfected with HCV genotype 1b and treated with hsa-miR-602, LNA-anti-miR-122 or both. Cytopathic effect (CPE) and cell aggregation were observed regularly after transfection. HCV load and localization in the un-transfected, transfected, and treated cells were detected by qRT-PCR and immunostaining, respectively. qRT-PCR detected the expression of p53, p73, and RASSF1A. The Cancer Cell Lines Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) were used to correlate gene copy number and expression for TP53, TP73 and RASSF1A in normal and hepatocellular carcinoma (HCC) patient tissues and HCC cell lines. The survival curve for patients with MIR- 602 expression was done using TCGA HCC clinical data sets. Results: HCV load decreased in all treated cells but was more pronounced with hsa-miR-602. HCV transfected Huh-7.5 cells treated with hsa-miR-602 showed no morphological changes whereas cells treated with LNA-anti-miR-122 or LNA-anti-miR-122 and hsa-miR-602 together showed morphological changes. p53, p73, RASSF1A were down-regulated in HCV transfected cells and all treatments restored expression to near normal levels. Similar down-regulated expression was seen in the HCC patient samples and cell lines. Conclusion: Hsa-miR-602 effectively reduces HCV load and restores p53, p73, and RASSF1A expression in HCV transfected cells.

Supporting Role for GTPase Rab27a in Hepatitis C Virus RNA Replication through a Novel miR-122-Mediated Effect.

The small GTPase Rab27a has been shown to control membrane trafficking and microvesicle transport pathways, in particular the secretion of exosomes. In the liver, high expression of Rab27a correlates with the development of hepatocellular carcinoma. We discovered that low abundance of Rab27a resulted in decreased hepatitis C virus (HCV) RNA and protein abundances in virus-infected cells. Curiously, both cell-associated and extracellular virus yield decreased in Rab27a depleted cells, suggesting that reduced exosome secretion did not cause the observed effect. Instead, Rab27a enhanced viral RNA replication by a mechanism that involves the liver-specific microRNA miR-122. Rab27a surrounded lipid droplets and was enriched in membrane fractions that harbor viral replication proteins, suggesting a supporting role for Rab27a in viral gene expression. Curiously, Rab27a depletion decreased the abundance of miR-122, whereas overexpression of miR-122 in Rab27a-depleted cells rescued HCV RNA abundance. Because intracellular HCV RNA abundance is enhanced by the binding of two miR-122 molecules to the extreme 5’ end of the HCV RNA genome, the diminished amounts of miR-122 in Rab27a-depleted cells could have caused destabilization of HCV RNA. However, the abundance of HCV RNA carrying mutations on both miR-122-binding sites and whose stability was supported by ectopically expressed miR-122 mimetics with compensatory mutations also decreased in Rab27a-depleted cells. This result indicates that the effect of Rab27a depletion on HCV RNA abundance does not depend on the formation of 5’ terminal HCV/miR-122 RNA complexes, but that miR-122 has a Rab27a-dependent function in the HCV lifecycle, likely the downregulation of a cellular inhibitor of HCV gene expression. These findings suggest that the absence of miR-122 results in a vulnerability not only to exoribonucleases that attack the viral genome, but also to upregulation of one more cellular factor that inhibit viral gene expression.

A cross-talk between Hepatitis B virus and host mRNAs confers viral adaptation to liver.

Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide. The replication of HBV which genome is only 3.2 kb long relies heavily on host factors. Previous studies demonstrated that a highly expressed liver-specific microRNA (miRNA) miR-122 suppresses HBV expression and replication in multiple ways. In this study, we found that the miR-122 response elements in viral genome facilitate HBV expression and replication in miR-122 highly-expressed hepatocytes. Moreover, mutations in miR-122 response elements are correlated with viral loads and disease progression in HBV-infected patients. We next found that HBV mRNA with miR-122 response elements alone could lead to altered expression of multiple host genes by whole genome expression analysis. HBV mRNA-mediated miR-122 down-regulation plays a major role in HBV mRNA-induced differential gene expression. HBV mRNA could enhance viral replication via miR-122 degradation and the up-regulation of its target cyclin G1. Our study thereby reveals that under the unique condition of high abundance of miR-122 and viral mRNAs and much lower level of miR-122 target in HBV infection, HBV may have evolved to employ the miRNA-mediated virus and host mRNAs network for optimal fitness within hepatocytes.

Interaction of host cell microRNAs with the HCV RNA genome during infection of liver cells.

It has remained an enigma how hepatitis C viral (HCV) RNA can persist in the liver of infected patients for many decades. With the recent discovery of roles for microRNAs in gene expression, it was reported that the HCV RNA genome subverts liver-specific microRNA miR-122 to protect its 5' end from degradation by host cell exoribonucleases. Sequestration of miR-122 in cultured liver cells and in the liver of chimpanzees by small, modified antisense RNAs resulted in dramatic loss of HCV RNA and viral yield. This finding led to the first successful human trial in which subcutaneous administration of antisense molecules against miR-122 lowered viral yield in HCV patients, without the emergence of resistant virus. In this review, the authors summarize the molecular mechanism by which miR-122 protects the HCV RNA genome from degradation by exoribonucleases Xrn1 and Xrn2 and discuss the application of miR-122 antisense molecules in the clinic.

Hepatitis C virus subverts liver-specific miR-122 to protect the viral genome from exoribonuclease Xrn2.

The abundant, liver-specific microRNA miR-122 forms extensive base-pairing interactions with the 5' noncoding region of the hepatitis C virus (HCV) RNA genome, protecting the viral RNA from degradation. We discovered that the 5'-3' exoribonuclease Xrn2, which plays a crucial role in the transcription termination of RNA polymerase II, modulates HCV RNA abundance in the cytoplasm, but is counteracted by miR-122-mediated protection. Specifically, Xrn2 depletion results in increased accumulation ofviral RNA, while Xrn2 overexpression diminishes viral RNA abundance. Depletion of Xrn2 did not altertranslation or replication rates of HCV RNA, but affected viral RNA stability. Importantly, during sequestration of miR-122, Xrn2 depletion restored HCV RNA abundance, arguing that Xrn2 depletion eliminates the miR-122 requirement for viral RNA stability. Thus, Xrn2 has a cytoplasmic, antiviral function against HCV that is counteracted by HCV's subversion of miR-122 to form a protective oligomeric complex at the 5' end of the viral genome.

Transient Replication of Hepatitis C Virus Sub-Genomic RNA in Murine Cell Lines Is Enabled by miR-122 and Varies with Cell Passage

Hepatitis C Virus (HCV) is a serious global health problem, infecting almost 3% of the world’s population. The lack of model systems for studying this virus limit research options in vaccine and therapeutic development, as well as for studying the pathogenesis of chronic HCV infection. Herein we make use of the liver-specific microRNA miR-122 to render mouse cell lines permissive to HCV replication in an attempt to develop additional model systems for the identification of new features of the virus and its life cycle. We have determined that some wild-type and knockout mouse cell lines – NCoA6 and PKR knockout embryonic fibroblasts – can be rendered permissive to transient HCV sub-genomic RNA replication upon addition of miR-122, but we did not observe replication of full-length HCV RNA in these cells. However, other wild-type and knockout cell lines cannot be rendered permissive to HCV replication by addition of miR-122, and in fact, different NCoA6 and PKR knockout cell line passages and isolates from the same mice demonstrated varying permissiveness phenotypes and eventually complete loss of permissiveness. When we tested knockdown of NCoA6 and PKR in Huh7.5 cells, we saw no substantial impact in sub-genomic HCV replication, which we would expect if these genes were inhibitory to the virus’ life cycle. This leads us to conclude that along with the influence of specific gene knockouts there are additional factors within the cell lines that affect their permissiveness for HCV replication; we suggest that these may be epigenetically regulated, or modulated by cell line immortalization and transformation.

The impact of chronic hepatitis C infection on the circadian clock and sleep

The impact of chronic hepatitis C infection on the circadian clock and sleep

Enhancement of hepatitis C viral RNA abundance by precursor miR-122 molecules

The hepatitis C viral RNA genome forms a complex with liver-specific microRNA (miR-122) at the extreme 5' end of the viral RNA. This complex is essential to stabilize the viral RNA in infected cultured cells and in the liver of humans. The abundances of primary and precursor forms of miR-122, but not the abundance of mature miR-122, are regulated in a circadian rhythm in the liver of animals, suggesting a possible independent function of precursor molecules of miR-122 in regulating viral gene expression. Modified precursor molecules of miR-122 were synthesized that were refractory to cleavage by Dicer. These molecules were found to enhance the abundance of HCV RNA. Furthermore, they diminished the expression of mRNAs that contained binding sites for miR-122 in their 3' noncoding regions. By use of duplex and precursor miR-122 mimetic molecules that carried mutations in the passenger strand of miR-122, the effects on viral and reporter gene expression could be pinpointed to the action of precursor miR-122 molecules. Targeting the circadian expression of precursor miR-122 by specific compounds likely provides novel therapeutic strategies.

MicroRNA-122-dependent and -independent replication of Hepatitis C Virus in Hep3B human hepatoma cells

The study of Hepatitis C Virus (HCV) has benefitted from the use of the Huh7 cell culture system, but until recently there were no other widely used alternatives to this cell line. Here we render another human hepatoma cell line, Hep3B, permissive to the complete virus life cycle by supplementation with the liver-specific microRNA miR-122, known to aid HCV RNA accumulation. When supplemented, Hep3B cells produce J6/JFH-1 virus titres indistinguishable from those produced by Huh7.5 cells. Interestingly, we were able to detect and characterize miR-122-independent replication of di-cistronic replicons in Hep3B cells. Further, we show that Argonaute-2 (Ago2) is required for miR-122-dependent replication, but dispensable for miR-122-independent replication, confirming Ago2's role in mediating the activity of miR-122. Thus Hep3B cells are a model system for the study of HCV, and miR-122 independent replication is a model to identify proteins involved in the function of miR-122.

MicroRNA Regulation of Oncolytic Adenovirus 6 for Selective Treatment of Castration-Resistant Prostate Cancer

Almost all patients with advanced prostate cancer progress to castration-resistant stage with limited treatment options. Oncolytic adenoviruses have been actively pursued as potential agents for cancer treatment. Virtually all clinical trials on oncolytic adenovirus are based on serotype 5. However, viral replication in hepatocytes induces severe liver toxicity and limits its systemic administration for metastatic disease. Moreover, rapid clearance of viral particles injected intravenously further hinders the anticancer efficacy. Adenovirus 6 (Ad6) was previously reported to exhibit less liver toxicity and escape Kupffer cells absorption after systemic administration. To further improve its safety, we generated a novel oncolytic adenovirus Ad6miR, in which four copies of binding sites of a liver-specific microRNA miR122 were incorporated into E1A gene of Ad6. miR122 regulation significantly decreased Ad6 replication in hepatocytes and consequently hepatotoxicity because of the negative regulation of miR122. Cytotoxicity assay using primary or established prostate cancer cell lines showed robust oncolytic activity of Ad6miR. Systemic treatment of established tumors with Ad6miR showed strong antitumor activity, comparable with that of Ad6 or Ad5. Although Ad6 evaded Kupffer cells, its blood clearance rate was as rapid as Ad5. The vast majority of Ad6 particles intravenously injected localized in liver sinusoidal endothelial cells rather than previously reported Kupffer cells. Elevating Ad6miR injection dose increased circulating Ad6miR concentration and its antitumor efficacy. miR122 regulation of Ad6 significantly improves its safety profile after systemic administration, which allows increasing therapeutic doses leading to improved anticancer efficacy of systemic treatment of castration-resistant prostate cancer.

Combating Hepatitis C Virus by Targeting MicroRNA-122 Using Locked Nucleic Acids

MicroRNAs have been predicted to regulate the stability and translation of many target mRNAs that are involved in modulating disease outcome. Thus, valuable strategies to enhance or to diminish the function of microRNAs are needed to manipulate microRNA-mediated target gene expression. Recently, it has become apparent that one class of antisense oligonucleotides, locked nucleic acids, can be used to sequester microRNAs in the liver of a variety of animals including humans, opening the possibility of applying locked nucleic acid-mediated gene therapy. This review summarizes the success of sequestration of liver-specific microRNA miR-122 by antisense locked nucleic acids and their use in combating hepatitis C virus in clinical trials.

High-Throughput Luciferase Reporter Assay for Small-Molecule Inhibitors of MicroRNA Function

MicroRNAs (miRNAs) are endogenous, single-stranded, noncoding RNAs of 21 to 23 nucleotides that regulate gene expression, typically by binding the 3′ untranslated regions of target messenger RNAs. It is estimated that miRNAs are involved in the regulation of 30% of all genes and almost every genetic pathway. Recently, the misregulation of miRNAs has been linked to various human diseases including cancer and viral infections, identifying miRNAs as potential targets for drug discovery. Thus, small-molecule modifiers of miRNAs could serve as lead structures for the development of new therapeutic agents and be useful tools in the elucidation of detailed mechanisms of miRNA function. As a result, we have developed a high-throughput screen for potential small-molecule regulators of the liver-specific microRNA miR-122, which is involved in hepatocellular carcinoma development and hepatitis C virus infection. Our small-molecule screen employs a Huh7 human hepatoma cell line stably transfected with a Renilla luciferase sensor for endogenous miR-122. The assay was optimized and validated using an miR-122 antisense agent and a previously identified small-molecule miR-122 inhibitor. The described reporter assay will enable the high-throughput screening of small-molecule miR-122 inhibitors and can be readily extended to other miRNAs.

Mechanisms Involved in the Development of Chronic Hepatitis C as Potential Targets of Antiviral Therapy

At present, about 3% of the human population are infected with hepatitis C virus (HCV). The first, acute stage of the disease is usually asymptomatic. However, only 15-25% of the infected eliminate the virus, while the remaining patients develop chronic hepatitis C (CHC). After 10-30 years of CHC, cirrhosis occurs in 20-30% of patients; 5-10% of this group eventually suffer from hepatocellular carcinoma. Unfortunately, up till now no effective methods protecting against HCV or allowing for efficient CHC treatment have been elaborated. This is primarily because not much is known about the mechanism of CHC emergence and the factors affecting anti-HCV therapy. There are several lines of evidence that some specific features of the virus, especially its high genetic variability might be responsible for the maintenance of HCV infection. Moreover, a few mechanisms which affect host-virus interactions and can additionally support CHC development have recently been identified. Hybridization between the host-encoded, liver-specific microRNA (miR-122) and the 5'-untranslated region of HCV genome was shown to be required for effective viral RNA replication. It was also postulated that HCV proteins mimic some of the human ones; that is why the virus is not eliminated. Another hypothesis assumes that interactions between HCV E2 glycoprotein and CD81 receptor modulate various cellular pathways, thus supporting viral propagation. There is no doubt that a better understanding of the mechanisms described above is of great importance for designing new therapeutic strategies and anti-HCV drugs.

HepG2 Cells Expressing MicroRNA miR-122 Support the Entire Hepatitis C Virus Life Cycle

The liver-specific microRNA miR-122 is required for efficient hepatitis C virus (HCV) RNA replication both in cell culture and in vivo. In addition, nonhepatic cells have been rendered more efficient at supporting this stage of the HCV life cycle by miR-122 expression. This study investigated how miR-122 influences HCV replication in the miR-122-deficient HepG2 cell line. Expression of this microRNA in HepG2 cells permitted efficient HCV RNA replication and infectious virion production. When a missing HCV receptor is also expressed, these cells efficiently support viral entry and thus the entire HCV life cycle.

The Control of Iron Homeostasis: microRNAS Join the Party

Castoldi M, Spasic MV, Altamura S, et al. (Department of Pediatric Hematology, Oncology, and Immunology, University of Heidelberg, Heidelberg, Germany; others). The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice. J Clin Invest 2011;121:1386–1396. Our understanding of the regulation of iron homeostasis has increased rapidly in the last decade. The identification of the iron regulatory hormone hepcidin, and the genes underlying iron disorders such as hereditary hemochromatosis have been instrumental in identifying the molecules necessary for the control of iron balance....