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Unravelling the importance of microRNAs during hepatitis C virus infection in the human liver

2009; Elsevier BV; Volume: 51; Issue: 3 Linguagem: Inglês

10.1016/j.jhep.2009.03.018

1600-0641

Sébastien Pfeffer, Thomas F. Baumert,

Circular RNAs in diseases

Decreased levels of microRNA miR-122 in individuals with hepatitis C responding poorly to interferon therapy. Sarasin-Filipowicz M, Krol J, Markiewicz I, Heim MH, Filipowicz W.Several microRNAs (miRNAs), including liver-specific miR-122, have been implicated in the control of hepatitis C virus (HCV) RNA replication and its response to interferon (IFN) in human hepatoma cells. Our analysis of liver biopsies from subjects with chronic hepatitis C (CHC) undergoing IFN therapy revealed no correlation of miR-122 expression with viral load and markedly decreased pretreatment miR-122 levels in subjects who had no virological response during later IFN therapy; other investigated miRNAs showed only limited changes. These data have implications for the prospect of targeting miRNAs for CHC therapy.[Abstract reproduced by permission of Nat Med 2009;15:31–33] Decreased levels of microRNA miR-122 in individuals with hepatitis C responding poorly to interferon therapy. Sarasin-Filipowicz M, Krol J, Markiewicz I, Heim MH, Filipowicz W. Several microRNAs (miRNAs), including liver-specific miR-122, have been implicated in the control of hepatitis C virus (HCV) RNA replication and its response to interferon (IFN) in human hepatoma cells. Our analysis of liver biopsies from subjects with chronic hepatitis C (CHC) undergoing IFN therapy revealed no correlation of miR-122 expression with viral load and markedly decreased pretreatment miR-122 levels in subjects who had no virological response during later IFN therapy; other investigated miRNAs showed only limited changes. These data have implications for the prospect of targeting miRNAs for CHC therapy. [Abstract reproduced by permission of Nat Med 2009;15:31–33] Antiviral defense was one of the first elucidated roles played by small non-coding RNAs during RNA silencing processes in plants. The genetic requirements of this response against viruses are now well defined in plants but also in insect organisms [[1]Ding S.W. Voinnet O. Antiviral immunity directed by small RNAs.Cell. 2007; 130: 413-426Abstract Full Text Full Text PDF PubMed Scopus (1020) Google Scholar]. Hence, Dicer or Dicer-like enzymes will recognize the invading viral RNA, or its replication intermediate, to cleave it into small interfering (si) RNAs. These siRNAs are then loaded into the RNA-induced silencing complex (RISC), invariably containing a member of the Argonaute family, which in turn will target the viral genome for degradation. This mechanism does not seem to have been preserved as such in mammals [[2]Cullen B.R. Is RNA interference involved in intrinsic antiviral immunity in mammals?.Nat Immunol. 2006; 7: 563-567Crossref PubMed Scopus (120) Google Scholar], probably due to the development of a more robust innate immune response involving interferon and PKR. Some viruses even take advantage of the cellular machinery to express their own micro (mi) RNAs [[3]Pfeffer S. Zavolan M. Grasser F.A. Chien M. Russo J.J. Ju J. et al.Identification of virus-encoded microRNAs.Science. 2004; 304: 734-736Crossref PubMed Scopus (1226) Google Scholar]. However, several studies have shown that miRNAs, an endogenous class of small non-coding RNAs, can to some extent recognize viral transcripts and regulate their expression. The biogenesis of miRNA shares some components of the RNAi machinery [[4]Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (27032) Google Scholar] (Fig. 1A). Similarly to siRNAs, miRNAs assemble into RISC complexes and guide them on target transcripts mostly by inhibiting translation after imperfect binding in their 3′ UTR. In 2005, a first report described that the primate foamy virus could be negatively regulated by the cellular miRNA miR-32 [[5]Lecellier C.H. Dunoyer P. Arar K. Lehmann-Che J. Eyquem S. Himber C. et al.A cellular microRNA mediates antiviral defense in human cells.Science. 2005; 308: 557-560Crossref PubMed Scopus (749) Google Scholar], although it was not clear whether this recognition was fortuitous or played a real role during infection. This initial observation has been confirmed for other viruses, and a definite proof of the antiviral role of cellular miRNAs during viral infection was shown in a mouse model of vesicular stomatitis virus infection [[6]Otsuka M. Jing Q. Georgel P. New L. Chen J. Mols J. et al.Hypersusceptibility to vesicular stomatitis virus infection in Dicer1-deficient mice is due to impaired miR24 and miR93 expression.Immunity. 2007; 27: 123-134Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar]. The relationship between hepatitis C virus (HCV) and the miRNA machinery is more complex. Hence, the first miRNA that was described to interact with HCV, the liver specific miR-122, exerts a positive effect on the virus replication in cell culture after imperfect binding in the viral 5′ UTR [[7]Jopling C.L. Yi M. Lancaster A.M. Lemon S.M. Sarnow P. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA.Science. 2005; 309: 1577-1581Crossref PubMed Scopus (2011) Google Scholar] (see Fig. 1B). This finding suggested that miR-122 is an important host factor for HCV replication and could at least partly explain why HCV replicates more efficiently in Huh 7 cells compared to other cell lines. Indeed, they are the only liver cell line that express a detectable amount of miR-122 [[7]Jopling C.L. Yi M. Lancaster A.M. Lemon S.M. Sarnow P. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA.Science. 2005; 309: 1577-1581Crossref PubMed Scopus (2011) Google Scholar]. Conversely, another study has shown that some miRNAs that were induced by interferon (IFN) β could inhibit HCV replication in cell culture [[8]Pedersen I.M. Cheng G. Wieland S. Volinia S. Croce C.M. Chisari F.V. et al.Interferon modulation of cellular microRNAs as an antiviral mechanism.Nature. 2007; 449: 919-922Crossref PubMed Scopus (716) Google Scholar] (Fig. 1B). In the same study, the authors had demonstrated that miR-122 was down-regulated following IFN-β treatment and postulated that this finding could contribute to the antiviral activity of IFN-based therapies. The current standard of therapy for chronic hepatitis C (CHC) consists of the combination of pegylated IFN-α and ribavirin and is characterized by viral resistance in a large fraction of treated patients. To address the functional relevance of miRNAs for the antiviral activity of IFN-α in HCV infection in vivo, Sarasin-Filipowicz and colleagues analyzed liver biopsies from subjects with CHC undergoing IFN therapy and quantified by qRT-PCR a number of miRNAs including miR-122 in these samples [[9]Sarasin-Filipowicz M. Krol J. Markiewicz I. Heim M.H. Filipowicz W. Decreased levels of microRNA miR-122 in individuals with hepatitis C responding poorly to interferon therapy.Nat Med. 2009; 15: 31-33Crossref PubMed Scopus (273) Google Scholar]. The authors then compared levels of these miRNAs between primary nonresponder (PNR) subjects or complete early virological responders (cEVR) subjects. To get an absolute quantification of each miRNA analyzed, they used chemically synthesized miRNAs as standards. This thorough approach enabled them to calculate the exact number of molecules per 10 pg of total RNA analyzed, or per cell and allowed for the first time to study the impact of miRNAs for HCV infection in the human liver in vivo. One of the key observations of the study is that the level of miR-122 was significantly lower in PNR subjects than in cEVR subjects regardless of the HCV genotype examined. Furthermore, the authors demonstrated that the level of HCV RNA in liver and serum of the HCV-infected patients did not correlate with the abundance of miR-122. These findings suggest that the impact of miR-122 for HCV replication may be less prominent in the HCV-infected liver in vivo than in cell culture model systems. The observed effect on miR-122 in nonresponders could be explained by the fact that it is a gene negatively regulated by IFN, as was observed by Pedersen et al. in Huh 7 cells [[8]Pedersen I.M. Cheng G. Wieland S. Volinia S. Croce C.M. Chisari F.V. et al.Interferon modulation of cellular microRNAs as an antiviral mechanism.Nature. 2007; 449: 919-922Crossref PubMed Scopus (716) Google Scholar]. However, the level of miR-122 in liver biopsies collected before and 4 h after pegylated IFN-α treatment, or in mouse livers treated with IFN for 49 h, was not affected significantly (Fig. 1B). The authors also measured the levels of the same miRNAs shown previously shown to be up-regulated by IFN-β in Huh 7 cells, i.e. miR-1, miR-196, miR-296, miR-351 and miR-448. All of them but miR-448 were induced two- to six-fold by IFN-β. Nonetheless, in terms of absolute number of miRNA copies per cell, most of them were present at very low levels jeopardizing a substantial biological effect. Similar observations were made in mouse liver, and in human biopsies, except for miR-296 that accumulated to significant levels. However, according to the authors and based on an analysis of perfused mouse liver, this miRNA originates from blood cells in the liver, and therefore cannot be accounted for any effect on HCV. In summary, the findings of this study provide a marked advancement in the understanding of the role of miRNAs in HCV infection and the antiviral activity of IFN in vivo. The absent modulation of miRNA levels in the human liver in vivo following administration of interferon suggests that it is unlikely that the antiviral effects of IFN during CHC therapy can be predominantly explained by changes in the levels of the investigated miRNAs. A second very important observation of the study is the absence of a correlation between intrahepatic miR-122 and HCV RNA levels, since one would expect that a reduction in miR-122 should decrease viral replication and increase the effects of the therapy. Thus, the findings of Sarasin-Filipowicz and colleagues suggest that observations on miRNA–HCV interactions in vitro cannot be directly extrapolated for HCV infection of the human liver in vivo. What are the clinical implications of these findings? The results obtained by Sarasin-Filipowicz and colleagues do not preclude the use of miR-122 as a therapeutic target. If it turns out that even low levels of the miRNA are necessary for facilitating HCV replication in vivo, its low abundance in PNR patients might be beneficial for future therapeutic interventions involving its inhibition by antisense oligonucleotides such as antagomirs [[10]Krutzfeldt J. Rajewsky N. Braich R. Rajeev K.G. Tuschl T. Manoharan M. et al.Silencing of microRNAs in vivo with ‘antagomirs’.Nature. 2005; 438: 685-689Crossref PubMed Scopus (3146) Google Scholar]. Furthermore, the finding that miR-122 expression is significantly lower in PNR subjects than in complete EVR subjects (Fig. 1C) suggests this could be a biomarker suitable for predicting the outcome of IFN therapy [[9]Sarasin-Filipowicz M. Krol J. Markiewicz I. Heim M.H. Filipowicz W. Decreased levels of microRNA miR-122 in individuals with hepatitis C responding poorly to interferon therapy.Nat Med. 2009; 15: 31-33Crossref PubMed Scopus (273) Google Scholar] in combination with other markers as described previously [11Asselah T. Bieche I. Narguet S. Sabbagh A. Laurendeau I. Ripault M.P. et al.Liver gene expression signature to predict response to pegylated interferon plus ribavirin combination therapy in patients with chronic hepatitis C.Gut. 2008; 57: 516-524Crossref PubMed Scopus (190) Google Scholar, 12Sarasin-Filipowicz M. Oakeley E.J. Duong F.H. Christen V. Terracciano L. Filipowicz W. et al.Interferon signaling and treatment outcome in chronic hepatitis C.Proc Natl Acad Sci USA. 2008; 105: 7034-7039Crossref PubMed Scopus (562) Google Scholar]. It might actually prove interesting to see whether any of the previously identified biomarkers do cluster with low miR-122 levels, in order to elucidate the link between the low pretreatment miR-122 levels and IFN-resistance. Future investigations of the molecular mechanisms underlying this observation will undoubtedly further advance our understanding of the pathogenesis of HCV infection and antiviral resistance and may ultimately pave the way for novel antiviral strategies. The authors thank J. Perot and G. Suffert for critical reading of the manuscript and acknowledge financial support from the Agence Nationale de la Recherche (ANR-07-MIME-012-01; S.P. and ANR-05-CEXC-008; T.F.B), CNRS (S.P.), Inserm (T.F.B.), the European Union (LSHM-CT-2004-503359; T.F.B.), the Else Kroener-Fresenius Foundation (A83/06 T.F.B) and the Ligue contre le Cancer (S.P., T.F.B.).