"Post/Trans/Hyper?: The Case for Humanism", in: Monitor ISH: Journal for the Humanities and the Social Sciences, Vol. 23, N°. 2, 2021, pp. 113-126. https://journal.almamater.si/index.php/monitorish/issue/view/33/PDF2302 Post/Trans/Hyper ? The Case for Humanism Klaus-Gerd Giesen Professor of Political Science Université Clermont Auvergne Clermont-Ferrand, France klaus@giesen.fr

The Covid-19 pandemic has been, and continues to be, a tremendous challenge for all humans. In the following pages I will first argue that the crisis has led to a humanist leap on a planetary scale. Secondly, I will examine the possible objections to my argument from three currents of thought (posthumanism, transhumanism, hyperhumanism) that believe, each in its own way, that the coronavirus pandemic demonstrates, on the contrary, that it is important to go beyond any humanist interpretation and, consequently, to give a completely different meaning to the coronavirus disruption.

I.

First, the thesis defended here can be summarized as follows: through the Covid-19 crisis humanism has perhaps just celebrated its greatest triumph of all times, if we understand humanism as any effort (moral, cultural, spiritual, educational, scientific, etc.) to make humans better, to raise them above their lower gregarious instincts. Indeed, against all utilitarian calculations based on economic consequences, which are undoubtedly extremely harmful in the short and medium term, the whole planet has mobilized to try to protect, by multiple and prolonged confinements and other spectacular measures (putting bodies at a distance, systematic wearing of masks, invention of vaccines and systematization of vaccination, etc.), above all the most vulnerable among us, those who run the greatest risk of dying from Covid-19 in times of limited medical resources.

The relationship not to death, but to inequality in the face of death, has thus been profoundly modified: on a global scale, the functioning of the economy and society has been slowed down considerably in an attempt to defend, albeit very imperfectly, first the most fragile, and then -and only then -all those who so desire. Unlike previous epidemics in the history of humanity, almost all the governments of the world have hastily and simultaneously organized what could be called a humanist planetary learning process, and a large majority of the populations willingly participated, not only out of interest but also often out of conviction and genuine concern for others. The scientific race for vaccines and the sharing of epidemiological data on a global scale is part of it. An earthquake, coming from the depths, which magnifies the human being, his capacity to rise beyond its biological condition and to organize itself as humanity, and which ultimately magnifies life itself. An immense hope for the future of homo sapiens sapiens.

Human life, which by definition has no price (Kant), nevertheless has a cost. The new imperative to preserve it at almost any economic cost indicates, once again, a sudden reversal of perspective: the primacy of life over the economy; health as a public good; the return to the precautionary principle (stocks of masks, tests, vaccines, hospital beds, respirators, etc.) and the sanctuarization of certain health spaces and functions; the astonishment of the former colonial and imperialist powers (Europe, USA) at the much more efficient management of the epidemic by many states considered to be "developing" (Vietnam, Taiwan, Vanuatu, etc.), a not inconsiderable cultural shock for any eurocentric attitude ; the role of a certain number of multilateral world organizations, such as the WHO, whose financing and competences should be reinforced in the future.

Never again will it be possible to say that it is not possible to stop, or at least to strongly slow down, the productivist mega-machine of triumphant capitalism. Since the beginning of 2020 this has been done several times, over long weeks, even months.

The consequences have proven to be in part very beneficial, both for the individual and humanity as a whole, as well as for nature: a considerable reduction in pollution of all kinds, simpler and healthier food, less noise, revitalization of some natural areas, a drastic reduction in car or subway journeys to work, replacement of business trips by plane with video conferences, a decrease in superfluous consumption, a renaissance of local leisure and tourism, in short: acceptance of our own vulnerability, deceleration of time lived and return to the essential and to simplicity -a true revelation for many citizens in affluent countries.

We may have caught a glimpse of tomorrow's world. In any case, ecological and lifestyle awareness has been singularly strengthened, with a lasting effect on a planetary scale. Here again, it is important to seize this historic moment to try to change the course of things in favor of an ever-increasing quality of life and the preservation of nature. Starting with our daily behaviors, and by the consequent reduction of the externalization of the environmental costs of production and consumption.

Almost overnight, many neoliberal dogmas have collapsed like houses of cards. We are witnessing the great return of the state to the economic game, beyond its traditional role as insurer of last resort. Gone, with a magic sweep, are the policies of austerity, public debt thresholds, limitation of central bank actions, autonomy of the financial sphere from the real economy, privatization of all individual risks. Instead we find gigantic recovery plans of the states and the EU, on a scale never seen before and partly in favor of investments in the green economy alone, a state socialization of the losses suffered by businesses and workers during periods of confinement, and an almost unlimited refinancing of private assets in some

Western countries, that is to say, everything that was considered until two years ago as real heresies by the dominant economic doctrine.

More generally, the crisis reveals that in affluent countries the search for well-being, rather than just the accumulation of consumer goods and the increase in wealth measured by purely quantitative indicators of growth, could become the cornerstone of Western societies (Boyer 2020, 90-94). « Progress » may be changing its definition. Thus, a humanist permutation of values, already begun before the appearance of the coronavirus, seems to accelerate, including through the objective of a controlled growth, or even degrowth.

We have the immense privilege of witnessing a major disruption. This is frightening, destabilizing, and remains a source of many serious dangers. A mourning is never easy to live. At the same time, a new world is also being born: it is our future. A revolutionary humanist situation is taking place, which remains for the moment still a little obscure. As always, a restoration, or even a reactionary movement, is naturally also possible. Everything depends on the political struggles that follow. 1 II.

At least three currents of thought can raise objections to such a radically humanist and rather optimistic interpretation of the Covid crisis. They have in common the view that humanism is no longer sufficient, and propose to grasp the historical moment of the crisis in order to outbid humanism. However, there are also important differences between them.

The first current is that of posthumanism. As its name indicates it wants to go beyond the classical humanism, paradoxically estimating that humanism itself would have gone too far, starting with its anthropocentric obsession. The thinkers attached to it, as for example the late Jacques Derrida, question the very concept of human nature, and underline the importance of non-human agents. Often close to the deep ecology movement, they advocate a radical change of thinking. Various "collapsologist" thinkers, such as Pablo Servigne, believe that the extreme ecological vulnerability of our consumerist societies, operating at a frantic pace and over vast distances, would have favored the pandemic crisis, and that this one heralds a far more serious collapse. The philosopher Dominique Bourg thinks that "the virus comes from bats, via the pangolin. It is because their habitats have been destroyed that bats have moved closer to human dwellings and domestic animals. To this phenomenon is added the disappearance of species. All this leads to zoonoses, diseases that pass from animals to humans. And he draws from this a liberticidal, even authoritarian, instruction for political action: "To repair the living is to refuse that each person can in his corner decide his way of life" (Le Temps, April 22, 2020). The Parisian sociologist Bruno Latour goes further by evoking, in a perfectly posthumanist mode, "the sudden and painful realization that the classical definition of society -humans among themselves -has no meaning. The state of the social depends at every moment on the associations between many actors, most of whom do not have human form. This is true of microbes -we have known this since Pasteur -but also of the Internet, the law, the organization of hospitals, the capacities of the State, as well as the 1 The previous paragraphs are partially translated from my personal French blog: Giesen 2020.

climate" (Latour 2020). In short, in his great impulse of deep ecology, tinged with catastrophism, Latour dissolves society into a great magma of all living beings where humans remain in the minority and must quickly adapt at all costs, under penalty of disappearing in a not too distant future.

The main counter-objection that one can formulate to such a posthumanist posture lies in the fact that, paradoxically, it neglects the responsibility of the human being with regard to the current state of the world. Indeed, in the course of its evolution it has acquired such a domination over the rest of "nature" that it is simply no longer possible to go back: now the human being must assume its overwhelming superiority and the fact that it has forever fundamentally altered "nature". As a result, he is and remains entirely responsible for what happens next. Such an overwhelming responsibility therefore still refers to an anthropocentric and humanist point of view. Minimizing it, by trying to "drown" the figure of mankind in a great magma of all living beings, means actually resigning in face of the gravity of the facts.

The pandemic demonstrates that, thanks to science and culture, humanity is capable of great and rapid mutations, for example by developing protective vaccines against the virus. Such humanist impulses to surpass oneself can, and must, also be implemented to "renaturalize" the world. As explained above, the Covid-19 crisis can be one of the propelling movements.

The second objection to any humanist interpretation of the pandemic situation comes from transhumanism. As a form of ultraliberalism it naturally remains very concerned with individual liberties, first of all with the freedom of circulation of information that would allow fighting effectively against the pandemic. And then, transhumanists naturally advocate the absolute freedom of research, including that which no longer differentiates between "repair" and "enhancement" of the human being in order to be biologically better armed against the virus, or to prevent future pandemics. Thus, the avant-gardist Ray Kurzweil pleads for a combination of Artificial Intelligence and biotechnologies and writes about it: « We are seeing the beginnings of a profound paradigm shift in health technology » [START_REF] Kurzweil | La crise sanitaire incite à se préparer à la mutation climatique[END_REF]. This is not surprising: science alone would make it possible to improve the human species in order to protect it from any present and future virological threat, and to raise it biologically to a higher level, in particular through the systematic use of NBIC technologies: the increasingly systematic interconnection of Nanotechnologies, Biotechnologies, Informatics and Cognitive Sciences which leads to a new "Great Convergence" producing the advanced and growing integration between the infinitely small (N), the manufacturing of the biological (B), the thinking machines (I) and the cognitive study of the human brain (C) [START_REF] Besnier | Demain les posthumains. Le futur a-t-il encore besoin de nous ?[END_REF].

Such materialism obviously has little to do with classical humanism. It aims at overcoming the latter, not by elevating the human being culturally, educationally and spiritually, but exclusively by technological means. Advocating a real overcoming of the human condition in order to gradually develop -in a "proactive" way and thus in opposition to the precautionary principle (Fuller and Lipinska 2014) -a posthuman creature. Indeed, transhumanism advocates a headlong rush: human beings would be too limited biologically to be able to effectively face the challenges of the world's growing complexity, especially as far as the increasing threat of pandemics is concerned. Therefore, their capacities have to be increased by integrating them with all kinds of emerging technologies. Transhumanists such as Oxford philosopher Nick Bostrom consider that the Darwinian evolution is proceeding too slowly and that the biological condition of human beings it has established remains largely unsatisfactory [START_REF] Harris | Enhancing Evolution: The Ethical Case for Making Better People[END_REF].

The political objective is therefore clearly stated. It is nothing less than the establishment of a new (post)human being and, consequently, an entirely new society, a grand plan that some political ideologies (communism, fascism, etc.) have pursued in the past in other, ultimately less radical. The normative core of transhumanism can thus be summarized around three main purposes (Giesen 2018):

1/ The "natural" human being is obsolete and must be improved by technology, which therefore becomes a vector for the artificial continuation of the hominization process.

Transhumanism thus brings the human species, as such, abruptly into the political game, and immediately recalls the formulation used by Michel Foucault: "What could be called society's 'threshold of biological modernity' lies in the moment when the species is at stake in its own political strategies…" (Foucault 1976, 187-188). In other words, according to the transhumanist authors, mankind must be replaced by a post-sapienssapiens species. If necessary, zoologically speaking, this will be a moment of true speciation, i.e. a truly exceptional situation where a new, artificially created human species appears in the world.

2/ The posthuman being and the machine must be fully merged, which goes far beyond the current human-machine interface (e. g. interaction with mobile phones or computers).

The spectacular figure of the human-machine is a form of hybridization, as proposed repeatedly for instance by Ray Kurzweil who believes that the human being must become an integral part of the machine [START_REF] Kurzweil | The Singularity is Near: When Humans Transcend Biology[END_REF]. It should become possible to program it like software, reprogramming if necessary, including to boost its resistance to biological virus. It is nothing less than subjecting the human being entirely to technical rationality to the detriment of human subjectivity: a point from which technology, considered as the new driving force of hominization, paradoxically also becomes the main vector of dehumanization. Thus, transhumanist machinism is fundamentally anti-humanist, even if only because the machine is by definition inhuman.

3/ Therefore, it is also necessary to go beyond humanism. While humanism focuses on a possible moral improvement of the individual through education, culture and spiritual activities ("the humanization of the human"), transhumanism proposes entirely new values by stipulating the need for a transition to a post-human creature that must constantly improve itself by integrating new technical artifacts. These are called on to replace the moral, educational and cultural effort.

All together, the project of the transhumanist philosophy perfectly embodies the anti-humanist programme once dissected and denounced by the philosopher Günther Anders: to achieve "the obsolescence of humankind" [START_REF] Anders | Die Antiquiertheit des Menschen[END_REF] 2002), and its extinction as a species.

Therefore, it should be rejected. As for Hervé Fischer, he explains: "I speak of hyperhumanism to emphasize that we have today a humanism based on hyperlinks and that, at the same time, it develops this augmented consciousness, so more humanism" (Le Devoir, October 5, 2013). According to him, the new communication technologies therefore produce, so to speak automatically, a surplus of humanism. Thus, hyperhumanism removes from transhumanism most of its machinist aspects to keep only an excessive belief in digital technologies and its algorithms. If it remains true that virtual communication has singularly increased during the various confinements and that it has undoubtedly contributed to alleviate the loneliness of some, as well as to circulate more quickly the relevant information related to the Covid-19 virus, it is even much more relevant that communication technology alone cannot elevate the human being, even in its numerical totality, if each individual human being does not make a personal humanist effort beforehand.

Hyperhumanism thus remains well and truly anchored in machinism; it even represents the culmination of the machinist fetishism of the cybernetic movement born after the Second World War, as embodied by Norbert Wiener and other mathematicians and philosophers [START_REF] Wiener | Cybernetics: Or Control and Communication in the Animal and the Machine[END_REF][START_REF] Wiener | The Human Use of Human Beings[END_REF].

With the hyperhumanist position we find ourselves not in a hyperhuman world, but in a hypohuman reality. Because here again, as in the transhumanist stance, the human is called to integrate, at least partially, with the machine, to let itself be led by cybernetic algorithms. An increasingly cold world, a hybridization opposite to the humanist dream of becoming more and more human on the moral, cultural and spiritual levels, including in the acceptance without any hesitation of nature and its nature.

The three mentioned objections to a humanist interpretation of the pandemic do not seem to lead to convincing perspectives, since each one finally reduces, in one way or the other, the human part. However, the lesson of the Covid crisis 2020-2021 is above all that the outbidding of humanism does not seem to be relevant and that we need, on the contrary, always more humanity and human vitalism.

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  The third objection comes from hyperhumanism. It criticizes the posthumanist and transhumanist approaches, but also proposes to go beyond classical humanism. Authors such as Joël de Rosnay and Hervé Fischer propose in particular to outbid the humanist focus on the individual alone, and to aim from the outset at humanity as such, which would be called upon to rise collectively on the moral, cultural and spiritual levels thanks to augmented collective intelligence. This one could be constituted progressively thanks to the new communication technologies, to lead to the cybionte, a new planetary collective being, nourished of reflections and spiritualties[START_REF] Rosnay | L'homme symbiotique[END_REF]. The Covid crisis of 2020/2021 would have demonstrated that "it will be a matter of existing in symbiosis with artificial intelligence [, and that] the future of humanity lies not in the individual, but in the organized community made possible by social networks, blogs and other sharing platforms."(Le Temps, July 4, 2021)


Angélique Gougelet

Sabine Colnot

Chapter 6: MicroRNAs linking cancer and inflammation: focus on liver cancer

Keywords: microRNA signature, liver disease, hepatocellular carcinoma, inflammation, miRNA-based therapies

Due to their great variety of targets, miRNAs play a key role in number of physiological processes and in oncogenesis. The identification of specific miRNA signatures in various types of tumours, including hepatocellular carcinoma (HCC), highlighted the dual role of miRNAs, both oncogenic and tumour suppressive. HCC is a cancer of poor prognosis that mainly develops on an injured liver. Here, we will review the current knowledge concerning the deregulation of miRNA expression at all stages of hepatocarcinogenesis, including the underlying liver disease, in particular steatohepatitis and fibrosis. We will develop the data concerning the identification of specific miRNA signatures in hepatocellular carcinoma, either in tumor or in sera. Since HCC appears in a context of chronic inflammation after repeted liver injury, it also constitutes one of the best example in which miRNAs are key mediators for the communication between tumor cells and tumor microenvironment. The role of miRNAs in liver inflammation will be detailed in this chapter.

To conclude, all studies focusing on miRNAs in HCC argue for their possible use as diagnostic and prognostic biomarkers. Promisingly, miRNAs appear as potent therapeutic targets to improve HCC treatment, for which surgery remains the most frequent therapeutic option.

Liver architecture

By its central position, liver plays a crucial metabolic function in the body. The liver is divided into lobules centered onto the central vein and surrounded by six portal triads, composed of a branch of the hepatic artery, the portal vein and a biliary duct (Figure 6.1).

Blood flow within the liver determines the organization around the sinusoids of the liver cell plate, the anatomical unit of the hepatic parenchyma. The hepatocyte population represents 60% to the total number of liver cells and assumes the main liver functions.

According to their localization near the portal vein or around the central vein, the hepatocytes show different metabolic properties due to the expression of different enzymes. This phenomenon was defined by Jungermann as the liver metabolic zonation [START_REF] Katz | Autoregulatory shift from fructolysis to lactate gluconeogenisis in rat hepatocyte suspensions. The problem of metabolic zonation of liver parenchyma[END_REF]. Nonparenchymal liver cells also populate liver in the sinusoidal compartment of the lobule. They regulate hepatocyte function and protect them from liver injuries through the secretion of various molecules ( [START_REF] Bouwens | Liver cell heterogeneity: functions of non-parenchymal cells[END_REF] for a review). Three main cell types composed the wall of hepatic sinusoid: the sinusoidal endothelial cells (SECs), the Kupffer cells, and the hepatic stellate cells (HSC, also known as Ito cells). Sinusoidal endothelial cells constitute the wall of the hepatic sinusoid, in which they exert filtrating and endocytic activity. SECs also present original markers (i.e. the immunoglobulin receptors or CD4), which facilitate their interaction with Kupffer cells to clear immune complexes and to favor a T-cell tolerance. SECs are also able to secrete cytokines, eicosanoids, and some components Liver parenchyma is organized in cell plates distributed around the sinusoids. The sinusoid wall is composed of endothelial cells, hepatic stellate cells and Kupffer cells. The hepatic parenchyma is divided into two distinct populations of hepatocytes according to their localization around the central vein or the portal triad composed of the portal vein, the hepatic artery and a bile duct.

of the extracellular matrix (ECM). Kupffer cells are the liver macrophages. Their phagocytic activity is required to control damages induced by dietary and bacterial metabolites arriving from the gut. They also secrete immune mediators in response to bacterial products like lipopolysaccharide (LPS) to promote an oral tolerance to bacterial antigens [START_REF] Crispe | The liver as a lymphoid organ[END_REF].

During liver injury, Kupffer cells are activated, secrete inflammatory mediators (i.e. tumor necrosis factor α (TNFα), interleukin-10, reactive oxygen species…) to control the early phase of liver inflammation and to favor innate immune defense [START_REF] Seki | Toll-like receptors and adaptor molecules in liver disease: update[END_REF].

Hepatic stellate cells are perisinusoidal cells involved in collagen synthesis and vitamin A storage. During liver injury, HSCs transdifferentiate into myofibroblast-like cells; this constitutes the crucial event for inflammatory fibrotic process. In response to hepatocyte damages, HSCs secrete cytokines, chemokines and growth factors, and participate to ECM remodeling [START_REF] Brenner | New aspects of hepatic fibrosis[END_REF]. Finally, liver immunosurveillance is also assumed by intrahepatic lymphocytes called Pit cells, which reside in the sinusoid lumen after they have crossed the sinusoid endothelium ( [START_REF] Lalor | Recruitment of lymphocytes to the human liver[END_REF] for a review). In response to different damages, a panel of inflammatory cytokines and chemokines are secreted in liver (i.e. IFNγ, IP-10, MIP-1α…), which results in the recruitment of various immune cells. Different types of lymphocytes can be recruited in liver: CD4 and CD8 T cells, dendritic cells, liver-specific natural killer (NK) or NKT cells. All these cells are required for a correct protection against viral infections and tumor cells ( [START_REF] Parker | Immune functioning in non lymphoid organs: the liver[END_REF] for a review). Following repeated insults to liver, an exacerbation of the inflammatory response can occur and promote tumor development.

Liver cancer

Liver cancer constitutes the third cause of cancer-related death worldwide. The most frequent form of primary liver cancer is hepatocellular carcinoma (HCC) with a prevalence of 500 000 new cases/year. It mainly affects men with cirrhosis. HCC frequently appears in a context of chronic liver disease caused by infection with hepatitis B and C viruses (HBV and HCV, respectively), alcohol consumption, obesity or genotoxic exposure [START_REF] El-Serag | Hepatocellular carcinoma: epidemiology and molecular carcinogenesis[END_REF] for a review). The classical view of carcinogenesis is that a normal cell is submitted to the first « gatekeeping » mutation (i.e. single-base substitutions, deletion, insertion, translocation…) leading to a selective growth advantage to survive and proliferate, and to finally become a microscopic clone. The following « driver » mutations facilitate cell growth, and result in tumor development [START_REF] Vogelstein | Cancer genome landscapes[END_REF]. HCC is a complex and heterogeneous disease due to different genetic alterations, whose sequential appearance during the evolution from benign to malignant lesions is still poorly known. At the end of the 2000's, the global transcriptomic analysis performed on HCC samples established a molecular classification of these tumors [START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF]). The most frequently signaling pathways altered in HCC are the β-catenin pathway (15-40%), the p53 pathway (26%), and the Ras pathway (5-10%) [START_REF] De | Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas[END_REF][START_REF] Guichard | Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma[END_REF]. In brief, the subgroups of HCC mutated for the β-catenin, appear in an etiological context of HCV virus infection or alcohol abuse. They present a lower proliferation rate, are more genetically stable, well differentiated, cholestatic and of relative better prognosis [START_REF] Audard | Cholestasis is a marker for hepatocellular carcinomas displaying beta-catenin mutations[END_REF]); inversely, the other subgroups of HCC appeared after hepatitis B virus infection. They are characterized by a genetic instability, are poorly differentiated, steatotic and of bad prognosis (Figure 6.2). But liver cancer is also a perfect example of the relationship between tumor microenvironment and tumor development and/or progression. Over the past decades, the studies focusing on cancer do not consider so far the tumor cells as an unique entity but as an organ, whose development is orchestrated by surrounding cells, which could be immune cells, inflammatory cells, endothelial cells or fibroblasts… The tumor microenvironment was in consequence defined as a hallmark of cancer by Hanahan and Weinberg, and the tumorpromoting inflammation was defined these recent years as one of its emerging characteristic [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF]. To prevent an exacerbation of liver chronic inflammation, immune cells dedicated to protect the hepatic parenchyma drain the adult liver and their recruitment and /or activation influence hepatocarcinogenesis (Anson et al. 2012, He and[START_REF] He | NF-kappaB and STAT3 -key players in liver inflammation and cancer[END_REF].

Although numerous efforts have been conducted to improve the comprehension and the treatment of liver cancer, the median life expectancy after HCC diagnosis does not go beyond six months. In consequence, it appears crucial to identify new molecular actors involved in liver tumor progression, which could be useful for its diagnosis and its treatment.

In consideration with the key role of microRNAs (miRNAs) in metabolism, immunity, inflammation and cancer, these small RNAs appear as promising targets. In this chapter, we review the main data concerning the impact of microRNAs in HCC progression and the therapies based on miRNAs, which proceed from this molecular deciphering.

miRNAs in liver

Since the discovery of lin-4 in Caenorhabditis Elegans in 1993 [START_REF] Lee | The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[END_REF], miRNAs emerge as promising prognostic and diagnostic biomarkers, in particular in cancer.

More than half of the genes encoding miRNAs are located in cancer-associated regions or fragile sites [START_REF] Calin | Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers[END_REF]. Their abnormal expression contributes to the progression of tumors, in which they play an ambivalent role of tumour suppressor or oncogene. miRNA expression is submitted to a great variety of genetic regulations, similarly to more « classical » genes, i.e. gene amplification or deletion, promoter hypermethylation, translocation, single nucleotide polymorphism… A number of works based on a global analysis of miRNA expression in different types of tumors highlighted specific miRNA signatures according to the cellular and tissular origin of the tumors [START_REF] Lu | MicroRNA expression profiles classify human cancers[END_REF][START_REF] Volinia | A microRNA expression signature of human solid tumors defines cancer gene targets[END_REF]). These recent years, a number of studies have shown the key role of microRNAs in liver organogenesis and also in liver tumor development and/or progression [START_REF] Ladeiro | MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations[END_REF][START_REF] Murakami | Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues[END_REF]) (Figure 6.3).

The role of microRNAs in liver physiology and pathology has been supported by two works of Sekine et al., which showed by a transgenic mouse model that the deletion of Dicer, the crucial protein involved in miRNA maturation, results in a loss of liver zonation and the appearance of tumors (Sekine et al. 2009, Sekine et al. 2009). Thereafter, a number of studies has highlighted the preponderant role of a panel of miRNAs in embryonic liver development, whose the great majority are deregulated in hepatic diseases like alcoholic liver disease (ALD), non-alcoholic steatohepatitis (NASH) and fibrotic processes, all pathologies predisposing to HCC, and in HCC themselves (Figure 6.3) ( [START_REF] Wang | MicroRNAs in liver disease[END_REF] for a review).

6.4 Main miRNAs involved in liver metabolism.

MicroRNA expression in the liver changes during its development: in the adult liver in addition to miR-122, other microRNAs such as miR-21, miR-29a, miR-194 and the let-7 family are also abundant, while miR-92a and miR-483 are expressed in the fetal liver [START_REF] Girard | miR-122, a paradigm for the role of microRNAs in the liver[END_REF]. Due to the central metabolic function of liver, it is not surprising that hepatic miRNAs mainly target metabolic enzymes and in consequence finely regulate these processes.

6.4.1 miR-122 miR-122 is the most abundant microRNA in liver (70% of liver miRNAs). It is a target of the transcription factor HNF-4α, a master regulator of hepatocyte differentiation. miR-122 is involved in iron and lipid metabolisms and cholesterol synthesis ( (Vaulont 2011, Wen and Friedman 2012) for reviews). This miRNA targets cyclin G1 [START_REF] Gramantieri | Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma[END_REF]), a protein involved in G2/M cell cycle arrest in response to DNA damages, and the anti-apoptotic molecule Bcl-w [START_REF] Lin | miR-122 targets an anti-apoptotic gene, Bcl-w, in human hepatocellular carcinoma cell lines[END_REF]). In addition, miR-122 is used by hepatitis C virus for its replication. That's the reason why the first therapeutic strategy based on miRNA inhibition in liver was the anti-miR-122, Miravirsen, which succeeds in the treatment of chronic hepatitis C virus in primates [START_REF] Lanford | Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection[END_REF] and in humans [START_REF] Janssen | MicroRNA expression, survival, and response to interferon in liver cancer[END_REF]).

miR-370

miR-370 has also been described as a key regulator of cholesterol and lipid metabolism.

Iliopoulos et al. firstly showed that the overexpression of a dominant negative form of c-Jun leads to an up-regulation of miR-370 correlated to a downregulation of carnitine palmitoyl transferase-1 (CPT-1), which ends to impair fatty acid oxidation [START_REF] Iliopoulos | MicroRNA-370 controls the expression of microRNA-122 and Cpt1alpha and affects lipid metabolism[END_REF]. The authors also demonstrate in hepatic cell culture that miR-370 up-regulates miR-122 and SREBP-1c expression.

miR-33

miR-33a/b are also key regulators of lipid metabolism and cholesterol trafficking in cooperation with SREBP. By different approaches, three studies showed that miR-33 regulates cholesterol efflux and high-density lipoprotein formation through the targeting of Abca1 and Abcg1 [START_REF] Marquart | miR-33 links SREBP-2 induction to repression of sterol transporters[END_REF][START_REF] Najafi-Shoushtari | MicroRNA-33 and the SREBP host genes cooperate to control cholesterol homeostasis[END_REF][START_REF] Rayner | MiR-33 contributes to the regulation of cholesterol homeostasis[END_REF]. miR33a/b also regulated the β-oxidation of fatty acids by the binding to CPT-1 [START_REF] Gerin | Expression of miR-33 from an SREBP2 intron inhibits cholesterol export and fatty acid oxidation[END_REF]. miR-33 can also target AMPKα and thus increases the level of cholesterol, fatty acids and lipids in hepatocytes [START_REF] Davalos | miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling[END_REF]).

Main miRNAs deregulated in liver pathologies

Due to their essential role in metabolic processes, it appears intuitive that the expression of the miRNAs described in the last paragraph is altered in chronic liver diseases.

In particular, a prolonged decrease in miR-122 expression is associated with non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and fibrosis [START_REF] Alisi | Mirnome analysis reveals novel molecular determinants in the pathogenesis of diet-induced nonalcoholic fatty liver disease[END_REF][START_REF] Cheung | Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression[END_REF][START_REF] Tsai | MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis[END_REF]. In addition, a miRNA signature has also been identified for the different forms of liver diseases (see figure 6.2). 6.5.1 miR-155 in alcoholic liver disease miR-155 is mainly expressed by Kupffer cells. Its overexpression in these cells, and also in hepatocytes, is observed in alcohol-fed mice. This induction is responsible for the highest production of TNFα by Kupffer cells observed in patients with ALD [START_REF] Bala | Up-regulation of microRNA-155 in macrophages contributes to increased tumor necrosis factor {alpha} (TNF{alpha}) 21 production via increased mRNA half-life in alcoholic liver disease[END_REF].

Recently, a study performed by the same team highlighted the presence of miR-155 into exosomes, a multiprotein structure secreted by mammalian cells, in a mouse model of ALD [START_REF] Bala | Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug-induced, and inflammatory liver diseases[END_REF]. This microRNA targets SOCS1, an inhibitor of signal transducer and activator of transcription 3 (STAT3)/matrix metalloproteinase 9 (MMP9) cascade [START_REF] Yan | Hepatocellular carcinoma-associated mesenchymal stem cells promote hepatocarcinoma progression: Role of the S100A4-miR155-SOCS1-MMP9 axis[END_REF]).

miRNA deregulation in NAFLD and NASH

NAFLD is the most prevalent chronic liver disease in industrialized countries in expansion these recent years with the increase of obesity, leading to hepatic accumulation of triglycerides in the absence of alcohol consumption. This pathology could be characterized by a simple steatosis but could also result to NASH. The impact of miRNAs in NAFLD has been firstly assessed on steatotic human L02 hepatocytes cultured with high concentration of free fatty acids by Zheng et al. They correlate miR-10b overexpression with the triglyceride levels in L02 cells due to the targeting of the peroxisome proliferator-activated receptor α (PPAR α) [START_REF] Zheng | Effect of miRNA-10b in regulating cellular steatosis level by targeting PPAR-alpha expression, a novel mechanism for the pathogenesis of NAFLD[END_REF]. In mice fed with high-fat diet, Ahn et al. shows that a loss of miR-476b is associated with an increase in the lipoprotein lipase level [START_REF] Ahn | High fat diet induced downregulation of microRNA-467b increased lipoprotein lipase in hepatic steatosis[END_REF]. Finally, a recent study performed on mice depleted for the low-density lipoprotein receptor fed with a high fat/western-type diet correlates the decrease in miR-216 and miR-302a expression with NAFLD development [START_REF] Hoekstra | Nonalcoholic fatty liver disease is associated with an altered hepatocyte microRNA profile in LDL receptor knockout mice[END_REF]. Concerning the alteration of microRNAs in NASH, a first study of Cheung et al. on human samples reveals that miR-146b and miR-34a are induced in the liver of NASH subjects [START_REF] Cheung | Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression[END_REF]. miR-34a is a well-documented miRNA, which is a target of p53 and a key regulator of the NAD-dependent deacetylase Sirtuin-1. Sirtuin-1 inhibition leads to AMP Kinase dephosphorylation, HMGCoA reductase phosphorylation and hepatic cholesterol accumulation [START_REF] Min | Increased hepatic synthesis and dysregulation of cholesterol metabolism is associated with the severity of nonalcoholic fatty liver disease[END_REF]. miR-34a and miR-122 deregulation is also confirmed in mice fed with methyl-deficient diet. In this model, miR-155, miR-200b and miR-221 are found overexpressed, while miR-29c, miR-192 and miR-203 are downregulated [START_REF] Pogribny | Difference in expression of hepatic microRNAs miR-29c, miR-34a, miR-155, and miR-200b is associated with strain-specific susceptibility to dietary nonalcoholic steatohepatitis in mice[END_REF]).

miRNA in fibrosis

NAFLD pathology can also progress to complications like liver fibrosis. Liver fibrosis consists to a remodeling of liver architecture mainly orchestrated by hepatic stellate cells.

HSCs switch from a quiescent stage to myofibroblast-like cells. Activated HSC secrete ECM proteins, collagen and inflammatory mediators in response to liver injury. HSC express two crucial miRNAs, miR-29 and miR-199. miR-29 is a target of two inflammatory pathways in liver, the NFkB and TGFβ pathway. It targets the anti-apoptotic bcl-2 and Mcl-1 molecules [START_REF] Law | Emerging roles of microRNA in the intracellular signaling networks of hepatocellular carcinoma[END_REF]. In a mouse model of liver fibrosis induced by carbon tetrachloride, the family miR-29a/b/c is lost [START_REF] Roderburg | Micro-RNA profiling reveals a role for miR-29 in human and murine liver fibrosis[END_REF]. miR-199 is found in HSCs, and also in sinusoidal endothelial cells. Its expression is upregulated during fibrosis. In particular, its expression and those of miR-200a/b are highly increased in human samples and in a mouse liver fibrosis model [START_REF] Murakami | The progression of liver fibrosis is related with overexpression of the miR-199 and 200 families[END_REF]). The miR-34 family is also a crucial actor of liver fibrosis. Its expression is downregulated in fibrotic liver induced by dimethylnitrosamine in rats (Li et al. 2011).

To conclude, these miRNAs deregulated in all hepatic disease predisposing to cancer, are also affected in liver tumors and in particular in hepatocellular carcinoma. These microRNAs could constitute potent early biomarkers for the initiation and progression of liver tumors. They could also be promising targets for the prevention and the treatment of HCC.

6.6 miRNA signature in liver tumors 6.6.1 miRNA alteration in hepatocellular carcinoma Besides the divergence in the results obtained by the authors due to the differences in samples, miRNA measurement and statistical analyses, the global analysis of the miRNAs expressed in liver tumors highlighted a miRNA signature common to all types of HCC. As mentioned previously, the great majority of miRNAs found deregulated in chronic liver diseases are also found deregulated in HCC. A loss of miR-122 [START_REF] Ladeiro | MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations[END_REF], miR-29 [START_REF] Xiong | Effects of microRNA-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma[END_REF], and miR-199 [START_REF] Hou | Identification of miRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular carcinoma[END_REF] has been identified in HCC (Figure 2). In addition, other miRNAs with a tumor suppressive role have also been largely described. We could cite: Let-7 [START_REF] Lan | Hsa-let-7g inhibits proliferation of hepatocellular carcinoma cells by downregulation of c-Myc and upregulation of p16(INK4A). International journal of cancer[END_REF], miR-101 [START_REF] Su | MicroRNA-101, down-regulated in hepatocellular carcinoma, promotes apoptosis and suppresses tumorigenicity[END_REF]) and miR-124 [START_REF] Furuta | miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma[END_REF]. Inversely, some oncogenic miRNAs have also been identified: miR-155 [START_REF] Yan | Hepatocellular carcinoma-associated mesenchymal stem cells promote hepatocarcinoma progression: Role of the S100A4-miR155-SOCS1-MMP9 axis[END_REF], miR-21 [START_REF] Meng | MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer[END_REF]), miR-221/miR-222 [START_REF] Pineau | miR-221 overexpression contributes to liver tumorigenesis[END_REF], miR-181 [START_REF] Wang | TGFbeta-mediated upregulation of hepatic miR-181b promotes hepatocarcinogenesis by targeting TIMP3[END_REF], miR-224 [START_REF] Wang | Profiling microRNA expression in hepatocellular carcinoma reveals microRNA-224 up-regulation and apoptosis inhibitor-5 as a microRNA-224-specific target[END_REF], and the miRNA cluster miR-17/92 [START_REF] Yang | miR-17-5p Promotes migration of human hepatocellular carcinoma cells through the p38 mitogen-activated protein kinase-heat shock protein 27 pathway[END_REF])(Figure 2) ( (Huang andHe 2011, Law and[START_REF] Law | Emerging roles of microRNA in the intracellular signaling networks of hepatocellular carcinoma[END_REF] for reviews).

All these miRNAs participate to apoptosis (Table 6.4). The mitochondrial apoptotic pathway is particularly induced through the increase expression of anti-apoptotic actors (i.e.

Mcl-1 targeted by miR-101 [START_REF] Su | MicroRNA-101, down-regulated in hepatocellular carcinoma, promotes apoptosis and suppresses tumorigenicity[END_REF], and miR-29 [START_REF] Xiong | Effects of microRNA-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma[END_REF], bcl-w targeted by miR-122 [START_REF] Lin | miR-122 targets an anti-apoptotic gene, Bcl-w, in human hepatocellular carcinoma cell lines[END_REF], bcl-2 by miR-29 [START_REF] Xiong | Effects of microRNA-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma[END_REF])), and inversely through the inhibition of proapoptotic mediators (i.e. Bmf inhibited by miR-221 [START_REF] Gramantieri | MicroRNA-221 targets Bmf in hepatocellular carcinoma and correlates with tumor multifocality[END_REF], and Bim by miR-25 (Li et al. 2009)). miR-221/222 can also confer resistance to the extrinsic apoptotic pathway dependent on TNF-related apoptosis induced ligand (TRAIL) [START_REF] Garofalo | miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation[END_REF]). These microRNAs also regulate the tumor growth through the modulation of many cell cycle actors, in particular miR-122 targets cyclin G1 [START_REF] Gramantieri | Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma[END_REF], miR-221 regulates p27 [START_REF] Le Sage | Regulation of the p27(Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation[END_REF]) and p57 [START_REF] Fornari | MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma[END_REF], miR-124 represses the cyclin-dependent kinase 6 [START_REF] Furuta | miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma[END_REF]. Finally, all these miRNAs can also alter the survival of tumor cells by the targeting of receptor tyrosine kinase pathways like the ERK pathway targeted by miR-101 (Li et al. 2009), and Let-7c [START_REF] Shah | Peroxisome proliferator-activated receptor alpha regulates a microRNA-mediated signaling cascade responsible for hepatocellular proliferation[END_REF]), the c-MET-activated pathway by miR-34a (Li et al. 2009), or the PI3K/AKT pathway repressed by miR-21 [START_REF] Meng | MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer[END_REF], and miR-221/222 [START_REF] Garofalo | miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation[END_REF][START_REF] Wong | MiR-222 overexpression confers cell migratory advantages in hepatocellular carcinoma through enhancing AKT signaling[END_REF].

miR-221 is one of the miRNAs, for which the role in the development and the progression of liver cancer is the best-described. As mentioned before, this miRNA is induced during fibrosis and chronic liver disease. Number of studies showed its induction in HCC samples [START_REF] Fornari | MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma[END_REF][START_REF] Garofalo | miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation[END_REF][START_REF] Gramantieri | MicroRNA-221 targets Bmf in hepatocellular carcinoma and correlates with tumor multifocality[END_REF][START_REF] Le Sage | Regulation of the p27(Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation[END_REF][START_REF] Pineau | miR-221 overexpression contributes to liver tumorigenesis[END_REF][START_REF] Wong | MiR-222 overexpression confers cell migratory advantages in hepatocellular carcinoma through enhancing AKT signaling[END_REF]. miR-221 is also reduced in cholangiocarcinoma, epithelial tumors emerging from biliary cells [START_REF] Karakatsanis | Expression of microRNAs, miR-21, miR-31, miR-122, miR-145, miR-146a, miR-200c, miR-221, miR-222, and miR-223 in patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma and its prognostic significance[END_REF]. The main targets of miR-221/222 in HCC are the tumor suppressor p27, p57, PTEN and the inhibitor of metalloproteinase TIMP-3. In vivo experiments are currently performed to test its invalidation in mice; this aspect will be developed at the end of this chapter. Another well-studied miRNA in HCC is miR-21. This microRNA is induced in tumor cells [START_REF] Karakatsanis | Expression of microRNAs, miR-21, miR-31, miR-122, miR-145, miR-146a, miR-200c, miR-221, miR-222, and miR-223 in patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma and its prognostic significance[END_REF][START_REF] Zhu | miR-21 promotes migration and invasion by the miR-21-PDCD4-AP-1 feedback loop in human hepatocellular carcinoma[END_REF], as well as in liver cancer stem cells (Li et al. 2010), and in cholangiocarcinoma samples [START_REF] Karakatsanis | Expression of microRNAs, miR-21, miR-31, miR-122, miR-145, miR-146a, miR-200c, miR-221, miR-222, and miR-223 in patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma and its prognostic significance[END_REF][START_REF] Selaru | MicroRNA-21 is overexpressed in human cholangiocarcinoma and regulates programmed cell death 4 and tissue inhibitor of metalloproteinase 3[END_REF]). Interestingly, miR-21 can be detected in patient sera, a part that we will detail in the next paragraph. It thus constitutes a potent biomarker for HCC diagnosis (Xu et al. 2011, Zhou et al. 2011); in particular, miR-21 is a marker of necroinflammation for HCC developed during hepatitis C virus infection [START_REF] Bihrer | Serum microRNA-21 as marker for necroinflammation in hepatitis C patients with and without hepatocellular carcinoma[END_REF].

Finally, Cairo et al. have identified two clusters of miRNAs (miR-100/let-7a-2/miR-125b-1 and the cluster miR-371-3), which are targets for the oncogene MYC, as a bad prognosis factor in liver tumors [START_REF] Cairo | Stem cell-like micro-RNA signature driven by Myc in aggressive liver cancer[END_REF]. By the measurement of the four miRNAs miR-100, miR-371, miR-373 and Let-7a, the authors are able to discriminate on a large cohort of patients, the tumor samples from HCC patients with tumor properties very closed to hepatoblastoma, the most common primitive liver tumor in child; this subtype of HCC are less differentiated and more invasive. This work supports the existence of a miRNA signature common to hepatoblastoma and HCC, and highlights the great interest of this signature of four miRNAs for the prognosis and the treatment of both tumors.

Despite the identification of a common miRNA signature for HCC samples, other miRNAs are specific of a class of tumor. A loss of miR-375 is characteristic of tumors mutated for the β-catenin, while miR-126* is lost in HCC developed after alcohol abuse, and miR-96 is associated to hepatitis B virus infection (Zucman-Rossi 2010). The microRNA miR-18a is also a very interesting marker in HCC. Indeed, HCC is more prevalent in men (by 2-6-fold), and the women with HCC have a longer life expectancy. This suggests the importance of sexdependent molecular mechanisms in HCC progression, which could be notably linked to estrogens. miR-18a is, in fact, a regulator of the estrogen receptor α (ERα), which is overexpressed in HCC samples from woman patients. It thus appears that the overexpression of miR-18a is associated with a loss of the protective effects of estrogen for these women [START_REF] Liu | MicroRNA-18a prevents estrogen receptor-alpha expression, promoting proliferation of hepatocellular carcinoma cells[END_REF]). In addition, this miRNA has been recently described as a serum biomarker for HCC due to HBV infection [START_REF] Li | Serum miR-18a: a potential marker for hepatitis B virus-related hepatocellular carcinoma screening[END_REF].

Finally, some works also showed that this miRNA panel could constitute a predictive tool to follow the tumor response to surgery or chemotherapeutic treatments. In this way, miR-122 sensitizes HCC cells to Adriamycin and vincristine (Xu et al. 2011), while miR-199a-3p affects cell sensitivity to doxorubicin and 5-fluorouracil [START_REF] Fornari | MiR-199a-3p regulates mTOR and c-Met to influence the doxorubicin sensitivity of human hepatocarcinoma cells[END_REF].

Table 6.4. Role of miRNAs deregulated in HCC

This table compiled all the data concerning the miRNAs described to interfere with proliferation, apoptosis, migration and invasion of HCC tumors. The miRNAs listed here are at least cited by two independent works and their targets are clearly identified. The miRNAs, (Li 2009) miR-122 (ADAM10,17) [START_REF] Tsai | MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis[END_REF] miR-124 (VIM) [START_REF] Furuta | miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma[END_REF] miR-199 (c-MET) [START_REF] Fornari | MiR-199a-3p regulates mTOR and c-Met to influence the doxorubicin sensitivity of human hepatocarcinoma cells[END_REF] miR-23b (uPA, c-MET) [START_REF] Salvi | MicroRNA-23b mediates urokinase and c-met downmodulation and a decreased migration of human hepatocellular carcinoma cells[END_REF] miR-34a (c-MET) (Li 2009) miR-375 (YAP) (Liu 2010) Let-7c (Li et al. 2010) miR-17-5p [START_REF] Zheng | High expression of serum miR-17-5p associated with poor prognosis in patients with hepatocellular carcinoma[END_REF] miR-18a (Li 2012) miR-122 (Xu 2011) miR-15b [START_REF] Liu | Circulating miR-15b and miR-130b in serum as potential markers for detecting hepatocellular carcinoma: a retrospective cohort study[END_REF] miR-130b [START_REF] Liu | Circulating miR-15b and miR-130b in serum as potential markers for detecting hepatocellular carcinoma: a retrospective cohort study[END_REF] miR-183 [START_REF] Liu | Circulating miR-15b and miR-130b in serum as potential markers for detecting hepatocellular carcinoma: a retrospective cohort study[END_REF] miR-21 [START_REF] Bihrer | Serum microRNA-21 as marker for necroinflammation in hepatitis C patients with and without hepatocellular carcinoma[END_REF], Xu 2011) miR-25 (Li 2010) miR-221 (Li et al. 2011) miR-223 (Xu 2011) miR-224 (Li 2011) miR-375 (Li 2010) miR-34a [START_REF] Cermelli | Circulating microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver disease[END_REF] which could be detected in the patient serum, are listed in the last column. The miRNAs written in bold are those for which a therapeutic strategy anti-miRNA have been tested in HCC; the miRNAs written in red are those for which a mimic strategy have been administered to animals.

miRNA expression in HCC patient sera

As we suggested before, microRNAs not only constitute potent biomarkers in tumor cells, but they could also be detectable in various biological fluids like serum, plasma, urine or saliva. Circulating miRNAs could be associated to proteins or encapsulated into exosomes.

Conde-Vancells et al. deciphered the proteome of exosomes secreted by hepatocytes [START_REF] Conde-Vancells | Characterization and comprehensive proteome profiling of exosomes secreted by hepatocytes[END_REF]. Many of the proteins were previously described to be present in exosomes secreted by various cells (i.e. CD81 or CD63), while others are only expressed in hepatocytesderived exosomes, and involved in the liver-specific functions of lipid, carbohydrate and xenobiotic metabolism. To date, there are few data concerning the microRNA content of exosomes derived from any type of liver cells, or the characterization of their functional impact on the recipient hepatic cells. In 2009, a work showed that exosomes purified from bile are able to stimulate the growth of SEC cells, due to the presence of hedgehog in these exosomes [START_REF] Witek | Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells[END_REF], Yang et al. 2008). For various types of tumors, it has been shown that miRNA expression could be altered in patient sera, and in particular for HCC. Although there are some divergence concerning the discriminating miRNAs identified in patient sera, some miRNAs deregulated in HCC are overexpressed in HCC patient sera: miR-21, miR-221/222 and miR-122 (Li 2011, Xu 2011, Zhou 2011). The reason why miR-122 is decreased in liver tumors and increased in patient sera is not elucidated yet. Recently, Liu et al. argue for the measurement of miR-15b and miR-130b in serum for the diagnosis of HCC [START_REF] Liu | Circulating miR-15b and miR-130b in serum as potential markers for detecting hepatocellular carcinoma: a retrospective cohort study[END_REF]. By this method, the authors could detect early stage HCC that could not be discriminated by αfoetoprotein measurement. The detection of miRNAs in body fluids could be a great advantage for the diagnosis and the treatment of HCC, and also for other pathologies, since it could constitute a non-invasive tool.

miRNA expression in metastatic HCC

In addition to the identification a specific miRNA signature in tumors, a number of works aimed to identify a miRNA signature in metastatic HCC. Divergent results were obtained probably due to divergence in the sampling and the data analysis. Although a recent work of Wong et al. is in favor of a more pronounced alteration of the same panel of miRNAs in metastases as compared to the tumors [START_REF] Wong | Sequential alterations of microRNA expression in hepatocellular carcinoma development and venous metastasis[END_REF]), a study of Budhu et al. showed the existence of a miRNA signature of twenty metastatic miRNAs [START_REF] Budhu | Identification of metastasisrelated microRNAs in hepatocellular carcinoma[END_REF]. Numbers of studies concerning only one miRNA have now enriched the deep-sequencing data, and it appears that metastatic HCC express some miRNAs found deregulated in HCC like miR-122 [START_REF] Tsai | MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis[END_REF], miR-124 [START_REF] Budhu | Identification of metastasisrelated microRNAs in hepatocellular carcinoma[END_REF], miR-17 (Yang 2010), let-7g [START_REF] Ji | Let-7g targets collagen type I alpha2 and inhibits cell migration in hepatocellular carcinoma[END_REF], miR-21 [START_REF] Zhu | miR-21 promotes migration and invasion by the miR-21-PDCD4-AP-1 feedback loop in human hepatocellular carcinoma[END_REF], miR-221 [START_REF] Wong | MiR-222 overexpression confers cell migratory advantages in hepatocellular carcinoma through enhancing AKT signaling[END_REF] or miR-143 [START_REF] Zhang | Up-regulated microRNA-143 transcribed by nuclear factor kappa B enhances hepatocarcinoma metastasis by repressing fibronectin expression[END_REF]), but the authors also identified an alteration of other miRNAs, i.e. miR-9-2 (Tan 2010), miR-148a [START_REF] Budhu | Identification of metastasisrelated microRNAs in hepatocellular carcinoma[END_REF], miR-125b [START_REF] Budhu | Identification of metastasisrelated microRNAs in hepatocellular carcinoma[END_REF], miR-30a [START_REF] Budhu | Identification of metastasisrelated microRNAs in hepatocellular carcinoma[END_REF], or miR-34a (Li 2009) (Table 6.4). Some of these miRNAs have been described as promoters of metastasis. As we mentioned previously, miR-221 and miR-222 target the tumor suppressor TIMP3, and thus activate the AKT pathway and metalloproteinases to induce HCC invasion and metastasis [START_REF] Garofalo | miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation[END_REF][START_REF] Wong | MiR-222 overexpression confers cell migratory advantages in hepatocellular carcinoma through enhancing AKT signaling[END_REF]. TIMP3

is also a target of miR-181b, which is induced by TGFβ in HCC (Wang 2010). miR-181b induction results in MMP2 and MMP9 activation, also induced by miR-21, another miRNA induced in metastatic HCC. More recently, it has been shown that miR-143 exerts a prometastatic activity in vitro and in vivo. miR-143b targets the fibronectin type III domain containing 3B (FNDC3B) [START_REF] Zhang | Up-regulated microRNA-143 transcribed by nuclear factor kappa B enhances hepatocarcinoma metastasis by repressing fibronectin expression[END_REF]). This microRNA is induced by NFκB in metastatic HBV-related HCC. Inversely, there are also anti-metastatic miRNAs, i.e. miR-122, which suppresses intrahepatic metastasis through the inhibition of disintegrin and metalloprotease ADAM10 and ADAM17 [START_REF] Bai | MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to sorafenib[END_REF]). Let-7g was also found impaired in metastastic HCC.

Transfection of let-7g inhibited cell migration and this antimetastatic effect is reduced by the addition of type I collagen alpha2 [START_REF] Ji | Let-7g targets collagen type I alpha2 and inhibits cell migration in hepatocellular carcinoma[END_REF]. Finally, the main signaling pathway targeted by miRNAs currently deregulated in HCC is the c-MET activated pathway. c-MET activation stimulates cell motility and invasion, and protects cells from apoptosis. c-MET is regulated by miR-1, miR-23b, miR-34a and miR-199a-3p, four miRNAs altered in HCC. Silencing of miR-1 inhibits HCC growth and invasion by the targeting of c-MET [START_REF] Datta | Methylation mediated silencing of MicroRNA-1 gene and its role in hepatocellular carcinogenesis[END_REF]. miR-34a can decrease c-MET mRNA and protein level and impair the invasive capacity of HepG2 cells (Li 2009). Finally, miR-23b is also able to decrease c-MET and the urokinase-type plasminogen activator (uPA) to reduce the proliferation and migration properties of HCC cells [START_REF] Salvi | MicroRNA-23b mediates urokinase and c-met downmodulation and a decreased migration of human hepatocellular carcinoma cells[END_REF]).

6.7 miRNAs and HCC microenvironment: role in inflammation.

The tumor microenvironment is an expending domain, which not only considers the behavior of cancer cells alone, but also integrates the surrounding cells. This dynamic system is largely orchestrated by inflammatory cells like immune cells, fibroblasts and myofibroblasts, the cytokines and chemokines they secrete and also the extracellular matrix.

Liver cancer constitutes a good example in which the microenvironment plays a crucial role in the progression of the disease due to its chronic inflammation. The HCC microenvironment is a complex and intricate system composed of various cells, i.e. cancer-associated fibroblasts, hepatic stellate cells, tumor-infiltrating leukocytes, endothelial cells, hepatoma cells and tumor associated macrophages [START_REF] Leonardi | The tumor microenvironment in hepatocellular carcinoma (review)[END_REF] for a review). All these cells secrete a cocktail of growth factors (HGF, EGF, FGF, TGFβ), cytokines (IL-6, IL-8, IL-4, IL-10, TNFα) and chemokines (CCL17, CCL22, CCL24), which all orchestrate immune response to liver injury (Figure 6.5).

Figure 6.5: miRNA network involved in liver inflammation

The transforming growth factor β plays a key and paradoxical role in carcinogenesis. In liver, it has been shown that TGFβ plays crucial roles at different steps of liver pathogenesis including cirrhosis [START_REF] Matsuzaki | Modulation of TGF-beta signaling during progression of chronic liver diseases[END_REF], HBV and HCV infection [START_REF] Marotta | The pathogenesis of hepatocellular carcinoma is multifactorial event. Novel immunological treatment in prospect[END_REF]) and tumorigenesis [START_REF] Massague | TGFbeta in Cancer[END_REF]. TGFβ exerts a tumor suppressive activity in the premalignant state but could also promote tumor progression, through the repression of immune surveillance and through increase in tumor cell proliferation, migration and invasion. As mentioned before, TGFβ induces miR-181b, a process blocked by SMAD4 silencing, and this promotes cell survival, mobility and invasion (Wang 2010). TGFβ also induces the cluster of miRNAs, miR-23a, miR-27a and miR-24 to promote cell proliferation and invasion [START_REF] Huang | Upregulation of miR-23a approximately 27a approximately 24 decreases transforming growth factor-beta-induced tumorsuppressive activities in human hepatocellular carcinoma cells. International journal of cancer[END_REF]. A recent work of [START_REF] Yang | TGF-beta-miR-34a-CCL22 signaling-induced Treg cell recruitment promotes venous metastases of HBV-positive hepatocellular carcinoma[END_REF] has shown that TGFβ represses the expression of miR-34a resulting in CCL22 production to recruit of lymphocyte T regulators leading to immune escape in HBV-related HCC [START_REF] Yang | TGF-beta-miR-34a-CCL22 signaling-induced Treg cell recruitment promotes venous metastases of HBV-positive hepatocellular carcinoma[END_REF]. The axis TGFβ/miR-34a/CCL22 thus promotes venous metastasis in this type of HCC. TGFβ is also required for HSC activation during fibrogenesis [START_REF] Liu | Therapeutic strategies against TGF-beta signaling pathway in hepatic fibrosis[END_REF]. Recently, miR-146a has been described as a modulator of TGFβ-1 dependent activation of HSC. The interleukin-6 is also a crucial mediator in liver cancer progression. Its level is increased during cirrhosis [START_REF] Tilg | Serum levels of cytokines in chronic liver diseases[END_REF], and a high serum level of IL-6 is a risk factor and a factor of bad prognosis for HCC [START_REF] Nakagawa | Serum IL-6 levels and the risk for hepatocarcinogenesis in chronic hepatitis C patients: an analysis based on gender differences. International journal of cancer[END_REF][START_REF] Wong | High serum interleukin-6 level predicts future hepatocellular carcinoma development in patients with chronic hepatitis B. International journal of cancer[END_REF]. A recent work of Iliopoulos' lab highlights a circuit orchestrated by microRNAs that results in liver tumorigenesis and inflammation. This feedback loop, governed by miR-24 and miR-629, promotes a HNF-4α transient inhibition resulting in miR-124 induction and STAT3 activation. The authors described the interconnection between the trio miR-24/miR-629/miR-124 and HNF-4α as a key regulator of HCC progression by limiting the tumor inflammatory environment through the inhibition of the IL-6/STAT3 pathway, predisposing a patient to liver cancer development and immune escape [START_REF] Hatziapostolou | An HNF4alpha-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis[END_REF]. The level of interleukin-6 has also been inversely correlated to miR-26 expression in HCC [START_REF] Janssen | MicroRNA expression, survival, and response to interferon in liver cancer[END_REF]. Patients with a low level of miR-26 and inversely a high level of IL-6 have a lower survival but are also better responders to IFNα treatment, suggesting that this miRNA could be a predictive marker for patient response to IFNα treatment. In this study, NFκB expression has also been inversely correlated to miR-26 level.

NFκB is a transcription factor induced in response to TNFα. It plays an essential role in liver homeostasis by preventing hepatocyte death. As mentioned before, NFκB regulates a number of miRNA deregulated in HCC i.e. miR-155 and miR-143 [START_REF] Zhang | Up-regulated microRNA-143 transcribed by nuclear factor kappa B enhances hepatocarcinoma metastasis by repressing fibronectin expression[END_REF]. NFκB has also been demonstrated as a regulator of miR-224, a miRNA upregulated in HCC. NFκB binding on miR-224 promoter is significantly induced in response to TNFα and LPS [START_REF] Scisciani | Transcriptional regulation of miR-224 upregulated in human HCCs by NFkappaB inflammatory pathways[END_REF].

The IKK inhibitor BMS-345541 blocks pre-miR-224-induced cellular migration and invasion.

NFκB is also a key regulator of HSC activation through the regulation of miR-222. miR-222 induction in HSC during liver fibrosis in response to TNFα and TGFβ is abrogated by a NFκB inhibitor [START_REF] Ogawa | MicroRNA-221/222 upregulation indicates the activation of stellate cells and the progression of liver fibrosis[END_REF]. Inversely, some microRNAs regulate NFκB expression or activity: the miRNA miR-491-5p controls NFκB level and sensitizes hepatoma cells to TNFαinduced apoptosis [START_REF] Yoon | Acute liver injury upregulates microRNA-491-5p in mice, and its overexpression sensitizes Hep G2 cells for tumour necrosis factoralpha-induced apoptosis[END_REF], while miR-140 suppresses NFκB activity through the inhibition of its coactivators NRP1 and NCoA1 [START_REF] Takata | MicroRNA-22 and microRNA-140 suppress NF-kappaB activity by regulating the expression of NF-kappaB coactivators[END_REF]. Last years, a work of Boesch-Saadatmandi shows that the anti-inflammatory molecule, querceptin, which reduces NFκB activity through the targeting of redox factor 1, induces miR-122 and miR-125b expression, resulting in the reduction of liver inflammation in mice (Boesch-Saadatmandi et al.

2012).

Finally, miR-199 appears as a central regulator of HCC progression but also of liver fibrosis and hypoxic stress. As mentioned before, miR-199 is decreased in HCC in correlation with its aggressiveness and invasiveness, and thus constitutes a potential predictive marker [START_REF] Fornari | MiR-199a-3p regulates mTOR and c-Met to influence the doxorubicin sensitivity of human hepatocarcinoma cells[END_REF][START_REF] Hou | Identification of miRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular carcinoma[END_REF]. As previously mentioned, miR-199 is also upregulated in HSC cells during fibrogenesis [START_REF] Murakami | The progression of liver fibrosis is related with overexpression of the miR-199 and 200 families[END_REF]. [START_REF] Lee | Farnesoid X receptor protects hepatocytes from injury by repressing miR-199a-3p, which increases levels of LKB1[END_REF] suggests that the farnesoid X receptor FXR decrease in liver during fibrotic process leads to miR-199 induction and LKB1 reduction [START_REF] Lee | Farnesoid X receptor protects hepatocytes from injury by repressing miR-199a-3p, which increases levels of LKB1[END_REF]. miR-199 also controls hypoxia, which affects tumor and stromal cells, and enhances proliferation, metastasis and resistance to treatment of HCC. The loss of miR-199b has been directly linked to hypoxia-induced factor 1α (HIF-1α) activation in HCC samples [START_REF] Wang | Underexpressed microRNA-199b-5p targets hypoxia-inducible factor-1alpha in hepatocellular carcinoma and predicts prognosis of hepatocellular carcinoma patients[END_REF]. miR-199b transfection into HCC cells inhibits cell proliferation during hypoxia and restores their radiosensitivity. Recently, the cluster miR-199a/miR-214 has also been identified as a key orchestrator of HCC progression in response to endoplasmic reticulum stress [START_REF] Duan | ER stress negatively modulates the expression of the miR-199a/214 cluster to regulates tumor survival and progression in human hepatocellular cancer[END_REF]. The authors showed that the expression of these miRNAs is suppressed in response to unfolded protein response-induced hypoxia, and this is repressed by NFκB inhibition.

To conclude, as supported by all these data, miRNAs constitute potent biomarkers, which can be very useful to improve the classification of HCC, its diagnosis and the prediction of its response to treatment. A non-invasive diagnostic tool based on miRNAs is also possible, by blood or urinary sampling, even if an improvement of their detection in body fluids is required. Finally, tumoral miRNAs also emerge as promising therapeutic targets, as we will develop in the next part of this chapter. Following the identification of a panel of microRNAs deregulated in HCC, a number of works have been conducted to restore the expression of these miRNAs, initially in vitro and then in vivo (Table 6.4). The in vitro experiments on cell lines gave encouraging results, which support the potent effect of an administration of miRNA-based therapies to mice. A number of mouse models mimicking liver disease, and particularly HCC, have been developed these recent years [START_REF] Heindryckx | Experimental mouse models for hepatocellular carcinoma research[END_REF]. Chemically induced tumor models, i.e.

following diethylnitrosamine [START_REF] Hacker | Histochemical profile of mouse hepatocellular adenomas and carcinomas induced by a single dose of diethylnitrosamine[END_REF] or carbon tetrachloride injection [START_REF] Weisburger | Carcinogenicity studies on halogenated hydrocarbons[END_REF] are frequently used. Some teams also work on xenograft models following ectopic or orthotopic implantation. Alternatively, various mouse models have been genetically created to model the different subtypes of HCC, following the introduction of a viral gene [START_REF] Araki | Expression and replication of hepatitis B virus genome in transgenic mice[END_REF], or the activation of the oncogenes myc [START_REF] Thorgeirsson | Transgenic mouse models in carcinogenesis: interaction of c-myc with transforming growth factor alpha and hepatocyte growth factor in hepatocarcinogenesis[END_REF] or catenin [START_REF] Colnot | Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas[END_REF]). More recently, these miRNA-based therapies have been successfully tested in primates and in patients.

As we mentioned at the beginning of this chapter, an anti-miR-122, called Miravirsen, based on the locked nucleic acid technology (LNA), was the first therapy to enter into clinical trials in 2008 to treat HCV. A LNA is a nucleic acid chemically modified by the addition of a methylene bridge in the ribose. This modification increases the LNA affinity for its target and also its stability. The LNA becomes resistant to nuclease degradation. The first works performed in mouse and primates infected with hepatitis C virus suggest an absence of toxicity for Miravirsen. This anti-miR-122 successfully impairs the level of viral RNAs in plasma, since HCV uses liver miR-122 for its replication [START_REF] Lanford | Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection[END_REF]. In 2012, encouraging data were obtained in patients infected with chronic HCV. Miravirsen successfully decreases the level of virus RNAs and of plasmatic cholesterol. [START_REF] Janssen | MicroRNA expression, survival, and response to interferon in liver cancer[END_REF]. Another type of inhibitors has also been tested in vivo, the antagomiRs. An inhibitor against miR-221 has been tested in a mouse model, and successfully delays HCC development [START_REF] Park | miR-221 silencing blocks hepatocellular carcinoma and promotes survival[END_REF]). An and in vivo as compared to LNAs [START_REF] Obad | Silencing of microRNA families by seed-targeting tiny LNAs[END_REF].

The second approach based on miRNA consists to restore miRNA expression thanks to a double strand RNA, mimicking the activity of the endogenous miRNA, and, so, called "mimic". The administration of a mimic of miR-26 with an adenovirus vehicle in a mouse model of HCC mutated for the oncogene MYC, successfully abrogates cancer-cell proliferation and tumor development in correlation with a loss of cyclin E 2 and D 2 in tumors without toxic effect in the remaining liver [START_REF] Kota | Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model[END_REF]). In the same way, the work of Hatziapostoulou et al., supporting the crucial role of the trio miR-24/miR-629/miR-124 in IL-6-dependent inflammation in HCC induced by diethylnitrosamine, shows the efficiency of an administration of a mimic of miR-124 encapsulated into liposomes in this mouse model of HCC [START_REF] Hatziapostolou | An HNF4alpha-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis[END_REF]. Finally, the miRNAtherapeutics company, dedicated to develop mimic-based therapies, has created liposomes containing a mimic of miR-34a, called miRX34. This mimic formulation is currently in clinical trials for solid tumors, and in particular for liver tumors. They showed that in a model of human HCC grafted to mouse, miRX34 significantly improved mouse survival. This company also begins the preclinical trials for a mimic Let-7g in liver tumors (www.mirnatherapeutics.com).

Despite promising results obtained for miRNA-based therapies, a number of challenges remained to date to improve the efficiency of this type of treatments. Their inconvenient are similar to those which have delayed the use of therapies based on small interfering RNAs: improvement of their stability, free administration or delivery encapsulated into nanoparticules or exosomes, problems of specificity, off-targets or targeting of non-identified mRNAs, tissue distribution, response persistence, secondary effects… 6.9 Conclusions

Since their discovery in 1993, the study of miRNAs is an expanding domain that allows the identification of a miRNA signature for a great variety of tumors and in particular for liver tumors. The better knowledge of the panel of miRNAs deregulated in liver disease and notably in hepatocellular carcinoma, is in favor of the use of miRNAs as potent tools for diagnosis, prognosis and treatment of HCC patients. Interestingly, miRNAs integrate all the counterparts of liver tumorigenesis, whether inflammation, hypoxia, immune escape as well as tumor cell proliferation, migration and invasion. Although a great number of studies help to decipher the different targets of one miRNA of interest, the main challenge for the future remains to better understand how the different targets of each miRNA interact in one pathology and how a mRNA, targeted by different miRNAs, behave in this context.

Figure

  Figure 6.1: liver lobule organization

Figure 6

 6 Figure 6.2: miRNA expression in physiological and pathological conditions ALD: alcohol-liver disease; NAFLD: non-fatty acid liver disease, HNAS: non alcoholic steatohepatitis; HBV: hepatitis B virus

Figure 6

 6 Figure 6.3: number of publications concerning liver cancer and miRNAs referenced in PUBMED in February 2013

6. 8

 8 miRNAs in liver: a new therapeutic approach? Although numerous efforts have been conducted to improve HCC treatment, surgery is still nowadays the most frequent therapeutic option. Sorafenib, an inhibitor of receptors tyrosine kinase, is the unique drug accepted by FDA for the treatment of liver cancer. It has only enhanced the patient life expectancy of three months. miRNAs appear as promising therapeutic targets since they could target different signaling pathways by their multiplicity of action. Above all, miRNA-based therapy could target in the same time the metabolic, inflammatory and immune counterparts of liver tumors responsible for the development and progression of HCC.

  antagomiR presents a sequence complementary to the whole sequence of the miRNA of interest, in contrast to a LNA, which only hybridizes with a part of the miRNA sequence. It induces the sequestration or the degradation of its miRNA target. To improve the antagomiR stability in vivo, some chemical modifications have been added, i.e. an addition of 2'-Omethyl or 2'-O-methoxyethyl (MOE) group, or creation of a phosphorothioate backbone. In 2010, Rosetta Genomics Ltd. developed a systemic 2'MOE-antagomiR against miR-191, frequently overexpressed in HCC. The orthotopic xenograft mouse model study demonstrates that a marked suppression of a microRNA, miR-191, leads to a significant two-fold decrease of tumor mass due to reduction of cancer-cell proliferation and enhanced apoptosis[START_REF] Elyakim | hsa-miR-191 is a candidate oncogene target for hepatocellular carcinoma therapy[END_REF]). These recent years, a new class of inhibitors appears, the tiny-LNAs. Due to their shorter sequence, these LNAs have a better tissue distribution and can inhibit an entire family of miRNAs. A tiny-LNA directed against miR-122 showed a better efficiency in liver cells

This work has been supported by the Ligue Nationale Contre le Cancer (Labellisation Programme 2011-2013) and the Agence Nationale de la Recherche (WNT-METABOLIV, 2011-2013).