<p>Human testis expressed 261 (TEX261) protein is predicted to be involved in essential cellular pathways such as proliferation, apoptosis, and COPII-mediated intracellular trafficking, yet has been scarcely researched in human cell models. Since TEX261 dysregulation has been observed in HCC, investigating the role of TEX261 in hepatocytes is essential. In this study we utilized molecular cloning and fluorescent protein tags to visualize the expression of TEX261 and associated proteins SAR1A and ALPL by confocal microscopy. We observed that TEX261 is closely associated with both proteins, indicating that TEX261 may be involved in ALPL packing into the COPII complex responsible for intracellular trafficking from the ER to the Golgi apparatus. We assessed TEX261 role in apoptosis by measuring caspase 3 activity and observed that TEX261 overexpression induced apoptosis at a similar rate as the positive control but did not significantly increase apoptosis compared to the negative control. We also recorded cell proliferation by overexpressing and silencing TEX261 in two cell lines. Our data showed that altered TEX261 expression did not impact Thle-2 proliferation, but TEX261 overexpression did significantly decrease proliferation in the HCC cell line Hep3B2.1-7. Overall, our results suggest that TEX261 does play a role in intracellular trafficking, apoptosis and proliferation, yet future studies need to be done to further define its role in cell regulatory mechanisms. Better control of the experimental error seems to be required to define the function of TEX261 in apoptosis.</p>

The mechanistic target of rapamycin (mTOR) is a key serine/threonine protein kinase that functions in complexes mTORC1 and mTORC2. mTORC1, originally discovered due to its sensitivity towards the mTOR inhibitor rapamycin, responds to extracellular growth factor signaling, WNT signaling, and nutrient abundance via glucose and amino acid-triggered signaling. Downstream effectors of mTORC1 include autophagy, mitochondrial metabolic function, protein synthesis, and ribosome biogenesis. mTORC2, initially discovered as a rapamycin-insensitive complex of mTOR, responds to insulin, growth factor signaling, and inflammatory signaling such as tumor necrosis factor-alpha, with its downstream effectors being Akt, a key serine/threonine kinase that functions in cell division and is frequently dysregulated in many types of cancer, the NFkB pathway, and cytoskeletal reorganization and protein synthesis. Much research has been devoted to mTORC1 signaling, with mTORC2 receiving significantly less attention, despite both complexes’ regulation of key cellular activities and response to rapamycin, as well as to other rapamycin-derived drugs (rapalogs). We have targeted both mTORC1 and mTORC2 for hepatocyte-specific deletion during the gestational period of mice, with the goal of describing mTORC1 and mTORC2 signaling and its perturbation in the adult mouse hepatocyte. Our model has shown that deletion of RAPTOR, the regulatory associated protein of mTOR, and RICTOR, the rapamycin insensitive component of mTOR, in mTORC1 and mTORC2 respectively, leads to widespread effects on the hepatocyte transcriptome. We have found that a subset of genes responds both to Raptor and Rictor knockout, and an analysis of these genes indicates their function in key disorders of the liver, such as non-alcoholic fatty liver disease and hepatocellular carcinoma. Bioinformatic analysis following hepatocyte RNA sequencing of mTORC1 and mTORC2 knockout mice has revealed an unexpected upregulation of genes known to be regulated by these respective complexes. We have also found that cross talk exists between both complexes, in which the knockout of one yields the activation of the other. We have additionally found translationally relevant enrichments following Ingenuity Pathway Analysis (IPA) of RNA sequencing data. These results provide a key mechanistic discovery of mTOR signaling activity, and allow for a better understanding of the potential physiological effects of mTOR inhibition in human patients.

Ce travail concerne le développement d’un système de vectorisation nanoparticulaire pour l’interférence ARN, constituant une nouvelle proposition thérapeutique applicable à des pathologies virales ou tumorales, et possiblement complémentaire aux traitements existants. La vectorisation de siRNA est ici basée sur l’enrobage multicouche de nanoparticules de phosphate de calcium, la multicouche étant constituée de dépôts alternés de PEI modifié et de siRNA. Ce système permet d’obtenir une efficacité de transfection des cellulaires cibles supérieure à celle des procédés conventionnels et une rémanence fonctionnelle in vitro jusqu’à neuf jours. Les résultats d’interférence ARN obtenus ont permis notamment d’inhiber l’infection par le virus de l’hépatite C jusqu’à 99,95%, l’inhibition de l’expression d’une protéine intrinsèque jusqu’à90,5%, et le ralentissement de la croissance cellulaire dans un modèle 3D mimant une tumeur hépatique jusqu’à 46,5%. Ces nanoparticules pourraient présenter un intérêt majeur, en offrant une action à long terme et en résolvant la plupart des difficultés rencontrées en utilisant des siRNA en thérapie. / This work concerns the development of a nanoparticle vector system for RNA interference,constituting a new therapeutic option applicable to viral and tumor pathologies,and possibly complementary to existing treatments.siRNA vectorisation is here based on multi layer coating of alcium phosphate nanoparticles,the multi layer being constituted of alternate coatings of modified PEI and siRNA.This system triggers a better transfection efficiency of target cells than classic techniques,as well as a functional persistence up to 9 days in vitro.RNA interference results using CPnp allowed inhibition of hepatitis C virus infection up to 99.95%,of intrinsic protein expression up to 90.5%,and of cell growth in a 3 D model mimicking an hepatic tumor up to 46.5%.These nanoparticles could be of major interest,by offering a long term action,and resolving most of the issues found in the use of siRNA in therapy.

Le premier objectif a été de sélectionner des promoteurs de gènes cellulaires actifs spécifiquement dans les HCC à l’aide d’une recherche bibliographique puis en utilisant la base de donnée UniGene. Leur activité a été vérifiée par RT-qPCR et CHIP dans des lignées modèles HCC et dans des hépatocytes. Ces promoteurs ont été clonés en amont de la luciférase dans la région intergénique 20 du génome HSV-1 afin d’étudier leur force d’activité, 2 types de cinétiques et leur activité différentielle en fonction du type cellulaire et dans le contexte d’une infection virale. Le deuxième objectif a été de construire des virus oncolytiques ciblés pour l’expression de la protéine Us3, une protéine virale impliquée dans le contrôle de la réponse apoptotique induite par HSV-1. L’expression de la protéine Us3 est placée sous contrôle d’un promoteur cellulaire spécifique d’HCC. L’hypothèse est qu’en l’absence d’activité du promoteur cellulaire dans les cellules non HCC, la protéine Us3 ne sera pas synthétisée et, par conséquent, l’apoptose qui ne sera pas réprimée, inhibera le cycle de réplication et par conséquent, la production virale dans les cellules saines. Dans les cellules HCC, le promoteur actif permettra la réplication virale aboutissant à la destruction de lamasse tumorale. Un virus HSV-1 Us3- a été construit en utilisant la technique de recombinaison en plasmide BAC (Bacterial artificial chromosome), puis 2 virus oncolytiques en réintroduisant le gène Us3 sous contrôle du promoteur ANGPTL3 ou du promoteur HRE (hypoxia responsive element). Leur comportement oncolytique a été étudié en réalisant des courbes de croissance sur lignées cellulaires d’HCC et cellules hepatocyte-like. / Our long-term purpose is to develop transcriptionally targeted oncolytic vectors, derived from herpes simplex virus type 1 (HSV-1), designed to eradicate hepatocellular carcinomas (HCC). We have identified several HCC-specific promoters, as well as other cancer-specific promoters, that maintain their specificity when expressed from the virus genome. More precisely, we have demonstrated that these promoters are able to drive reporter gene (luciferase) expression from the virus genome in HCC-derived cells, both in cultured cells and in nude mice, but not in fresh human hepatocytes or in the WRL38 hepatocyte-like cells. HSV-1 infection induces, but then inhibits, a cellular antiviral apoptotic response, and the early virus protein US3 is a key actor in inhibiting apoptosis. We have hypothesized that inhibition of US3 expression in hepatocytes should led to early apoptotic death of these cells, therefore precluding virus multiplication and spread. In contrast, expression of US3 in cancer cells is expected to block apoptosis, leading to the achievement of the virus life cycle, cell lysis, and virus spread within the tumours. We report in this communication the construction and properties of two different potentially oncolytic HSV-1 vectors. One of them expresses US3 protein under the control of the HCC-specific promoter ANGPTL3, while the second promoter contains 9 repeats of the hypoxia responsive elements of vascular-endothelial growth factor (VEGF) (9xHRE promoter). Growth curves of these viruses were performed on different HCC cell lines to show their oncolytic properties.

Non-alcoholic fatty liver disease (NAFLD) is a major chronic liver disease and thus a main reason for liver-related morbidities and mortality. NAFLD covers a wide range of diseases starting with steatosis and frequently progressing to non-alcoholic steatohepatitis (NASH), which is an independent predictor for the development of the hepatocellular carcinoma (HCC). Nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme of the mammalian NAD salvage pathway, recycles nicotinamide to nicotinamide mononucleotide (NMN), which is further converted to nicotinamide adenine dinucleotide (NAD). NAD is not only an important redox partner but also a crucial co-substrate for NAD-dependent enzymes such as sirtuin 1 (SIRT1). Thus, NAD metabolism might be involved in the progression of NAFLD by regulating many cellular processes, such as apoptosis, de novo lipogenesis, glycolysis and gluconeogenesis, in the liver. Interestingly, tumor cells have a high NAD turnover due to their rapid proliferation and high activity of NAD-dependent enzymes. For these reasons, I hypothesized that the NAD salvage pathway is dysregulated during the progression of non-alcoholic fatty liver disease. Therefore, the first study of the present work deals with the role of the NAD salvage pathway in a diet-induced mouse model of hepatic steatosis. In mice fed a high-fat diet for 11 weeks hepatic NAMPT mRNA, protein abundance and activity as well as NAD levels were increased. Additionally, SIRT1 protein abundance was upregulated indicating a higher SIRT1 activity. This could be confirmed by detecting decreased acetylation or transcription of SIRT1 targets. For example, p53 and nuclear factor κB (NF-κB) were less acetylated demonstrating lower activity of key regulators of apoptosis and inflammation, respectively. In the second study of this thesis NAMPT activity was inhibited by applying its specific inhibitor FK866 in hepatocarcinoma cells to investigate whether or not NAMPT inhibition could be a potential novel therapeutic approach in HCC treatment. Hepatocarcinoma cells were more sensitive to NAMPT inhibition by FK866 than primary human hepatocytes, presenting a high number of apoptotic cells after FK866 treatment. FK866 induced NAD and ATP depletion which was associated with activation of the key regulator of energy metabolism 5’-AMP-activated protein kinase (AMPK) and decreased activity of its downstream target mammalian target of rapamycin (mTOR). This thesis shows that the NAD salvage pathway is involved in hepatic steatosis and HCC. During hepatic steatosis NAD metabolism is upregulated to potentially protect against adverse effects of the massive hepatic lipid accumulation. To repress the progression to NASH it might be useful to maintain the hepatic NAD levels during early disease stages by administration of NAD precursors, such as NMN. However, hepatocarcinoma cells have a higher activity of NAMPT and NAD-dependent enzymes. NAMPT inhibition by FK866 could be a potential therapeutic approach in HCC, especially due to the fact that NAD depletion is selectively induced in hepatocarcinoma cells, but not in primary human hepatocytes.

The area of data imputation, which is the process of replacing missing data with substituted values, has been covered quite extensively in recent years. The literature on the practical impact of data imputation however, remains scarce. This thesis explores the impact of some of the state of the art data imputation methods on HCC survival prediction and classification in combination with data-level methods such as oversampling. More specifically, it explores imputation methods for mixed-type datasets and their impact on a particular HCC dataset. Previous research has shown that, the newer, more sophisticated imputation methods outperform simpler ones when evaluated with normalized root mean square error (NRMSE). Contrary to intuition however, the results of this study show that when combined with other data-level methods such as clustering and oversampling, the differences in imputation performance does not always impact classification in any meaningful way. This might be explained by the noise that is introduced when generating synthetic data points in the oversampling process. The results also show that one of the more sophisticated imputation methods, namely MICE, is highly dependent on prior assumptions about the underlying distributions of the dataset. When those assumptions are incorrect, the imputation method performs poorly and has a considerable negative impact on classification. / Forskningen kring data imputation, processen där man ersätter saknade data med substituerade värden, har varit omfattande de senaste åren. Litteraturen om den praktiska inverkan som data imputation metoder har på klassificering är dock otillräcklig. Det här kandidatexamensarbetet utforskar den inverkan som de nyare imputation metoderna har på HCC överlevnads klassificering i kombination med andra data-nivå metoder så som översampling. Mer specifikt, så utforskar denna studie imputations metoder för heterogena dataset och deras inverkan på ett specifikt HCC dataset. Tidigare forskning har visat att de nyare, mer sofistikerade imputations metoderna presterar bättre än de mer enkla metoderna när de utvärderas med normalized root mean square error (NRMSE). I motsats till intuition, så visar resultaten i denna studie att när imputation kombineras med andra data-nivå metoder så som översampling och klustring, så påverkas inte klassificeringen alltid på ett meningsfullt sätt. Detta kan förklaras med att brus introduceras i datasetet när syntetiska punkter genereras i översampling processen. Resultaten visar också att en av de mer sofistikerade imputation metoderna, nämligen MICE, är starkt beroende på tidigare antaganden som görs om de underliggande fördelningarna i datasetet. När dessa antaganden är inkorrekta så presterar imputations metoden dåligt och har en negativ inverkan på klassificering.

Die evolutionär konservierten morphogenen Signalwege Wnt/β-Catenin und Hedgehog (Hh) spielen vor allem in der Embryogenese, Zelldifferenzierung und Kanzerogenese eine große Rolle. Es ist bekannt, dass es zwischen Komponenten der beiden Signalkaskaden zu verschiedensten Wechselwirkungen und Hintergrundreaktionen in unterschiedlichsten Organismen und Geweben kommt. Ziel dieser Arbeit war die Aufklärung einzelner mole- kularer, transkriptioneller Prozesse hinter diesen Kreuzreaktionen in primären Hepatozyten und dem Hepatozellulären Karzinom. Dafür sind die beiden Signalwege durch verschiedenste Einflüsse, wie dem Einsatz von siRNAs, transgenen Mausmodellen und rekombinanten Proteinen gegen einzelne Faktoren der Hedgehog, aber auch der Wnt/β-Catenin Kaskade in ihrer Genexpression verändert und die Reaktionen der Signalwegskomponenten mittels der quantitativen Real-Time-PCR untersucht worden. Neben dem Organismus Maus haben einzelne vergleichende Experimente auch auf der humanen Ebene zum Erkenntnisgewinn beigetragen. Durch die Einflussnahme auf den Hedgehog Signalweg in murinen Hepatozyten wird deutlich, dass die Antworten auf die einzelnen Veränderungen in den Signalkaskaden sehr vielschichtig und umfangreich sind. Abhängig vom induzierten bzw. reprimierten Gen, aber auch vom gelenkten Signalweg variieren die Genexpressionen auf unterschiedlichste, und zum Teil gegenläufige Weise. Ferner wird deutlich, dass es zu Unterschieden in der Genantwort bezüglich der verschiedenen Organismen Maus und Mensch, aber auch zu Variationen der Interaktionen in den diversen Gewebe bzw. Zelltypen kommt.

Title of Dissertation: FUNCTION OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 7(IGFBP7) IN HEPATOCELLULAR CARCINOMA By Dong Chen. Purpose: Hepatocellular carcinoma (HCC) is a highly virulent malignancy with no effective treatment, thus requiring the development of innovative and effective targeted therapies. The oncogene Astrocyte Elevated Gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis and profoundly downregulates Insulin-like Growth Factor Binding Protein-7 (IGFBP7). The present study focuses on analyzing potential tumor suppressor functions of IGFBP7 in HCC and the relevance of IGFBP7 downregulation in mediating AEG-1 function. Experimental Design: IGFBP7 expression was detected by immunohistochemistry in HCC tissue microarrays by real-time PCR and ELISA in human HCC cell lines. Dual Fluorescence in situ hybridization was performed to detect loss of heterozygosity at the IGFBP7 locus. Stable IGFBP7- overexpressing clones were established in the background of AEG-1- overexpressing human HCC cells and were analyzed for in vitro proliferation, senescence, in vivo tumorigenesis and angiogenesis. HCC cell lines infected with an adenovirus expressing IGFBP7 (Ad.IGFBP7) were analyzed by using in vitro cell cycle, apoptosis, in vivo tumorigenesis assays. Results: IGFBP7 expression is significantly downregulated in both human HCC patients’ samples and cell lines compared to normal liver and hepatocytes. IGFBP7 expression was also found to inversely correlate with the stages and grade of HCC. Genomic deletion of IGFBP7 was identified in 26% of HCC patients. Forced overexpression of IGFBP7 in AEG-1 overexpressing HCC cells inhibited in vitro growth and induced senescence. When injected into nude mice, in vivo growth was profoundly suppressed, potentially as a result of inhibition of both angiogenesis and IGF1R activation by IGFBP7. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing Reactive Oxygen Species (ROS) and activating a DNA damage response. N-acetylcysteine could neutralize ROS and rescue the cells from apoptosis. In early phase after Ad.IGFBP7 infection, activation of cell cycle control proteins like Rb, p53, ATM, ATR, CHK1 and CHK2 were identified and G2/M cell cycle arrest was recorded by FACS. Ad.IGFBP7 infection resulted in the activation of p38 MAPK, and a p38 MAPK inhibitor SB 203580 could block the apoptotic process. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intra-hepatic metastasis. In a nude mouse subcutaneous model, xenografts from human HCC cells were established in both flanks and only left- side tumors received intratumoral injection of Ad.IGFBP7. Ad.IGFBP7 markedly inhibit growth of both left-sided injected tumors and right-sided un- injected tumors by profound suppression of angiogenesis. Conclusion: The present findings provide evidence that IGFBP7 functions as a novel putative tumor suppressor for HCC and establish the corollary that IGFBP7 downregulation can effectively modify AEG-1 function. Targeted overexpression of IGFBP7 may be a potential novel and effective therapy for HCC.

Student Number : 9903082V - MSc (Med) dissertation - Faculty of Health Sciences / Chronic infection with the hepatitis B virus (HBV) is endemic to sub-Saharan Africa and south-east Asia where it is a major risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). Currently available therapy is only effective in a small subset of chronic carriers. The development of novel treatment modalities for the management of HBV therefore remains an important global medical objective. Sequence plasticity of the HBV genome is limited by its small size and the overlapping nature of its open reading frames (ORFs). These features make HBV an ideal target for therapy based on nucleic acid hybridization. The use of ribozymes (RNA enzymes) and antisense molecules to inhibit gene expression is well documented. The recent discovery of RNA interference (RNAi) has added to the arsenal of therapy based on nucleic acid hybridization. RNAi is the process whereby short RNA duplexes (called short interfering RNA or siRNA) mediate the sequence-specific post-transcriptional silencing of genes homologous in sequence to the siRNA. siRNA function by guiding a protein complex (RNA Induced Silencing Complex or RISC) to target mRNA for degradation or translational repression. The protein X ORF (HBx ORF) is a conserved region of the HBV genome and is common to all viral transcripts. HBx is required for infection by the virus and plays an important role in the establishment of chronic infections in vivo as well as in the development of HCC. RNAi targeted against the HBx ORF may therefore prove useful as treatment of chronic HBV infection. Plasmid based expression cassettes capable of endogenously generating short hairpin RNA (shRNA) targeted to the HBx ORF were constructed. The shRNA function as substrates for the RNAi machinery and are processed into siRNA. The ability of the expression cassettes to knockdown markers of HBV gene expression was tested in a human hepatoma cell line. A panel of 10 U6 promoter-driven shRNA expression vectors was generated. The U6 promoter (an RNA polymerase III promoter) is normally involved in the transcription of small nuclear RNA and as such is ideal for the generation of shRNA of precisely defined length. Three cytomegalovirus (CMV) promoter-driven shRNA expression cassettes incorporating ribozymes that produce defined hairpin sequences were also generated. The CMV promoter (an RNA polymerase II) promoter is involved in the transcription of large messenger RNA. Two hammerhead ribozymes lying 5’ and 3’ of the shRNA encoding sequence were incorporated into the cassette. Cis-cleavage by the ribozymes releases a shRNA of defined length thereby overcoming the limitations imposed by extraneous sequences from CMV promoter-driven transcription. U6 promoter-driven shRNA expression vectors efficiently knocked down markers of HBV replication in liver cells. The CMV promoter-driven expression vectors were incapable of inhibiting HBV gene expression; however shRNA generated in vitro from these vectors mediated efficient knockdown of HBV replication. shRNA-mediated inhibition of gene expression therefore holds promise as a novel treatment strategy for the management of HBV and other mobile genetic elements.

Le carcinome hépatocellulaire (CHC) est la tumeur maligne primitive du foie la plus fréquente (90 % des cas). Le CHC est le cinquième cancer par ordre de fréquence à l'échelon mondial, la troisième cause de décès par cancer dans le monde entier et son incidence est actuellement croissante. Parmi Les principaux facteurs prédictifs de survenue de CHC ; les altérations génétiques, l'alcool et les infections par les virus des hépatites B (VHB) et C (VHC). Les travaux de thèse en cotutelle menés entre l’Universités Paris Sud 11-France et l’Université Mohammed V- Rabat –MAROC ont porté essentiellement sur l’étude de deux facteurs de risque impliqués dans le développement du carcinome hépatocellulaire ; le polymorphisme génétique et l’infection par le virus de l’hépatite C. Dans un premier temps, ce travail de recherche s’est focalisée sur l’éventuelle relation pouvant liée les polymorphismes génétiques des gènes PCAF, P300 au carcinome hépatocellulaire. Sur le plan génétique nous avons constaté que les génotypes Val/Val de EP300 et Ser/Ser de PCAF apparaissent comme les plus associés à l’hépatocarcinome. Par ailleurs, ces derniers se trouvent associés d’une manière significative au carcinome hépatocellulaire. Par la suite, notre travail a consisté à mieux caractériser la variabilité génétique du HCV et l’épidémiologie des souches virales chez la population marocaine pour lesquelles peu de données étaient disponibles. Les analyses phylogénétiques des régions NS5B et E2 et les données épidémiologiques recueillies ont permis de répondre à ces questions. Enfin, La dernière partie des travaux de cette thèse vise à identifier des facteurs cellulaires essentiels à la réplication du VHC, dans l’objectif de définir de nouvelles cibles thérapeutiques pour le traitement des hépatites virales C. Nous avons pu identifier la septine 9 comme facteur cellulaire impliqué dans le cycle de vie virale du HCV, ainsi que les protéines PIAS1 et SUMO1 comme protéines régulatrices de la septine 9. De plus nous avons pu établir la relation entre ces protéines cellulaires dont la présence est nécessaire à la fois à la production des protéines virales ( CORE et E2) et à la réplication du virus dans la cellule hôte. / Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer worldwide and the third most common cause of cancer mortality. HCC is one of the few cancers with welldefined major risk factors. Major causes of HCC include hepatitis B, hepatitis C, alcoholic liver disease, nonalcoholic, steatohepatitis, hereditary hemochromatosis, and geneticalteration. The multifactorial causes of HCC might explain its complex molecular pathogenesis. Detailed understanding of epidemiologic factors and molecular mechanisms associated with HCC ultimately could improve our current concepts for screening and treatment of this disease. With a view toward current concepts for screening of this disease, first, we analyzed a genetic susceptibility to hepatocellular carcinoma. The aim of the current study was to assess the impact of the Ile997Val in EP300 and Asn386Ser in PCAF polymorphisms on the risk of HCC. we found that the Val/Val of the EP300 at codon 997 and Ser/Ser of the PCAF at codon 386 were associated with an increased risk of HCC in the Moroccan population. A higher risk of HCC was observed in HCV-negative subjects. We also found that male with Ser/Ser in thePCAF gene and women with Val/Val genotype of the EP300 had a higher risk to develop HCC. It appears that variants of the transcriptional coactivator genes (EP300 and PCAF) may influence HCC risk in populations with low mutations or hromosomal instability rates. Additional surveys are warranted to confirm this first report. The incidence of HCC is increasing in Western countries, mostly because of the high prevalence of hepatitis C virus (HCV) infection. In this sense, we have tried to identify new host factors involved in replication of HCV. Hepatitis C virus (HCV) replicates in the liver,and chronic infection often leads to cirrhosis, steatosis, and hepatocellular carcinoma. There is no vaccine to protect against HCV infection. Major improvement has been recently achieved regarding treatment of HCV infection however there is already evidence for emergence of resistance due to the high genetic variability of the HCV RNA genome. Thus it should be important, to design therapies which avoid genotypic resistance by targeting cellular proteins. Understanding the mechanisms that allow proper assembly and intracellular trafficking of HCV proteins may have a profound impact on the treatment efficacy. An important hallmark of chronic HCV infection is liver steatosis associated with the accumulation of lipid droplets (LDs). The LDs accumulated in the HCV infected cells and the HCV core protein induces the redistribution of LDs, through the clustering of these organelles in the perinuclear area, providing a platform for virus assembly and in the production of infectious particles. Several host proteins have been proposed to facilitate the replication of HCV however much is needed to elucidate the implicated mechanisms. Here we report that HCV infection increases expression and formation of septin 9 filament surrounding HCV core. Data of our study revealed the highest expression of septin 9 in samples from hepatocellular carcinoma (HCC) from patients with chronic HCV infection. Indeed, we brought newperception of septin 9 function regarding both microtubule and lipid structure organization. We proposed that HCV infection conducts to assembly of septin 9 in filaments through PIAS1. This may provide scaffolds to organize the microtubules network and LDs redistribution required for HCV assembly and replication. This study illuminates a new function of septin 9 in HCV life cycle through its association with lipids and microtubules. These events might depend on septin 9 SUMO1 modification by PIAS1. These findings also provide a step forwards in understanding the relationship between HCV and its host and open new therapeutic directions.