NOVEL ROLES OF MICRORNAS IN HEPATITIS C

Approximately, 170 million people are infected by hepatitis C virus (HCV) worldwide, and of these, nearly 85% will develop chronic hepatitis C (CHC). Despite finding new anti-viral treatment that increase response rate from 45 % to 65-70 %, investigations continue to find more effective treatments for hepatitis C because of side effects and limitations of current treatment. It is known that miR-122 enhances HCV replication by binding to two closely spaced target sites in the 5’-UTR of the viral genome, which leads to an increase in abundance of HCV RNA. We found that miR-122 down- regulates Occludin (OCLN), one of the key HCV receptors, by directly targeting 3’-UTR of OCLN mRNA. We also found that interaction of miR-122 with 3’-UTR of OCLN mRNA eventually results in a decrease in HCV entry. In accordance with our in vitro study, we found an inverse correlation between pre-treatment levels of miR-122 and HCV RNA levels in patients with CHC. This is a new finding of our study which is consonant with our hypothesis that miR-122 may play an antiviral role in uninfected hepatocytes and early stages of HCV infection. Protein Kinase R (PKR), a double- stranded RNA-dependent protein kinase, is among the well-known members of cellular antiviral proteins transcriptionally induced by IFNs in response to viral infection. We found that miR-122 down- regulates PRKRA expression by targeting 3’-UTR of PRKRA mRNA in uninfected Huh7.5 cells. This down-regulation led to decrease in phosphorylation of PKR. Our results are consonant with the notion that, in infected hepatocytes, miR-122 preferentially binds to 5’-UTR of HCV RNA rather than to the3’-UTR of PRKRA, and this is the main factor that increases HCV replication. Based on our findings, both in vitro and in CHC patients, we speculate that miR-122 could play a dual role in HCV infection; in uninfected hepatocytes miR-122 plays an antiviral role through down-regulation of OCLN while, in infected hepatocytes, miR-122 increases HCV replication through binding to the 5’-UTR of HCV RNA. Our results suggest that miR-122 mimics may be more beneficial than miR-122 inhibitors in the earlier stages of infection or as a prophylactic approach when few or no hepatocytes are infected with HCV. Both responses to treatment as well as spontaneous outcome of HCV infection are critically affected by host genetic factors. We found that pre-treatment levels of hepatic miR-29b were significantly lower in CHC patients with early viral response (EVR) than those without EVR. This novel finding could be very important both for predicting the outcome of disease as well as suggesting new treatment approaches for chronic hepatitis C. Low levels of miR-29b in early responders to HCV therapy might potentially benefit future therapeutic interventions involving the use of miR-29 antagomirs. We also showed that miR-29b level serves as an independent factor for predicting advanced stages of fibrosis in patients with CHC. These findings are unexpected, because miR-29b has been shown to exhibit anti-fibrotic effects in vitro. Hence, caution should be exercised in extrapolating in vitro observations to subjects with CHC. Higher levels of mir-29b in these patients may suggest a role of over-expression of miR-29b as an anti-fibrotic factor in advanced degree of liver fibrosis as a healing process for liver. Broader translational and in vitro studies are needed to unravel the importance of miR-29b in prognosis and treatment of hepatitis C

MOLECULAR PATHOGENESIS OF HEPATITIS B VIRUS AND HEPATITIS C VIRUS INFECTIONS

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections cause a wide range of liver diseases including hepatocellular carcinoma (HCC) worldwide. Because these two viruses have the same modes of transmission, HBV HCV co-infections are found in approximately 7 to 20 million people globally. HBV HCV co-infections are associated with more severe liver diseases and higher risk of HCC. Previous studies have established that HBV and HCV mono-infection can cause hepatic steatosis, and steatosis is a confirmed risk factor for HCC. However, whether and how HBV HCV co-infections synergistically increase the risk of HCC development through modulating lipid metabolism is not well understood. PTEN is a phosphatase which contains both lipid and protein phosphatase activities. PTEN acts as a tumor suppressor by down-regulating the phosphoinositide-3-kinase (PI3K)/Akt/ sterol regulatory element-binding protein (SREBP) signaling pathway. It has been reported that PTEN is frequently mutated or deleted in tumors including HCC. PTEN-Long, a longer isoform of PTEN, which is able to be exported into the extracellular compartments, enter neighboring cells, and induce signaling events in recipient cells. Both HBV and HCV infections can inhibit PTEN and activate SREBP. However, how regulation of PTEN/SREBP pathway affects HBV and HCV infections is not fully understood. Therefore, the effect of the PTEN/SREBP pathway on HBV and/or HCV infections is worthy to study. In this study, we showed that both HBV X protein (HBx) and HCV core protein regulate PTEN/SREBP pathway. We established that HBx activates SREBP-1a and SREBP-1c through different mechanisms. This process is involved in up-regulating of HBV enhancer I or HBV enhancer II. We demonstrated multiple mechanisms of how HBx regulates HBV replication. We also showed that HCV core interacts with PTEN and PTEN-Long which is involved in regulating of HCV life cycle. In an HBV HCV co-infection cellular model, HBx and HCV core have similar regulatory effects on the PTEN/SREBP pathway as in mono-infections. However, PTEN and SREBP differentially regulate HBV and HCV replication in HBV HCV co-infection

An investigation of genome-wide promoter region cytosine-phosphate-guanine (CpG) Island methylation profiles in patients with chronic hepatitis B virus infection

Includes bibliographical references.Hepatitis B virus (HBV) is oncogenic and a major cause of hepatocellular carcinoma (HCC) in the developing world. It integrates parts of its genome such as the HBx gene, core and surface antigens into the human genome. The integrated viral DNA disrupts gene function resulting in physiological changes that cause liver disease. The viral inserts are inactivated through methylation. This is a protective innate response driven by human DNA methyltransferases triggered by the presence of viral DNA inserts. This thesis investigates the hypothesis that during the innate response to methylate integrated HBV DNA, there is unintended methylation of genomic DNA around the intercalated viral DNA that could be adjacent host promoter Cytosine-phosphateGuanine (CpG) islands. This would activate or silence genes including tumour suppressors and result in the clinical disease phenotypes of hepatic inflammation, fibrosis and HCC that characterise chronic HBV infection. Genome-wide microarray analysis was used to investigate for the presence of promoter CpG island methylation in a cohort of patients with liver disease due to HBV infection, HCC, autoimmune hepatitis which is a non-viral liver disease and normal cases with no liver disease. The study identified hypermethylation in promoter regions, transcription start sites, gene exons and introns. Only sites in the promoter region and within 100bp upstream of a transcription start site were analysed for this thesis presentation. Using an extended cohort of patients with chronic HBV infection and normal controls, bisulfite DNA sequencing was used to validate and confirm the presence of DNA methylation in a selection of some of genes identified. HBV infected patients were shown to have hypermethylation in the promoter CpG island regions of several genes that regulate hepatic metabolism, tumour suppression, ribonucleic acid splicing, vitamin D receptor binding, protein ubiquitination and the cell cycle. Many of these genes have transcriptional binding factors that are known to be affected by the transcriptional transactivator HBx protein, suggesting that HBx protein is important in the pathogenesis of liver disease. Amongst the most hypermethylated core promoter regions identified were those for cyclin kinases genes such as Cyclin D3 (CCND3). CCND3 gene is important in liver regeneration and wound healing and its abnormal function has been linked to the development of liver fibrosis and HCC. Increased methylation of CCND3 gene was associated with HBV e antigen positive status and genotype D, supporting the hypothesis that increased methylation is associated with host and viral factors. Methylation induced alteration in the function of the identified gene promoters would affect cellular signalling with effects on cell growth, differentiation, proliferation and apoptosis. These changes would explain the development of hepatic inflammation, apoptosis, fibrosis and malignant transformation seen in chronic HBV infection. Further investigation of these genes will provide new insights on mechanisms of HBV induced liver disease and the development of new molecular diagnostic tools or therapeutic interventions

MicroRNA profiling of human hepatocytes induced by HBx in hepatocarcinogenesis.

Yip, Wing Kit.Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.Includes bibliographical references (leaves 100-119).Abstract also in Chinese.Abstract (English version) --- p.iAbstract (Chinese version) --- p.iiiAcknowledgments --- p.vTable of Contents --- p.viiList of Tables --- p.xList of Figures --- p.xiList of Abbreviations --- p.xiiiChapter CHAPTER 1 --- INTRODUCTION --- p.1Chapter 1.1 --- Hepatocellular Carcinoma --- p.1Chapter 1.1.1 --- Epidermiology --- p.1Chapter 1.1.2 --- Etiology --- p.1Chapter 1.2 --- Hepatitis B Virus --- p.3Chapter 1.2.1 --- The Epidermiology of Hepatitis B Virus Infection --- p.3Chapter 1.2.2 --- The Morphology and Genome of Hepatitis B Virus --- p.4Chapter 1.2.3 --- HBV Genotypes and Their Significance --- p.8Chapter 1.3 --- Hepatitis B Virus X Protein --- p.9Chapter 1.3.1 --- HBx Alters Various Signal Transduction Pathways --- p.10Chapter 1.3.2 --- HBx Interacts with Various Transcription Factors and Co-activators --- p.12Chapter 1.3.3 --- HBx Induces Epigenetic Alterations --- p.14Chapter 1.3.4 --- Identification of COOH-terminal Truncated HBx in Liver Tumors --- p.15Chapter 1.4 --- MicroRNAs --- p.17Chapter 1.4.1 --- Transcriptional Regulation and Biogenesis of MicroRNAs --- p.18Chapter 1.4.2 --- MicroRNAs and Cancer --- p.21Chapter 1.4.3 --- MicroRNAs and HCC --- p.25Chapter 1.5 --- Hypothesis and Aims of the Study --- p.29Chapter CHAPTER 2 --- MATERIALS and METHODS --- p.30Chapter 2.1 --- Patients --- p.30Chapter 2.2 --- Cell Lines --- p.30Chapter 2.3 --- Cloning of Various HBx Constructs --- p.32Chapter 2.3.1 --- PCR Amplification of HBx Fragments --- p.32Chapter 2.3.2 --- Cloning of HBx Fragments into TA-vectos --- p.33Chapter 2.3.3 --- Heat Shock Transformation --- p.33Chapter 2.3.4 --- Sub-cloning of HBx Fragments into Lentiviral Vectors --- p.34Chapter 2.4 --- Generation of Lentivirus --- p.37Chapter 2.4.1 --- Lentivirus Infection --- p.37Chapter 2.5 --- RNA Extraction --- p.38Chapter 2.6 --- Western Blot Analysis --- p.39Chapter 2.7 --- MiRNA Microarray --- p.40Chapter 2.7.1 --- Cyanine3-pCp Labeling of RNA Samples --- p.40Chapter 2.7.2 --- Sample Hybridization --- p.41Chapter 2.7.3 --- Microarray Wash --- p.41Chapter 2.7.4 --- Array Slide Scanning and Processing --- p.41Chapter 2.8 --- Detection of HBx Gene Deletion by PCR --- p.43Chapter 2.9 --- Immunohistochemistry --- p.44Chapter 2.10 --- Quantitative Real-time PCR --- p.45Chapter 2.11 --- Proliferation Assay --- p.47Chapter 2.12 --- Cell Cycle Analysis --- p.48Chapter 2.13 --- Annexin V Apoptosis Assay --- p.49Chapter 2.14 --- Colony Formation Assay --- p.50Chapter 2.15 --- Statistical Analysis --- p.51Chapter CHAPTER 3 --- RESULTS --- p.52Chapter 3.1 --- Detection of Full-length and COOH-terminal Truncated HBx in HCC Tissues --- p.52Chapter 3.2 --- Confirmation of HBx Expression in HCC Tissues --- p.55Chapter 3.3 --- Comparison of HBx from Different HBV Genotypes for Study --- p.61Chapter 3.4 --- Functional Characterization of COOH-tterminal Truncated HBx --- p.64Chapter 3.4.1 --- Selection of COOH-terminal Truncated HBx --- p.64Chapter 3.4.2 --- Generation of Various HBx-expressing Hepatocyte Cell Lines --- p.66Chapter 3.4.3 --- Effect of Full-length and COOH-terminal Truncated HBx on Cell Proliferation --- p.69Chapter 3.4.4 --- Effect of Full-length and COOH-terminal Truncated HBx Cell Cycle --- p.34Chapter 3.4.5 --- Effect of Full-length and COOH-terminal Truncated HBx on Apoptosis --- p.45Chapter 3.5 --- MicroRNA Profiling of Various HBx-expressing Hepatocyte Cell Lines --- p.76Chapter 3.5.1 --- Identification of Deregulated MicroRNAs by Microarray --- p.76Chapter 3.5.2 --- Validation of Deregulated MicroRNAs by Real-time PCR Analysis --- p.80Chapter 3.5.3 --- Confirmation of Deregulated MiRNAs in HCC and Adjacent Non-tumor Tissues --- p.84Chapter 3.5.4 --- Potential Downstream Targets of the HBx-deregulated MiRNAs --- p.87Chapter CHAPTER 4 --- DISCUSSION --- p.91Chapter 4.1 --- The Impact of COOH-terminal Truncated HBx in HCC --- p.91Chapter 4.2 --- The Biological Significance of COOH-terminal Truncated HBx Induced MiRNAs --- p.94Chapter 4.3 --- Limitations of the Present Study --- p.97Chapter 4.4 --- Future Studies --- p.98Chapter CHAPTER 5 --- CONCLUSION --- p.99REFERENCES --- p.10

Lipid Nanoparticles as Carriers for RNAi against Viral Infections: Current Status and Future Perspectives

The efforts made to develop RNAi-based therapies have led to productive research in the field of infections in humans, such as hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), herpetic keratitis, human papillomavirus, or influenza virus. Naked RNAi molecules are rapidly digested by nucleases in the serum, and due to their negative surface charge, entry into the cell cytoplasm is also hampered, which makes necessary the use of delivery systems to exploit the full potential of RNAi therapeutics. Lipid nanoparticles (LNP) represent one of the most widely used delivery systems for in vivo application of RNAi due to their relative safety and simplicity of production, joint with the enhanced payload and protection of encapsulated RNAs. Moreover, LNP may be functionalized to reach target cells, and they may be used to combine RNAi molecules with conventional drug substances to reduce resistance or improve efficiency. This review features the current application of LNP in RNAi mediated therapy against viral infections and aims to explore possible future lines of action in this field.This work was supported by the Basque Government's Department of Education, Universities and Investigation (IT-341-10)

Papel de los microARNs en el diagnóstico del carcinoma hepatocelular relacionado con el virus de la hepatitis B y su papel en la regulación del ADN circular covalente cerrado en pacientes con hepatitis B crónica en la zona gris

Antecedentes: La infección por el virus de la hepatitis B (VHB) sigue siendo un problema de salud pública mundial, destacando como el principal factor de riesgo del carcinoma hepatocelular (CHC). La mayoría de los pacientes con hepatitis B crónica (HBC) se encuentran en alguna de las 5 fases de la historia natural del VHB definidas por la Asociación Europea para el Estudio del Hígado (EASL) en 2017, pero algunos individuos no encajan en ninguno de los estados inmunes habituales y se consideran estar en la “zona gris (ZG)”. El ADN circular covalente cerrado (ADNccc) juega un papel clave en el ciclo de vida del virus y no puede ser eliminado por las terapias actuales. Además, la cuantificación del ADNccc no es fácil de generalizar debido a la naturaleza invasiva de la biopsia hepática. La creciente evidencia indica que los microARNs (miARNs) podrían regular la expresión del ADNccc intrahepático, participar en el diagnóstico y la historia natural de la enfermedad. Objetivos: (1) Realizar un metaanálisis para evaluar la precisión diagnóstica de miARN circulante como biomarcador no invasivo en el diagnóstico precoz del CHC relacionado con el VHB (CHC-VHB). (2) Explorar los marcadores subrogados no invasivos para ADNccc intrahepático en pacientes con hepatitis B crónica en la zona gris (HBC-ZG). (3) Explorar el papel regulador de los miARNs en la expresión del ADNccc en pacientes con HBC-ZG, establecer una red de interacción miARN-ARNm y proporcionar una base teórica para encontrar nuevas dianas terapéuticas. Materiales y métodos: Después de una revisión sistemática de los estudios relevantes, se combinaron la sensibilidad, la especificidad, la razón de verosimilitud positiva (PLR) y negativa (NLR), la odds ratio de diagnóstico (DOR) y el área bajo la curva (AUC) para diagnosticar el CHC-VHB. Se realizó un análisis de metarregresión y un análisis de subgrupos para explorar la heterogeneidad y se utilizó el gráfico en embudo de Deeks para evaluar el sesgo de publicación. Se incluyeron un total de 13 pacientes con HBC-ZG. Se cuantificaron el ADNccc intrahepático y los marcadores séricos de actividad virológica (incluido el ARNpg-VHB, ADN-VHB, cuantificación del HBsAg, ALT y AST). Las correlaciones entre el ADNccc intrahepático y los marcadores séricos de actividad virológica se analizaron mediante la prueba de correlación de Spearman. Se consideró estadísticamente significativa cuando p < 0,05. Según el nivel de expresión de ADNccc intrahepático, 13 pacientes con HBC-ZG se dividieron en grupo de referencia (n = 7) con ADNccc log1,5 y el valor p < 0,05 se establecieron como valor de corte. A continuación, se realizaron los análisis de enriquecimiento de las vías GO, KEGG y GSEA. Además, las interacciones miARN-ARNm se adquirieron a partir de miRTarBase, y se empleó cytoscape para construir la red de miARN-ARNm. Se calcularon el coeficiente de correlación de Pearson (r) y su valor p. Se consideró que la correlación entre miARN-ARNm diana era fuerte cuando el coeficiente era r < - 0,7 y el valor p < 0,01. Resultados: En el metaanálisis actual se incluyeron 19 artículos que incluyen 32 estudios. La sensibilidad, especificidad, PLR, NLR, DOR y AUC generales fueron 0,83 (IC 95%: 0,79 - 0,87), 0,78 (IC 95%: 0,73- 0,83), 3,9 (IC 95%: 3,0 - 4,9), 0,21 (IC 95%: 0,16 - 0,27), 18 (IC 95%: 12 - 27) y 0,88 (IC 95%: 0,85 - 0,91), respectivamente. El análisis de subgrupos muestra que el uso de los clusters de miARN junto con un aumento del tamaño de muestra pueden mejorar la precisión diagnóstica. Además, no hay sesgo de publicación. En pacientes con HBC-ZG, se vieron correlaciones significativas entre los niveles séricos de ARNpg-VHB, ADN-VHB y cuantificación del HBsAg con ADNccc intrahepático (r = 0,790, p = 0,001, r = 0,660, p = 0,013, y r = 0,730, p = 0,004, respectivamente). Se cribaron un total de 19 ED miARNs que consisten en 7 ED miARNs up-regulados y 12 down-regulados, y 340 ED ARNm que consisten en 180 up-regulados y 160 down-regulados. Los términos GO más enriquecidos estaban relacionados con el proceso biológico del virus, como la regulación del ciclo de vida viral, la regulación del proceso viral y la regulación de la replicación del genoma viral. El análisis de enriquecimiento de la vía KEGG sugirió que estos objetivos predichos estaban vinculados con la hepatitis B. Finalmente, se estableció una posible red reguladora de miARN-ARNm basada en los resultados del análisis de correlación de expresión, entre los cuales miR-4295 - ZNF224 mostró una correlación relativamente alta. Conclusión: Los miARNs circulantes (especialmente el miR-125b) podrían servir como un biomarcador no invasivo potencial en el diagnóstico precoz de pacientes con CHC-VHB. Existe una correlación significativa entre el ARNpg-VHB sérico y el ADNccc intrahepático en pacientes con HBC-ZG, lo que indica que el ARNpg-VHB sérico es un potencial biomarcador subrogado fiable del ADNccc intrahepático. La disminución de la expresión de miR-4295 en pacientes con HBC-ZG con bajo nivel de ADNccc está relacionada con el aumento de la expresión de ZNF224, que está involucrado en el proceso biológico de la replicación del virus y la regulación del ciclo de vida. Nuestros hallazgos pueden proporcionar nuevos objetivos potenciales para el tratamiento de HBC. En el futuro, se necesitan más experimentos para verificar nuestros hallazgos

Exploiting and exploring the interactions between microRNA-122 and Hepatitis C virus

Hepatitis C virus (HCV) is a single-stranded plus-sense RNA virus that is transmitted by blood-to-blood contact, and infects the human liver. HCV has a unique dependence on the liver-specific microRNA miR-122, where miR-122 binds the 5´ un-translated region of the viral RNA at two tandem sites and increases viral RNA abundance. The mechanisms of augmentation are not yet fully understood, but the interaction is known to stabilize the viral RNA, increase translation from the viral internal ribosomal entry site (IRES), and result in increased viral yield. In an attempt to create a small animal model for HCV, we added miR-122 to mouse cell lines previously thought non-permissive to HCV, which rendered these cells permissive to the virus, additionally showing that miR-122 is one of the major determinants of HCV hepatotropism. We found that some wild-type and knockout mouse cell lines – NCoA6 and PKR knockout embryonic fibroblasts – could be rendered permissive to transient HCV sub-genomic, but not full-length, RNA replication upon addition of miR-122, and that other wild-type and knockout cell lines cannot be rendered permissive to HCV replication by addition of miR-122. These knockout cell lines demonstrated varying permissiveness phenotypes between passages and isolates and eventually completely lost permissiveness, and we were unable to achieve sub-genomic RNA replication in PKR knockout primary hepatocytes. Knockdown of NCoA6 and PKR in Huh7.5 cells did not substantially impact sub-genomic replication, leading us to conclude that there are additional factors within the cell lines that affect their permissiveness for HCV replication such as epigenetic regulation during passage or transformation and immortalization. We also added miR-122 to Hep3B cells, a human hepatoma cell line lacking expression of miR-122 and previously thought to be non-permissive to HCV replication. Added miR-122 rendered the cells as highly permissive to HCV replication as the Huh7-derived cell lines commonly used to study the virus. In these cells, we were also able to observe miR-122-independent replication of sub-genomic HCV RNA. This was verified by use of a miR-122 antagonist that had no impact on the putative miR-122-independent replication, and by mutating the miR-122 binding sites to make them dependent on a single nucleotide-substituted microRNA. This replication in the absence of miR-122 was not detected in full-length HCV RNA, but was detectable using a bi-cistronic full-length genomic replicon, suggesting that the addition of a second IRES in sub-genomic and full-genomic replicons altered replication dynamics enough to allow detectable RNA replication without miR-122 binding. Because miR-122 has been implicated in protecting the viral RNA from destabilization and degradation by Xrn1, the main cytoplasmic 5´ to 3´ RNA exonuclease, we employed our miR-122-independent system to test this miR-122-mediated protection. We verified that miR-122 functions to protect the viral RNA from Xrn1, but this was insufficient to account for the overall impact of miR-122 on replication, meaning that miR-122 has further functions in the virus’ life cycle. We showed that the effect of miR-122 on translation is due to stabilization of the RNA by protecting it from Xrn1, through binding at both sites. We further evaluated the role of each miR-122 binding site (S1 and S2) in the virus life cycle, and found that binding at each site contributes equally to increasing viral RNA replication, while binding at both sites exerts a co-operative effect. Finally, we determined that binding of miR-122 at site S2 is more important for protection from Xrn1, suggesting that miR-122 binding at S1 is more important for the additional functions of miR-122 in enhancing HCV RNA accumulation. Altogether, we have shown that miR-122 is partially responsible for the hepatotropic nature of Hepatitis C virus, and that supplementation with this microRNA can render non-permissive cells permissive to viral replication. We have also identified and confirmed replication of both sub-genomic and full-length HCV RNA in the absence of miR-122. Finally, we have characterized the impact of the host RNA exonuclease Xrn1 on the HCV life cycle, and determined the roles of each miR-122 binding site in shielding the viral RNA from this host restriction factor

MicroRNA-Based Therapeutics in Hepatocellular Carcinoma: In Vitro and in Vivo Studies

Hepatocellular carcinoma (HCC) is a serious public health problem, without an effective cure. It has been demonstrated that the deregulation of microRNAs expression contribute to tumorigenesis. In HCC, miR-221 was shown to be up-regulated in more than 70% of the cases and was associated with higher tumor stage, metastasis and a shorter time to recurrence after surgery. A tumor promoting function of miR-221 was proved in a transgenic mouse model, which was predisposed to the development of liver cancers. These findings suggested that miR-221 could represent a potential target for anti-tumor approaches. Conversely, miR-199a is of interest because its level was shown to be reduced in almost 100% of HCC, it was significantly correlated with poor prognosis and was shown to target c-Met, an oncogene involved in invasion and metastasis. In the present thesis, novel Adenoviruses and Adeno-Associated viral vector (AAVVs) were developed. They were genetically modified to drive the expression of multiple binding sites for miR-221, the “miR-221 sponge”, as well as miRNA precursor for miR-199a. Analysis of miR-221 sponge in HCC cells revealed the capability to reduce miR-221 endogenous levels, which was accompanied by an increase in CDKN1B/p27 protein, a known target of miR-221. An increase in apoptosis was detected in Hep3B cells after infection with any of adeno or AAVs in comparison with control viruses. Moreover, restoring the level of miR-199a could also reduce viability and increase apoptosis of HepG2 cells. Therapeutic efficacy of miR-221 antisense oligonucleotides and a tumor suppressive role of miR-199a alone or in combination with sorafenib were also evaluated. We showed that combining an antimiR-221/mimic miR-199a with sorafenib improves the response of HCC cells to molecular targeted treatment through enhancing the inhibition of cell proliferation and induction of apoptosis and arresting the cell cycle in G1. To validate the therapeutic potential of miR-199a, we demonstrated that in vivo delivery of miR-199a oligonucleotide leads to a reduction of the number and size of tumor nodules as sorafenib, without any apparent toxicity. This study showed that methods for modulating microRNA activities could elicit measurable anti-tumor effects, that deserve further study to improve existing therapies