Based on matching analysis between different EBV strains, we found two nucleotide variations in miR-BART21 and four nucleotide changes in miR-BART22. Interestingly, two nucleotide variations upstream of mature miR-BART22 likely favor its biogenesis by Drosha/DGCR8 processing and we experimentally confirmed this augmentation by in-vitro Drosha digestion, and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. / Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1 and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remain largely unclear. / MicroRNAs (miRNAs) are a class of small, non-coding RNAs with a size around 18--24 nucleotides with significant roles in regulating gene expression by either transcriptional silencing or translational suppression. As gene regulators, recent miRNA studies have emphasized the contribution of aberrant miRNA expression in cancer development. The recent discovery of EBV encoded viral miRNAs (ebv-miRNAs) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNAs. In this study, we have systematically examined the NPC associated EBV genome for viral-encoded miRNA expression. By constructing small cDNA libraries from a native EBV positive NPC cell line (C666-1) and a xenograft (X2117), we screened about 3000 clones and detected several small EBV fragments, within which two novel ebv-miRNAs in the BARTs region were identified. These two newly identified miRNAs, now named miR-BART21 and miR-BART22, were proven to be abundantly expressed in most NPC samples by both Northern blot and QRT-PCR analysis. / Taken together, this thesis shows that two newly identified EBV-encoded miRNAs are highly expressed in latent EBV infection in NPC. Frequent expression of miR-BART22 can be explained partially by a specific EBV strain that is associated with NPC in our locality. Our findings emphasize the role of miR-BART22 in modulating LMP-2A expression. Because LMP-2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells (CTLs), down-modulation of LMP-2A expression by mir-BART22 may permit escape of EBV-infected cells from host immune surveillance. / We attempted to predict the potential viral and cellular targets of miR-BART21 and miR-BART22 by public available computer programs, miRanda and RNAhybrid. A number of potential cellular mRNA targets were suggested, although many failed to be validated by luciferase reporter assay. However, we found a putative miR-BART22 binding site in the LMP2A-3'UTR. Although the LMP-2A transcript is consistently detected in NPC, only 6 out of 26 (23%) primary NPC tumors show weak LMP-2A expression by immunohistochemistry (IHC). The expression levels of miR-BART22 and LMP-2A mRNA have also been determined in eleven of these tumors. Interestingly, the LMP-2A mRNA expression level did not directly correlate with protein expression, and relatively low expression levels of miR-BART22 miRNA were observed in all 3 LMP-2A positive-primary tumors. The suppressive effect of miR-BART22 on LMP-2A was also experimentally validated by a series of dual luciferase reporter assays using reporter constructs containing the putative or mutated recognition site at the LMP-2A 3'UTR. By co-transfection of different amounts of miR-BART22 with the LMP-2A-3'UTR expression vector in reporter assay, we confirmed that miR-BART22 suppressed the LMP-2A protein level in a dose-dependent manner. Furthermore, transfection of miR-BART22 into HEK293 cells that had been stably transfected with pcDNA3.1-LMP-2A, which contains a complete LMP-2A ORF and 3'UTR, readily suppressed levels of the LMP-2A protein. / Lung, Wai Ming Raymond. / Adviser: To Ka Fai. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 197-226). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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

<p>Vaccination with DNA encoding the encephalitogenic autoantigen myelin oligodendrocyte glycoprotein (MOG), pMOG91-108, induce a protective immunity against experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. By injection of a DNA vaccine that contains a DNA region encoding short interfering RNA specific for IFNβ (pMOG-IFNβ) the protective effect of the DNA vaccination is totally inhibited. This demonstrates that IFN-β is directly involved in the protective mechanism against EAE.</p><p>The objective of this project was to study how molecules involved in the inflammatory process in EAE are regulated by suppressive DNA vaccination. mRNA expression of IL-1β, TGF β, IL-23p40 and Axl receptor tyrosine kinas did not show any significant differences between the groups vaccinated with these DNA vaccines. IL-6 and IFNγ mRNA expression after MOG stimulation in rats treated with pCI, a control vaccine was significantly higher compared to the group vaccinated with vaccine containing pMOG-IFNβ. IL-17 m RNA expression after MOG stimulation in pCl-treated rats was significantly higher compared to the group vaccinated with vaccine containing pMOG-91-108. Of these results the mRNA expression of IL-17 and IL-6 were of interest for the project.</p><p>The immune system normally protects the body against infections and T-cells have an important role in this defence system. In MS and EAE, the immune system attacks the myelin and this process is caused by a dysregulation of the T-cells. IL-17-producing Th17 cells mediate EAE. Naïve CD4 T-cells in the presence of IL-6 and TGFβ are differentiated to Th17 cells instead of differentiating into T-helper or regulatory T-cells. These IL-17-producing T-cells are highly pathogenic and essential for the development of EAE. The results showed that pMOG IFNβ vaccine had an effect at the immune response, which resulted in an inhibition of the IL-6 production and that vaccination with pMOG91-108 impairs differentiation of IL-17-producing T-cells.</p>

Vaccination with DNA encoding the encephalitogenic autoantigen myelin oligodendrocyte glycoprotein (MOG), pMOG91-108, induce a protective immunity against experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. By injection of a DNA vaccine that contains a DNA region encoding short interfering RNA specific for IFNβ (pMOG-IFNβ) the protective effect of the DNA vaccination is totally inhibited. This demonstrates that IFN-β is directly involved in the protective mechanism against EAE. The objective of this project was to study how molecules involved in the inflammatory process in EAE are regulated by suppressive DNA vaccination. mRNA expression of IL-1β, TGF β, IL-23p40 and Axl receptor tyrosine kinas did not show any significant differences between the groups vaccinated with these DNA vaccines. IL-6 and IFNγ mRNA expression after MOG stimulation in rats treated with pCI, a control vaccine was significantly higher compared to the group vaccinated with vaccine containing pMOG-IFNβ. IL-17 m RNA expression after MOG stimulation in pCl-treated rats was significantly higher compared to the group vaccinated with vaccine containing pMOG-91-108. Of these results the mRNA expression of IL-17 and IL-6 were of interest for the project. The immune system normally protects the body against infections and T-cells have an important role in this defence system. In MS and EAE, the immune system attacks the myelin and this process is caused by a dysregulation of the T-cells. IL-17-producing Th17 cells mediate EAE. Naïve CD4 T-cells in the presence of IL-6 and TGFβ are differentiated to Th17 cells instead of differentiating into T-helper or regulatory T-cells. These IL-17-producing T-cells are highly pathogenic and essential for the development of EAE. The results showed that pMOG IFNβ vaccine had an effect at the immune response, which resulted in an inhibition of the IL-6 production and that vaccination with pMOG91-108 impairs differentiation of IL-17-producing T-cells.

Over 500 million people worldwide suffer from disease associated with intestinal inflammation, including gastric cancer, inflammatory bowel disease, h. pylori infections, and numerous viral and bacterial infections. Although potentially effective therapeutics exist for many of these pathologies, delivery challenges thwart their clinical viability. The objective of this work was to develop drug delivery platforms that could target toxic immunomodulatory therapeutics to diseased intestinal tissues. To meet this objective, we developed an oral delivery vehicle for siRNA and an NF-κB inhibiting nanoparticle that reduces drug-resistance. Small interfering RNA (siRNA) represents a promising treatment strategy for numerous gastrointestinal (GI) diseases; however, the oral delivery of siRNA to inflamed intestinal tissues remains a major challenge. In this presentation, we describe a delivery vehicle for siRNA, termed thioketal nanoparticles (TKNs), that can orally deliver siRNA to sites of intestinal inflammation, and thus inhibit gene expression in diseased intestinal tissue. Using a murine model of ulcerative colitis, we demonstrate that orally administered TKNs loaded with TNFα-siRNA (TNFα-TKNs) diminish TNFα messenger RNA (mRNA) levels in the colon and protect mice from intestinal inflammation. Activation of nuclear factor-κB (NF-κB) results in the expression of numerous prosurvival genes that block apoptosis, thus mitigating the efficacy of chemotherapeutics. Paradoxically, all conventional therapeutics for cancer activate NF-κB, and in doing so initiate drug resistance. Although adjuvant strategies that block NF-κB activation could potentiate the activity of chemotherapeutics in drug resistant tumors, clinical evidence suggests that current adjuvant strategies also increase apoptosis in non-malignant cells. In this presentation, we present a nanoparticle, formulated from a polymeric NF-κB-inhibiting prodrug, that target the chemotherapeutic irinotecan (CPT-11) to solid tumors, and thus abrogates CPT-11-mediated drug resistance and inhibits tumor growth. In order to maximize the amount of NF-κB inhibitor delivered to tumors, we synthesized a novel polymeric prodrug, termed PCAPE, that releases the NF-κB inhibitor caffeic acid phenethyl ester (CAPE) as its major degradation product. Using a murine model of colitis-associated cancer, we demonstrate that when administered systemically, CPT-11-loaded PCAPE-nanoparticles (CCNPs) are three time more effective than a cocktail of the free drugs at reducing both tumor multiplicity and tumor size.

Small silencing RNAs function in almost every aspect of cellular biology. Argonaute proteins bind small RNA and execute gene silencing. The number of Argonaute paralogs range from 5 in Drosophila melanogaster , 8 in Homo sapiens to an astounding 27 in Caenorhabditis elegans. This begs several questions: Do Argonaute proteins have different small RNA repertoires? Do Argonaute proteins behave differently? And if so, how are they functionally and mechanistically distinct? To address these questions, we examined the thermodynamic, kinetic and functional properties of fly Argonaute1 (dAgo1), fly Argonaute2 (dAgo2) and mouse Argonaute2 (mAGO2). Our studies reveal that in fly, small RNA duplexes sort into Argonaute proteins based on their intrinsic structures: extensively paired siRNA duplex is preferentially sorted into dAgo2 while imperfectly paired miRNA duplex is channeled into dAgo1. The sorting of small RNA is uncoupled from its biogenesis. This is exemplified by mir-277, which is born a miRNA but its extensive duplex structure licenses its entry into dAgo2. In the Argonaute protein, the small RNA guide partitions into functional domains: anchor, seed, central, 3' supplementary and tail. Of these domains, the seed initiates binding to target. Both dAgo2 and mAGO2 (more closely related to and a surrogate for dAgo1 in our studies) bind targets at astonishing diffusion-limited rates (~107–108 M−1s−1). The dissociation kinetics between dAgo2 and mAGO2 from their targets, however, are different. For a fully paired target, dAgo2 dissociates slowly (t½ ~2 hr) but for a seed-matched target, dAgo2 dissociates rapidly (t½ ~20 s). In comparison, mAGO2 does not discriminate between either targets and demonstrates an equivalent dissociation rate (t½ ~20 min). Regardless, both dAgo2 and mAGO2 demonstrate high binding affinity to perfect targets with equilibrium dissociation constants, KD ~4–20 pM. Functionally, we also showed that dAgo1 but not dAgo2 silence a centrally bulged target. By contrast, dAgo2 cleaved and destroyed perfectly paired targets 43-fold faster than dAgo1. In target cleavage, dAgo2 can tolerate mismatches, bulged and internal loop in the target but at the expense of reduced target binding affinities and cleavage rates. Taken together, our studies indicate that small RNAs are actively sorted into different Argonaute proteins with distinct thermodynamic, kinetic and functional behaviors. Our quantitative biochemical analysis also allows us to model how Argonaute proteins find, bind and regulate their targets.

piRNAs guide PIWI proteins to silence transposons in animal germ cells. In Drosophila, the heterochromatic piRNA clusters transcribe piRNA precursors to be transported into nuage, a perinuclear structure for piRNA production and transposon silencing. At nuage, reciprocal cycles of piRNA-directed RNA cleavage—catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila—destroy the sense transposon mRNA and expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate. My thesis research mainly focuses on two fundamental questions about the piRNA production: How does the germ cell differentiate piRNA precursor from mRNAs for piRNA biogenesis? And what is the mechanism to impose Aub Ping-Pong with Ago3? For the first question, we show that the HP1 homolog protein Rhino marks the piRNA cluster regions in the genome for piRNA biogenesis. Rhino seems to anchor a nuclear complex that suppresses cluster transcript splicing, which may differentiate piRNA precursors from mature mRNAs. Moreover, LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger de novo piRNA production from a trans combination of sense and antisense transgenes. For the second question, we show that Qin, a new piRNA pathway factor contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, enforces heterotypic Ping- Pong between Aub and Ago3. Loss of qinleads to less Ago3 binding to Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, DNA damage accumulates in the germ cells and flies are sterile.

Selective gas-phase dissociation strategies have been developed for the characterization of cross-linked peptides and proteins in quadrupole ion trap mass spectrometers. An infrared chromogenic cross-linker (IRCX) containing a phosphotriester afforded rapid differentiation of cross-linked peptides from unmodified ones in proteolytic digests of cross-linked proteins by selective infrared multiphoton dissociation (IRMPD). Only the cross-linked peptides containing the chromogenic phosphate underwent IRMPD and unmodified peptides were not affected by IR irradiation. IRMPD of IRCX-cross-linked peptides yielded uncross-linked y-ion sequence tags of the constituent peptides due to secondary dissociation of all primary product ions which contained the chromophore, thus allowing successful de novo sequencing of the cross-linked peptides. Peptides cross-linked via a two-step conjugation strategy through the formation of a bis-arylhydrazone (BAH) bond were selectively dissociated by ultraviolet radiation at 355 nm. The BAH-cross-linked peptides could be distinguished from not only unmodified peptides but also dead-end modified peptides based on the selectivity of ultraviolet photodissociation. In a complementary approach, electron transfer dissociation of BAH-cross-linked peptides resulted in preferential cleavage of the hydrazone bond which produced two modified peptides. These modified peptides were subsequently interrogated by CID which allowed for the original site of cross-linking to be pinpointed. IRMPD was implemented in a dual pressure linear ion trap to demonstrate successful photodissociation of peptides having modest absorptivities. Peptides were observed to efficiently dissociation by IR irradiation exclusively in the low pressure cell whereas no dissociation was observed in the high pressure cell due to extensive collisional cooling. IRMPD provided greater sequence coverage of the peptides than CID and yielded product ion mass spectra which were predominantly composed of singly charged product ions which simplified spectral interpretation. IRMPD was further applied for the sequencing of small-interfering RNA. Complete sequence coverage was obtained and the results were compared to CID. / text

Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for research and treatment of numerous diseases. However, siRNA cannot easily cross the cell membrane due to its inherent instability, large molecular weight and anionic nature. For this reason, a carrier that protects, delivers and unloads siRNA is required for successful gene silencing. The goal of this research was to develop a potential siRNA delivery system for in vitro and in vivo applications using cationic polymers, chitosan and polyethylenimine (PEI), poly(ethylene glycol) (PEG), mannose, and poly(D,L-lactic-co-glycolic acid) (PLGA). Furthermore, the delivery system was constructed in two different ways to explore the effect of mannose location in the structure. In the first approach, mannose and PEG were directly conjugated to the chitosan/PEI backbone, while mannose was connected to the chitosan/PEI backbone through PEG spacer in the second approach. First, the ability of modified chitosan polymers to complex and deliver siRNA for gene silencing was investigated. Despite the modified chitosan polymers successfully formed nanoplexes with siRNA, entered target cells and reduced cytotoxicity of unmodified chitosan, they showed limited gene silencing efficiency. For this reason, modified PEIs were examined to improve in vitro gene knockdown. The modified PEI polymers also complexed with siRNA and facilitated endocytosis of the nanoplexes. In addition, the modifications reduced inherent cytotoxicity of unmodified PEI without compromising the gene silencing efficiency on both mRNA and protein levels. Interestingly, we found that complexation of siRNA with PEI-PEG-mannose resulted in higher cell uptake and gene silencing than complexes made with mannose-PEI-PEG. Finally, the effect of sustained release of the mannosylated pegylated PEI/siRNA nanoplexes on gene silencing was tested by encapsulating the nanoplexes within PLGA microparticles. The modified PEIs enhanced the entrapment efficiency of siRNA into the particles and resulted in reduced initial burst followed by sustained release. Incorporating the modified PEIs increased cellular uptake of siRNA, whereas it did not enhance in vitro gene knockdown efficiency due to the sustained release properties. The modified PEIs reduced the in vitro cytotoxicity and in vivo hepatotoxicity of the PLGA microparticles. In addition, encapsulating the nanoplexes into PLGA microparticles further reduced the cytotoxicity of PEI. Throughout the study, the second structure was proven more efficacious than the first structure in cellular uptake, gene silencing, siRNA encapsulation, and sustained release. We have developed novel polymeric siRNA delivery systems that enhance delivery efficiency and cellular uptake of siRNA. They have great potential for utility as a long-acting siRNA delivery system in biomedical research.

Das Harnblasenkarzinom (BCa) stellt in Deutschland die vierthäufigste Tumorneuerkrankung und die zehnthäufigste krebsbedingte Todesursache bei Männern dar. Nichtmuskelinvasive BCa werden organerhaltend aus der Blasenwand entfernt und zur Rezidiv- und Progressionsprophylaxe mittels intravesikaler Chemo- oder Immuntherapien behandelt. Trotz dieser adjuvanten Therapien, die mit starken Nebenwirkungen verbunden sein können, ist nur eine bedingte Minimierung des Rezidivrisikos möglich. Besonders im fortgeschrittenen Stadium weisen Harnblasenkarzinome eine schlechte Prognose auf. Obwohl das BCa eine chemosensitive Erkrankung darstellt, wird das Ansprechen auf lokale oder systemische Chemotherapien häufig durch auftretende Resistenzmechanismen limitiert. Daher stehen sowohl die Verbesserung konventioneller Chemotherapien als auch die Suche nach neuartigen Behandlungsstrategien im Fokus der experimentellen BCa-Forschung. Die Apoptose, eine Form des programmierten Zelltodes, ist ein essenzieller, streng regulierter biologischer Prozess, welcher der Aufrechterhaltung der Gewebshomöostase und der gezielten, entzündungsfreien Eliminierung geschädigter Zellen dient. Fehlregulationen in den Apoptosesignalwegen stellen ein zentrales Ereignis in der Tumorgenese dar und tragen außerdem zur Entstehung von Chemo- und Radiotherapieresistenzen bei. Eine wichtige Rolle in der Apoptoseregulation spielen die Mitglieder der BCL2- und der Inhibitor of Apoptosis Protein (IAP)-Familien, deren wichtigste antiapoptotische Vertreter BCL2, BCL-XL, XIAP und Survivin häufig in Tumoren, einschließlich des BCa, überexprimiert sind. Unter Verwendung von small interfering RNAs (siRNAs), synthetischen Nukleinsäurekonstrukten zur selektiven Geninhibition, wurde im Rahmen der Arbeit in vitro und in vivo untersucht, ob die Hemmung der Apoptoseinhibitoren BCL2, BCL-XL, XIAP und Survivin – allein und in Kombination mit Chemotherapie – eine Therapieoption zur Behandlung des BCa darstellen könnte. Da zur Tumorentstehung und -progression eine Vielzahl von genetischen Veränderungen beitragen, erscheint der Angriff eines einzelnen Zielgens unzureichend für eine effektive Tumortherapie. Aufgrund dessen wurde untersucht, ob durch simultane Reduktion der ausgewählten Apoptoseinhibitoren in BCa-Zellen stärkere wachstumsinhibitorische Effekte erzielt werden können. In der vorliegenden Arbeit wurde gezeigt, dass insbesondere die siRNA-vermittelte Hemmung von BCL-XL und Survivin in den BCa-Zelllinien EJ28 und J82 antiproliferative Effekte hervorruft und diese Tumorzellen gegenüber einer nachgeschalteten Chemotherapie mit Mitomycin C oder Cisplatin sensitiviert. Hingegen bewirkte sowohl die transiente als auch die stabile RNAi-induzierte Hemmung von BCL2 und XIAP in den untersuchten BCa-Monolayerzellkulturen, möglicherweise infolge kontinuierlicher Versorgung der Tumorzellen mit Sauerstoff und Nährstoffen, keine Reduktion des Tumorwachstums. Eine gegenüber den Einzelbehandlungen deutliche Verstärkung der antitumoralen und insbesondere der chemosensitivierenden Effekte in den BCa-Zelllinien wurde durch simultane Hemmung von BCL-XL und Survivin erzielt. Beispielsweise stieg der Anteil apoptotischer Zellen von 64 % nach Survivin-siRNA+Cisplatin-Behandlung auf 94 % nach gleichzeitiger BCL-XL+Survivin-Inhibition in Kombination mit Cisplatin. Folglich stellt die simultane Inhibition von BCL-XL und Survivin in Kombination mit Chemotherapeutika eine äußert viel versprechende BCa-Therapieoption dar. Tierexperimentelle Studien belegen die wachstumsinhibitorische Wirkung der Survivin-Reduktion und der kombinierten BCL-XL-siRNA+Chemotherapie-Behandlung, so wurde das Tumorendvolumen im Vergleich zur Kontrollbehandlung um 43 % bzw. um 48 % reduziert.