Exprese miRNA u nádorů hlavy a krku asociovaných a neasociovaných s HPV

Nádory hlavy a krku představují skupinu nádorů dvojí etiologie. První skupinou jsou nádory asociované s infekcí HPV, druhou skupinou jsou nádory nevirové etiologie, které jsou spojeny s dvěma hlavními rizikovými faktory, a tím je kouření a konzumace alkoholu. Z publikovaných studií je zřejmé, že HPV-pozitivní nádory se vyskytují častěji u mladších pacientů a zároveň tito pacienti vykazují lepší prognózu a celkové přežívání. Z tohoto důvodu je zvažována modifikace léčby na základě etiologie nádorového onemocnění. Nicméně je důležité zajistit specifický, citlivý a klinicky významný biomarker pro přesné určení etiologie nádoru. Vhodným kandidátem pro takové biomarkery jsou miRNAs, jakožto malé nekódující regulační molekuly, které jsou stabilní i v archivních vzorcích, a jejichž rozdílná exprese byla detekována v řadě lidských nádorů a je specifická pro nádory různého původu. Předkládaná práce je zaměřena na miRNA profilování v HPV-pozitivních a HPV-negativních nádorech krčních mandlí a nádorech děložního hrdla s cílem najít rozdíly v regulaci důležitých karcerogenních drah nádorů virové a nevirové etiologie. Naše výsledky ukazují velkou heterogenitu v expresních profilech miRNAs u těchto nádorů. I přes využití velmi dobře charakterizovaného a jednotného souboru vzorků jsme detekovali malý překryv...Head and neck cancers represent a group of tumors with two different etiologies. The first type is associated with the viral HPV infection, the second one is virus-independent and it is associated with smoking and alcohol consumption as two main risk factors. Numerous studies show that HPV-positive tumors are more frequent in younger patients, as well as that the prognosis and overall survival of these patients is remarkably better. Therefore, the modification of the treatment is considered. For this, however, specific, sensitive and clinically relevant biomarkers for accurate identification of tumor etiology is needed. Suitable candidates for such biomarkers are miRNAs, small non-coding regulatory molecules stable in archived samples, that have been shown as differentially expressed in human cancers and the expression pattern seems specific for tumors of different origin. The submitted thesis focuses on miRNA profiling in HPV-positive and HPV-negative tonsillar tumors and cervical carcinomas with the aim to find out the differences between regulation of important carcinogenetic pathways of tumors of viral and non-viral etiology. Our data have shown very large heterogeneity of the miRNA expression profiles of these tumors. Despite the well characterized and uniform samples collection, we have found...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyFaculty of SciencePřírodovědecká fakult

Oncogene and Cancer

This book describes a course of cancer growth starting from normal cells to cancerous form and the genomic instability, the cancer treatment as well as its prevention in form of the invention of a vaccine. Some diseases are also discussed in detail, such as breast cancer, leucaemia, cervical cancer, and glioma. Understanding cancer through its molecular mechanism is needed to reduce the cancer incidence. How to treat cancer more effectively and the problems like drug resistance and metastasis are very clearly illustrated in this publication as well as some research result that could be used to treat the cancer patients in the very near future. The book was divided into six main sections: 1. HER2 Carcinogenesis: Etiology, Treatment and Prevention; 2. DNA Repair Mechanism and Cancer; 3. New Approach to Cancer Mechanism; 4. New Role of Oncogenes and Tumor Suppressor Genes; 5. Non Coding RNA and Micro RNA in Tumorigenesis; 6. Oncogenes for Transcription Factor

Exploring the interaction between the human microbiota and infections

The microbiota is a living ecosystem that is influenced by a variety of host and environmental factors. Distinct microbiota colonizes various body sites, such as the gastrointestinal system and vaginal tract, corresponding to the unique microenvironment. A healthy gut microbiota contains a stable, balanced, and highly diverse reservoir of microbes. Commensal microorganisms co-evolved with the host have conferred pathogen colonization resistance. Lactobacillus species usually dominate the vaginal microbiota of majority healthy women. The vaginal microbiota has also been linked to sexually transmitted diseases, such as the human papillomavirus (HPV) infection. MicroRNA expression was found to be associated with microbiota composition. We studied the interactions of microbiota gut and vagina with metabolites, miRNA expression, and HPV infection in our investigations. In vitro three- dimensional (3D) cell-culture methods were also analyzed and developed for mechanistic investigations of the microbiota-host interaction. Study I is a cross-sectional study investigating the microbiota features in HPV-related diseases. We defined the HPV-related microbial composition in a high-vaccination coverage population of 345 young Swedish women. The associations of microbial composition and the infection of 27 HPV types were analyzed. HPV infection, especially for the infection of oncogenic HPV types, was characterized by a higher microbial alpha-diversity with non- Lactobacillus-dominant vaginal microbiota composition. The prevalence of bacterial vaginosis-associated bacteria (BV AB), Sneathia, Prevotella, and Megasphaera were significantly higher in women with HPV infection than in uninfected individuals. Study II investigated the associations among miRNA, HPV infection, and vaginal microbiota composition. A global miRNA expression increase was identified in non-Lactobacillus- dominate women compared with Lactobacillus-dominated women. The top two differently expressed miRNA, miR-23a-3p and miR-130a-3p, showed a prediction accuracy of > 97% for distinguishing Lactobacillus-dominated and non- Lactobacillus-dominated samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the target genes of miR-23a-3p and miR-130a-3p found that many pathways involved in cancer and infection were enriched, such as the mitogen-activated protein kinase (MAPK) signaling pathway, and the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. Study III explored the interactions between commensal bacteria and pathogens. We cocultured Salmonella with commensal bacteria isolations from an anaerobically cultivated human intestinal microflora (ACHIM). Commensal bacteria isolations belonging to the Clostridium and Eubacterium family showed significant inhibition of Salmonella growth. Following metabolite extraction and liquid chromatography-mass spectrometry analysis of the commensal bacteria and Salmonella metabolites in the coculture system of the commensal bacteria and Salmonella, adenine and adenosine were significantly higher in the coculture systems that inhibited Salmonella growth compared with the expressive systems. Functional assays of metabolite activity further validated the inhibition effect of adenine and adenosine on the growth of Salmonella and other antibiotic-resistant pathogens. Study IV established an anaerobic gut 3D model to study bacteria-host interactions. The Caco2 cells showed good cell viability and formed intestinal villi-like structures in our model. Anaerobic culture for 12 hours did not show a significant effect on the viability of cells, with only six genes differently expressed between cells cultured under anaerobic and aerobic conditions in RNA sequencing. RNA sequencing of Salmonella infected Caco2 cells cocultured in anaerobic conditions showed a large set of genes differentially expressed compared to aerobic conditions with the pathways associated with cell cycle, homologous recombination, and DNA replication. This supports our gut 3D model could be used for investigating host-microbe direct interactions under the anaerobic condition, which is essential for obligate anaerobic gut microbes

Role of Exosomes in the Transfer of Viral Nucleic Acids to Recipients Cells: in Vitro Studies with Cell Line Supernatant and Patient-Derived Exosomes

Human PapillomaVirus (HPV) is a main cause of cervical cancer in which HPV DNA is frequently found integrated into human genome at fragile sites. Meta analytic studies reported association between HPV and Breast Cancer (Bae JM, 2016). We found 30% of HPV DNA in breast cancer tissues, confirmed by ISH assay. HPV DNA is also found in other extra-genital tissues, including oro-pharingeal, anal, colon and skin (Kim SM, 2016; Pérez LO, 2010). Owing to the lack of viremia, HPV DNA presence in other districts, is still a conundrum. We hypothesized that the presence of HPV DNA in extra-genital tissues could be in relation with exosomes. Exosomes are extracellular vesicles, involved in cellular communication, tumor progression and biological molecules carriers. We isolated exosomes from 59 serum specimens from breast cancer patients by differential ultracentrifugation and we identified 5 HPV DNA positive samples. Subsequently we isolated exosomes, from HeLa and Caski supernatants (HPV18 and HPV16 positive, respectively) and from HPV DNA positive serum, urine and liquor patient by CD9-immunobeads isolation kit. Exosomes obtained were used to perform viral transfer in different cell lines. Finally, the literature reports association between SV40 and certain types of cancer, including bone tumors (Vilchez RA, 2003). In line with literature, we found 7/70 SV40 DNA in bone tumors (unpublished). We isolated exosomes, from COS7 supernatant (SV40 positive cell line) and we tested acid nucleic viral transfer to osteosarcoma cell lines. Exposed cells were analyzed by PCR, Real-time PCR and Digital PCR, demonstrating the viral nucleic acids transfer in receiving cells. The viral nucleic acids discovery in receiving cells, confirm the role of exosomes as biological molecules carriers. The mechanism through which the viral content is released, is yet to be clarified, as well as if it is sufficient to trigger infection

Proteomic study of the therapeutic effect of a DNA vector on cervical cancer cells

Cancer is a major global health problem, accounting for more than 8 million deaths/year worldwide. In particular, cervical cancer is the 3rd most common malignancy and 4th cause of death among women globally, being a persistent High Risk-HPV infection the main factor for cervical cancer development. E6 and E7 HPV oncoproteins are responsible for cancer development by degrading and inhibiting p53 and pRb tumour suppressor proteins, respectively. Moreover, microRNAs (miRs) have been found to regulate tumorigenesis. In fact, miR-375 has the ability to silence HPV E6 and E7 oncoproteins. Gene therapy is a promising strategy to treat acquired diseases and/or genetic disorders, aiming to deliver genetic material into target cells or tissue, expressing it to induce a therapeutic effect. Minicircle DNA (mcDNA) is a new biopharmaceutical product only composed by the eukaryotic transcription unit, improving its safety and therapeutic effect, obtained through intramolecular recombination of the parental plasmid. Thus, this work aims to produce and purify a mcDNA vector encoding primiR-375 and p53 genes to silence E6 and E7 oncoproteins and re-establish p53 and pRb tumour suppressor levels on cervical cancer cells. The purification of mcDNA vectors was performed using size exclusion chromatography sepharose columns (Sephacryl S-1000 SF), showing that for the smallest vector, mcDNAprimiR-375, a 106 mL column allowed the efficient recovery of chromatographic fractions composed only with mcDNA. On the other hand, for the biggest mcDNA vectors, it was necessary to exploit the parameters affecting the sample molecules separation, choosing to use a column with 180 mL of volume, allowing, similarly to the mcDNA-primiR-375 purification, recover fraction only composed with mcDNA. Simultaneously, cytotoxicity assays on human fibroblasts and CaSki cells were performed, confirming that none of the vectors were toxic to the fibroblasts and the mcDNA primiR-375+p53 presented the lower cell viability for CaSki cells. In addition, the proliferation assay results performed on CaSki cells show that number of viable cells decreases for the mcDNA vectors transfected cells, corroborating the cytotoxicity assays results for this cell line. Western-Blot confirmed the re-establishment of tumour suppressor proteins levels after 48h of transfection using mcDNA-p53 and mcDNAprimiR-375+p53. Overall, these results suggest that the use of DNA vectors encoding for more than one therapeutic gene, for example, the mcDNA-primiR-375+p53, allowed to achieve, on in vitro assays, results suggesting a possible faster therapeutic action, thus demonstrating that they may revolutionize their application in targeted therapies.O cancro é um dos principais problemas de saúde, sendo responsável por mais de 8 milhões de mortes todos os anos, a nível mundial. O cancro do colo do útero é a 3ª malignidade mais frequente, sendo a 4ª causa de morte nas mulheres a nível mundial. A infeção persistente por HPV de alto risco é o fator principal para o desenvolvimento de cancro do colo do útero, dado que as oncoproteínas E6 e E7 são capazes de promover o desenvolvimento cancerígeno através da degradação e inibição das proteínas supressoras de tumor, p53 e pRb, respetivamente. A terapia génica é uma estratégia promissora no tratamento de doenças adquiridas e/ou desordens genéticas, tendo como objetivo a entrega de material genético em células ou tecidos alvo, de forma a induzir um efeito terapêutico. O DNA minicircular (mcDNA) é um novo produto biofarmacêutico, que se caracteriza por ser apenas constituído pela unidade de transcrição eucariota, aumentando a sua segurança e efeito terapêutico, sendo obtido através da recombinação intramolecular do plasmídeo parental. Além disso, tem-se atribuído aos microRNAs (miRs) um papel regulador da carcinogénese, por exemplo, está descrito que o miR-375 possui a capacidade de silenciar as oncoproteínas E6 e E7. Desta forma, este trabalho tem como objetivo a produção e purificação de vetores de mcDNA que codifiquem para os genes primiR-375 e p53, de forma a silenciar as oncoproteínas E6 e E7 e reestabelecer os níveis das proteínas supressoras de tumor p53 e pRb nas células do cancro do colo do útero. A purificação dos vetores de mcDNA foi efetuada utilizando colunas de sefarose (Sephacryl S1000 SF) de cromatografia de exclusão molecular, demonstrando que para o vetor de tamanho mais pequeno, mcDNA-primiR-375, uma coluna de 106 mL permitiu uma recuperação eficiente de frações cromatográficas compostas apenas por mcDNA. No entanto, para os vetores de maior tamanho, mcDNA-p53 e mcDNA-primiR-375+p53, foi necessário explorar os parâmetros que afetam a separação das moléculas das amostras a purificar, optando-se por utilizar uma coluna com o volume de 180 mL, permitindo assim, à semelhança do mcDNA-primiR-375, recuperar frações compostas apenas por mcDNA. Paralelamente foram realizados ensaios de citotoxicidade em fibroblastos humanos e células CaSki, revelando que nenhum dos vetores é tóxico nos fibroblastos humanos e que o mcDNA-primiR-375+p53 apresentou a menor viabilidade celular em células CaSki. Para além disso, os resultados do ensaio de proliferação realizado em células CaSki demonstraram que o número de células viáveis diminui nas células transfectadas com os vetores de DNA, corroborando os ensaios de citotoxicidade realizados nesta mesma linha celular cancerígena. A realização do ensaio de Western-Blot confirmou o restabelecimento dos níveis da proteína supressora de tumor p53 após 48 horas de transfecção com mcDNA-p53 e mcDNA-primiR-375+p53 em células CaSki. Os resultados obtidos nos em ensaios in vitro desenvolvidos neste trabalho, sugerem que o uso de vetores de DNA que codifiquem para mais do que um gene com finalidade terapêutica, como é exemplo o mcDNA-primiR-375+p53, permitiram observar efeitos mais significativos, sugerindo assim que este tipo de vetores podem ter uma ação terapêutica mais eficiente

Regulation of the long non-coding RNA FAM83H-AS1 by human papillomavirus in cervical cancer

Non-coding RNAs (NcRNAs), such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), have been found to be involved in a variety of critical biological processes, and dysregulation of ncRNAs have been involved with several human diseases including cancer. High-risk human papillomavirus (HPV) infection is one of the first events in the process of carcinogenesis in cervical and a subset of head and neck cancers. The expression of the viral oncoproteins E6 and E7 is essential in this process by inactivating the tumor suppressor proteins p53 and Rb, respectively, in addition to their interactions with other host proteins and regulation of ncRNAs. Our group identified novel regulation of host lncRNAs by HPV oncoprotein E6. More specifically, we discovered that a lncRNA known as FAM83H-AS1 is involved with proliferation, migration, and apoptosis in cervical cells, and high expression of this lncRNA correlates with poor overall cervical cancer patient survival. FAM83H-AS1 is a nuclear RNA, and mechanistically it is regulated through the E6-p300 pathway in a p53-independent manner. These findings provide knowledge of a specific lncRNA that could be studied further as a biomarker and/or therapeutic target not only in HPV-related cancers but also in other types of cancers where FAM83H-AS1 expression is dysregulated. In parallel with these studies, our group identified a specific subgroup of miRNAs that are induced during quiescence and processed by a non-canonical biogenesis pathway by using primary human cells. miRNA expression is dysregulated when cells undergo a reversible state of growth arrest known as quiescence. These primary (pri-)miRNAs are modified with a 2,2,7-trimethylguanosine (TMG)-cap such that they are processed downstream in an Exportin-1 (XPO1)-dependent manner, independent of the canonical Exportin-5 (XPO5) protein used for exportation to the cytoplasm. The discovery of a new alternative miRNA pathway in quiescent primary human cells opens the door to future studies in other types of cells, such as stem cells and cancer stem cells, where the state of quiescence is important in their biological functions

Detection and characterisation of enteroviruses and the gut virome in islet autoimmunity and type 1 diabetes using novel molecular methods

Type 1 diabetes (T1D) is a chronic autoimmune condition affecting over 8.4 million individuals globally. Usually preceded by islet autoimmunity (IA), T1D results from a complex interplay between host genetics and environmental factors, with virus infections, especially enteroviruses (EV) identified as a prominent candidate factor. Advances in molecular techniques including next generation sequencing (NGS) have enabled sensitive and high-throughput virus detection, improving on cell culture and serology techniques. There are over 110 human/primate EVs identified, classified within four species (A-D). Elucidation of which subtypes precipitate IA/T1D would inform the design of primary prevention studies. The project aimed to 1) develop a novel method of EV amplification using NGS; 2) optimise capture-based sequencing of viruses in stools of children at-risk of T1D; 3) characterise viruses in stools of participants in the Viruses in the Genetically at Risk (VIGR) and Environmental Determinants of Islet Autoimmunity (ENDIA) Australian longitudinal cohorts; and 4) update our previous systematic review examining the association between EVs and IA/T1D. Near full-length EV was detected in 67% of 113 VIGR stools and 96% of 23 EV prototypes examined. Coxsackievirus (CV)B3, ECHO18/30, and infection with multiple EV subtypes were associated with IA in VIGR stools. Polymorphisms frequently mapped to VP2/VP1 capsid regions in cases and to 2C/VP2/VP3/3A in controls. Children in the VIGR study had 129 differentially abundant gut viruses, with EV-As CVA2/5/6/8/14 and EV-Bs CVB3 and ECHO6/18/30 more abundant in cases. Infants of mothers with T1D enrolled in ENDIA were more likely to have a virus infection, with bocavirus/rotavirus more abundant and CVA6/RV-C/torque teno viruses less abundant. Systematic review of 3,266 publications revealed 60 eligible studies (40 T1D, 9 IA, 11 both) comprising 12,077 individuals. Meta-analysis demonstrated significant associations between EV and IA (odds ratio 2.1; 95% confidence interval 1.3 to 3.3; P=0.002), T1D (8.0; 4.9 to 13.0; P<0.00001); within one month of T1D (16.2; 8.6 to 30.5; P <0.00001). Substantial heterogeneity was observed between studies. These results strengthen the rationale for EV-targeted vaccines for primary prevention of IA/T1D. Future studies will examine virus infection during pregnancy and early life in populations with and without genetic risk for T1D

Circulating Biomarkers for Cancer Immunoprofiling

abstract: Biomarkers find a wide variety of applications in oncology from risk assessment to diagnosis and predicting and monitoring recurrence and response to therapy. Developing clinically useful biomarkers for cancer is faced with several challenges, including cancer heterogeneity and factors related to assay development and biomarker performance. Circulating biomarkers offer a rapid, cost-effective, and minimally-invasive window to disease and are ideal for population-based screening. Circulating immune biomarkers are stable, measurable, and can betray the underlying antigen when present below detection levels or even no longer present. This dissertation aims to investigate potential circulating immune biomarkers with applications in cancer detection and novel therapies. Over 600,000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers. A key challenge in understanding HPV immunobiology and developing immune biomarkers is the diversity of HPV types and the need for multiplexed display of HPV antigens. In Project 1, nucleic acid programmable protein arrays displaying the proteomes of 12 HPV types were developed and used for serum immunoprofiling of women with cervical lesions or invasive cervical cancer. These arrays provide a valuable high-throughput tool for measuring the breadth, specificity, heterogeneity, and cross-reactivity of the serologic response to HPV. Project 2 investigates potential biomarkers of immunity to the bacterial CRISPR/Cas9 system that is currently in clinical trials for cancer. Pre-existing B cell and T cell immune responses to Cas9 were detected in humans and Cas9 was modified to eliminate immunodominant epitopes while preserving its function and specificity. This dissertation broadens our understanding of the immunobiology of cervical cancer and provides insights into the immune profiles that could serve as biomarkers of various applications in cancer.Dissertation/ThesisDoctoral Dissertation Molecular and Cellular Biology 201