Transcription profiling in papillary thyroid carcinoma reveals potential diagnostic markers and drug targets

Financial support FAPESP and CAPES

Gene Expression Profile Induced by Two Different Variants of Street Rabies Virus in Mice

Pathogenicity and pathology of rabies virus (RABV) varies according to the variant, but the mechanisms are not completely known. In this study, gene expression profile in brains of mice experimentally infected with RABV isolated from a human case of dog rabies (V2) or vampire bat-acquired rabies (V3) were analyzed. In total, 138 array probes associated with 120 genes were expressed differentially between mice inoculated with V2 and sham-inoculated control mice at day 10 post-inoculation. A single probe corresponding to an unannotated gene was identified in V3 versus control mice. Gene ontology (GO) analysis revealed that all of the genes upregulated in mice inoculated with V2 RABV were involved in the biological process of immune defense against pathogens. Although both variants are considered pathogenic, inoculation by the same conditions generated different gene expression results, which is likely due to differences in pathogenesis between the dog and bat RABV variants. This study demonstrated the global gene expression in experimental infection due to V3 wild-type RABV, from the vampire bat Desmodus rotundus, an important source of infection for humans, domestic animals and wildlife in Latin America

Genome-wide methylation and transcriptome analysis in penile carcinoma: uncovering new molecular markers

Background: Despite penile carcinoma (PeCa) being a relatively rare neoplasm, it remains an important public health issue for poor and developing countries. Contrary to most tumors, limited data are available for markers that are capable of assisting in diagnosis, prognosis, and treatment of PeCa. We aimed to identify molecular markers for PeCa by evaluating their epigenomic and transcriptome profiles and comparing them with surrounding non-malignant tissue (SNT) and normal glans (NG).Results: Genome-wide methylation analysis revealed 171 hypermethylated probes in PeCa. Transcriptome profiling presented 2,883 underexpressed and 1,378 overexpressed genes. Integrative analysis revealed a panel of 54 genes with an inverse correlation between methylation and gene expression levels. Distinct methylome and transcriptome patterns were found for human papillomavirus (HPV)-positive (38.6%) and negative tumors. Interestingly, grade 3 tumors showed a distinct methylation profile when compared to grade 1. In addition, univariate analysis revealed that low BDNF methylation was associated with lymph node metastasis and shorter disease-free survival. CpG hypermethylation and gene underexpression were confirmed for a panel of genes, including TWIST1, RSOP2, SOX3, SOX17, PROM1, OTX2, HOXA3, and MEIS1.Conclusions: A unique methylome signature was found for PeCa compared to SNT, with aberrant DNA methylation appearing to modulate the expression of specific genes. This study describes new pathways with the potential to regulate penile carcinogenesis, including stem cell regulatory pathways and markers associated to a worse prognosis. These findings may be instrumental in the discovery and application of new genetic and epigenetic biomarkers in PeCa.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

miR-22 and miR-205 Drive Tumor Aggressiveness of Mucoepidermoid Carcinomas of Salivary Glands

ObjectivesTo integrate mRNA and miRNA expression profiles of mucoepidermoid carcinomas (MECs) and normal salivary gland (NSGs) tissue samples and identify potential drivers. Material and MethodsGene and miRNA expression arrays were performed in 35 MECs and six NSGs. ResultsWe found 46 differentially expressed (DE) miRNAs and 3,162 DE mRNAs. Supervised hierarchical clustering analysis of the DE transcripts revealed two clusters in both miRNA and mRNA profiles, which distinguished MEC from NSG samples. The integrative miRNA-mRNA analysis revealed a network comprising 696 negatively correlated interactions (44 miRNAs and 444 mRNAs) involving cell signaling, cell cycle, and cancer-related pathways. Increased expression levels of miR-205-5p and miR-224-5p and decreased expression levels of miR-139-3p, miR-145-3p, miR-148a-3p, miR-186-5p, miR-338-3p, miR-363-3p, and miR-4324 were significantly related to worse overall survival in MEC patients. Two overexpressed miRNAs in MEC (miR-22 and miR-205) were selected for inhibition by the CRISPR-Cas9 method. Cell viability, migration, and invasion assays were performed using an intermediate grade MEC cell line. Knockout of miR-205 reduced cell viability and enhanced ZEB2 expression, while miR-22 knockout reduced cell migration and invasion and enhanced ESR1 expression. Our results indicate a distinct transcriptomic profile of MEC compared to NSG, and the integrative analysis highlighted miRNA-mRNA interactions involving cancer-related pathways, including PTEN and PI3K/AKT. ConclusionThe in vitro functional studies revealed that miR-22 and miR-205 deficiencies reduced the viability, migration, and invasion of the MEC cells suggesting they are potential oncogenic drivers in MEC

Interplay Between Immune and Cancer-Associated Fibroblasts: A Path to Target Metalloproteinases in Penile Cancer

Extracellular matrix (ECM) remodeling and inflammation have been reported in penile carcinomas (PeCa). However, the cell types and cellular crosstalk involved in PeCa are unexplored. We aimed to characterize the complexity of cells and pathways involved in the tumor microenvironment (TME) in PeCa and propose target molecules associated with the TME. We first investigated the prognostic impact of cell types with a secretory profile to identify drug targets that modulate TME-enriched cells. The secretome analysis using the PeCa transcriptome revealed the enrichment of inflammation and extracellular matrix pathways. Twenty-three secreted factors were upregulated, mainly collagens and matrix metalloproteinases (MMPs). The deregulation of collagens and MMPs was confirmed by Quantitative reverse transcription - polymerase chain reaction (RT-qPCR). Further, the deconvolution method (digital cytometry) of the bulk samples revealed a high proportion of macrophages and dendritic cells (DCs) and B cells. Increased DCs and B cells were associated with better survival. A high proportion of cancer-associated fibroblasts (CAFs) was observed in low-survival patients. Patients with increased CAFs had decreased immune cell proportions. The treatment with the MMP inhibitor GM6001 in CAF cells derived from PeCa resulted in altered cell viability. We reported a crosstalk between immune cells and CAFs, and the proportion of these cell populations was associated with prognosis. We demonstrate that a drug targeting MMPs modulates CAFs, expanding the therapeutic options of PeCa

Integrative methodology in penile carcinomas

O desenvolvimento de metodologias sobre integração de dados na área de biologia de sistemas é de grande importância devido ao aumento contínuo de dados resultantes de análises globais que são depositados em bancos de dados públicos. Poucas metodologias e ferramentas de bioinformática levam em consideração a diferenciação entre drivers e passengers, fundamental para a identificação de genes importantes para o desenvolvimento e progressão tumoral. Os perfis de expressão gênica têm possibilitado a identificação de assinaturas genéticas em uma grande variedade de tumores humanos. Além disso, as alterações epigenéticas, como a expressão de microRNAs (miRNA) e a metilação do DNA, também contribuem para o desenvolvimento de diversos tipos de doenças. Entretanto, a grande maioria destes estudos não mostra integração dos resultados obtidos pelas diferentes estratégias utilizadas, o que teria maior impacto na identificação de drivers moleculares. Neste estudo foi realizada a integração de quatro níveis de alterações em 31 amostras de câncer de pênis (CaPe): alteração do número de cópias do DNA, metilação de ilhas CpGs, expressão de miRNAs e expressão de transcritos codificadores. O conhecimento das alterações genéticas e epigenéticas relacionadas ao desenvolvimento de câncer de pênis é bastante limitado, devido principalmente a sua rara incidência. Uma parcela significativa dos casos de CaPe tem sido associada com a infecção pelo Papilomavírus Humano (HPV). A metodologia para integração de dados foi aplicada utilizando duas abordagens: (1) estudo das alterações em câncer de pênis (tumor e normal) independente da infecção pelo HPV e (2) estudo das alterações relacionadas à infecção pelo HPV. A análise foi dividida em duas etapas, com a seleção de genes alvos específicos da doença e inferência de módulos a partir desses alvos. Destacam-se na metodologia a seleção de genes candidatos a driver utilizando a atribuição de pesos para cada alteração seguindo critérios pré-determinados, e.g. se o gene estava presente em uma região rara, após classificação pelo DGV (Database of Genomic Variants) e a utilização desses genes como alvos para identificação das possíveis relações entre eles e os módulos. Também foi realizada a adaptação da metodologia de redes em módulos, com a inclusão de genes passengers e interação proteína-proteína (PPI) como um critério para seleção dos módulos. Essa análise se mostrou eficaz na identificação de módulos gênicos bem relacionados com os drivers, resultando na escolha de vias biológicas potencialmente responsáveis pelo desenvolvimento do tumor. Os genes identificados após a comparação entre amostras tumorais e normais (SOX17, TWIST1, CAV1, PPARG, FLI1 e TNFSF10) e no estudo entre amostras positivas e negativas para infecção pelo HPV (PCNA, SOX14 e RFC4) foram validados in situ por técnicas independentes. Para validação in silico das alterações encontradas na metodologia de integração de dados e para validação da metodologia de redes em módulos foram utilizadas 255 amostras de glioblastoma multiforme obtidas no banco de dados TCGA (The Cancer Genome Atlas). Foram identificadas vias biológicas importantes relacionadas ao processo tumoral, como regulação do crescimento celular (GO:0001558, p=0,0062), homeostase (GO:0048872, $p=0,0082) e regulação da transcrição (GO:0003700, p=0,00089). Também foi realizada uma meta-análise utilizando amostras do TCGA, que encontrou um perfil similar de expressão para os genes CAV1, DLC1,FLI1, MSX1, NRN1, PML, PPARG e SOX17 (T vs N) e PCNA e RFC4 (HPV+ vs HPV-). Para o nosso conhecimento, esse é o primeiro estudo em CaPe utilizando análise integrada de quatro níveis de alteração. Além disso, foram encontradas alterações não randômicas capazes de modificar transcritos específicos e contribuir para o conhecimento da patobiologia dos tumores de pênis.Methodologies for data integration in systems biology area have great importance due to continuous increase of public data resulting from large-scale analysis, which are deposited in public databases. Few methodologies and bioinformatics tools take into consideration the differentiation between drivers and passengers, fundamental for the identification of important genes for tumor development and progression. The gene expression profiles have allowed the identification of genetic signatures in a wide variety of human tumors. In addition, epigenetic changes, such as the expression of microRNA (miRNA) and DNA methylation, also contribute to the development of a veriety of diseases. However, most of these studies did not show integration of results obtained by different strategies used, which would have increased impact to identify molecular drivers. This study provides a methodology for integration of four levels of changes in 31 samples of penile cancer (PeCa): copy number alterations, DNA methylation of CpG islands, miRNA expression and gene expression of coding transcripts. Knowledge about genetic and epigenetic changes related to the development of penile cancer is very limited, mainly due to its rare incidence. A significant portion of PeCa samples has been associated with infection by Human papillomavirus (HPV). The methodology for integrative data was applied using two approaches: (1) the study considering alterations in penile carcinoma (tumor and normal), independent of HPV infection and (2) the study considering alteration related to HPV infection in PeCa. In each study, the methodology was divided into two stages, with the selection of target genes and the inference of disease specific modules from these targets. It is highlighted in the methodology the selection of candidate genes using the driver assigning weights to each change following predetermined criteria, e.g if the gene was present in a rare region after classification using the DGV database (Database of Genomic Variants) and the use of these genes as seeds for identification of possible relationships between them and the modules. For this, another contribution of this study was the adaptation of module network methodology, with the inclusion of passengers genes and protein-protein interaction (PPI) as a criteria to select the modules. This analysis was effective in identifying gene modules and related drivers, resulting in the choice of biological pathway potentially responsible for the tumor development. The genes identified after comparing tumor and normal samples (SOX17, TWIST1, CAV1, PPARG, FLI1 and TNFSF10) and the genes identified in the study of positive and negative samples for HPV infection (PCNA, SOX14 and RFC4) were validated in situ by independent techniques. For in silico validation of the changes found in the integrative methodology and the modules network were used 255 samples of glioblastoma multiforme obtained at TCGA database (The Cancer Genome Atlas). Biological pathways have been identified related to the tumoral process, such as cell growth regulation (GO:0001558, p=0,0062), homeostasis (GO:0048872, p=0,0082) and transcription regulation (GO:0003700, p=0,00089). Also, a meta-analysis was performed using samples from TCGA, who found a similar expression profile for CAV1, DLC1, FLI1, MSX1, NRN1, PML, PPARG and SOX17 (T vs N) and PCNA and RFC4 genes (HPV + vs HPV-). To our knowledge, this is the first integrative analysis in PeCa using a four-level of gene alterations. In addition, it was found non-random alterations capable to modifying specific transcripts and contribute to the knowledge about the pathobiology of penile tumors

Informática no ensino fundamental e médio: investigando o uso de computadores na escola

Com o passar do tempo e com a maior evolução da informática devido à criação de computadores cada vez mais potentes foram feitos diversos investimentos em ferramentas para transmissão de informações a distância. Dentre essas ferramentas estão softwares para interação usuário-máquina, programas que auxiliam a realização de processos de maneira eficaz e automatizada e a internet, que possibilitou conectar vários usuários em todo o mundo. O uso da informática é cada vez mais necessário em todas as áreas e isso não é diferente na educação. Este trabalho teve como objetivo investigar as condições de 10 escolas localizadas em três cidades do Estado de São Paulo: Campinas, Leme e Botucatu, com relação à utilização da informática no processo de aprendizagem dos alunos e como recurso aos professores e funcionários para o auxilio a informatização do ambiente escolar e desenvolvimento de novas práticas de ensino. Por meio das respostas foi possível fazer um levantamento das condições do uso da informática nas escolas visitadas identificando as deficiências ainda encontradas com relação à utilização dos computadores. Nota-se ainda nas escolas, um equívoco sobre a real função do computador que muitas vezes é confundido como simples entretenimento ao invés de um grande mecanismo para o processo de desenvolvimento cognitivo e aprendizagem

Epigenetic mechanisms in penile carcinoma

Penile carcinoma (PeCa) represents an important public health problem in poor and developing countries. Despite its unpredictable behavior and aggressive treatment, there have only been a few reports regarding its molecular data, especially epigenetic mechanisms. The functional diversity in different cell types is acquired by chromatin modifications, which are established by epigenetic regulatory mechanisms involving DNA methylation, histone acetylation, and miRNAs. Recent evidence indicates that the dysregulation in these processes can result in the development of several diseases, including cancer. Epigenetic alterations, such as the methylation of CpGs islands, may reveal candidates for the development of specific markers for cancer detection, diagnosis and prognosis. There are a few reports on the epigenetic alterations in PeCa, and most of these studies have only focused on alterations in specific genes in a limited number of cases. This review aims to provide an overview of the current knowledge of the epigenetic alterations in PeCa and the promising results in this field. The identification of epigenetically altered genes in PeCa is an important step in understanding the mechanisms involved in this unexplored disease. © 2013 by the authors; licensee MDPI, Basel, Switzerland

DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53 mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq