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Tese Priscila Cirilo Corrigida Universidade Estadual Paulista “Júlio de Mesquita Filho” Instituto de Biociências PRISCILA DANIELE RAMOS CIRILO ALTERAÇÕES GENÔMICAS E PERFIL DE EXPRESSÃO GÊNICA GLOBAL EM LEIOMIOMAS UTERINOS Tese apresentada ao Instituto de Biociências de Botucatu, Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, como requisito á obtenção do título de Doutor em Ciências Biológicas – Área de concentração: Genética. Orientadora: Profa. Dra. Silvia Regina Rogatto Botucatu 2011 Dedico este trabalho, Ao meu Deus, que sempre cuidou de mim em todas as etapas da minha vida. Obrigada Senhor, por mais esta conquista. “Porque dele e por ele, e para ele, são todas as coisas.” Romanos 11:36 AGRADECIMENTOS À minha orientadora Profa. Dra. Silvia Regina Rogatto, Obrigada pela oportunidade de trabalhar em sua equipe, pelo carinho, confiança e ensinamentos que me acompanharão por toda a vida. Sempre serei grata por toda a atenção e tempo dedicados a mim para realização deste trabalho. Muito Obrigada! À Dra. Anaglória Pontes, À Dra. Maria Aparecida Custódio Domingues, À Dra. Cláudia Aparecida Rainho, Agradeço todas vocês pelas colaborações essenciais neste trabalho. À Biblioteca e Pós-graduação - IBB, UNESP, Botucatu, Obrigada por toda ajuda e atenção durante estes anos. À todos do Centro Internacional de Pesquisa e Ensino (CIPE), Obrigada por nos receber e pela contribuição necessária para execução deste trabalho. À FAPESP, CAPES e CNPq pelo apoio financeiro. À todos integrantes e ex-integrantes do Laboratório NeoGene, Ana, Anderson, André, Ariane, Carlos, Carol, Cássia, Deborah, Eliane, Eloísa, Fábio, Fabíola, Fernanda, Francine, Georgia, Graziela, Gustavo, Haydée, Hellen, Juan, Julia, Lívia, Luciana, Márcia, Mateus, Miriam, Renata Bueno, Renata Canevari, Renée, Rodrigo, Rogério, Sabrina, Sandra, Sandra Regina e Sara. e do CIPE – Hospital A. C. Camargo, Alex, Carol, Eloísa, Felipe, Gustavo, Rodrigo, Nair, Vera e Yukie. Cada um, de alguma forma, marcou a minha vida. Obrigada pelos momentos felizes, tristes, pelas ajudas nos experimentos, correções de relatório, tese e etc….enfim, pela história construída durante este tempo. Obrigada! “E amigos são prá sempre como eterno é nosso Deus. Como amigos nós diremos até breve, nunca adeus.” Á minha família, meus pais Jair e Gumercinda e minha irmã Rafaela, pelo amor, carinho e por sempre acreditarem nos meus sonhos. Vocês sabem que não foi fácil, mas com vocês ao meu lado, tudo se tornou mais simples. Amo vocês. Obrigada. À minha avó Cida, mesmo com diagnóstico de Mal de Alzheimer há sete anos, sou muito grata por ainda se lembrar de mim. Saiba que todas ás vezes que eu ouço da sra “A vó sempre ora pedindo prá Deus te guardar lá em São Paulo” isso me dá forças prá continuar. Ao Marcelo, por sua paciência e compreensão durante a realização deste trabalho. Sua presença, amor, carinho e senso de humor peculiar me ajudaram muito! Te amo! Aos meus amigos Fábio, Lucas e Virginei, por todas as vezes que eu volto para Araçatuba e me fazerem sentir como se eu nunca tivesse saído de lá! Mesmo longe, obrigada por existirem em minha vida. Á minha amiga Bruna, por sempre me fazer acreditar que a vida “é mais do que isso”. Obrigada pelas risadas e pelos conselhos! À todos da Igreja Metodista em Araçatuba e Botucatu pelas orações e amizade construída durante todo este tempo. Deus abençõe vocês! RESUMO Os Leiomiomas Uterinos (LU) são os tumores benignos mais comuns do trato genital feminino afetando entre 25-30% das mulheres em idade reprodutiva. Embora com etiologia pouca conhecida, estes tumores constituem um importante problema de saúde pública, sendo a principal indicação para histerectomia. Aproximadamente 40- 50% dos LU apresentam anormalidades citogenéticas não randômicas, portanto, a outra metade pode conter alterações submicroscópicas, como as alterações no número de cópias de DNA (CNVs). Estudos de expressão gênica globais têm revelado o envolvimento de genes que participam das vias de proliferação e ciclo celular, do ácido retinóico, sinalização TGF-beta e IGF-1 em resposta ao estrógeno e a progesterona. Entretanto, poucos genes mapeados em regiões de CNVs foram diretamente associados com o desenvolvimento dos LU. O objetivo deste estudo foi investigar alterações no número de cópias de DNA em pacientes com LU múltiplos e únicos. Utilizando a técnica de CGH array, foram avaliadas 80 amostras de LU obtidas de 56 pacientes. O perfil de expressão gênica global foi investigado em 51 amostras com o objetivo de identificar genes diferencialmente expressos em relação ao miométrio adjacente (MM). Posteriormente, os dados genômicos e transcriptômicos foram integrados com o objetivo de identificar CNVs funcionais que possam estar associadas ao fenótipo tumoral, sendo esta análise importante para a descrição de novos marcadores moleculares e possíveis alvos terapêuticos. Amostras de DNA e RNA foram obtidas dos LU e MM de pacientes nas fases proliferativa e secretora do ciclo menstrual. As 80 amostras de DNA tumoral e DNA referência universal foram digeridas, marcadas e hibridadas em lâminas Agilent Human 4x44K CGH Microarrays . Os dados foram analisados pelo programa Nexus v5.0 com o algoritmo Rank Segmentation e limiar de significância de 1,00E-4. A classificação de CNVs não casuais ( P≤0,05) foi baseada na presença em mais de10% dos casos e exclusão de CNVs presentes em mais de 5% da população controle. Cinquenta e uma amostras de RNA foram convertidas em cRNA marcados e foram hibridados em lâminas Agilent Whole Human Genome 4x44K . Foram aplicados os testes SAM, FDR<5%, 1000 permutações e agrupamento hierárquico não- supervisionado (HCL - correlação de Pearson ) pelo programa TMeV v.4.5, para identificar os genes diferencialmente expressos em relação ao MM. A classificação dos genes com aumento ou diminuição de expressão foi baseada em valores de fold-change com cut-off de 1.5. A integração dos dados foi realizada pelo algoritmo CONEXIC, com utilização da análise de enriquecimento de genes (GSEA) para a classificação de genes moduladores. Foram identificadas 45 CNVs significativas em 77/80 amostras (96%), sendo 25% dos casos com CNVs não casuais; 29 CNVs (64%) ainda não haviam sido descritas. Os ganhos genômicos mais frequentes foram em 2q35, 6p21.32, 7q22.1 e 11q12.3-q13.2 e perdas em 4q28.3, 7q22.1-q22.2 e 7q31.33. A análise de agrupamento hierárquico (teste exato de Fisher) revelou uma tendência de separação de dois grupos de amostras com tumores múltiplos (Grupo 1) e não múltiplos (Grupo 2). Foram observados ganhos em 2q35, 7q22.1, 19p13.12-p13.11 e 19q13.32-q13.33 ( P<0,01, tumores múltiplos) e perdas em 7q22.1-q22.2 ( P<0,01, tumores não-múltiplos). A análise funcional de redes e vias dos genes mapeados nas regiões significativas realizadas pelo sistema Ingenuity Pathway Analysis (IPA) revelou funções associadas i com proliferação celular, processos fibróides e expressão gênica. A análise funcional revelou que genes do Grupo 1 (ganhos) foram associados com processos de transcrição e proliferação celular e genes do Grupo 2 (perdas), com processos de organização e ciclo celular. A análise de expressão gênica global revelou 206 genes diferencialmente expressos em relação ao MM, no qual 34% apresentaram expressão aumentada (incluindo COL3A1, COL4A1, COL17A1, IGF1 ) e 66% diminuída (incluindo CDKN2A , JUN , FOS , PCNA ). Genes com expressão aumentada atuam principalmente na proliferação e matriz extracelular e genes com expressão diminuída participam dos processos de recombinação cromossômica e reparo a danos no DNA. A análise integrada dos dados revelou 75 genes moduladores, entre os quais 26 tiveram correlação positiva e 3 correlação negativa. Trinta e um genes com correlação inversa apresentaram regulação por miRNAs. A análise GSEA revelou 32/75 genes associados a módulos de genes de câncer. Entre estes genes, FGFR1, DBN1, NUPR1, COL3A1, AOC3, IFITM1, CALCRL, NOVA2, MVP, MFAP5, DIP2C apresentaram correlação positiva e CENPF , RHOH e BICD1 apresentaram correlação negativa. A análise funcional dos 75 genes moduladores revelou que eles atuam principalmente no ciclo celular e proliferação e crescimento celular, confirmando os achados independentes de CGH array e microarrays de expressão. Os genes FGFR1 e IGFBP5 foram associados com vias de proliferação e crescimento celular, enquanto que o COL3A1 foi associado com organização celular. Estes genes estavam localizados na periferia das redes e ligados indiretamente á fatores de transcrição, indicando que podem atuar indiretamente na transcrição de genes alvos. Em adição, os genes COL3A1 , IGFBP5 e FGFR1 foram associados à via canônica de proliferação da fibrose hepática e podem ser potenciais alvos terapêuticos. Os achados deste estudo permitiram descrever novas regiões de CNVs, além de confirmar o envolvimento de outras já descritas em LU. Estas CNVs envolvem genes associados principalmente com mecanismos transcricionais e ciclo celular. A análise de expressão gênica global revelou a diminuição da atividade de genes que controlam os processos de recombinação cromossômica e reparo a danos no DNA e genes com atividade aumentada associados com estímulos de proliferação e acúmulo da matriz extracelular. A nova estratégia de integração dos dados genômicos e transcriptômicos revelou novos marcadores moleculares com uso potencial para o tratamento do Leiomiomas Uterinos, uma doença frequente e com etiologia pouco conhecida. ii ABSTRACT Uterine leiomyomas (UL) are the most common benign tumors affecting between 25- 30% of women in reproductive age. Although little is known about its etiology, these tumors represent a major problem in public health being the main indication for hysterectomy. About 40-50% have nonrandom cytogenetic abnormalities, thus half of these tumors may have submicroscopic alterations, as well as copy number variations (CNVs). Global gene expression studies have shown genes that act in proliferation and cell cycle process, retinoic acid, TGF-beta and IGF-1 signaling in response to estrogen and progesterone. However, few genes mapped at CNVs regions were directly associated with the UL development. The aim of this study was to investigate DNA copy number alterations in patients with multiple and unique UL.
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