Perfil transcricional do câncer de mama em cadelas por sequenciamento de nova geração
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Data
2022-05-27
Autores
Santos, Driéle Bretones dos
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Universidade Estadual Paulista (Unesp)
Resumo
As neoplasias mamárias são frequentes em cadelas não castradas, representando até 46% dos casos de câncer nesses animais no Brasil. Cães podem ser um ótimo modelo de estudos sobre cânceres de mama já que estes animais e humanos compartilham características fisiológicas, histológicas e genômicas, além de apresentarem semelhanças quanto ao desenvolvimento, comportamento clínico e vias moleculares da doença. Análises comparativas do transcriptoma de tecidos tumorais e normais podem identificar genes e vias moleculares desreguladas, permitindo, a médio prazo, o desenvolvimento de ensaios laboratoriais para uso no diagnóstico, ou, a longo prazo, indicar potenciais alvos terapêuticos para medicina humana e canina. Nesse contexto, o objetivo principal do presente estudo foi, a partir do sequenciamento de nova geração (NGS), determinar o perfil transcricional de amostras de carcinomas ductais mamários em cadelas, buscando esclarecer a relevância dos transcritos desregulados nas vias moleculares envolvidas na doença. Para tanto, utilizou-se amostras de carcinoma ductal mamário e do tecido mamário não tumoral adjacente, provenientes da mastectomia radical de seis (6) cadelas, de diferentes raças e idades, atendidas pelo serviço de Cirurgia de Pequenos Animais do Hospital Veterinário da FMVZ-UNESP em Botucatu. O RNA total de cada amostra foi extraído, quantificado e submetido a análise de qualidade. Posteriormente, essas amostras foram reunidas em pools correspondendo a quatro grupos (dois tumorais e dois normais). Utilizou-se cada pool para a construção de uma biblioteca de cDNA, que, então foi sequenciada na plataforma NextSeq 500 System (Illumina, EUA). O resultado do sequenciamento foi encaminhado para análises de qualidade, limpeza e alinhamento ao genoma de referência, assim, identificando-se os transcritos diferencialmente expressos. Foram encontrados 11.278 genes dentre os, aproximadamente, 36.930 genes do genoma de referência de cachorro. Ao serem comparadas as amostras tumorais e o tecido normal adjacente, foram encontrados 1.206 genes diferencialmente expressos, sendo 633 regulados negativamente (downregulados) e 573 regulados positivamente (upregulados), os quais foram capazes de diferenciar os grupos pela PCA (Principal Component Analysis). A partir da análise de ontologia genética foram identificadas vias inflamatórias, de diferenciação e adesão celular e manutenção da matriz como as principalmente desreguladas nessa casuística. A maioria dos genes upregulados mais destacados estão envolvidos na organização da matriz extracelular, através da deposição de colágeno, vasculogênese mimética e da capacidade de invasão das células por meio da transição epitélio-mesenquimal. Ainda, os principais genes diferencialmente expressos observados no presente estudo, indicam maior agressividade da doença e pior prognóstico, destacando-se os dez genes mais upregulados. Finalmente, o estudo do transcriptoma canino parece indicar ser este um ótimo modelo para gerar informações relevantes à oncologia nas duas espécies.
Breast neoplasms are frequent in spayed female dogs, representing up to 46% of cancer cases in these animals in Brazil. Thus, dogs can be an excellent model for studies of breast cancer, because these animals and humans share physiological, histological, and genomic characteristics; in addition to having similarities in the development, clinical behavior, and molecular pathways of the disease. Therefore, comparative analyzes of the transcriptome of tumor and normal tissues can identify dysregulated genes and molecular pathways, allowing, in the medium term, the development of laboratory assays for use in the diagnosis, or, in the long term, indicate potential therapeutic targets for human and canine medicine. In this context, the main objective of this study was, from next-generation sequencing (NGS), to determine the transcriptional profile of samples of mammary ductal carcinomas in female dogs, seeking to clarify the relevance of deregulated transcripts in the molecular pathways involved in the disease. For this purpose, samples of tumor tissue from mammary ductal carcinoma and adjacent non-tumor breast tissue were used, from the radical mastectomy of 6 female dogs of different breeds and ages, attended by the Small Animal Surgery Service of the Veterinary Hospital of FMVZ- UNESP in Botucatu. The total RNA of each sample was extracted, quantified, and submitted to quality analysis. Subsequently, these samples were pooled corresponding to four groups (two tumoral and two normal). Each pool was used to build a cDNA library, which was then sequenced on the NextSeq 500 System platform (Illumina, USA). The sequencing result was sent for quality analysis, cleaning, and alignment with the reference genome, thus identifying the differentially expressed transcripts. 11,278 genes were found among approximately 36,930 genes in the reference dog genome. When comparing tumor and normal tissue samples, 1,206 genes were found to be differentially expressed, with 633 downregulated and 573 upregulated, which were able to differentiate the groups by PCA (Principal Component Analysis). From the analysis of gene ontology, inflammatory pathways, cell differentiation and adhesion, and matrix maintenance were identified to be mainly deregulated in this sample. Most of the most prominent upregulated genes are involved in the organization of the extracellular matrix, through collagen deposition, mimetic vasculogenic, and the invasiveness of cells through the epithelial-mesenchymal transition. Furthermore, the main differentially expressed genes observed in the present study indicate greater disease aggressiveness and a worse prognosis, highlighting the ten most upregulated genes. Finally, the study of the canine transcriptome seems to indicate that this is an excellent model to generate information relevant to oncology in both species
Breast neoplasms are frequent in spayed female dogs, representing up to 46% of cancer cases in these animals in Brazil. Thus, dogs can be an excellent model for studies of breast cancer, because these animals and humans share physiological, histological, and genomic characteristics; in addition to having similarities in the development, clinical behavior, and molecular pathways of the disease. Therefore, comparative analyzes of the transcriptome of tumor and normal tissues can identify dysregulated genes and molecular pathways, allowing, in the medium term, the development of laboratory assays for use in the diagnosis, or, in the long term, indicate potential therapeutic targets for human and canine medicine. In this context, the main objective of this study was, from next-generation sequencing (NGS), to determine the transcriptional profile of samples of mammary ductal carcinomas in female dogs, seeking to clarify the relevance of deregulated transcripts in the molecular pathways involved in the disease. For this purpose, samples of tumor tissue from mammary ductal carcinoma and adjacent non-tumor breast tissue were used, from the radical mastectomy of 6 female dogs of different breeds and ages, attended by the Small Animal Surgery Service of the Veterinary Hospital of FMVZ- UNESP in Botucatu. The total RNA of each sample was extracted, quantified, and submitted to quality analysis. Subsequently, these samples were pooled corresponding to four groups (two tumoral and two normal). Each pool was used to build a cDNA library, which was then sequenced on the NextSeq 500 System platform (Illumina, USA). The sequencing result was sent for quality analysis, cleaning, and alignment with the reference genome, thus identifying the differentially expressed transcripts. 11,278 genes were found among approximately 36,930 genes in the reference dog genome. When comparing tumor and normal tissue samples, 1,206 genes were found to be differentially expressed, with 633 downregulated and 573 upregulated, which were able to differentiate the groups by PCA (Principal Component Analysis). From the analysis of gene ontology, inflammatory pathways, cell differentiation and adhesion, and matrix maintenance were identified to be mainly deregulated in this sample. Most of the most prominent upregulated genes are involved in the organization of the extracellular matrix, through collagen deposition, mimetic vasculogenic, and the invasiveness of cells through the epithelial-mesenchymal transition. Furthermore, the main differentially expressed genes observed in the present study indicate greater disease aggressiveness and a worse prognosis, highlighting the ten most upregulated genes. Finally, the study of the canine transcriptome seems to indicate that this is an excellent model to generate information relevant to oncology in both species
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Palavras-chave
Cadelas, Carcinoma ductal mamário, Transcriptoma, Female dogs, Transcriptome