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Alterações transcricionais induzidas pelo vírus da hepatite delta numa perspectiva genómica
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O vírus da hepatite delta é o mais pequeno vírus conhecido dos que infectam o ser humano. Possui um genoma de RNA, numa molécula circular com cerca de 1700 nucleótidos. A partícula viral é composta por RNA genómico e por ambas as formas do antigénio delta (codificadas por uma única grelha de leitura aberta), rodeados por um invólucro formado por antigénios de superfície do vírus da hepatite B. Devido a essa relação entre os dois vírus, o vírus da hepatite delta apenas infecta indivíduos que se encontrem infectados pelo vírus da hepatite B. Apesar disto, é capaz de induzir a forma mais agressiva de hepatite viral, estimando-se que afecte cerca de 20 milhões de pessoas globalmente.
Apesar das células do mesmo organismo partilharem o seu genoma, essas possuem diferentes fenótipos e funções, muitas vezes devido a diferenças transcriptómicas. Uma das formas de analisar essas diferenças é utilizando a técnica de RNA-seq que permite, através de next generation sequencing aplicada a DNA complementar, perceber como se encontra a população de RNAs de um conjunto de células num determinado momento ou condição.
Neste trabalho estabeleceu-se um modelo de infecção pelo vírus da hepatite delta, através da transfecção de um plasmídeo (pSVL(D3)) que contém cDNA do genoma do HDV, em células de uma linha celular (HuH-7) derivada de carcinoma hepatocelular humano. A partir daí, procedeu-se à extracção do RNA total e à sequenciação do mesmo. Diversos genes foram identificados pelo RNA-seq como diferencialmente expressos na presença de HDV. Um dos genes identificado como sobre-expresso com a infecção por HDV foi o gene c-fos. Este integra o factor de transcrição AP-1, encontrando-se envolvido em diversas vias de sinalização celular e interagindo com vários outros genes.
Uma vez que o factor AP-1 é um dímero constituído por proteínas de diferentes famílias, foi analisada, por PCR, a expressão de outros genes da família FOS e da família JUN, concluindo-se que apenas o gene c-fos se encontrava alterado com a presença de HDV. Também a ciclina D1 (um dos genes regulados por c-fos/AP-1) apresentou-se mais expressa após a infecção por HDV.
Em conclusão, este trabalho permitiu obter uma visão das alterações transcricionais globais induzidas pela infecção do vírus da hepatite delta. Em particular, permitiu identificar um aumento de expressão do proto-oncogene c-fos, e de genes por si regulados, sugerindo que o gene c-fos pode desempenhar um papel importante no efeito patogénico do HDV. Estes resultados poderão contribuir para uma melhor compreensão do processo de desenvolvimento de carcinoma hepatocelular desencadeado pelo vírus da hepatite delta.
The hepatitis delta virus is the smallest virus known to infect humans. It has an RNA genome,in a circular molecule with about 1700 nucleotides. The viral particle is composed of genomic RNAand both forms of delta antigen (encoded by a single open reading frame), surroundedby an envelope formed by surface antigens of the hepatitis B virus. Due to therelationship between bothviruses, the hepatitis delta virus only infects individuals who are infected with the hepatitis B virus. Despite this, it is able to induce the most aggressive form of viral hepatitis and is estimated to affect about 20 million people globally.Although the cells of the same organism share their genomes, they have different phenotypes and functions, often due to transcriptomic differences. One way to analyze these differences is by using the RNA-seq technique which allows,through next generation sequencing applied to complementary DNA,to perceive how the population of RNAs from a set of cells is found at a given time or condition.In this work we establisheda model of infection by the hepatitis delta virus, transfecting a plasmid (pSVL (D3)) containing cDNA of HDV genome, into cells of a cell line (HuH-7) derived from human hepatocellular carcinoma. Afterwards, thetotalRNA wasextracted and sequenced.Several genes were identified through RNA-seq as differentially expressed on the presence of HDV.One of the genes identified as over-expressed with the infection of HDV was c-fos. It integrates the AP-1 transcription factor, is involved in several cell pathways, and interacts with several other genes.Since the AP-1 factor is a dimer made up of proteins from different families, other genes from the FOS family and from the JUN family expression were analyzed by PCR, concluding that only c-fos was altered with the presence of HDV. Also cyclin D1 (one of the c-fos / AP-1 regulated genes) was most expressed after HDV infection. In conclusion, this work provided an overview of the global transcriptional changes induced by hepatitis delta virus infection. In particular, it identifiedan overexpressionof the c-fos proto-oncogene, and genes regulated by it, suggesting that the c-fos gene may play an important role in the pathogenic effect of HDV. These results may contribute to a better understanding of the development process of hepatocellular carcinoma causedby hepatitis delta virus.
The hepatitis delta virus is the smallest virus known to infect humans. It has an RNA genome,in a circular molecule with about 1700 nucleotides. The viral particle is composed of genomic RNAand both forms of delta antigen (encoded by a single open reading frame), surroundedby an envelope formed by surface antigens of the hepatitis B virus. Due to therelationship between bothviruses, the hepatitis delta virus only infects individuals who are infected with the hepatitis B virus. Despite this, it is able to induce the most aggressive form of viral hepatitis and is estimated to affect about 20 million people globally.Although the cells of the same organism share their genomes, they have different phenotypes and functions, often due to transcriptomic differences. One way to analyze these differences is by using the RNA-seq technique which allows,through next generation sequencing applied to complementary DNA,to perceive how the population of RNAs from a set of cells is found at a given time or condition.In this work we establisheda model of infection by the hepatitis delta virus, transfecting a plasmid (pSVL (D3)) containing cDNA of HDV genome, into cells of a cell line (HuH-7) derived from human hepatocellular carcinoma. Afterwards, thetotalRNA wasextracted and sequenced.Several genes were identified through RNA-seq as differentially expressed on the presence of HDV.One of the genes identified as over-expressed with the infection of HDV was c-fos. It integrates the AP-1 transcription factor, is involved in several cell pathways, and interacts with several other genes.Since the AP-1 factor is a dimer made up of proteins from different families, other genes from the FOS family and from the JUN family expression were analyzed by PCR, concluding that only c-fos was altered with the presence of HDV. Also cyclin D1 (one of the c-fos / AP-1 regulated genes) was most expressed after HDV infection. In conclusion, this work provided an overview of the global transcriptional changes induced by hepatitis delta virus infection. In particular, it identifiedan overexpressionof the c-fos proto-oncogene, and genes regulated by it, suggesting that the c-fos gene may play an important role in the pathogenic effect of HDV. These results may contribute to a better understanding of the development process of hepatocellular carcinoma causedby hepatitis delta virus.
Descrição
Palavras-chave
Ciências biomédicas Biologia molecular Doenças tropicais Hepetite delta RNA-seq. c-fos Carcinoma hepatocelular
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical