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O papel do supressor tumoral RBM5 em hepatocarcinoma celular induzido por HDV
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O vírus da hepatite delta (HDV) é um vírus hepatotrópico responsável pela forma mais severa e mortal de hepatite viral. O HDV co-infeta ou super-infeta hepatócitos previamente infetados pelo vírus da hepatite B (HBV), aumentando o risco de desenvolvimento de cirrose, carcinoma hepatocelular (HCC) e hepatite fulminante. A associação clínica entre estes dois vírus explica-se pelo facto do invólucro externo do HDV ser constituído pelas proteínas de superfície do HBV (HBsAgs). O HDV é assim considerado um vírus satélite do HBV.
O genoma do HDV, com cerca de 1700 nucleótidos, consiste numa molécula de RNA circular de cadeia simples com polaridade negativa que codifica uma única proteína, o antigénio delta. Devido à sua simplicidade, presume-se que as etapas de transcrição e replicação do HDV estejam intimamente dependentes da maquinaria da célula infetada. Consequentemente, a análise das interações estabelecidas entre o genoma e/ou proteínas do HDV e as proteínas do hospedeiro, assume um elevado interesse dada a possibilidade de virem a ser utilizadas como potenciais novos alvos terapêuticos, que são essenciais dado que até à data não existe nenhuma terapia específica para HDV.
Recentemente, o nosso laboratório utilizou o sistema de três híbrido em levedura para identificar proteínas do hospedeiro que interagem com o RNA genómico do HDV. Um dos fatores celulares identificados foi o fator de splicing SF3B155, uma proteína que faz parte do complexo U2snRNP e que é essencial para as etapas iniciais de reconhecimento de locais de splicing nos pre-mRNAs de genes humanos. Partindo da hipótese experimental que o RNA genómico do HDV funciona como um transcrito tóxico que sequestra SF3B155 e desregula o normal funcionamento do splicing alternativo, fomos avaliar o efeito da expressão de HDV no splicing de genes dependentes de SF3B155.
Utilizando a linha celular Huh7 como modelo celular humano de infeção de HDV, mostramos que a expressão de HDV promove um conjunto de alterações de splicing no transcrito do gene RBM5 com consequente redução dos níveis proteicos desse fator. O RBM5 é um gene supressor tumoral que codifica um regulador transcricional implicado no controlo da expressão de genes envolvidos em progressão de ciclo celular e apoptose. Dada a redução dos níveis proteicos de RBM5 observados, fomos analisar os níveis de expressão de genes regulados por RBM5, tanto no modelo de células Huh-7 como em hepatócitos primários humanos (PHH) infetados com HDV. Em ambos os modelos utilizados, os resultados por nós obtidos mostraram alterações de expressão num conjunto de genes que incluem oncogenes com papel bem documentado no desenvolvimento e progressão de HCC, como STAT3, CDK2 e DNMT3B por exemplo.
Cumulativamente, os resultados descritos neste trabalho mostram que a expressão de HDV em células humanas tem a capacidade de modular o splicing e os níveis transcricionais de diversos genes humanos, alterações essas que podem funcionar como biomarcadores de HCC induzido por HDV e vir a ser alvos favoráveis para futuras intervenções terapêuticas.
The hepatitis delta virus (HDV) is a hepatotropic virus responsible for the most severe and deadly form of viral hepatitis. HDV co-infects or super-infects hepatocytes previously infected with the hepatitis B virus (HBV), increasing the risk of developing cirrhosis, hepatocellular carcinoma and fulminant hepatitis. The clinical association between these two viruses is explained by the fact that the outer envelope of HDV consists of HBV surface proteins (HBsAgs). HDV is thus considered a satellite virus of HBV. The HDV genome is composed of approximately 1700 nucleotides and exists as a negative sense, single stranded, circular RNA molecule which encodes a single protein, the delta antigen. Due to its simplicity, it is assumed that HDV hijacks the host cell machinery to complete its transcription and replication steps. As such, the analysis of the interactions established between the HDV genome and/or proteins and host proteins is of high interest given their potential use as therapeutic targets, which are essential given that there is currently no specific therapy for HDV. Recently, our laboratory used the yeast three-hybrid system to identify host proteins that interact with the HDV genomic RNA. One of the cellular factors we identified was the splicing factor SF3B155, a protein that is part of the U2snRNP complex which is essential for the initial recognition steps of splicing sites in the pre-mRNA of human genes. Based on the experimental hypothesis that HDV genomic RNA functions as a toxic transcript that sequesters SF3B155 and deregulates the normal functioning of alternative splicing, we were able to evaluate the effect of HDV expression on the splicing of SF3B155 dependant genes. Using the Huh-7 cell line as a human cell model for HDV infection, we showed that HDV expression promotes a set of splicing changes in the RBM5 gene transcript with consequent reduction of the protein levels of this factor. RBM5 is a tumour suppressor gene that encodes a transcriptional regulator that controls the expression of genes involved in cell cycle progression and apoptosis. Given the reduction in RBM5 protein levels observed, we analysed the expression levels of RBM5 regulated genes, both in the Huh-7 cell model and in human primary hepatocytes (PHH) infected with HDV. In both models, the results obtained by us have shown expression changes in a set of genes that include well documented oncogenes involved in the development and progression of HCC, such as STAT3, CDK2 and DNMT3B for example. Cumulatively, the results described in this work show that HDV expression in human cells has the ability to modulate splicing and the transcriptional levels of several human genes, which can function as biomarkers for HDV induced HCC and become favourable targets for future therapeutic interventions.
The hepatitis delta virus (HDV) is a hepatotropic virus responsible for the most severe and deadly form of viral hepatitis. HDV co-infects or super-infects hepatocytes previously infected with the hepatitis B virus (HBV), increasing the risk of developing cirrhosis, hepatocellular carcinoma and fulminant hepatitis. The clinical association between these two viruses is explained by the fact that the outer envelope of HDV consists of HBV surface proteins (HBsAgs). HDV is thus considered a satellite virus of HBV. The HDV genome is composed of approximately 1700 nucleotides and exists as a negative sense, single stranded, circular RNA molecule which encodes a single protein, the delta antigen. Due to its simplicity, it is assumed that HDV hijacks the host cell machinery to complete its transcription and replication steps. As such, the analysis of the interactions established between the HDV genome and/or proteins and host proteins is of high interest given their potential use as therapeutic targets, which are essential given that there is currently no specific therapy for HDV. Recently, our laboratory used the yeast three-hybrid system to identify host proteins that interact with the HDV genomic RNA. One of the cellular factors we identified was the splicing factor SF3B155, a protein that is part of the U2snRNP complex which is essential for the initial recognition steps of splicing sites in the pre-mRNA of human genes. Based on the experimental hypothesis that HDV genomic RNA functions as a toxic transcript that sequesters SF3B155 and deregulates the normal functioning of alternative splicing, we were able to evaluate the effect of HDV expression on the splicing of SF3B155 dependant genes. Using the Huh-7 cell line as a human cell model for HDV infection, we showed that HDV expression promotes a set of splicing changes in the RBM5 gene transcript with consequent reduction of the protein levels of this factor. RBM5 is a tumour suppressor gene that encodes a transcriptional regulator that controls the expression of genes involved in cell cycle progression and apoptosis. Given the reduction in RBM5 protein levels observed, we analysed the expression levels of RBM5 regulated genes, both in the Huh-7 cell model and in human primary hepatocytes (PHH) infected with HDV. In both models, the results obtained by us have shown expression changes in a set of genes that include well documented oncogenes involved in the development and progression of HCC, such as STAT3, CDK2 and DNMT3B for example. Cumulatively, the results described in this work show that HDV expression in human cells has the ability to modulate splicing and the transcriptional levels of several human genes, which can function as biomarkers for HDV induced HCC and become favourable targets for future therapeutic interventions.
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Ciências biomédicas Biologia molecular Medicina tropical Hepatite delta Vírus
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Instituto de Higiene e Medicina Tropical