4 research outputs found
Virus-encoded microRNA contributes to the molecular profile of EBV-positive Burkitt lymphomas
Burkitt lymphoma (BL) is an aggressive neoplasm characterized by consistent morphology and phenotype, typical clinical behavior and distinctive molecular profile. The latter is mostly driven by the MYC over-expression associated with the characteristic translocation (8;14) (q24; q32) or with variant lesions. Additional genetic events can contribute to Burkitt Lymphoma pathobiology and retain clinical significance. A pathogenetic role for Epstein-Barr virus infection in Burkitt lymphomagenesis has been suggested; however, the exact function of the virus is largely unknown.In this study, we investigated the molecular profiles (genes and microRNAs) of Epstein-Barr virus-positive and -negative BL, to identify specific patterns relying on the differential expression and role of Epstein-Barr virus-encoded microRNAs.First, we found significant differences in the expression of viral microRNAs and in selected target genes. Among others, we identified LIN28B, CGNL1, GCET2, MRAS, PLCD4, SEL1L, SXX1, and the tyrosine kinases encoding STK10/STK33, all provided with potential pathogenetic significance. GCET2, also validated by immunohistochemistry, appeared to be a useful marker for distinguishing EBV-positive and EBV-negative cases. Further, we provided solid evidences that the EBV-encoded microRNAs (e.g. BART6) significantly mold the transcriptional landscape of Burkitt Lymphoma clones.In conclusion, our data indicated significant differences in the transcriptional profiles of EBV-positive and EBV-negative BL and highlight the role of virus encoded miRNA
Proposal of microRNA signature profile in EBV+ diffuse large Bcell lymphoma of the elderly and potential therapeutic targets
Introdução: O linfoma difuso de grandes celulas B do idoso Epstein-Barr positivo (EBV+DLBCLe) afeta individuos com mais de 50 anos sem imunodefiCiência previa documentada, e apresenta evolucao clinica desfavoravel. Atualmente nao existe um padrao caracteristico da expressao de microRNAs (miRNAs) nesta doenca. Portanto, este estudo tem por objetivo caracterizar um perfil de assinatura para esta nova entidade e explorar miRNAs como biomarcadores e potenciais alvos terapeuticos alternativos para EBV+DLBCLe. Metodos: Setenta e um pacientes com mais de 50 anos e diagnostico de LDGCB foram considerados potenciais candidatos a serem EBV+DLBCLe. A deteccao do EBV (EBER-1) foi realizada por hibridacao in situ. Quatro amostras EBV+ e quatro amostras EBV negativas foram analisadas atraves de PCR quantitativo em tempo real (qPCR). O RNA foi extraido a partir de blocos de parafina e o cDNA inserido em duas plataformas contendo, cada uma, 384 miRNAs humanos. Consideramos miRNAs diferencialmente expressos aqueles que apresentaram expressao com valor acima ou abaixo de 1,5. Resultados: 8,5% dos pacientes com LDGCB foram considerados EBV positivos. A sobrevida global dos pacientes com EBV+DLBCLe foi inferior a dos pacientes EBV negativos (p= 0,0201, teste Log-rank). Foram encontrados 10 miRNAs diferencialmente expressos entre os dois grupos, mas apenas sete alcancaram diferencas estatisticamente significantes a serem validadas em uma coorte multicentrica (29 EBV + DLBCLe versus 65 LDGCB). Confirmamos o aumento de expressao de hsa-miR-126, hsa-miR-146a, hsa-miR-146b, hsa-miR-150 e hsa-miR-222, e tambem a diminuicao de expressao de hsa-miR-151 nos casos EBV+DLBCLe quando casos EBV+ foram comparados com EBV negativos. Utilizando o cutoff de 1,5, o hsa-miR-146b mostrou especificidade de 91,4 %, para identificar os casos EBV+, AUC = 0,8849. Embora o hsa-miR-222 tenha presentado aumento de expressao em menos de 1/3 dos casos, tambem mostrou alta especificidade (98,5%) e valor preditivo positivo (90%), AUC = 0,8180, no grupo EBV + quando comparado com o grupo EBV negativo, com o mesmo cutoff. Conclusoes: O merito do presente estudo foi propor uma assinatura de miRNA para uma doenca recentemente descrita e destacar o hsa-miR-146b e hsa-miR-222 como possiveis biomarcadores e alvos terapeuticos para EBV+DLBCLe. Antagomirs para hsa-miR-146b e hsa-miR-222 (este ultimo em teste em alguns tipos de cancer) tambem poderiam ser utilizados como terapia adjuvante ao R-CHOP nos casos de EBV+DLBCLe, nos quais confirmamos pior prognosticoCoordenação de Aperfeiçoamento de Pessoal de NÃvel Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP: 2010/17668-6BV UNIFESP: Teses e dissertaçõe
Characterisation and functional analysis of the murine gammaherpesvirus-68-encoded microRNAs
All mammalian cells encode microRNAs (miRNAs), which are small non-coding RNAs
(~ 22 nucleotides) that control numerous physiological processes via regulation of gene
expression. A number of viruses, in particular herpesviruses, also encode miRNAs.
Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi’s sarcoma
associated herpesvirus (KSHV) are associated with lymphoproliferative disorders and
some types of cancer in humans. Gammaherpesvirus-encoded miRNAs are predicted to
contribute to pathogenesis and virus life cycle by suppressing host and viral target genes.
However, the exact functions of these miRNAs during virus infection in the natural host
are largely unknown. Strict species specificity has limited research on the human
gammaherpesviruses mainly to in vitro studies.
Murine gammaherpesvirus 68 (MHV-68) encodes at least 15 miRNAs and provides a
unique tractable small animal model to investigate in vivo gammaherpesvirus
pathogenic features that are difficult to assess in humans. Following intranasal infection
of lab mice, the virus undergoes primary lytic infection in the lung epithelial cells and
then spreads to the spleen establishing latent infection in splenic B lymphocytes,
macrophages, and dendritic cells. The peak of the latent viral load occurs in the spleen at
14 dpi and then it decreases over time, but the virus is not completely eliminated and the
latent viral genomes remain in the host cells for lifetime and can reactivate to produce
infectious virus under certain conditions.
The aims of my project were to: (1) establish and develop quantitative reverse
transcription polymerase chain reaction (qRT-PCR) assays for quantification of the
MHV-68 miRNAs, (2) determine the miRNAs expression profiles during the two stages
of virus infection (lytic and latent infection), (3) investigate the kinetics of the miRNAs
expression during latency in vivo, (4) construct an MHV-68 miRNA mutant virus
lacking 9 miRNAs (designated MHV-68.ΔmiRNAs), and (5) carry out thorough
phenotypic characterisation of this mutant virus in order to determine the possible
functions MHV-68 miRNAs in the context of natural host infection.
It was found that the MHV-68 miRNAs expression pattern varied during different stages
of infection, suggesting a differential regulation of the expression of these miRNAs
depending on the phase of infection. In order to investigate the kinetics of miRNAs
expression during latency in vivo, BALB/c mice were infected intranasally with MHV-
68 virus and spleens were harvested at days 10, 14, 21, and 32 post infection. The levels
of miRNAs expression were determined by qRT-PCR in the splenocytes from infected
mice. Interestingly, in contrast to the lytic MHV-68 protein coding genes, the expression
of the miRNAs increased over time after 21 dpi, suggesting that the MHV-68-encoded
miRNAs may play more fundamental roles during later stages of latent infection.
In order to determine the potential roles of the MHV-68 miRNAs in virus pathogenesis,
a miRNA mutant virus lacking the expression of 9 miRNAs, named MHV-
68.ΔmiRNAs, was constructed. The miRNA mutant virus replicated with the same
kinetics as wild type virus in vitro and in vivo demonstrating that the deleted MHV-68
miRNAs are dispensable for virus lytic replication. To examine the roles of the miRNAs
during virus latency, the MHV-68.ΔmiRNAs virus was characterised throughout a 49-
day course of infection.
Although the level of ex vivo reactivation of the MHV-68.ΔmiRNAs virus was
comparable to that of the WT virus during the establishment of latency and as late as 28
dpi, the reactivation of the MHV-68.ΔmiRNAs virus was approximately 18-times higher
than that of the WT virus at 49 dpi despite the similar levels of the genomic viral DNA
loads at the same time-point. This suggests that the MHV-68 miRNAs suppress virus
reactivation and promote maintenance of long-term latency. Moreover, the lytic viral
gene expression levels were higher in splenocytes from the MHV-68.ΔmiRNAs-infected
mice than the basal expression levels in the splenocytes from WT MHV-68-infected
mice, suggesting that the MHV-68 miRNAs may suppress viral lytic gene expression
during long-term latency in vivo and thus help the virus lay low
Role of miRNAs in Cancer
MicroRNAs are the best representatives of the non-coding part of the genome and their functions are mostly linked to their target genes. During the process of carcinogenesis, both dysregulation of microRNAs and their target genes can explain the development of the disease. However, most of the target genes of microRNAs have not yet been elucidated. In this book, we add new information related to the functions of microRNAs in various tumors and their associated targetome