RésuméDuring latency, herpesvirus infection results in the establishment of a dormant state in which a highly restricted set of viral genes are expressed. This is associated with extensive methylation of CpG motifs in non-expressed viral genes. Together with these alterations of the viral genome, several host genes undergo epigenetic modifications during the latent infection. In some of the human and animal herpesvirus infections, these epigenetic dysregulations of cellular genes are involved in the development of cancer. This PhD program was carried out using an animal model of virus induced lymphoma causing the Marek’s disease (MD) in chicken. This lymphoproliferative disease is the ultimate consequence of chicken infection with virulent strains of gallid herpesvirus-2 (GaHV-2). This Alphaherpesvirinae actually shares several properties with Gammaherpesvirinae (such as human herpesvirus-4 and -8) which are associated with the development of tumors under specific conditions in latently infected cells. GaHV-2 was shown to modulate the expression of several cellular miRNAs in chicken. Altered expressions of host-encoded miRNAs were analyzed in vitro and in vivo in several studies. Although only few of the cellular miRNA dysregulations triggered by GaHV-2 were analyzed in depth, these studies suggested that altered expressions of host miRNAs are involved in the molecular pathways of GaHV-2 oncogenicity.
Therefore, we decided to focus on a host miRNA, miR-126 since several pieces of evidence suggested it might be downregulated during GaHV-2 tumorigenesis. Originally described as a miRNA mediating proper angiogenesis and vascular integrity, miR-126 has been reported to impair cancer progression through signaling pathways that control tumor cell proliferation, migration, invasion and survival. MiR-126 was shown to be downregulated in several human cancers such as in oral, stomach, colon, lung, breast, cervix, bladder and prostate carcinoma. MiR-126 is an intronic miRNA integrated in the intron 7 of a cellular gene, the epidermal growth factor like domain 7 (egfl-7). In human, the latter gene is controlled by three promoters generating three transcripts, a long, an alternative and a short one. It was shown that epigenetic modifications have an impact on the alternative transcript expression by remodeling the alternative promoter.
The core issue of this PhD program is to know whether miR-126 downregulation is a key event of GaHV-2 oncogenicity. Three study were carried out in order to bring some insights.
The First part of this work, was to set up a thorough quantification method of miR-126 expression. This method is essential to compare, in the most accurate way, miR-126 expression level in any situation corresponding to the different stages of GaHV-2 infection. The quantification method was tested during an in vivo challenge with a chicken line highly sensitive to MD (B13/B13 strain). Chickens were infected with a very virulent strain of GaHV-2 (RB-1B). The method relied on the use of several reference genes (RGs): gapdh, beta-actin, small nuclear RNA U6 (U6), 18SrRNA, Hydroxymethylbilan synthase (hmbs) and succinyl dehydrogenase (sdha). The three RGs that showed the most stable expression were selected with an Excelbased software (GeNorm) and used to normalize the expression level of three cellular genes (miR-126, cd4 and cd8) and one viral one (meq, the major oncogene of GaHV-2). The results showed that depending on the RG used, different conclusions have been obtained. The conclusion of this part is that combining several RGs to normalize gene expression is crucial for drawing accurate conclusions from quantification data.
The second part of this work determined miR-126 expression level at the key steps of GaHV-2 infection (in vitro and in vivo) and looked out for epigenetic modifications involved in the control of its expression. The miR-126 expression level was evaluated by using the reliable quantification method set up in the first part of the study. The cellular miRNA was found to be repressed during the viral-induced oncogenesis phase. In order to determine whether miR-126 low expression level was associated with specific epigenetic signatures and with peculiar transcription profiles, DNA methylation patterns and transcriptional isoforms were established at miR-126 genomic locus. Repression was found to be associated with hypermethylation at a CpG island located in the miR-126 host gene (egfl-7). Moreover, transcriptional isoforms analysis showed the existence of alternative promoters during chicken development. This analysis also permitted revealing the existence of two main initiation sites associated each with a CpG islands. Since miR-126 repression is likely to play a pivotal role in altering gene expression patterns during cell transformation, we investigated the impact of miR-126 restoration. A strategy was developed to overexpress miR-126 and control miRNAs in transformed CD4+ T cells propagated from MD lymphoma. To this end, a conditional expression system (Tet-on inducible expression) was developed to counteract miR-126 shutdown. The preliminary functional analysis showed that miR-126 inhibition might participate to the cancerous process induced by GaHV-2 by supporting cell proliferation.
To complete the in vitro functional analysis, the third part of this work concentrated on the creation of a recombinant GaHV-2 that constitutively expresses the miRNA lost during lymphoma development. The aim was to interfere with miR-126 silencing during the natural course of GaHV-2 infection. Recombinant viruses expressing either wild-type or mutated versions of miR-126 were generated from a very virulent GaHV-2 strain (RB-1B) cloned as an infectious Bacterial Artificial Chromosome (BAC). Infectious viruses were amplified and characterized in vitro to evaluate if the recombinant viruses replicate to similar levels as the original strain and if this strategy is adapted to overexpress miR-126 together with viral infection.
Altogether, these results brought new insights on the expression and the regulation of a cellular miRNA in the context of MD.
|la date de réponse||19 juin 2018|
|Sponsors||Fund for Research Training in Industry and Agriculture (FRIA)|
|Superviseur||Benoit Muylkens (Promoteur), NICOLAS GILLET (Président), Benjamin Le Calve (Jury), Benjamin Dewals (Jury), Carine Van Lint (Jury), Sylvie Laurent (Jury) & Benjamin Dewals (Jury)|