Inflammatory bowel disease (IBD) is a chronic immune-mediated condition of the gastrointestinal tract that includes Crohn disease and ulcerative colitis. Pediatric IBDs are of particular interest since their incidence is rising and, even if different pharmacological strategies are used, the optimal treatment is far from being achieved. Glucocorticoid (GCs) are prescribed for inducing remission but there is a high risk of adverse effects especially in subjects that poorly respond to these agents and require long treatments. The long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5) interacts with the activated glucocorticoid receptor (GR), inhibiting the transcription of GCs responsive genes. The first part of my thesis project is focused on the study of GAS5 as a molecular marker of GCs resistance. We evaluated the association between the lncRNA and the efficacy of steroids, in terms of inhibition of proliferation, in two immortalized cell lines from colon and ovarian cancers, a GC-resistant and GC-sensitive model, respectively. After GCs treatment in the GC-resistant cells GAS5 upregulation was observed and, in response to the drug, the lncRNA accumulated more in the cytoplasm compared to the nucleus. Furthermore, we evaluated GAS5 levels in the peripheral blood mononuclear cells of pediatric IBD patients at diagnosis and after 4 weeks of GCs administration. Gene expression analysis have shown an upregulation of the lncRNA in patients with unfavourable steroid response. These preliminary results suggest that GAS5 could be considered a novel pharmacogenomic marker useful for the personalization of GC therapy. GAS5 expression was also measured in IBD patients’ colon biopsies and its levels have been evaluated with respect to the gene and protein expression of two metalloproteinases (MMP-2, MMP-9) involved in tissue damage in IBDs. The GAS5 downregulation observed in inflamed tissues compared with the non-inflamed one is inversely related to MMPs expression suggesting a role of this lncRNA in controlling the activity of these molecules. In the second part of my thesis project we evaluated the role of the tristetraprolin (TTP) protein in IBDs. TTP is a zinc finger protein able to interact and inhibit pro-inflammatory cytokines through the binding with AU-rich elements on the 3’ untranslated region on mRNA. The role of phosphorylation on TTP activity was also evaluated, since this post-translational modification could impair protein activity and consequently the stabilization of cytokines levels. TTP protein expression was studied in pediatric IBDs patients’ colon tissues and in macrophages differentiated from peripheral blood mononuclear cells. An upregulation of TTP expression in both inflamed colon tissues and in macrophages of IBD patients was observed, and was closely related to the phosphorylation of the protein. These preliminary results, if confirmed with further experiments, could open new perspectives in the study of IBDs and in the investigation of new target therapy based on the modulation of TTP phosphorylation by phosphatases to favour pro-inflammatory cytokines degradation.

Le malattie infiammatorie corniche intestinali (MICI) sono un gruppo di malattie infiammatorie immunomediate che comprendono il morbo di Crohn e la rettocolite ulcerosa. Nella popolazione pediatrica le MICI sono di particolare interesse a causa della aumentata incidenza della malattia e, sebbene siano stati sviluppati diversi approcci terapeutici, è molto difficile individuare il trattamento ottimale. I glucocorticoidi (GC) sono farmaci prescritti per indurre la remissione ma alcuni pazienti risultano resistenti al trattamento o richiedono terapie prolungate e tali pazienti sono soggetti a numerosi reazioni avverse. Il long non-coding RNA (lncRNA) growth-arrest specific 5 (GAS5) interagisce con il complesso GC-recettore dei glucocorticoidi (GR) inibendo l’attività trascrizionale dei geni responsivi ai GC. La prima parte del mio progetto di tesi si occupa di studiare il ruolo di GAS5 come marker molecolare della resistenza farmacologica ai GC. L’associazione tra il lncRNA e l’efficacia degli steroidi, espressa in termini di inibizione della proliferazione, è stata valutata su due linee cellulari tumorali di colon e ovaio che sono state identificate rispettivamente come modello di resistenza e sensibilità farmacologica ai GC. Inoltre, il ruolo di GAS5 è stato osservato nelle cellule mononucleate del sangue periferico di pazienti pediatrici affetti da MICI sia alla diagnosi che dopo 4 settimane di trattamento con GC; una maggiore espressione di GAS5 è stata osservata nei pazienti con una risposta sfavorevole agli steroidi. Questi risultati preliminari indicano che GAS5 potrebbe essere considerato un nuovo biomarker di resistenza farmacologica ai GC. I livelli di espressione di GAS5 sono stati valutati anche nelle biopsie di colon di pazienti pediatrici affetti da MICI anche rispetto ai livelli di espressione proteica e genica di due metalloproteasi (MMP) coinvolte nel danno tissutale. La downregolazione di GAS5 osservata nei tessuti infiammati rispetto ai tessuti non infiammati è inversamente correlata all’espressione delle MMP suggerendo che il lncRNA potrebbe controllare l’attività di queste proteine. Nella seconda parte del mio progetto di tesi abbiamo valutato i livelli di espressione proteica della tristetraprolin (TTP) nelle MICI. La TTP e una proteina zinc finger capace di interagire e inibire le citochine pro-infiammatorie attraverso il legame con gli elementi ricchi di AU sul 3’ UTR degli mRNA target. Abbiamo considerato anche il ruolo della sua fosforilazione, poiché questa modificazione post-traduzionale interferisce con l’attività della TTP che in questo stato è responsabile della stabilizzazione delle citochine d’interesse. L’espressione proteica della TTP è stata valutata nei tessuti di colon e nei macrofagi dei pazienti pediatrici affetti da MICI. L’espressione della TTP risulta upregolata sia nei tessuti di colon che nei macrofagi. I risultati inoltre confermano il coinvolgimento della fosforilazione nell’attività della TTP. Questi risultati preliminari, se confermati con ulteriori esperimenti, potrebbero aprire nuove prospettive nello studio delle IBD e nella formulazione di una nuova terapia farmacologica mirata in grado di modulare la fosforilazione della TTP attraverso l’uso di fosfatasi e favorire così la degradazione delle citochine pro-infiammatorie.

Identification of new molecular targets for personalized therapy in pediatric patients with inflammatory bowel disease (IBD) / DI SILVESTRE, Alessia. - (2018 Mar 19).

Identification of new molecular targets for personalized therapy in pediatric patients with inflammatory bowel disease (IBD)

DI SILVESTRE, ALESSIA
2018-03-19

Abstract

Inflammatory bowel disease (IBD) is a chronic immune-mediated condition of the gastrointestinal tract that includes Crohn disease and ulcerative colitis. Pediatric IBDs are of particular interest since their incidence is rising and, even if different pharmacological strategies are used, the optimal treatment is far from being achieved. Glucocorticoid (GCs) are prescribed for inducing remission but there is a high risk of adverse effects especially in subjects that poorly respond to these agents and require long treatments. The long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5) interacts with the activated glucocorticoid receptor (GR), inhibiting the transcription of GCs responsive genes. The first part of my thesis project is focused on the study of GAS5 as a molecular marker of GCs resistance. We evaluated the association between the lncRNA and the efficacy of steroids, in terms of inhibition of proliferation, in two immortalized cell lines from colon and ovarian cancers, a GC-resistant and GC-sensitive model, respectively. After GCs treatment in the GC-resistant cells GAS5 upregulation was observed and, in response to the drug, the lncRNA accumulated more in the cytoplasm compared to the nucleus. Furthermore, we evaluated GAS5 levels in the peripheral blood mononuclear cells of pediatric IBD patients at diagnosis and after 4 weeks of GCs administration. Gene expression analysis have shown an upregulation of the lncRNA in patients with unfavourable steroid response. These preliminary results suggest that GAS5 could be considered a novel pharmacogenomic marker useful for the personalization of GC therapy. GAS5 expression was also measured in IBD patients’ colon biopsies and its levels have been evaluated with respect to the gene and protein expression of two metalloproteinases (MMP-2, MMP-9) involved in tissue damage in IBDs. The GAS5 downregulation observed in inflamed tissues compared with the non-inflamed one is inversely related to MMPs expression suggesting a role of this lncRNA in controlling the activity of these molecules. In the second part of my thesis project we evaluated the role of the tristetraprolin (TTP) protein in IBDs. TTP is a zinc finger protein able to interact and inhibit pro-inflammatory cytokines through the binding with AU-rich elements on the 3’ untranslated region on mRNA. The role of phosphorylation on TTP activity was also evaluated, since this post-translational modification could impair protein activity and consequently the stabilization of cytokines levels. TTP protein expression was studied in pediatric IBDs patients’ colon tissues and in macrophages differentiated from peripheral blood mononuclear cells. An upregulation of TTP expression in both inflamed colon tissues and in macrophages of IBD patients was observed, and was closely related to the phosphorylation of the protein. These preliminary results, if confirmed with further experiments, could open new perspectives in the study of IBDs and in the investigation of new target therapy based on the modulation of TTP phosphorylation by phosphatases to favour pro-inflammatory cytokines degradation.
19-mar-2018
DECORTI, GIULIANA
30
2016/2017
Settore BIO/14 - Farmacologia
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/2922616
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