P084 Investigation of non-coding RNAs as molecular markers during glucocorticoid treatment in children with inflammatory bowel disease

Abstract

Background: Despite the introduction of novel biological therapies, glucocorticoids (GCs) remain widely used for inducing remission in inflammatory bowel diseases (IBD), in particular for ulcerative colitis. Given the high incidence of suboptimal response, associated with a significant number of side effects, that are particularly severe in paediatric patients, the identification of subjects that are most likely to respond poorly to GCs is extremely important. In this context, recent results obtained in our laboratory suggest a role for the long noncoding RNA growth arrest-specific 5 (GAS5) in modulating GC response, suggesting that it could be considered a marker of GC resistance. To address this issue, we evaluated the association between the lncRNA GAS5 and the efficacy of steroids in IBD paediatric patients and by in vitro models. Methods: For the clinical studies, seventeen IBD paediatric patients treated with prednisone 1 to 2 mg/kg/day for 30 days, according to standard clinical protocol, were enrolled at the Paediatric Clinic of IRCCS Burlo Garofolo in Trieste. Peripheral blood was obtained from these patients at diagnosis (T0) and after 4 weeks of steroid treatment (T4). RNA was extracted from patients' peripheral blood mononuclear cells at T0 and T4, and used to analyze the levels of the lncRNA. Patients were classified on the basis of their clinical response into 3 groups: steroid resistant (SR), steroid sensitive (SS) and steroid dependent (SD). For the in vitro studies, the effect of methylprednisolone on the proliferation of HeLa and LoVo cells was determined by labelling metabolically active cells with [methyl-3H] thymidine, and the expression of GAS5 was determined by TaqMan® Assay. The localization of GAS5 during GC treatment was evaluated by subcellular fractionation and the functions of the lncRNA were assessed by silencing this lncRNA in vitro. Results: Among the 17 patients enrolled, 3 were SR, 8 SD and 6 SS; patients with unfavorable steroid response (SD + SR) presented higher GAS5 levels in comparison with SS group, supporting a contribution of GAS5 to steroid ineffectiveness. GAS5 was upregulated in GC resistant cells and accumulated more in the cytoplasm compared to the nucleus in response to the drug; in addition, GAS5 knock-down reduced the proliferation during GC treatment. Conclusions: We hypothesize that higher levels of GAS5 can result in the suppression of GC activity, and if these results are confirmed in a larger number of subjects, GAS5 should be considered a novel biomarker useful for the personalization of GC therapy in paediatric IBD patients and for elucidating the molecular pathways that underline GC resistance.