INTRODUCTION: Alterations in the expression levels of RNAs in the pathogenesis of sporadic ALS (sALS) are becoming increasingly relevant, with RNA-seq data highlighting numerous deregulated long non-coding RNAs (lncRNAs) in tissues derived from sALS patients. Among these, the oncogenic lncRNA ZEB1-AS1 emerged as strongly downregulated in peripheral blood mononuclear cells (PBMCs) of sALS patients. In cancer-derived cell lines, ZEB1-AS1 has been shown to act in a feedback negative loop with mir200c, acting as a molecular sponge for this miRNA. Furthermore, ZEB1-AS1’s interaction with mir200c results in the upregulation of the downstream molecule BMI1. BMI1 has also been found downregulated in another neurodegenerative disorder, Alzheimer’s Disease, resulting in impaired activation of p53 and GSK3b. Both these molecules have been correlated with neuronal death in ALS pathogenesis. METHODS: Total RNA was extracted using TRIZOL reagent and the genes’ expression levels were determined by Real Time PCR. Nuclear and cytoplasmic RNA was extracted using the Cytoplasmic & Nuclear RNA Purification Kit and genes’ expression levels were measured by ddPCR. RIP analysis was performed following standard protocol using antibodies against IgG and FUS. RNA was subsequently extracted and quantified via Real Time PCR RESULTS: We created an in vitro model silencing ZEB1-AS1 in SH-SY5Y, mimicking the observation in sALS patients. This downregulation does not influence ZEB1’s levels, and on the contrary, we observed an increase of mir200c and a decrease of BMI1, in an opposite pattern to what is observed in cancer, suggesting a possible sALS involved pathway. Furthermore, we observed an upregulation of BMI1’s downstream mediators p53 and GSK3b, both involved in neuronal death in ALS. Concordantly, we found that ZEB1-AS1, ZEB1, BMI1, p53 and GSK3b present the same deregulations in PBMCs of sALS patients. Our results show an implication of the ZEB1-AS1 pathway in sALS, and an interesting prospective with respect to oncogenic cellular signalling. We demonstrated that ZEB1-AS1 can bind the ALS-implicated RNA binding protein FUS, and we demonstrated this both in SH-SY5Y cells and in PBMCs. Interestingly, we saw that there is a reduction in the amount of ZEB1-AS1 bound to FUS in sALS patients, suggesting the mechanism connecting ZEB1-AS1 to sALS pathology. DISCUSSION & CONCLUSIONS: Taken together these results demonstrated an important role for the lncRNA ZEB1-AS1 in ALS pathogenesis, possibly through the implication of the RNA-binding protein FUS.

Role of the oncogenic lncRNA ZEB1-AS1 in sporadic ALS : at a cross-road between neurodegeneration and cancer / F. Rey, T. Giallongo, A. Balsari, S. Gagliardi, C. Pandini, A.M. DI GIULIO, C. Cereda, S. Carelli. ((Intervento presentato al 5. convegno Congresso DISS tenutosi a Milano nel 2019.

Role of the oncogenic lncRNA ZEB1-AS1 in sporadic ALS : at a cross-road between neurodegeneration and cancer

F. Rey
Primo
;
T. Giallongo;C. Pandini;A.M. DI GIULIO;S. Carelli
2019

Abstract

INTRODUCTION: Alterations in the expression levels of RNAs in the pathogenesis of sporadic ALS (sALS) are becoming increasingly relevant, with RNA-seq data highlighting numerous deregulated long non-coding RNAs (lncRNAs) in tissues derived from sALS patients. Among these, the oncogenic lncRNA ZEB1-AS1 emerged as strongly downregulated in peripheral blood mononuclear cells (PBMCs) of sALS patients. In cancer-derived cell lines, ZEB1-AS1 has been shown to act in a feedback negative loop with mir200c, acting as a molecular sponge for this miRNA. Furthermore, ZEB1-AS1’s interaction with mir200c results in the upregulation of the downstream molecule BMI1. BMI1 has also been found downregulated in another neurodegenerative disorder, Alzheimer’s Disease, resulting in impaired activation of p53 and GSK3b. Both these molecules have been correlated with neuronal death in ALS pathogenesis. METHODS: Total RNA was extracted using TRIZOL reagent and the genes’ expression levels were determined by Real Time PCR. Nuclear and cytoplasmic RNA was extracted using the Cytoplasmic & Nuclear RNA Purification Kit and genes’ expression levels were measured by ddPCR. RIP analysis was performed following standard protocol using antibodies against IgG and FUS. RNA was subsequently extracted and quantified via Real Time PCR RESULTS: We created an in vitro model silencing ZEB1-AS1 in SH-SY5Y, mimicking the observation in sALS patients. This downregulation does not influence ZEB1’s levels, and on the contrary, we observed an increase of mir200c and a decrease of BMI1, in an opposite pattern to what is observed in cancer, suggesting a possible sALS involved pathway. Furthermore, we observed an upregulation of BMI1’s downstream mediators p53 and GSK3b, both involved in neuronal death in ALS. Concordantly, we found that ZEB1-AS1, ZEB1, BMI1, p53 and GSK3b present the same deregulations in PBMCs of sALS patients. Our results show an implication of the ZEB1-AS1 pathway in sALS, and an interesting prospective with respect to oncogenic cellular signalling. We demonstrated that ZEB1-AS1 can bind the ALS-implicated RNA binding protein FUS, and we demonstrated this both in SH-SY5Y cells and in PBMCs. Interestingly, we saw that there is a reduction in the amount of ZEB1-AS1 bound to FUS in sALS patients, suggesting the mechanism connecting ZEB1-AS1 to sALS pathology. DISCUSSION & CONCLUSIONS: Taken together these results demonstrated an important role for the lncRNA ZEB1-AS1 in ALS pathogenesis, possibly through the implication of the RNA-binding protein FUS.
8-nov-2019
Settore BIO/11 - Biologia Molecolare
Dipartimento di Scienze della Salute- Università dedli Studi di Milano
Role of the oncogenic lncRNA ZEB1-AS1 in sporadic ALS : at a cross-road between neurodegeneration and cancer / F. Rey, T. Giallongo, A. Balsari, S. Gagliardi, C. Pandini, A.M. DI GIULIO, C. Cereda, S. Carelli. ((Intervento presentato al 5. convegno Congresso DISS tenutosi a Milano nel 2019.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/764974
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