Zanobio, Mariateresa
(2018)
14-3-3θ, AF4 and MLL-AF4 are targets of MiR-27a in t(4;11) acute lymphoblastic leukemia: implication for new targeted therapy options.
[Tesi di dottorato]
Tipologia del documento: |
Tesi di dottorato
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Lingua: |
English |
Titolo: |
14-3-3θ, AF4 and MLL-AF4 are targets of MiR-27a in t(4;11) acute lymphoblastic leukemia: implication for new targeted therapy options |
Autori: |
Autore | Email |
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Zanobio, Mariateresa | mt.zanobio@gmail.com |
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Data: |
11 Dicembre 2018 |
Numero di pagine: |
66 |
Istituzione: |
Università degli Studi di Napoli Federico II |
Dipartimento: |
Medicina Molecolare e Biotecnologie Mediche |
Dottorato: |
Medicina molecolare e biotecnologie mediche |
Ciclo di dottorato: |
31 |
Coordinatore del Corso di dottorato: |
nome | email |
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Avvedimento, Vittorio Enrico | vittorioenrico.avvedimento@unina.it |
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Tutor: |
nome | email |
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Esposito, Gabriella | [non definito] |
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Data: |
11 Dicembre 2018 |
Numero di pagine: |
66 |
Parole chiave: |
Leukemia, MLL-AF4, MiR-27a |
Settori scientifico-disciplinari del MIUR: |
Area 05 - Scienze biologiche > BIO/12 - Biochimica clinica e biologia molecolare clinica |
[error in script]
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Informazioni aggiuntive: |
Cell. 3894720935 |
Depositato il: |
09 Gen 2019 13:29 |
Ultima modifica: |
23 Giu 2020 09:36 |
URI: |
http://www.fedoa.unina.it/id/eprint/12676 |
Abstract
The t(4;11)(q21;q23) chromosomal translocation results in the KMT2A/AFF1 fusion gene, which encodes the mixed-lineage leukemia (MLL)-AF4 oncogenic chimera, a hallmark of an aggressive form of acute lymphoblastic leukemia (ALL). In t(4;11) ALL, MLL-AF4 recruits the endogenous co-activator AF4 and aberrantly triggers transcription of MLL target genes, including HOXA9 and MEIS1, thereby driving transformation of hematopoietic progenitors. We previously demonstrated that the scaffold protein 14-3-3θ is a direct interactor of AF4 and promotes AF4 binding to the HOXA9 promoter. Notably, 14-3-3θ is a target of MiR-27a, which acts as tumor suppressor in various human leukemias; moreover, expression levels of MiR-27a correlate with relapse free survival in childhood ALL. This PhD thesis aims to assess the potential role of MiR-27a in t(4;11) ALL pathogenesis. In different leukemia cell lines, we found an inverse correlation between levels of MiR-27a and 14-3-3θ, which was particularly relevant in t(4;11) cell lines. In t(4;11) leukemia cells, transient transfection of MiR-27a led to a decrease in 14-3-3θ protein amount and HOXA9, HOXA7 and MEIS1 transcription. Interestingly, our bioinformatic analysis predicted that AFF1-3’UTR, which is shared with KMT2A/AFF1, contains a putative MiR-27a seed sequence. Consistently, MiR-27a overexpression strongly reduced AF4 and MLL-AF4 protein levels, as well as protein level of RUNX1, a known target of MiR-27a with a key role in t(4;11) leukemia context. We therefore cloned AFF1-3’-UTR in an opportune plasmid vector and performed a luciferase reporter assay. The decreased luciferase activity we measured after co-transfection of MiR-27a and the recombinant plasmid proved that AFF1-3’UTR is a direct target of MiR-27a. Accordingly, transfection of anti-MiR-27a enhanced the expression of AF4 protein, in RS4;11 cells. Moreover, ChIP experiments gave direct proof that MiR-27a overexpression impaired MLL-AF4 binding to HOXA9 promoter. Consistently, MiR-27a overexpression decreased viability, proliferation and clonogenicity of t(4;11) cells, whereas enhanced their apoptotic rate. Lastly, we found that relative expression of MiR-27a was significantly lower in 9 patients affected by severe t(4;11) ALL compared with 9 patients affected by t(12;21) leukemia, which has a benign prognosis. Similarly to the t(4;11) cell lines, in our ALL patients we found an inverse relationship between MiR-27a and 14-3-3θ transcript levels. Overall, we demonstrate that MiR-27a has a pivotal role in t(4;11) ALL molecular pathogenesis and therefore it is a promising novel target for the therapy of this aggressive form of leukemia.
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