CREB engages C/EBPδ to initiate leukemogenesis.

cAMP response element binding protein (CREB) is frequently overexpressed in acute myeloid leukemia (AML) and acts as a proto-oncogene; however, it is still debated whether such overactivation alone is able to induce leukemia as its pathogenetic downstream signaling is still unclear. We generated a zebrafish model overexpressing CREB in the myeloid lineage, which showed an aberrant regulation of primitive hematopoiesis, and in 79% of adult CREB-zebrafish a block of myeloid differentiation, triggering to a monocytic leukemia akin the human counterpart. Gene expression analysis of CREB-zebrafish revealed a signature of 20 differentially expressed human homologous CREB targets in common with pediatric AML. Among them, we demonstrated that CREB overexpression increased CCAAT-enhancer-binding protein-δ (C/EBPδ) levels to cause myeloid differentiation arrest, and the silencing of CREB-C/EBPδ axis restored myeloid terminal differentiation. Then, C/EBPδ overexpression was found to identify a subset of pediatric AML affected by a block of myeloid differentiation at monocytic stage who presented a significant higher relapse risk and the enrichment of aggressive signatures. Finally, this study unveils the aberrant activation of CREB-C/EBPδ axis concurring to AML onset by disrupting the myeloid cell differentiation process. We provide a novel in vivo model to perform high-throughput drug screening for AML cure improvement

The lncRNA CASC15 regulates SOX4 expression in RUNX1-rearranged acute leukemia

Abstract Background Long non-coding RNAs (lncRNAs) play a variety of cellular roles, including regulation of transcription and translation, leading to alterations in gene expression. Some lncRNAs modulate the expression of chromosomally adjacent genes. Here, we assess the roles of the lncRNA CASC15 in regulation of a chromosomally nearby gene, SOX4, and its function in RUNX1/AML translocated leukemia. Results CASC15 is a conserved lncRNA that was upregulated in pediatric B-acute lymphoblastic leukemia (B-ALL) with t (12; 21) as well as pediatric acute myeloid leukemia (AML) with t (8; 21), both of which are associated with relatively better prognosis. Enforced expression of CASC15 led to a myeloid bias in development, and overall, decreased engraftment and colony formation. At the cellular level, CASC15 regulated cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4. Differentially regulated genes following CASC15 knockdown were enriched for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. Interestingly, we found that CASC15 enhances YY1-mediated regulation of the SOX4 promoter. Conclusions Our findings represent the first characterization of this CASC15 in RUNX1-translocated leukemia, and point towards a mechanistic basis for its action

The LiberAction Project: Implementation of a Pediatric Liberation Bundle to Screen Delirium, Reduce Benzodiazepine Sedation, and Provide Early Mobilization in a Human Resource-Limited Pediatric Intensive Care Unit

Background: Delirium, bed immobilization, and heavy sedation are among the major contributors of pediatric post-intensive care syndrome. Recently, the Society of Critical Care Medicine has proposed the implementation of daily interventions to minimize the incidence of these morbidities and optimize children functional outcomes and quality of life. Unfortunately, these interventions require important clinical and economical efforts which prevent their use in many pediatric intensive care units (PICU). Aim: First, to evaluate the feasibility and safety of a PICU bundle implementation prioritizing delirium screening and treatment, early mobilization (<72 h from PICU admission) and benzodiazepine-limited sedation in a human resource-limited PICU. Second, to evaluate the incidence of delirium and describe the early mobilization practices and sedative drugs used during the pre- and post-implementation periods. Third, to describe the barriers and adverse events encountered during early mobilization. Methods: This observational study was structured in a pre- (15th November 2019-30th June 2020) and post-implementation period (1st July 2020-31st December 2020). All patients admitted in PICU for more than 72 h during the pre and post-implementation period were included in the study. Patients were excluded if early mobilization was contraindicated. During the pre-implementation period, a rehabilitation program including delirium screening and treatment, early mobilization and benzodiazepine-sparing sedation guidelines was developed and all PICU staff trained. During the post-implementation period, delirium screening with the Connell Assessment of Pediatric Delirium scale was implemented at bedside. Early mobilization was performed using a structured tiered protocol and a new sedation protocol, limiting the use of benzodiazepine, was adopted. Results: Two hundred and twenty-five children were enrolled in the study, 137 in the pre-implementation period and 88 in the post-implementation period. Adherence to delirium screening, benzodiazepine-limited sedation and early mobilization was 90.9, 81.1, and 70.4%, respectively. Incidence of delirium was 23% in the post-implementation period. The median cumulative dose of benzodiazepines corrected for the total number of sedation days (mg/kg/sedation days) was significantly lower in the post-implementation period compared with the pre-implementation period: [0.83 (IQR: 0.53-1.31) vs. 0.74 (IQR: 0.55-1.16), p = 0.0001]. The median cumulative doses of fentanyl, remifentanil, and morphine corrected for the total number of sedation days were lower in the post-implementation period, but these differences were not significant. The median number of mobilizations per patient and the duration of each mobilization significantly increased in the post-implementation period [3.00 (IQR: 2.0-4.0) vs. 7.00 (IQR: 3.0-12.0); p = 0.004 and 4 min (IQR: 3.50-4.50) vs. 5.50 min (IQR: 5.25-6.5); p < 0.0001, respectively]. Barriers to early mobilization were: disease severity and bed rest orders (55%), lack of physicians' order (20%), lack of human resources (20%), and lack of adequate devices for patient mobilization (5%). No adverse events related to early mobilization were reported in both periods. Duration of mechanical ventilation and PICU length of stay was significantly lower in the post-implementation period as well as the occurrence of iatrogenic withdrawal syndrome. Conclusion: This study showed that the implementation of a PICU liberation bundle prioritizing delirium screening and treatment, benzodiazepine-limited sedation and early mobilization was feasible and safe even in a human resource-limited PICU. Further pediatric studies are needed to evaluate the clinical impact of delirium, benzodiazepine-limited sedation and early mobilization protocols on patients' long-term functional outcomes and on hospital finances

A sex-informed approach to improve the personalised decision making process in myelodysplastic syndromes: a multicentre, observational cohort study

Background Sex is a major source of diversity among patients and a sex-informed approach is becoming a new paradigm in precision medicine. We aimed to describe sex diversity in myelodysplastic syndromes in terms of disease genotype, phenotype, and clinical outcome. Moreover, we sought to incorporate sex information into the clinical decision-making process as a fundamental component of patient individuality. Methods In this multicentre, observational cohort study, we retrospectively analysed 13 284 patients aged 18 years or older with a diagnosis of myelodysplastic syndrome according to 2016 WHO criteria included in the EuroMDS network (n=2025), International Working Group for Prognosis in MDS (IWG-PM; n=2387), the Spanish Group of Myelodysplastic Syndromes registry (GESMD; n=7687), or the Dusseldorf MDS registry (n=1185). Recruitment periods for these cohorts were between 1990 and 2016. The correlation between sex and genomic features was analysed in the EuroMDS cohort and validated in the IWG-PM cohort. The effect of sex on clinical outcome, with overall survival as the main endpoint, was analysed in the EuroMDS population and validated in the other three cohorts. Finally, novel prognostic models incorporating sex and genomic information were built and validated, and compared to the widely used revised International Prognostic Scoring System (IPSS-R). This study is registered with ClinicalTrials.gov, NCT04889729. Findings The study included 7792 (58middot7%) men and 5492 (41middot3%) women. 10 906 (82middot1%) patients were White, and race was not reported for 2378 (17middot9%) patients. Sex biases were observed at the single-gene level with mutations in seven genes enriched in men (ASXL1, SRSF2, and ZRSR2 p<0middot0001 in both cohorts; DDX41 not available in the EuroMDS cohort vs p=0middot0062 in the IWG-PM cohort; IDH2 p<0middot0001 in EuroMDS vs p=0middot042 in IWG-PM; TET2 p=0middot031 vs p=0middot035; U2AF1 p=0middot033 vs p<0middot0001) and mutations in two genes were enriched in women (DNMT3A p<0middot0001 in EuroMDS vs p=0middot011 in IWG-PM; TP53 p=0middot030 vs p=0middot037). Additionally, sex biases were observed in co-mutational pathways of founding genomic lesions (splicing-related genes, predominantly in men, p<0middot0001 in both the EuroMDS and IWG-PM cohorts), in DNA methylation (predominantly in men, p=0middot046 in EuroMDS vs p<0middot0001 in IWG-PM), and TP53 mutational pathways (predominantly in women, p=0middot0073 in EuroMDS vs p<0middot0001 in IWG-PM). In the retrospective EuroMDS cohort, men had worse median overall survival (81middot3 months, 95% CI 70middot4-95middot0 in men vs 123middot5 months, 104middot5-127middot5 in women; hazard ratio [HR] 1middot40, 95% CI 1middot26-1middot52; p<0middot0001). This result was confirmed in the prospective validation cohorts (median overall survival was 54middot7 months, 95% CI 52middot4-59middot1 in men vs 74middot4 months, 69middot3-81middot2 in women; HR 1middot30, 95% CI 1middot23-1middot35; p<0middot0001 in the GEMSD MDS registry; 40middot0 months, 95% CI 33middot4-43middot7 in men vs 54middot2 months, 38middot6-63middot8 in women; HR 1middot23, 95% CI 1middot08-1middot36; p<0middot0001 in the Dusseldorf MDS registry). We developed new personalised prognostic tools that included sex information (the sex-informed prognostic scoring system and the sex-informed genomic scoring system). Sex maintained independent prognostic power in all prognostic systems; the highest performance was observed in the model that included both sex and genomic information. A five-to-five mapping between the IPSS-R and new score categories resulted in the re-stratification of 871 (43middot0%) of 2025 patients from the EuroMDS cohort and 1003 (42middot0%) of 2387 patients from the IWG-PM cohort by using the sex-informed prognostic scoring system, and of 1134 (56middot0%) patients from the EuroMDS cohort and 1265 (53middot0%) patients from the IWG-PM cohort by using the sex-informed genomic scoring system. We created a web portal that enables outcome predictions based on a sex-informed personalised approach. Interpretation Our results suggest that a sex-informed approach can improve the personalised decision making process in patients with myelodysplastic syndromes and should be considered in the design of clinical trials including low-risk patients. Copyright (c) 2022 Published by Elsevier Ltd. All rights reserved

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5–11 December, to 17.5% (25/143 samples) in the week 12–18, to 65.9% (89/135 samples) in the week 19–25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased fromone in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool

Identification of functional miRNA interactions in Malignant Melanoma progression

According to the World Health Organization (WHO), Malignant Melanoma is the most aggressive form of skin cancer. Melanoma accounts for only about 4% of skin cancer cases but for as many as 74% of all deaths caused by skin cancers. The development of regional and/or distant metastases and the lack of promising therapies for the treatment of Melanoma are the principal causes of extremely poor survival of Melanoma patients. Recently, enormous efforts are taken to unravel the molecular mechanisms that lead to tumour development and metastasis. A new class of small non-coding RNAs, the microRNAs (miRNAs), has been involved in tumour progression and metastasis. MiRNAs are able to modulate the expression of specific target genes binding to their 3’-UTRs through Ago proteins complex and usually causing transcript degradation. The ability of miRNAs to achieve simultaneous fine-tuning of numerous different targets makes them a fundamental system for cell regulation but, despite their biological importance, the identification of their targets is still a challenging research task. The aim of my project is the identification of real functional miRNA-target interactions involved in pathways that control Melanoma metastasis. The experiments were performed using the metastasis model developed by Xu and colleagues (Xu et al., 2008) composed by a low metastatic Melanoma cell line (LMCs), its derivative high metastatic cell lines (HMCs) and the lung metastases (Mets) arisen after these cell lines are injected into immunodeficient mice. This model mimics the behaviour of cancer cells during metastasis progression. To reach proposed aims, I have set up a meta-analysis approach first and a genome wide biochemical approach later. The combination of the two approaches have been allowed the experimental validation of results obtained by meta-analysis and the identification of new miRNA interactors. I first performed miRNA microarray experiments on LMCs, HMCs and Mets samples observing that miRNA expression profiles are able to distinguish the three different grade of metastasis. Since the evidence of miRNAs involvement in metastasi, I used a meta-analysis approach to evidence the influence of miRNA in pathways involved in the extravasation processes: WNT signalling pathway, Adherens Junction pathway, and VEGF signalling pathway. Moreover, I identified important miRNA clusters (e.g. miR-106a~363 cluster) involved in the regulation of metastatic hallmarks such as the cell protrusion formation, cell-cell signaling, and the tissue vascularization. In order to validate all the interactions identified by meta-analysis, I set up conditions for the simultaneous experimental identification of miRNA-target interactions through the high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HiTS-CLiP). This method uses ultraviolet irradiation to covalently crosslink RNA–protein complexes, in our case Ago proteins, that are in direct contact (approximately over single ängstrom distances) within cells. Through the immunoprecipitation complexes are purified allowing the identification of interacting RNA through high-throughput sequencing methods. This technique validated several miRNA-mRNA interactions identified by meta-analysis and by literature. In particular, has been observed the involvement of miR-106a in Adherens junction and VEGF signaling pathways where seems to act as metastasis suppressor inhibiting pro-metastatic genes as WAVE3 and VEGFA. Moreover, sequencing analyses have confirmed the metastatic powerful for miR-214 and let-7c in Melanoma metastasis. The importance of miR-214 was confirmed by results obtained by our collaborator Dr. Taverna of University of Torino, while I used let-7c inhibition to confirm that it interacts with two targets (FNDC3B and FOXN) identified by AGO-Hits-CLIP experiment and that can be studied in future functional studies. Finally, I have also discussed the involvement of a new class of miRNA regulators: the long non-coding RNAs (lncRNAs). I identified a lot of lncRNAs with the ability to regulate miRNA availability. Taken together, these findings demonstrate the strong involvement of miRNAs in metastasis. In fact, several functional miRNA-transcript interactions could regulate extravasation pathways during the Melanoma cells spreading. My experimental approach successfully guides us towards important biological results with interesting therapeutic implications in Melanoma.Il Melanoma è una delle forme più aggressive di tumore cutaneo. Pur rappresentando soltanto il 4% dei tumori della pelle causa quasi il 74% dei decessi dei pazienti che presentano neoplasie cutanee. Lo sviluppo di metastasi e la mancanza di terapie efficaci sono la causa di una mortalità così elevata. Recentemente, grazie a metodiche di medicina molecolare, i ricercatori hanno cercato di capire i meccanismi che portano allo sviluppo della massa tumorale e delle metastasi individuando nei microRNA (miRNA) dei possibili regolatori coinvolti nella progressione tumorale del Melanoma. Questi corti trascritti non codificanti hanno la capacità di regolare l’espressione di molti mRNA degradandoli o bloccandone la traduzione. Il processo di regolazione guidato dai miRNA è basato sul legame degli stessi a specifiche sequenze presenti nei trascritti target grazie all’ausilio di un complesso proteico formato prevalentemente dalle proteine della famiglia Argonaute (AGO). Lo scopo di questa tesi è di identificare queste interazioni miRNA-mRNA coinvolte nelle vie di segnale che controllano lo sviluppo di metastasi nel Melanoma. Gli esperimenti presentati in questo lavoro sono stati effettuati sfruttando un modello cellulare che mima lo sviluppo metastatico (Xu et al., 2008). Il sistema vede l’utilizzo di una linea cellulare a basso potenziale metastatico (LMC), delle linee cellulari derivate dalla stessa ma con maggiore potenziale metastatico (HMC) e delle metastasi polmonari originate dall’iniezione di queste linee cellulari in topi immuno-deficienti. Per raggiungere lo scopo di questa tesi ho messo a punto due approcci che mirano al raggiungimento del medesimo risultato permettendo così anche di validare gli stessi. Il primo approccio, prettamente di tipo bioinformatico, sfrutta i dati di espressione dei miRNA e degli mRNA ottenuti da esperimenti di microarray per individuare le coppie miRNA-mRNA coinvolte nello sviluppo del Melanoma. Il secondo approccio, invece, si basa su una nuova metodica (AGO-Hits-CLIP) per l’identificazione genome-wide di interazioni miRNA-mRNA. Questa metodica sfrutta l’immunoprecipitazione delle proteine AGO e il recupero delle molecole di RNA ad esse associate (in questo caso i miRNA e i messaggeri bersaglio a cui erano legati). Successivamente, l’RNA precipitato viene sequenziato per permetterne l’identificazione. L’integrazione dei dati ottenuti dall’analisi con i microarray (meta-analisi) e dall’AGO-Hits-CLIP mi hanno permesso di individuare delle interazioni miRNA-bersaglio coinvolte nello sviluppo di metastasi generate dal Melanoma. Innanzitutto mi ha permesso di individuare una serie di vie di segnale strettamente correlate all’extravasazione delle cellule dal torrente circolatorio. Queste vie sono probabilmente regolate da alcuni membri di un cluster policistronico di miRNA: il miR-106a~363. In particolare grazie alla metodica AGO-Hits-CLIP ho validato due interazioni del miR-106; una con il trascritto WAVE3 e l’altra con VEGFA. Questi sono rispettivamente coinvolti nella formazione delle protrusioni che la cellula utilizza per invadere i tessuti (l’invadopodium) e nella permeabilizzazione della parete dei vasi sanguigni,. Inoltre è stata confermata l’importanza del miR-214 e di let-7c nello sviluppo metastatico. L’effetto di miR-214 è stato confermato anche dagli studi di una nostra collaboratrice, la Professoressa Daniela Taverna dell’Università di Torino mentre per let-7c ho validato due interattori (FNDC3B e FOXN) identificati mediante la metodica AGO-Hits-CLIP. Oltre alla relazione miRNA:mRNA, la tecnica AGO-Hits-CLIP ha permesso di evidenziare una nuova classe di interattori: i lncRNA. Questi sono dei trascritti non codificanti lunghi più di 200 nt che sembrano funzionare da “spugna” per i microRNA, sequestrandoli e impedendone la loro funzione regolativa. Concludendo, i risultati ottenuti sostengono l’importanza dei miRNA nello sviluppo del Melanoma e nella sua metastatizzazione evidenziandone il ruolo cardine per un futuro sviluppo di nuovi farmaci che consentano la riduzione del rischio di sviluppare metastasi e, di conseguenza, di diminuire l’alta mortalità

Identification of functional miRNA interactions in Malignant Melanoma progression

According to the World Health Organization (WHO), Malignant Melanoma is the most aggressive form of skin cancer. Melanoma accounts for only about 4% of skin cancer cases but for as many as 74% of all deaths caused by skin cancers. The development of regional and/or distant metastases and the lack of promising therapies for the treatment of Melanoma are the principal causes of extremely poor survival of Melanoma patients. Recently, enormous efforts are taken to unravel the molecular mechanisms that lead to tumour development and metastasis. A new class of small non-coding RNAs, the microRNAs (miRNAs), has been involved in tumour progression and metastasis. MiRNAs are able to modulate the expression of specific target genes binding to their 3’-UTRs through Ago proteins complex and usually causing transcript degradation. The ability of miRNAs to achieve simultaneous fine-tuning of numerous different targets makes them a fundamental system for cell regulation but, despite their biological importance, the identification of their targets is still a challenging research task. The aim of my project is the identification of real functional miRNA-target interactions involved in pathways that control Melanoma metastasis. The experiments were performed using the metastasis model developed by Xu and colleagues (Xu et al., 2008) composed by a low metastatic Melanoma cell line (LMCs), its derivative high metastatic cell lines (HMCs) and the lung metastases (Mets) arisen after these cell lines are injected into immunodeficient mice. This model mimics the behaviour of cancer cells during metastasis progression. To reach proposed aims, I have set up a meta-analysis approach first and a genome wide biochemical approach later. The combination of the two approaches have been allowed the experimental validation of results obtained by meta-analysis and the identification of new miRNA interactors. I first performed miRNA microarray experiments on LMCs, HMCs and Mets samples observing that miRNA expression profiles are able to distinguish the three different grade of metastasis. Since the evidence of miRNAs involvement in metastasi, I used a meta-analysis approach to evidence the influence of miRNA in pathways involved in the extravasation processes: WNT signalling pathway, Adherens Junction pathway, and VEGF signalling pathway. Moreover, I identified important miRNA clusters (e.g. miR-106a~363 cluster) involved in the regulation of metastatic hallmarks such as the cell protrusion formation, cell-cell signaling, and the tissue vascularization. In order to validate all the interactions identified by meta-analysis, I set up conditions for the simultaneous experimental identification of miRNA-target interactions through the high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HiTS-CLiP). This method uses ultraviolet irradiation to covalently crosslink RNA–protein complexes, in our case Ago proteins, that are in direct contact (approximately over single ängstrom distances) within cells. Through the immunoprecipitation complexes are purified allowing the identification of interacting RNA through high-throughput sequencing methods. This technique validated several miRNA-mRNA interactions identified by meta-analysis and by literature. In particular, has been observed the involvement of miR-106a in Adherens junction and VEGF signaling pathways where seems to act as metastasis suppressor inhibiting pro-metastatic genes as WAVE3 and VEGFA. Moreover, sequencing analyses have confirmed the metastatic powerful for miR-214 and let-7c in Melanoma metastasis. The importance of miR-214 was confirmed by results obtained by our collaborator Dr. Taverna of University of Torino, while I used let-7c inhibition to confirm that it interacts with two targets (FNDC3B and FOXN) identified by AGO-Hits-CLIP experiment and that can be studied in future functional studies. Finally, I have also discussed the involvement of a new class of miRNA regulators: the long non-coding RNAs (lncRNAs). I identified a lot of lncRNAs with the ability to regulate miRNA availability. Taken together, these findings demonstrate the strong involvement of miRNAs in metastasis. In fact, several functional miRNA-transcript interactions could regulate extravasation pathways during the Melanoma cells spreading. My experimental approach successfully guides us towards important biological results with interesting therapeutic implications in Melanoma.Il Melanoma è una delle forme più aggressive di tumore cutaneo. Pur rappresentando soltanto il 4% dei tumori della pelle causa quasi il 74% dei decessi dei pazienti che presentano neoplasie cutanee. Lo sviluppo di metastasi e la mancanza di terapie efficaci sono la causa di una mortalità così elevata. Recentemente, grazie a metodiche di medicina molecolare, i ricercatori hanno cercato di capire i meccanismi che portano allo sviluppo della massa tumorale e delle metastasi individuando nei microRNA (miRNA) dei possibili regolatori coinvolti nella progressione tumorale del Melanoma. Questi corti trascritti non codificanti hanno la capacità di regolare l’espressione di molti mRNA degradandoli o bloccandone la traduzione. Il processo di regolazione guidato dai miRNA è basato sul legame degli stessi a specifiche sequenze presenti nei trascritti target grazie all’ausilio di un complesso proteico formato prevalentemente dalle proteine della famiglia Argonaute (AGO). Lo scopo di questa tesi è di identificare queste interazioni miRNA-mRNA coinvolte nelle vie di segnale che controllano lo sviluppo di metastasi nel Melanoma. Gli esperimenti presentati in questo lavoro sono stati effettuati sfruttando un modello cellulare che mima lo sviluppo metastatico (Xu et al., 2008). Il sistema vede l’utilizzo di una linea cellulare a basso potenziale metastatico (LMC), delle linee cellulari derivate dalla stessa ma con maggiore potenziale metastatico (HMC) e delle metastasi polmonari originate dall’iniezione di queste linee cellulari in topi immuno-deficienti. Per raggiungere lo scopo di questa tesi ho messo a punto due approcci che mirano al raggiungimento del medesimo risultato permettendo così anche di validare gli stessi. Il primo approccio, prettamente di tipo bioinformatico, sfrutta i dati di espressione dei miRNA e degli mRNA ottenuti da esperimenti di microarray per individuare le coppie miRNA-mRNA coinvolte nello sviluppo del Melanoma. Il secondo approccio, invece, si basa su una nuova metodica (AGO-Hits-CLIP) per l’identificazione genome-wide di interazioni miRNA-mRNA. Questa metodica sfrutta l’immunoprecipitazione delle proteine AGO e il recupero delle molecole di RNA ad esse associate (in questo caso i miRNA e i messaggeri bersaglio a cui erano legati). Successivamente, l’RNA precipitato viene sequenziato per permetterne l’identificazione. L’integrazione dei dati ottenuti dall’analisi con i microarray (meta-analisi) e dall’AGO-Hits-CLIP mi hanno permesso di individuare delle interazioni miRNA-bersaglio coinvolte nello sviluppo di metastasi generate dal Melanoma. Innanzitutto mi ha permesso di individuare una serie di vie di segnale strettamente correlate all’extravasazione delle cellule dal torrente circolatorio. Queste vie sono probabilmente regolate da alcuni membri di un cluster policistronico di miRNA: il miR-106a~363. In particolare grazie alla metodica AGO-Hits-CLIP ho validato due interazioni del miR-106; una con il trascritto WAVE3 e l’altra con VEGFA. Questi sono rispettivamente coinvolti nella formazione delle protrusioni che la cellula utilizza per invadere i tessuti (l’invadopodium) e nella permeabilizzazione della parete dei vasi sanguigni,. Inoltre è stata confermata l’importanza del miR-214 e di let-7c nello sviluppo metastatico. L’effetto di miR-214 è stato confermato anche dagli studi di una nostra collaboratrice, la Professoressa Daniela Taverna dell’Università di Torino mentre per let-7c ho validato due interattori (FNDC3B e FOXN) identificati mediante la metodica AGO-Hits-CLIP. Oltre alla relazione miRNA:mRNA, la tecnica AGO-Hits-CLIP ha permesso di evidenziare una nuova classe di interattori: i lncRNA. Questi sono dei trascritti non codificanti lunghi più di 200 nt che sembrano funzionare da “spugna” per i microRNA, sequestrandoli e impedendone la loro funzione regolativa. Concludendo, i risultati ottenuti sostengono l’importanza dei miRNA nello sviluppo del Melanoma e nella sua metastatizzazione evidenziandone il ruolo cardine per un futuro sviluppo di nuovi farmaci che consentano la riduzione del rischio di sviluppare metastasi e, di conseguenza, di diminuire l’alta mortalità