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• W2284179904 abstract "The DNA information appears nowadays more stratified than it was supposed to be a decade before. In this scenario, non coding RNAs have been introduced in the fraction of functional RNA, carrying information and underpinning regulatory circuits of complex genetic phenomena in eukaryotes. microRNAs are endogenous single stranded ~22 nt long transcripts among that unveiled non coding RNA with regulatory functions, detected both in animals and plants. Increasing evidence shows that deregulation of microRNAs (miRNAs) plays an important role in both solid and hematologic malignancies. In this work we considered microRNA non canonical functions and involvement in tumours, integrating computational analyses of genome-wide datasets and targeted experimental results, with a critical approach to the specific adopted computational tools.First, we studied miRNAs role in myeloproliferative disorders, more specifically in primary myelofibrosis, considering also other small RNAs detected in RNA-seq data. Myeloproliferative neoplasms are chronic myeloid cancers involving CD34+ hematopoietic stem cells alterations, evolving to acute leukemia in the most severe forms. This study deals indeed with an Illumina sequencing of small RNAs samples of CD34+ hematopoietic stem cells of patients affected by primary myelofibrosis and of controls, in order to characterize miRNAs profile and find relevant differentially expressed elements, as putative effectors of a disrupted post-transcriptional regulation involved in PMF initiation and progression.Then, in order to have a better understanding of each step of a computational analysis of RNA-seq data, we studied the impact on small RNAs differential expression analysis of normalization methods developed for long RNA. We evaluated five commonly used normalization methods to pinpoint a procedure to perform a robust RNA-seq analysis. We estimated statistical distribution parameters from a real microRNA numerous dataset and we simulated a huge number of small RNAs dataset. We controlled datasets characteristics in order to generate 9 different testing scenarios and measure the normalization impact on differentially expressed elements recognition, through ROC and AUC curves. We ascertain that normalization methods still need strong efforts in developing new algorithms in order to fill the wide room for improvement.Thereafter we evaluated the implication of microRNAs in the gene expression changes observed after H-ferritin silencing. We explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and we studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562shFHC) comparing it with K562 transduced with scrambled RNA (K562shRNA). The remarkable up-regulation of four miRNAs, hsa-let-7g-5p, hsa-let-7f-5p, hsa-let-7i-5p and hsa-miR-125b-5p, in silenced cells and their down-regulation when FHC expression was rescued supported a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network. Our data, confirmed by an experimental validation, indicate that, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and they add new insights to the relationship among iron homeostasis and miRNAs.We further explored a putative non canonical role of microRNAs, more specifically, in the context of the always more evident complex cross talk between protein-coding and non-protein coding RNAs. We worked on a preliminary study that deals with the involvement of microRNAs in the regulation of alternative translation (AT) and thus of protein isoform equilibrium. There is an increasing appreciation of the high prevalence of alternative translation in mammals. Complex and regulated translation pattern are achieved thanks to multiple Open Reading Frame (ORFs) and Translation Initiation Sites (TISs) in the same mRNA that can influence each other in different ways. miRNAs were recently demonstrated to be involved in modulation of protein isoform equilibrium binding to TISs. We provided novel data on the overlap of active TISs of mRNAs, experimentally defined using GTI-seq, to miRNA-binding sites, experimentally determined using CLASH technique. The genes whose sites were recognized are supposed to be involved in miRNA-modulated AT and we modelled the interaction mechanism. The miRNA-based regulation of mRNA alternative translation surely deserves further investigation to clarify if and how it impacts on cell processes and on disease." @default.
• W2284179904 created "2016-06-24" @default.
• W2284179904 creator A5060151528 @default.
• W2284179904 date "2015-01-31" @default.
• W2284179904 modified "2023-09-27" @default.
• W2284179904 title "MicroRNAs impact in cancer: from non canonical biogenesis and functions to methodological aspects" @default.
• W2284179904 hasPublicationYear "2015" @default.
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