Integrative Analysis of Gene and miRNA expression profiles in Primary Myelofibrosis CD34+ cells

Ph-negative myeloproliferative neoplasms (MPNs) are characterized by many somatic mutations which have already been shown useful in the prognostic assessment of MPN patients. Moreover, aberrant microRNA (miRNA) expression seems to add to the molecular complexity of MPNs, as specific miRNA signatures capable of discriminating MPN cells from those of normal donors were previously reported. In order to have a comprehensive picture of miRNA deregulation and its relationship with differential gene expression in primary myelofibrosis (PMF) cells, we obtained gene- (GEP) and miRNA expression profiles (miEP) of CD34+ cells from 31 healthy donors and 42 PMF patients using Affymetrix technology (HG-U219 and miRNA 2.0 arrays). Differentially expressed genes (DEG) and miRNAs (DEM) were sorted out by means of Partek Genomic Suite vs 6.6. Since each miRNA can target many mRNAs while a single mRNA can be targeted by multiple miRNAs, we performed Integrative Analysis (IA) by means of Ingenuity Pathway Analysis (IPA) to untangle this combinatorial complexity. In particular, IPA points out DEM-DEG pairs among experimentally validated interactions from TarBase, miRecords and Ingenuity Expert Findings as well as predicted microRNA-mRNA interactions from TargetScan. IPA microRNA Target Filter was then employed to select only the DEM-DEG pairs showing an anti-correlated expression pattern and to build regulatory networks. Finally, 3'UTR luciferase reporter assays were performed to validate IPA predicted miRNA-mRNA interactions.

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Norfo R, Zini R, Pennucci V, Bianchi E, Salati S, Guglielmelli P, Bogani C, Fanelli T, Mannarelli C, Rosti V, Pietra D, Salmoiraghi S, Bisognin A, Ruberti S, Rontauroli S, Sacchi G, Prudente Z, Barosi G, Cazzola M, Rambaldi A, Bortoluzzi S, Ferrari S, Tagliafico E, Vannucchi AM, Manfredini R, Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative Investigators.