title: Artificial MiRNA Knockdown of Platelet Glycoprotein lbα: A Tool for Platelet Gene Silencing.
abstract: In recent years, candidate genes and proteins implicated in platelet function have been identified by various genomic approaches. To elucidate their exact role, we aimed to develop a method to apply miRNA interference in platelet progenitor cells by using GPIbα as a proof-of-concept target protein. After in silico and in vitro screening of siRNAs targeting GPIbα (siGPIBAs), we developed artificial miRNAs (miGPIBAs), which were tested in CHO cells stably expressing GPIb-IX complex and megakaryoblastic DAMI cells. Introduction of siGPIBAs in CHO GPIb-IX cells resulted in 44 to 75% and up to 80% knockdown of GPIbα expression using single or combined siRNAs, respectively. Conversion of siGPIBAs to miGPIBAs resulted in reduced silencing efficiency, which could however be circumvented by tandem integration of two hairpins targeting different regions of GPIBA mRNA where 72% GPIbα knockdown was achieved. CHO GPIb-IX cells transfected with the miGPIBA construct displayed a significant decrease in their ability to aggregate characterized by lower aggregate numbers and size compared to control CHO GPIb-IX cells. More importantly, we successfully silenced GPIbα in differentiating megakaryoblastic DAMI cells that exhibited morphological changes associated with actin organization. In conclusion, we here report the successful use of miRNA technology to silence a platelet protein in megakaryoblastic cells and demonstrate its usefulness in functional assays. Hence, we believe that artificial miRNAs are suitable tools to unravel the role of a protein of interest in stem cells, megakaryocytes and platelets, thereby expanding their application to novel fields of basic and translational research.
authors.affiliation: Laboratory for Thrombosis Research, Interdisciplinary Research Facility Life Sciences, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: Tim
authors.initials: T
authors.lastname: Thijs
authors.affiliation: Laboratory for Thrombosis Research, Interdisciplinary Research Facility Life Sciences, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: Katleen
authors.initials: K
authors.lastname: Broos
authors.affiliation: Biomedical MRI, Department of Imaging and technology, KU Leuven, Leuven, Belgium.
authors.forename: Stefaan J
authors.initials: SJ
authors.lastname: Soenen
authors.affiliation: Laboratory for Thrombosis Research, Interdisciplinary Research Facility Life Sciences, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: Aline
authors.initials: A
authors.lastname: Vandenbulcke
authors.affiliation: Laboratory for Thrombosis Research, Interdisciplinary Research Facility Life Sciences, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: Karen
authors.initials: K
authors.lastname: Vanhoorelbeke
authors.affiliation: Laboratory for Thrombosis Research, Interdisciplinary Research Facility Life Sciences, KU Leuven Kulak, Kortrijk, Belgium.
authors.forename: Hans
authors.initials: H
authors.lastname: Deckmyn
authors.affiliation: Laboratory for Thrombosis Research, Interdisciplinary Research Facility Life Sciences, KU Leuven Kulak, Kortrijk, Belgium; Centre for Haematology, Imperial College London, London, United Kingdom.
authors.forename: Isabelle I
authors.initials: II
authors.lastname: Salles-Crawley
mesh_terms: D000818:Animals; D001792:Blood Platelets; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
chemical_list: D035683:MicroRNAs; D019038:Platelet Glycoprotein GPIb-IX Complex; D034741:RNA, Small Interfering; C094990:adhesion receptor