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.