科研之友
微信
新浪微博
Facebook
分享链接
摘要
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 GPIb alpha as a proof-of-concept target protein. After in silico and in vitro screening of siRNAs targeting GPIba (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 GPIba 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% GPIba knockdown was achieved. CHO GPIb-IX cells transfected with the miG-PIBA 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 GPIba 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.
- 出版日期2015-7-15
- 单位KU Leuven
