Neetu Dahiya and Chintamani D. Atreya* Pages 1 - 7 ( 7 )
Aim: Since RAP1B is critical for platelet functions including hemostasis, this study was conducted to identify RAP1B regulating microRNAs (miRNAs) in ex vivo stored platelets.
Background: Previous studies with platelets identified factors affecting RAP1B activity but regulatory miRNAs that affect RAP1B protein expression has not been reported.
Objective: To understand the functional significance of miRNA mediated regulation of RAP1B in stored platelets, using microRNA, miR-181a as an example.
Methods: A tagged RNA affinity approach (MS2-TRAP) was employed to identify miRNAs that bound to the 3` untranslated region (3`UTR) of the RAP1B mRNA first in HeLa cells as an assay system and subsequently the mRNA 3’UTR:miRNA interactions were verified in platelets through ectopic expression of miR-181a mimic and appropriate controls. Interaction of such miRNAs with RAP1B mRNA were also validated by qRT-PCR and Western analysis.
Results: Sixty-two miRNAs emerged through MS2 assay were then compared with already known 171 platelet abundant miRNAs to identify a common set of miRNAs. This analysis yielded six miRNAs (miR-30e, miR-155, miR-181a, miR-206, miR-208a and miR-454), which are also predicted to target RAP1B mRNA. From this pool miR-181a was selected for further study since RAP1B harbors two binding sites for miR-181a in its 3´UTR. Ectopic expression of miR-181a mimic in platelets resulted in lowering the endogenous RAP1B at both the mRNA and protein levels. Further, miR-181a ectopic expression reduced the surface expression of platelet activation marker, P-selectin.
Conclusion: MicroRNA-181a can target RAP1B and this interaction has the potential to regulate platelet activation during storage.
microRNA, RAP1, platelets, miR-181, platelet activation, MS2-TRAPmicroRNA, RAP1, platelets, miR-181, platelet activation, MS2-TRAP
Laboratory of Cellular Hematology, Division of Blood Components and Devices, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, Laboratory of Cellular Hematology, Division of Blood Components and Devices, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring