MicroRNA-223 Regulates Septin-2 and Septin-6 in Stored Platelets.

BACKGROUND: Septins have been identified to play important roles in platelets, but their regulation in platelets is unknown. Human platelet being an enucleated and terminally differentiated cell, mRNA downregulation by miRs is one of the posttranscriptional mechanisms operative in platelets.

OBJECTIVE: Since platelets are known to have miR-223 in abundance, the objective of this study is to test whether a) platelet septins have miR-223 interacting target sites in their mRNA 3'UTRs, b) septin mRNAs and miR-223 form complexes with Argonaute 2 (AGO2) protein in platelets, which is the catalytic component of an RNA Induced Silencing Complex (RISC), c) a reporter gene with septin mRNA 3' untranslated region (UTR) is subjected to downregulation by miR-223 and d) anti-miR-223 can suppress miR-223 activity and enhance septin-2 expression in platelets.

METHOD: Bioinformatics tools were used to screen mRNA 3'UTRs of septin-2 and septin-6 for miR- 223 target sites. Subsequently, platelet extracts were immunoprecipitated by AGO2 antibodies to identify that the two septin mRNAs and miR-223 were in complex with AGO2. A luciferase reporter chimeric- gene expression system was utilized to monitor miR-223 mediated downregulation luciferase gene containing the 3'UTR of either septin-2 or septin-6. Further, anti-miR-223 was utilized in platelets to directly demonstrate the role of miR-223 on the expression of septin-2.

RESULTS: Our results demonstrate that in stored platelets a) septine-2 and septin-6 mRNAs have miR- 223 target sites, b) septin-2 and septin-6 are in complex with Ago-2, c) in luciferase reporter gene system, the interaction of miR-223 with 3' UTRs of septin-2 and septin-6 leads to downregulation of luciferase expression and d) anti-miR-223 downregulated miR-223 activity and thereby the expression of septin-2 is upregulated.

CONCLUSION: The results demonstrate that like in nucleated cells, enucleated platelets also have miRbased mechanisms for the regulation of their septins.