RAP1 Downregulation by miR-320c Reduces Platelet Activation in Ex-vivo Storage

Author(s): Neetu Dahiya , Chintamani D. Atreya* .

Journal Name: MicroRNA

Volume 8 , Issue 1 , 2019

Become EABM
Become Reviewer

Graphical Abstract:


Abstract:

Background: A small GTPase Protein, the Ras-related Protein 1 (RAP1), abundant in platelets is known to be activated following agonist-induced platelet activation, suggesting that RAP1 downregulation could, in turn, reduce platelet activation in storage. Our objective of this study is to identify RAP1 regulating miRNAs and their role in platelet activation during storage.

Methods: We applied MS2-TRAP (tagged RNA affinity purification) methodology to enrich miRNAs that target the 3’ untranslated region (3’UTR) of RAP1 mRNA in two mammalian cell lines followed by miRNA identification by microarray of total RNA samples enriched for miRNAs. Data analyses were done using different bioinformatics approaches. The direct miR:RAP1 3’UTR interaction was confirmed by using a dual luciferase reporter gene expression system in a mammalian cell line. Subsequently, platelets were transfected with one selected miR to evaluate RAP1 downregulation by this miRNA and its effect on platelet activation.

Results: Six miRNAs (miR-320c, miR-181a, miR-3621, miR-489, miR-4791 and miR-4744) were identified to be enriched in the two cell lines tested. We randomly selected miR-320c for further evaluation. The luciferase reporter assay system confirmed the direct interaction of miR-320c with RAP1 3′UTR. Further, in platelets treated with miR-320c, RAP1 protein expression was decreased and concomitantly, platelet activation was also decreased.

Conclusion: Overall, the results demonstrate that miRNA-based RAP1 downregulation in ex vivo stored platelets reduces platelet activation.

Keywords: Bioinformatics, mammalian cell, microRNA, platelets, RAP1, storage.

[1]
Lova P, Paganini S, Hirsch E, et al. A selective role for phosphatidylinositol 3,4,5-triphosphate in the Gi-dependent activation of platelet Rap1B. J Biol Chem 2003; 278(1): 131-8.
[2]
Woulfe D, Jiang H, Mortensen R, et al. Activation of Rap1B by Gi family members in platelets. J Biol Chem 2002; 277: 23382-90.
[3]
Schubert P, Thon JN, Walsh GM, et al. A signaling pathway contributing to platelet storage lesion development: targeting PI3-kinase-dependent Rap1 activation slows storage-induced platelet deterioration. Transfusion 2009; 49: 1944-55.
[4]
Franke B, Akkerman JW, Bos JL. Rapid Ca2+- mediated activation of Rap1 in human platelets. EMBO J 1997; 16: 252-9.
[5]
Franke B, van Triest M, de Bruijn KM, et al. Sequential regulation of the small GTPase Rap1 in human platelets. Mol Cell Biol 2000; 20: 779-85.
[6]
Jung SM, Ohnuma M, Watanabe N, et al. Analyzing the mechanism of Rap1 activation in platelets: Rap1 activation is related to the release reaction mediated through the collagen receptor GPVI. Thromb Res 2006; 118: 509-21.
[7]
Dalmay T. Mechanism of miRNA-mediated repression of mRNA translation. Essays Biochem 2013; 54: 29-38.
[8]
Landry P, Plante I, Ouellet DL, et al. Existence of a microRNA pathway in anucleate platelets. Nat Struct Mol Biol 2009; 16: 961-6.
[9]
Yoon JH, Srikantan S, Gorospe M. MS2-TRAP (MS2-tagged RNA affinity purification): tagging RNA to identify associated miRNAs. Methods 2012; 58: 81-7.
[10]
Dahiya N, Sarachana T, Kulkarni S, et al. miR-570 interacts with mitochondrial ATPase subunit g (ATP5L) encoding mRNA in stored platelets. Platelets 2017; 28: 74-81.
[11]
Balduini A, Pecci A, Lova P, et al. Expression, activation, and subcellular localization of the Rap1 GTPase in cord blood-derived human megakaryocytes. Exp Cell Res 2004; 300: 84-93.
[12]
Osman A, Fälker K. Characterization of human platelet microRNA by quantitative PCR coupled with an annotation network for predicted target genes. Platelets 2011; 22: 433-41.
[13]
Plé H, Landry P, Benham A, et al. The repertoire and features of human platelet microRNAs. PLoS One 2012; 7: e50746.
[14]
Dahiya N, Kulkarni S, Atreya CD. Ingenuity pathway analysis of miRNAs and mRNAs in stored platelets identifies the potential of miRNAs in regulating platelet functions relevant to storage lesions. J Hematol Blood Transfus Disord 2016, 3: 012.
[15]
Safran M, Shmueli O, Rosen N, et al. et al. NAR Molecular Biology database collection entry number 350. http://www.genecards.org/
[16]
Kozomara A, Griffiths-Jones S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 2014; 42: D68-73.
[17]
Cottrell KA, Szczesny P, Djuranovic S. Translation efficiency is a determinant of the magnitude of miRNA-mediated repression. Sci Rep 2017; 7(1): 14884.


Rights & PermissionsPrintExport Cite as

Article Details

VOLUME: 8
ISSUE: 1
Year: 2019
Page: [36 - 42]
Pages: 7
DOI: 10.2174/2211536607666180521094532
Price: $58

Article Metrics

PDF: 31
HTML: 7