Thromb Haemost. 2017 Feb 28;117(3):529-542.

Hyperglycemia suppresses microRNA expression in platelets to increase P2RY12 and SELP mRNA levels in type 2 diabetes mellitus

Zsolt Fejes1, Szilárd Póliska2, Zsolt Czimmerer2, Miklós Káplár3, András Penyige4,5, Gabriella Gál Szabó1, Ildikó Beke Debreceni1, Satya P. Kunapuli6, János Kappelmayer1, Béla Nagy Jr1,*

1Department of Laboratory Medicine, 2Department of Biochemistry and Molecular Biology, Genomic Medicine and Bioinformatics Core Facility, 3Institute of Internal Medicine, 4Department of Human Genetics, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 5Faculty of Pharmacy, University of Debrecen, Debrecen, Hungary; 6Department of Physiology and Sol Sherry Thrombosis Center, Temple University School of Medicine, Philadelphia, PA, USA

Abstract 

Megakaryocyte (MK)-derived miRNAs have been detected in platelets. Here, we analyzed the expression of platelet and circulating miR-223, miR-26b, miR-126 and miR-140 that might be altered with their target mRNAs in type 2 diabetes mellitus (DM2). MiRNAs were isolated from leukocyte-depleted platelets and plasma samples obtained from 28 obese DM2, 19 non-DM obese and 23 healthy individuals. The effect of hyperglycemia on miRNAs was also evaluated in MKs using MEG-01 and K562 cells under hyperglycemic conditions after 8 hours up to 4 weeks. Quantitation of mature miRNA, pre-miRNAs and target mRNA levels (P2RY12 and SELP) were measured by RT-qPCR. To prove the association of miR-26b and miR-140 with SELP (P-selectin) mRNA level, overexpression or inhibition of these miRNAs in MEG-01 MKs was performed using mimics or anti-miRNAs, respectively. The contribution of calpain substrate Dicer to modulation of miRNAs was studied by calpain inhibition. Platelet activation was evaluated via surface P-selectin by flow cytometry. Mature and pre-forms of investigated miRNAs were significantly reduced in DM2, and platelet P2RY12 and SELP mRNA levels were elevated by two-fold at increased platelet activation compared to controls. Significantly blunted miRNA expressions were observed by hyperglycemia in MEG-01 and K562-MK cells versus baseline values, while the manipulation of miR-26b and miR-140 expression affected SELP mRNA level. Calpeptin pretreatment restored miRNA levels in hyperglycemic MKs. Overall, miR-223, miR-26b, miR-126 and miR-140 are expressed at a lower level in platelets and MKs in DM2 causing upregulation of P2RY12 and SELP mRNAs that may contribute to adverse platelet function.

Keywords: platelet activation, microRNA, megakaryocyte, diabetes mellitus, hyperglycemia

PMID: 27975100

DOI: 10.1160/TH16-04-0322

 

Supplement:

Increased activation level of platelets was previously described in type 2 diabetes mellitus (DM2) [1]. Among several mechanisms, one proposed explanation may be that megakaryocytes (MKs) produce abnormal platelets with increased numbers of glycoprotein receptors on cell surface in diabetic conditions [2]. In parallel, dysfunction of bone marrow stromal cells and endothelial cells induced by hyperglycemia can be also responsible for vascular complications in DM2 [3,4]. Platelets carry messenger RNAs (mRNAs), a large number of microRNAs (miRNAs) and several proteins involved in miRNA processing (e.g. Dicer), which are delivered from MKs [5]. Platelets can synthesize proteins with important biological activities in response to various extracellular signals [6], and the expression of platelet miRNAs correlates with platelet reactivity [7]. As such, platelet miR-223 regulates the expression of ADP-receptor P2Y12, which modulates platelet sensitivity to various stimuli [5].

Here our aim was to investigate the background of altered platelet function in DM2 via measuring the level of some of the most abundant platelet miRNAs that might be involved in platelet activation. Mature and precursor miRNAs and their major target mRNAs were measured by RT-qPCR in leukocyte-depleted platelets, in plasma samples and two independent MK cell cultures (MEG-01 and mature MKs differentiated from K562 cells). The following findings may explain the elevated platelet activation status in DM2:

1) Platelet and circulating level of mature miR-223, miR-26b, miR-126 and miR-140 were downregulated in diabetes versus healthy controls and BMI-matched non-DM obese subjects.

2) Lower expression of pre-miR-223 and pre-miR-26b was detected in diabetic platelets compared to normal controls, thus DM2 platelets contain a smaller amount of pre-miRNAs for the maturation process.

3) Decreasing level of mature miR-223, miR-26b and miR-140 was observed in MK cells among in vitro hyperglycemic conditions with augmented level of these pre-miRNAs due to lowered Dicer activity studied by calpain inhibitor calpeptin.

4) Increased levels of P2RY12-specific mRNA and SELP mRNA were identified in DM2 platelets versus normal individuals implicating elevated expression/function of P2Y12-receptor and P-selectin in DM2 with a higher risk for the development of abnormal platelet reactivity. Similar tendency was seen in hyperglycemic MKs.

5) For the first time, we proved that platelet miR-26b and miR-140 target the SELP mRNA based on the results of those experiments using miRNA specific mimics and inhibitors in transfected MKs.

In conclusion, hyperglycemia suppresses miRNA expression in MKs and platelets to increase platelet P2RY12 and SELP mRNA levels in DM2 contributing to adverse platelet function in diabetics (Figure 1). Accordingly, platelet miRNAs may represent a novel therapeutic target to prevent adverse thrombotic events in diabetic patients.

 

 

Figure 1. Biogenesis of MK and platelet miRNAs among normal (A) and diabetic conditions (B). Platelets contain MK-derived mRNAs, miRNAs and different proteins, such as Dicer to process miRNA maturation and protein synthesis (A). Hyperglycemia can modulate platelet miRNA expression via affecting the level of pre-miRNAs, Dicer activity and mature miRNAs in MKs in the bone marrow. These genetic alterations are transferred into developing platelets that are released into the circulation. In addition, high glucose may regulate the RNA content of circulating platelets via lowered level of platelet Dicer activity in diabetics observed by others [8]. Overall, changes in altered platelet miRNA and mRNA expression may generate elevated platelet function via abnormal protein synthesis and function (B).

 

Acknowledgements:

This study was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/2-11/1-2012-0001 ‘National Excellence Program’, and the TÁMOP-4.2.2.A-11/1/KONV-2012-0045 project. Béla Nagy Jr. was supported by the Lajos Szodoray Grant of the University of Debrecen.

 

Correspondence to:

Béla Nagy Jr, MD, PhD

Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Nagyerdei krt. 98. H-4032, Debrecen, Hungary

Email: nagyb80@gmail.com

 

References:

[1] Nagy B Jr, Csongrádi E, Bhattoa HP, et al. Investigation of Thr715Pro P-selectin gene polymorphism and soluble P-selectin levels in type 2 diabetes mellitus. Thromb Haemost 2007;98:186-191.
[2] Tschoepe D. The activated megakaryocyte-platelet-system in vascular disease: focus on diabetes. Semin Thromb Hemost 1995;21:152-160.
[3] Fadini GP, Ferraro F, Quaini F, et al. Concise review: diabetes, the bone marrow niche, and impaired vascular regeneration. Stem Cells Transl Med 2014;3:949-957.
[4] Olivieri F, Bonafè M, Spazzafumo L, et al. Age- and glycemia-related miR-126-3p levels in plasma and endothelial cells. Aging (Albany NY) 2014;6:771-787.
[5] Landry P, Plante I, Ouellet DL, et al. Existence of a microRNA pathway in anucleate platelets. Nat Struct Mol Biol 2009;16:961-966.
[6] Zimmerman GA, Weyrich AS. Signal-dependent protein synthesis by activated platelets: new pathways to altered phenotype and function. Arterioscler Thromb Vasc Biol 2008; 28: s17-24.
[7] Nagalla S, Shaw C, Kong X, et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood 2011;117:5189-5197.
[8] Elgheznawy A, Shi L, Hu J, et al. Dicer cleavage by calpain determines platelet microRNA levels and function in diabetes. Circ Res 2015;117:157-165.