title: Hyperglycaemia suppresses microRNA expression in platelets to increase P2RY12 and SELP levels in type 2 diabetes mellitus.
abstract: Megakaryocyte (MK)-derived miRNAs have been detected in platelets. Here, we analysed 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 hyperglycaemia on miRNAs was also evaluated in MKs using MEG-01 and K562 cells under hyperglycaemic conditions after 8 hours up to four 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 hyperglycaemia 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 hyperglycaemic 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.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Fejes
authors.forename: Szilárd
authors.initials: S
authors.lastname: Póliska
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Czimmerer
authors.forename: Miklós
authors.initials: M
authors.lastname: Káplár
authors.forename: András
authors.initials: A
authors.lastname: Penyige
authors.forename: Gabriella
authors.initials: G
authors.lastname: Gál Szabó
authors.forename: Ildikó
authors.initials: I
authors.lastname: Beke Debreceni
authors.forename: Satya P
authors.initials: SP
authors.lastname: Kunapuli
authors.forename: János
authors.initials: J
authors.lastname: Kappelmayer
authors.affiliation: Béla Nagy Jr, MD, PhD, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Nagyerdei krt. 98. H-4032, Debrecen, Hungary, Tel.: +36 52 340 006, Fax: +36 52 417 631, E-mail: nagyb80@gmail.com.
authors.forename: Béla
authors.initials: B
authors.lastname: Nagy
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000097:blood; D001786:Blood Glucose / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism; D016022:Case-Control Studies; D000074166:Circulating MicroRNA / Q000097:blood* / Q000235:genetics; D003430:Cross-Sectional Studies; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000097:blood* / Q000175:diagnosis / Q000235:genetics; D004151:Dipeptides / Q000494:pharmacology; D015536:Down-Regulation; D005260:Female; D006801:Humans; D020014:K562 Cells; D008297:Male; D008533:Megakaryocytes / Q000187:drug effects / Q000378:metabolism; D008875:Middle Aged; D019007:P-Selectin / Q000097:blood*; D015539:Platelet Activation; D058925:Receptors, Purinergic P2Y12 / Q000097:blood*; D043244:Ribonuclease III / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
chemical_list: D015415:Biomarkers; D001786:Blood Glucose; D000074166:Circulating MicroRNA; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D019007:P-Selectin; C548576:P2RY12 protein, human; D058925:Receptors, Purinergic P2Y12; C546937:SELP protein, human; C071482:calpeptin; C408087:DICER1 protein, human; D043244:Ribonuclease III; D002154:Calpain; D053487:DEAD-box RNA Helicases
keywords: Platelet activation; diabetes mellitus; hyperglycaemia; megakaryocyte; microRNA