title: Mapping the methylation status of the miR-145 promoter in saphenous vein smooth muscle cells from individuals with type 2 diabetes.
abstract: Type 2 diabetes mellitus prevalence is growing globally, and the leading cause of mortality in these patients is cardiovascular disease. Epigenetic mechanisms such as microRNAs (miRs) and DNA methylation may contribute to complications of type 2 diabetes mellitus. We discovered an aberrant type 2 diabetes mellitus-smooth muscle cell phenotype driven by persistent up-regulation of miR-145. This study aimed to determine whether elevated expression was due to changes in methylation at the miR-145 promoter. Smooth muscle cells were cultured from saphenous veins of 22 non-diabetic and 22 type 2 diabetes mellitus donors. DNA was extracted, bisulphite treated and pyrosequencing used to interrogate methylation at 11 CpG sites within the miR-145 promoter. Inter-patient variation was high irrespective of type 2 diabetes mellitus. Differential methylation trends were apparent between non-diabetic and type 2 diabetes mellitus-smooth muscle cells at most sites but were not statistically significant. Methylation at CpGs -112 and -106 was consistently lower than all other sites explored in non-diabetic and type 2 diabetes mellitus-smooth muscle cells. Finally, miR-145 expression per se was not correlated with methylation levels observed at any site. The persistent up-regulation of miR-145 observed in type 2 diabetes mellitus-smooth muscle cells is not related to methylation at the miR-145 promoter. Crucially, miR-145 methylation is highly variable between patients, serving as a cautionary note for future studies of this region in primary human cell types.
authors.affiliation: 1 Division of Cardiovascular and Diabetes Research, Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM), University of Leeds, Leeds, UK.|2 Faculty of Life Sciences, University of Bradford, Bradford, UK.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Riches
authors.affiliation: 3 Division of Reproduction and Early Development, Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM), University of Leeds, Leeds, UK.
authors.forename: John
authors.initials: J
authors.lastname: Huntriss
authors.affiliation: 4 Division of Epidemiology & Biostatistics, Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM), University of Leeds, Leeds, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Keeble
authors.affiliation: 5 School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Wood
authors.affiliation: 6 Multidisciplinary Cardiovascular Research Centre (MCRC), University of Leeds, Leeds, UK.|7 Department of Cardiac Surgery, The Yorkshire Heart Centre, Leeds General Infirmary, Leeds, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: O'Regan
authors.affiliation: 1 Division of Cardiovascular and Diabetes Research, Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM), University of Leeds, Leeds, UK.|6 Multidisciplinary Cardiovascular Research Centre (MCRC), University of Leeds, Leeds, UK.
authors.forename: Neil A
authors.initials: NA
authors.lastname: Turner
authors.affiliation: 1 Division of Cardiovascular and Diabetes Research, Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM), University of Leeds, Leeds, UK.|6 Multidisciplinary Cardiovascular Research Centre (MCRC), University of Leeds, Leeds, UK.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Porter
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016022:Case-Control Studies; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D003924:Diabetes Mellitus, Type 2 / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D044127:Epigenesis, Genetic; D005260:Female; D020022:Genetic Predisposition to Disease; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012501:Saphenous Vein / Q000378:metabolism; D015854:Up-Regulation
chemical_list: C541010:MIRN145 microRNA, human; D035683:MicroRNAs
keywords: DNA methylation; miR-145; pyrosequencing; saphenous vein; smooth muscle cell; type 2 diabetes