Browsing by Author "Alley, Philbe-Jeanne"
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- ItemInvestigation of differentially methylated microRNA genes in Type 2 diabetes(2017-03) Alley, Philbe-Jeanne; Carmen, Pheiffer; Sian, Hemmings; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Type 2 diabetes is a major source of morbidity and mortality worldwide, accounting for approximately 90% of the 415 million individuals who have Diabetes mellitus globally. The prevalence of T2D in South Africa is currently estimated at 9% and is expected to increase significantly due to the concomitant high prevalence of obesity, a major risk factor for the development of T2D. These metabolic disorders pose a major health burden to an already under-resourced and over-burdened health care system, and create an urgent need to identify therapeutics capable of preventing or treating T2D. Epigenetic mechanisms, such as microRNAs (miRNAs), reflect the interaction between genetic and environmental factors and are attracting considerable interest as strategies to elucidate the pathophysiology of disease. Investigation of miRNAs could lead to the development of high risk biomarkers for disease prevention, or to therapeutic targets. MiRNAs are dysregulated during T2D, however, the mechanisms regulating miRNAs are largely unknown. The aim of this study was to explore the role of DNA methylation, another important epigenetic mechanism, in the regulation of miRNA genes. Furthermore, we aimed to investigate the relationship between miRNA gene methylation and miRNA expression in an in vitro model relevant to T2D. To identify miRNA genes potentially regulated by DNA methylation, miRNAs that were differentially expressed in the whole blood of n=9 South African women of mixed ethnic ancestry during T2D were integrated with miRNA genes that were differentially methylated in the whole blood of a different subset of n=9 South African women of mixed ethnic ancestry during T2D. The methylation status of the identified miRNA genes were verified using the MassARRAY® EpiTYPER® system. To investigate the relationship between DNA methylation and miRNA gene expression under defined conditions, 3T3-L1 pre-adipocytes were differentiated in glucose conditions that mimic normoglycaemia and hyperglycaemia. Data integration showed that 43 miRNAs were differentially expressed during T2D compared to normoglycaemia, 21 miRNAs that were differentially expressed during impaired glucose tolerance compared to normoglycaemia, and 32 miRNAs that were differentially expressed during T2D compared to impaired glucose tolerance, were under the potential regulation of DNA methylation. Primers were designed for 26 randomly selected miRNA genes, of which 21 miRNA genes were successfully analysed by MassARRAY® EpiTYPER®. The methylation of two of these, mir-98 and mir-150, correlated with DNA methylation data conducted using methylated DNA immunoprecipitation sequencing. Differentiation of 3T3-L1 pre-adipocytes in glucose concentrations that mimic normoglycaemia and hyperglycaemia resulted in mature adipocytes that differed in metabolic activity and oxidative stress. In conclusion, this study provides support for DNA methylation as a mechanism regulating miRNA expression during T2D and demonstrates that 3T3-L1 pre-adipocytes differentiated in different glucose concentrations offer a viable model for investigating the relationship between miRNA expression and DNA methylation during hyperglycaemia, altered metabolic activity and oxidative stress. These findings pave the way for future studies to delineate the effect of DNA methylation on miRNA expression during conditions characteristic of T2D.