Investigation of differentially methylated microRNA genes in Type 2 diabetes

Alley, Philbe-Jeanne (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

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.

AFRIKAANSE OPSOMMING: Tipe 2 diabetes (T2D) is van die grootste oorsake van morbiditeit and mortaliteit wereldwyd, met ‘n voorkoms van meer as 90% van die 415 miljoen individue wat wereldwyd diagnoseer was met Diabetes mellitus in 2015. Huidiglik word die voorkoms van T2D in Suid Afrika beraam op 9% en dit word geskat dat hierdie voorkoms aansienlik gaan verhoog as gevolg van die gepaardgaande hoë voorkoms van vetsug en die verhoogde kans wat dit n individu bied vir die ontwikkeling van T2D. Ons gesondheidsstelsel is onder geweldige druk en die hoë voorkoms van hierdie metaboliese afwykings dra verder daartoe by. Dit beklemtoon dus die behoefte om terapeutiese middels te identifiseer wat die vermoë het om T2D te verhoed of effektief te behandel. Epigenetiese meganismes, byvoorbeeld mikroRNAs (miRNAs), weerspieel die interaksie tussen omgewings- en genetiese faktore wat groot belangstelling begin werf het as ‘n strategie om die onderliggende patofisiologie van T2D toe te lig, om ten einde biomerkers te identifiseer wat gebruik kan word om T2D te voorkom en as terapeutiese middels. MikroRNAs word disreguleer gedurende T2D, maar die meganismes waarby hierdie disregulasie plaasvind, is steeds grootendeels onbekkend. Die doel van hierdie studie was om te sien of DNA metilasie, nog ‘n belangrike epigenetiese meganisme, een van die maniere is waarop miRNA gene reguleer word. Verder, het ons gemik daarop om die verhouding tussen miRNA geen metilasie en miRNA geen uitdrukking in ‘n in vitro model te ondersoek. Ons het miRNAs wat differensieel uitgedruk was in die bloed van n=9 Suid Afrikaanse kleurling vroue met T2D van gemengde etniese afkoms, wat identifiseer was deur geenvolgordebepaling (miRNA-Seq), met miRNA gene wat identifiseer was om differensieel metileer te wees, deur DNA metilasie immunopresipitasie geenvolgordebepaling (DMIGB), in die bloed van n afsonderlike n=9 Suid Afrikaanse kleurling vroue met T2D van gemengde etniese afkoms, vergelyk om miRNA gene te identifiseer wat moontlik deur metilasie reguleer word. Die metilasie van die ge-identifiseerde miRNAs was bevestig deur die MassARRAY® EpiTYPER® analise. Om die verhouding tussen DNA metilasie en miRNA geen uitdrukking te bepaal onder spesifieke sellulere kondisies, was 3T3-L1 adiposiete differensieer in kondisies wat die patologie van T2D naboots. Gedurende die vergelykingsproses, was 43 miRNA gene (T2D vs. normoglisemies), 21 miRNA gene (prediabetes vs. normoglisemies) en 32 miRNA gene (T2D vs. prediabetes), respektiewelik, identifiseer wat moontlik deur DNA metilasie reguleer word. Inleiers was was ontwerp vir 26 lukraak gekiesde miRNA gene, waarvan 21 suksesvol geanaliseer was deur MassARRAY® EpiTYPER®. Die metilasie vlak van twee van die 21 miRNA gene, miR-98 en miR-150, het ooreengestem met die metilasie data gegenereer deur DMIGB. Differensiasie van 3T3-L1 pre-adiposiete in verskillende glukose konsentrasies, het gelei na volwasse adiposiete wat verskillende metabolise aktiwiteite en oksidatiewe stres vlakke het. Ten slotte, hierdie studie toon dat DNA metilasie wel n meganisme is van miRNA geen uitdrukking regulasie tydens T2D en dit demonstreer dat 3T3-L1 pre-adiposiete gedifferensieerd in verskillende glucose konsentrasies, ‘n praktiese en gepasde model is vir die ondersoek van die verhouding tussen miRNA uitdrukking en DNA metilasie gedurende hiperglisemie en die verandering in metabolise aktiwiteit en oksidatiewe stres is. Hierdie bevindings le grond vir toekomstige studies om die effek van DNA metilasie op miRNA uitdrukking in T2D patologie af te baken.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/101453
This item appears in the following collections: