MicroRNA expression profiling in peripheral blood mononuclear cells and serum of type 2 diabetic, pre-diabetic and normo-glycaemic individuals

Dias, Stephanie (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNAs that play a fundamental role in cellular function by regulating messenger RNA gene expression. Alterations in miRNA expression are implicated in metabolic dysregulation, with several studies reporting the involvement of miRNAs in the pathophysiology of Type 2 diabetes (T2D). Recently, circulating miRNAs have attracted considerable interest as biomarkers to identify individuals at risk for T2D, thus we hypothesised that circulating miRNA could be used as markers for T2D progression. The aim of this study was to determine whether miRNA expression profiles differ between diabetic, pre-diabetic and normo-glycaemic individuals. Individuals were recruited from local communities and classified as diabetic, pre-diabetic or normo-glycaemic according to World Health Organization criteria, whereafter miRNAs were extracted from peripheral blood mononuclear cells (PBMCs) and serum of age-, gender-, ethnicity- and BMI-matched diabetic (n=4), pre-diabetic (n=4) and normo-glycaemic (n=4) individuals. MiRNAs extracted from PBMCs were sequenced using the Illumina HiSeq 2500 platform, and validated by quantitative real time PCR (qRT-PCR) in PBMCs and serum of these individuals. Moreover, bioinformatics was conducted using various target prediction programs (TargetScan, DIANA and PITA) and the DAVID functional gene annotation tool to assign biological significance to the differentially expressed miRNAs identified by sequencing. Sequencing showed that 267 (pre-diabetics vs. normo-glycaemics), 277 (diabetics vs. normo-glycaemics) and 267 (pre-diabetics vs. diabetics) miRNAs were differentially expressed between groups. Of these, five differentially expressed miRNAs (miR-27b, miR-379, miR-21, miR-98 and miR-143) were selected for validation by qRT-PCR in PBMCs. Only miR-143 and miR-27b were significantly differentially expressed using qRT-PCR, although the results for miR-143 were different compared to the sequencing data. MiR-143 was upregulated in pre-diabetics compared to normo-glycaemic individuals (1.40-fold, p≤0.01), whereas sequencing showed upregulation of miR-143 in diabetics compared to pre-diabetics (1.75-fold, p≤0.05). The differential expression of miR-27b was consistent between qRT-PCR (1.55-fold; p=0.07) and sequencing (1.15-fold; p<0.01), where both methods showed upregulation in pre-diabetics compared to normo-glycaemic individuals. The expression of miR-27b was similarly upregulated in serum of pre-diabetics compared to normo-glycaemic individuals (2.0-fold; p≤0.05). Furthermore, five novel miRNAs identified by sequencing were successfully validated in PBMCs of diabetic, pre-diabetic and normo-glycaemic individual. Sequencing and qRT-PCR showed that miR-27b was upregulated in PBMCs and serum of pre-diabetics compared to normo-glycaemic individuals. Bioinformatics identified peroxisome proliferator-activated receptor gamma (Pparg) as a target for miR-27b. PPARG is an insulin sensitizing agent, thus we speculate that increased miR-27b expression in pre-diabetes suppresses Pparg, thereby inhibiting insulin signaling and subsequently decreasing glucose uptake. The increased insulin and glucose levels observed in the pre-diabetic individuals support this idea, although further work is required to confirm this hypothesis. In conclusion, we showed that miRNA profiles differ during T2D progression, and are able to discriminate between diabetic, pre-diabetic and normo-glycaemic individuals. To our knowledge, this is the first study to report differential expression of miR-27b during T2D, suggesting its potential as a biomarker that could be incorporated into predictive models for the identification of high risk individuals. However, miRNA profiling in a larger sample

AFRIKAANSE OPSOMMING: MikroRNAs (miRNAs ) is klein nie-koderende RNAs wat 'n fundamentele rol in sellulêre funksie speel deur regulering van boodskapperRNA geenuitdrukking. Verskeie studies ïmpliseer veranderings in miRNA ekspresie met metaboliese disregulering en in die patofisiologie van Tipe 2-diabetes (T2D). Onlangs het sirkulerende miRNAs groot belangstelling uitgelok as biomerkers om individue te identifiseer wat „n verhoogde risiko vir T2D het. Ons hipotese stel dus voor dat sirkulerende miRNA gebruik kan word as merkers vir T2D siekteprogressie. Die doel van hierdie studie was om vas te stel of miRNA geenuitdrukkings profiele verskil tussen diabete, prediabete en normoglisemiese individue. Individue wat uit plaaslike gemeenskappe gewerf is, is volgens die Wêreld Gesondheid Organisasie riglyne geklassifiseer as diabete, pre diabete of normoglisemiese individue. Hierna is miRNAs uit die perifere bloed mononukleêreselle (PBMS) en serum van ouderdom, geslag, etniesiteit en liggaamsmassa-indeks vergelykbare diabete (n=4), prediabete (n=4) en normoglisemiese individue (n=4), geïsoleer. Die geenvolgordebepaling van die geïsoleerde miRNAs is bepaal deur „n Illumina HiSeq 2500 platform, en bevestig deur kwantitatiewe “real time PCR” (qRT-PCR). Verder, is bioinformatika uitgevoer met behulp van verskeie teikenvoorspellings programme (TargetScan, Diana en PITA) asook David se funksionele geenannotasie instrument om biologiese betekenis aan die differensieel uitgedrukte miRNAs, te koppel. Geenvolgordebepaling het getoon dat 267 (prediabete vs. normoglisemies), 277 (diabete vs. normoglisemies) and 267 (prediabete vs. diabete) miRNAs differensieel uitgedruk word. Hiervan is vyf differensieel uitgedrukte miRNAs (miR-27b, miR-379, miR-21, miR-98 en miR-143) gekies vir bevestiging deur qRT-PCR in PBMS. MiR-143 en miR-27b differensiasie was deur qRT-PCR bevestig, hoewel die qRT-PCR resultate vir miR-143 verskil het met die geenvolgordebepaling data. Met qRT-PCR is miR-143 opgereguleer in die prediabete teenoor normoglisemiese individue (1,40-voudig, p≤0.01), terwyl met geenvolgordebepaling miR-143 in diabete teenoor prediabete (1,75-voudig, p≤0.05) opgereguleer was. Daar was ooreenstemming in die differensiële uitdrukking van miR-27b tussen die qRT-PCR (1,55-voudig; p=0,07) en geenvolgordebepaling (1,15-voudig; p<0,01), waar albei metodes opregulering gewys het in die prediabete teenoor normoglisemiese individue. In die serum monsters was die uitdrukking van miR-27b soortgelyk opgereguleer in prediabete (2,0-voudig; p≤0.05). Verder is vyf unieke miRNAs geïdentifiseer deur geenvolgordebepaling wat suksesvol bevestig is in PBMS van diabete en prediabete. Bioinformatika het Pparg geïdentifiseer as 'n teiken vir miR-27b. PPARG is 'n insuliensensiterings agent, dus spekuleer ons dat hoër miR-27b ekspresie, in prediabete Pparg onderdruk, wat die insuliensein demp en tot verlaagde glukose opname lei. Die verhoogde insulien en glukose vlakke wat in prediabete voorkom ondersteun hierdie idee, alhoewel verdere werk nodig is om hierdie hipotese te bevestig. Ten slotte, het ons getoon dat miRNA profiele tydens die T2D siekteprogressie verskil, en in staat is om tussen diabete, prediabete en normoglisemiese individue te diskrimineer. Tot ons kennis, dit is die eerste studie wat differensiele uitgedrukking van miR-27b in T2D rapporteer, en die potensiële toepassing as 'n nie-indringende biomerker uitwys. Dit kan moontlik in voorspellende modelle geïnkorporeer kan word vir die identifisering van hoë risiko individue. Maar verdere studies met groter monster getalle en prospektiewe longitudinale studies is nodig om die kliniese toepaslikheid te evalueer.

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