A unique plasma microRNA profile defines type 2 diabetes progression

Abstract

A major unmet medical need to better manage Type 2 Diabetes (T2D) is the accurate disease prediction in subjects who show glucose dysmetabolism, but are not yet diagnosed as diabetic. We investigated the possibility to predict/monitor the progression to T2D in these subjects by retrospectively quantifying blood circulating microRNAs in plasma of subjects with i) normal glucose tolerance (NGT, n = 9); ii) impaired glucose tolerance (IGT, n = 9), divided into non-progressors (NP, n = 5) and progressors (P, n = 4) based on subsequent diabetes occurrence, and iii) newly diagnosed T2D (n = 9). We found that impaired glucose tolerance associated with a global increase of plasma circulating microRNAs. While miR-148 and miR-222 were specifically modulated in diabetic subjects and correlated with parameters of glucose tolerance, the most accentuated microRNA dysregulation was found in NP IGT subjects, with increased level of miR-122, miR-99 and decreased level of let-7d, miR-18a, miR-18b, miR-23a, miR-27a, miR-28 and miR-30d in comparison with either NGT or T2D. Interestingly, several of these microRNAs significantly correlated with parameters of cholesterol metabolism. In conclusion, we observed the major perturbation of plasma circulating microRNA in NP pre-diabetic subjects and identified a unique microRNA profile that may become helpful in predicting diabetic development.

Fig 1. Dysregulation of plasma circulating miRNAs in glucose tolerance impaired subjects.

(A) Boxplots (10–90 percentile) for the number of detected miRNAs (left) and miRNA Ct values (right) in the three indicated sample groups. Non-parametric Kruskal-Wallis p-values are reported at the bottom of the graph, while Dunn’s multiple comparison test (versus NGT group) p-values are reported on single group plots (* <0.05; ** <0.01; *** <0.001). (B) Scatter plots showing the correlation between miRNA global means (for the 27 individuals belonging to the three groups of NGT, IGT and T2D) expressed as reversed Ct values and either oral glucose tolerance test (OGTT, that refers to glucose level measured 2 hours post load, left) or glycated hemoglobin (HbA1c, right). Spearman correlation coefficient r and p-values are also reported. (C) Boxplot for single miRNA non-parametric correlation with the global mean values, averaged per group. Non-parametric Kruskal-Wallis p-value is reported at the bottom of the graph. Dunn’s multiple comparison test (versus IGT group) p-values are shown (* <0.05; *** <0.001). (D) Heatmap reporting the correlation index map (non parametric Spearman r values) of each co-expressed miRNA versus all the others. The map is divided per group as indicated. (E) Vertical scatter plots for Log10 transformed global mean normalized miRNA values in the three groups as indicated. The IGT group is divided in two further groups: progressors (P IGT) and non-progressors (NP IGT), based on their clinical history (see text). miRNAs were found differentially expressed in at least one group comparisons (t Test p-value<0.05, see Table 2). (F) Hierarchical clustering of the four groups (the IGT group being divided as in panel E) using the Log10 transformed normalized values of differentially expressed miRNAs (as for panel E).

Fig 2. Analysis of miRNA correlation with metabolic parameters.

(A) Heatmap reporting the correlation index map (Spearman r values, red if positive, blue if negative) of differentially expressed miRNAs and the reported clinical parameters. BMI = body mass index; OGTT, 2h oral glucose tolerance test, at 2 hours; HOMA = homeostatic model assessment; HbA1c = glycated hemoglobin; LDL = low density lipoproteins; HDL = high density lipoproteins. Only miRNAs significantly correlated with at least two clinical parameters are shown. * p-values≤0.05. (B) Scatter plots showing the correlation between BMI (upper), waist (middle) and OGTT (lower) and miR-27a-3p normalized relative quantities (NRQs) for all 27 subjects under study. Spearman r and p-values are reported. (C) Scatter plots showing the correlation between miR-27a-3p Ct values and miRNA global mean for all 27 subjects divided into the three groups of NGT, IGT and T2D as indicated. Slope values are also reported.