MOESM1 of Glucose-sensing microRNA-21 disrupts ROS homeostasis and impairs antioxidant responses in cellular glucose variability

Additional file 1: Figure S1. Transfection efficiencies of the use of anti-miR-21 inhibitor evaluated by (A) viability assay using Trypan blue exclusion dye (dil 1:10), and by (B) q-PCR of miR-21 expression levels

Additional file 2: Figure S2. of A donor splice site mutation in CISD2 generates multiple truncated, non-functional isoforms in Wolfram syndrome type 2 patients

Chromatograms of the 400 nt-5′-RACE products not subjected to subcloning. (TIFF 155 kb

Clinical data.

<p>Clinical data.</p

A unique plasma microRNA profile defines type 2 diabetes progression

<div><p>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.</p></div

Dysregulation of plasma circulating miRNAs in glucose tolerance impaired subjects.

<p>(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 (<i>versus</i> 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 (<i>versus</i> 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 <i>versus</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188980#pone.0188980.t002" target="_blank">Table 2</a>). (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).</p

Densitometric analysis of native CD72 protein expression relative to β-actin in normal pregnancy and in miscarriage.

<p>a) CD72 is more abundant in first and second miscarriages and absent in term placenta. Significant differences in CD72 expression are detected only in first trimester vs first miscarriage (p<0.05) and in first vs third miscarriage (p<0.01). b) In first trimester and in first, second and third miscarriage, CD72 expression shows a significant increase from the 8^th to the 10^th week of gestation and subsequently a significant reduction after the 10^th week (both p<0.001). Similar CD72 expression levels are found in first trimester and in first, second and third miscarriage between the 8≤ w.g. <9 and the w.g. >10 subgroups. c) Trend of CD72 expression in first trimester and in the four miscarriage groups at approximately the same gestational week. Before the 9^th and after the 10^th week, CD72 expression is significantly greater in first miscarriage vs first trimester (p<0.001) and significantly lower in second, third and fourth miscarriage vs first miscarriage (p<0.001). Between the 9^th and the 10^th week, CD72 expression is significantly lower in first trimester vs first and second miscarriage (both p<0.001) and significantly higher in second miscarriage vs third and fourth miscarriage (both p<0.001). MC = miscarriage. Asterisks indicate the significance level: low (*), medium (**), high (***). 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10).</p

Tissue from a representative first miscarriage, 9^th week of gestation.

<p>Paraffin serial sections. A subpopulation of Hofbauer cells residing in the villous stroma, identified by CD68 (panel b,d,f), are positive for CD100 (panel a, red asterisks), CD72 (panel c, red asterisk) and CD45 (panel e, red asterisks). a,b,c,d,e,f,: Bar = 60 µm.</p

Antibodies used in the study.

*<p>mAb, monoclonal antibody; <b>^†</b>pAb, polyclonal antibody; <b>^‡</b>IH, immunohistochemistry; <b>^§</b>IP, immunoprecipitation; <b>^∥</b>WB, western blotting.</p

Positive and negative control tissue.

<p>Tonsil tissue was used as a positive control for CD100 (panel a), CD45 (panel b), CD72 (panel c) and CD68 (panel d). A positive reaction for CD100 (panel a, see inset), CD45 (panel b, see inset) and CD72 (panel c, see inset) was detected in lymphocytes, and a positive reaction for CD68 was seen in macrophages (panel d, see inset). Negative control is shown in panel e. a,b,c,d,e: Bar = 30 µm; insets: Bar = 300 µm.</p

Native CD100 protein expression in normal pregnancy and in miscarriage.

<p>Representative immunoblot. MC = miscarriage. 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10). β-actin: housekeeping protein. nCD100: native CD100.</p