Placental exosomes isolated from urine of patients with gestational diabetes exhibit a differential profile expression of microRNAs across gestation

Abstract

Placenta‑derived exosomes play an important role in cellular communication both in the mother and the fetus. Their concentration and composition are altered in several pregnancy disorders, such as gestational diabetes mellitus (GDM). The isolation and characterization of placental exosomes from serum, plasma and tissues from patients with GDM have been previously described; however, to the best of our knowledge, to date, there is no study available on placental exosomes isolated from urine of patients with GDM. In the present study, placental exosomes were purified from urine the 1st, 2nd and 3rd trimester of gestation. Placental exosomes were characterized by transmission electron microscopy in cryogenic mode and by western blot analysis, confirming the presence of exosomal vesicles. The expression profile of five microRNAs (miR‑516‑5p, miR‑517‑3p, miR‑518‑5p, miR‑222‑3p and miR‑16‑5p) was determined by RT‑qPCR. In healthy pregnant women, the expression of the miRNAs increased across gestation, apart from miR‑516‑5p, which was not expressed at the 2nd trimester. All the miRNAs examined were downregulated in patients with GDM at the 3rd trimester of gestation. The downregulated miRNAs affected several metabolic pathways closely associated with the pathophysiology of GDM. This provides further evidence of the regulatory role of miRNAs in the GDM. This also suggests that the of urinary exosomes may be an excellent source of biomarkers and therapeutic targets.

Figure 1

Characterization of urinary exosomes of placental origin in women with gestational diabetes mellitus. (A) Transmission electron microscopy in cryogenic mode. Arrows indicate the presence of exosomes and the size (B) Identification of exosomal markers by western blot. Loaded samples belong to urinary exosomes isolated from GDM patients. Loading control: C+ (vesicles isolated from HepG2 cells). Lanes 1 and 2, CD63 (30 kDa) and CD9 (25 kDa) markers; lanes 3 and 4, CD81 (25 kDa) marker; lanes 5 and 6, PLAP (52 kDa) marker.

Figure 2

Expression of (A) miR-16-5p and (B) miR-222-3p in exosomes of healthy pregnant women and with GDM through pregnancy. A non-parametric Kruskall-Wallis statistical test was used with a Dunn's post hoc test was performed. ^*P<0.05, ^**P<0.01.

Figure 3

Expression of (A) miR-516-5p, (B) miR-517-3p and (C) miR-518-5p in urinary exosomes of placental origin of healthy pregnant women and gestational diabetics during the three trimesters of gestation. A non-parametric Kruskall-Wallis statistical test was used with a Dunn's post hoc test was performed. ^*P<0.05, ^**P<0.01.

Figure 4

Common targets between the five studied microRNAs. The evaluation for the interaction and target genes networks was performed using the miRTargetLink program. Validated (red) and predicted (green) target genes are highlighted.

Figure 5

Heatmap showing the signaling pathways involved in regulation by the five studied microRNAs. Using the bioinformatics tool, The Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/), signaling pathways related to the study of microRNAs were evaluated, considering a value of P<0.05 to indicate statistically significant differences (the color scale represents statistical significance).