Upregulation of endothelial cell-derived exosomal microRNA-125b-5p protects from sepsis-induced acute lung injury by inhibiting topoisomerase II alpha

Abstract

Objective: Emerging evidence has revealed that exosomal microRNAs (miRNAs) are implicated in human diseases. However, role of exosomal miR-125b-5p in sepsis-induced acute lung injury (ALI) remains further explored. We focused on the effect of exosomal miR-125b-5p on ALI progression via targeting topoisomerase II alpha (TOP2A).

Methods: The ALI mouse models were established by cecal ligation and perforation, which were then treated with miR-125b-5p agomir or overexpressed TOP2A. Next, the pathological structure of ALI mouse lung tissues were observed, miR-125b-5p, TOP2A and vascular endothelial growth factor (VEGF) expression was determined, and the lung water content, inflammatory response, protein content in bronchoalveolar lavage fluid (BALF) and cell apoptosis in ALI mouse lung tissues were assessed. Exosomes were extracted from endothelial cells (ECs) and identified, which were then injected into the modeled mice to observe their roles in ALI. The targeting relationship between miR-125b-5p and TOP2A was confirmed.

Results: MiR-125b-5p was downregulated while TOP2A was upregulated in ALI mice. MiR-125b-5p elevation or ECs-derived exosomes promoted VEGF expression, improved pathological changes and restrained lung water content, inflammatory response, protein content in BALF and cell apoptosis in lung tissues ALI mice. TOP2A overexpression reversed the repressive role of miR-125b-5p upregulation in ALI, while downregulated miR-125b-5p abrogated the effect of ECs-derived exosomes on ALI. TOP2A was confirmed as a direct target gene of miR-125b-5p.

Conclusion: Our study indicates that ECs-derived exosomes overexpressed miR-125b-5p to protect from sepsis-induced ALI by inhibiting TOP2A, which may contribute to ALI therapeutic strategies.

Keywords: Endothelial cells-derived exosome; MicroRNA-125b-5p; Sepsis-induced acute lung injury; Topoisomerase II alpha; Vascular endothelial growth factor.

Fig. 1

miR-125b-5p and VEGF are reduced while TOP2A is elevated in lung tissues of ALI mice. a miR-125b-5p expression, and mRNA expression of TOP2A and VEGF in lung tissues of mice; b protein expression of TOP2A and VEGF in lung tissues of mice; c protein bands of TOP2A and VEGF in lung tissues of mice; *P < 0.05 vs the sham group, ^P < 0.05 vs the CLP + agomir NC group, ^&P < 0.05 vs the CLP + agomir NC + OE-TOP2A group. The measurement data were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA

Fig. 2

Elevated miR-125b-5p restricts W/D value in lung tissues and protein content in BALF of ALI mice and also restrains inflammatory response in BALF and regulates VEGF expression in BALF and serum of ALI mice. a W/D values in lung tissues of mice; b protein contents in BALF; c concentrations of TNF-α, IL-1β and IL-6 in BALF; d VEGF expression in BALF and serum; * P < 0.05 vs the sham group, ^ P < 0.05 vs the CLP + agomir NC group, ^&P < 0.05 vs the CLP + agomir NC + OE-TOP2A group. The measurement data were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA

Fig. 3

Elevated miR-125b-5p improves pathological changes and decelerates cell apoptosis in lung tissues of ALI mice. a pathological structure of lung tissues of mice; b representative images of TUNEL staining in lung tissues of mice and TUNEL positive cells in lung tissues of mice; *P < 0.05 vs the sham group, ^P < 0.05 vs the CLP + agomir NC group, ^&P < 0.05 vs the CLP + agomir NC + OE-TOP2A group. The measurement data were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA

Fig. 4

Identification of EC-Exos, and EC-Exos promotes expression of miR-125b-5p and VEGF, while inhibiting expression of TOP2A in lung tissues of ALI mice. a Morphology of EC-Exos was observed under a TEM; b exosome marker proteins in serum were assessed by Western blot analysis; c effects of EC-Exos on the expression of miR-125b-5p, TOP2A and VEGF in lung tissues of mice; d protein bands of TOP2A and VEGF in lung tissues of mice with exosome treatment; e effects of EC-Exos on the protein expression of TOP2A and VEGF in lung tissues of mice; *P < 0.05 vs the sham group, ^P < 0.05 vs the CLP group, ^&P < 0.05 vs the CLP + Exos-antagomir NC group. The measurement data were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA

Fig. 5

EC-Exos decrease W/D value in lung tissues and protein content in BALF and also restrain inflammatory response in BALF and modulate VEGF expression in BALF and serum of ALI mice. a W/D values in lung tissues of mice; b protein contents in BALF; c concentrations of TNF-α, IL-1β and IL-6 in BALF; d VEGF expression in BALF and serum; *P < 0.05 vs the sham group, ^P < 0.05 vs the CLP group, ^&P < 0.05 vs the CLP + Exos-antagomir NC group. The measurement data were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA

Fig. 6

EC-Exos improve pathological changes and decelerate cell apoptosis in lung tissues of ALI mice. a Pathological structure of lung tissues of mice; b representative images of TUNEL staining in lung tissues of mice and TUNEL positive cells in lung tissues of mice; *P < 0.05 vs the sham group, ^P < 0.05 vs the CLP group, ^&P < 0.05 vs the CLP + Exos-antagomir NC group. The measurement data were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA

Fig. 7

TOP2A is the target gene of miR-125b-5p. a Binding sites of miR-125b-5p and TOP2A were predicted by an online software; b target relation between miR-125b-5p and TOP2A was confirmed by dual luciferase reporter gene assay; *P < 0.01 vs the mimic-NC group. The measurement data conforming to the normal distribution were expressed as mean ± standard deviation and the unpaired t-test was performed for comparisons between two groups

Fig. 8

The EC-derived exosomal miR-125b-5p inhibits TOP2A expression to suppress VEGF level, thus repressing apoptosis in lung tissue and ALI development