Mesenchymal stem cells-derived extracellular vesicles carrying microRNA-17 inhibits macrophage apoptosis in lipopolysaccharide-induced sepsis

Abstract

Objective: Sepsis, as a disease affecting the microcirculation and tissue perfusion, results in tissue hypoxia and multiple organ dysfunctions. Bone mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) have been demonstrated to transfer trivial molecules (proteins/peptides, mRNA, microRNA and lipids) to alleviate sepsis. We sought to define the function of microRNA (miR)-17 carried in BMSC-EVs in sepsis.

Methods: The purity of the extracted BMSCs was identified and confirmed by detection of the surface markers by flow cytometry, followed by osteoblastic, adipogenic, and chondrocyte differentiation experiments. Subsequently, EVs were collected from the medium of BMSCs. The uptake of PKH-67-labeled BMSC-EVs or EVs carrying cy3-miR-17 by RAW264.7 cells was observed under laser confocal microscopy. Furthermore, a series of gain- and loss-of-function approaches were conducted to test the effects of LPS, miR-17 and BRD4 on the inflammatory factors (IL-1β, IL-6 and TNF-α), number of M1 macrophages and M2 macrophages, inflammatory-related signal pathway factors (EZH2, c-MYC and TRAIL), macrophage proliferation, and apoptosis in sepsis. The survival rates were measured in vivo.

Results: BMSC-EVs was internalized by the RAW264.7 cells. BDR4 was verified as a target of miR-17, while the expression pattern of miR-17 was upregulated in BMSC-EVs. MiR-17 carried by BMSC-EVs inhibited LPS-induced inflammation and apoptosis of RAW264.7 cells, but improved the viability of RAW264.7 cells. Next, in vitro experiments supported that miR-17 inhibited LPS-induced inflammation in RAW264.7 cells through BRD4/EZH2/TRAIL axis. BRD4 overexpression reversed the effects of miR-17. Moreover, the therapeutic function of BMSC-EVs carried miR-17 was verified by in vivo experiments.

Conclusions: MiR-17 derived from BMSCs-EVs regulates BRD4-mediated EZH2/TRAIL axis to essentially inhibit LPS-induced macrophages inflammation.

Keywords: BRD4; Bone mesenchymal stem cell; EZH2; Extracellular vesicle; Macrophages; MicroRNA-17; Sepsis; TRAIL.