Vitamin D modulates the content of inflammatory microRNAs in extracellular vesicles from human adipocyte cells in inflammatory context

Abstract

Inflammation of adipose tissue is a contributing factor to many chronic diseases associated with obesity. We previously showed that micronutrients such as vitamin D (VD) limited this metabolic inflammation by decreasing inflammatory markers expression including miR-155 (microRNA-155) or miR-146a in different in vitro and in vivo models. These miRNAs could be incorporated into extracellular vesicles (EVs) in order to modulate the activity of target cells. Nevertheless, the role of VD on the miRNAs contained in EVs from adipose tissue in inflammatory conditions remains unclear. In this study, we used a human model of SGBS (Simpson-Golabi-Behmel syndrome) adipocytes preincubated with 1,25(OH)₂D (the active form of VD) before an inflammatory stress with tumor necrosis factor α (TNFα). First, we confirmed by quantitative PCR that the expression of classical inflammatory factors (TNFα and chemokine ligand 2 [CCL2/MCP1]), miR-146a, and miR-155 was increased significantly under inflammatory conditions in SGBS cells and that VD prevented this up-regulation. Secondly, transmission electron microscope imaging of EVs preparations in supernatant allowed visualization of small and large vesicles under these conditions. Then, EVs were obtained with isolation kit and the expression of miR-155 and miR-146a were measured. The expression of miR-155 under TNFα effect was increased in EVs while miR-146a was not detected. Moreover, we also showed that the TNFα-mediated expression of miR-155 in EVs was significantly reduced by a VD pre-incubation of cells. Using miRNA PCR array, we also identified 33 miRNAs, organized in 5 clusters that were differentially regulated by TNFα and VD. Bioinformatic analysis of biological pathways revealed that the different miRNAs targeting genes that are involved in important cell process such as the regulation of transcription or protein phosphorylation. In conclusion, these results support for the first time that VD modulated the expression of miRNAs in EVs from adipocytes, which could represent a new mechanism of regulation of inflammation by micronutrients.

Keywords: Vitamin D; extracellular vesicles; human adipocyte; inflammation; microRNAs.