Lipopolysaccharide differently modulates microRNA profiles in both colon epithelial cells and their extracellular vesicles

Abstract

Introduction: Extracellular vesicles (EVs) play an essential role in cell and infection biology and have the potential to act as biomarkers for novel diagnostic tools. EVs are membrane-enclosed nanoparticles secreted by the majority of cells. They contain various types of biomolecules, including proteins, lipids, and non-coding RNAs such as miRNAs. It has been indicated that miRNAs found in EVs affect mRNA stability of recipient cells, thereby playing a crucial role in diseases progression. However, there is no clear consensus as to whether the packaging of miRNAs into EVs occurs randomly or selectively. Lipopolysaccharide (LPS) is an endotoxin of gram-negative bacteria known to initiate the response of immune system and was associated with pathogenesis of inflammatory bowel diseases and cancer. Fundamental mechanisms of molecular packaging into EVs and specificity remain unclear and need to be studied further. Aims & Methods: The aim of this study was to assess the miRNA profile of extracellular vesicles compared to the miRNA profile of their host cells (colon adenocarcinoma and healthy colon cells), after stimulation with lipopolysaccharide.Methods: Experiments were conducted using primary human colonic epithelial cells (HCoEpiC) and adenocarcinoma cells (Caco-2, HCT116) (5 pas-sages per cell line). Cells were treated with bacterial LPS from Salmonella enterica (Serotype typhimurium) (Sigma-Aldrich) (10 μg/ml) 24 hours and after 24, 48, 72 hours their EVs were collected using polyethylene-glycol-based method. RNA was purified from cells and EVs using commercial kits. Small RNA sequencing was performed using HiSeq 2500 platform (Illumina, CA, USA). Bioinformatic analysis performed using R package and algorithms for small sequencing data (DEseq2 and etc.). Results: After 24 hours LPS treatment, miR-146 and miR-451 were overexpressed in HCT116, Caco-2 and HCoEpic cells. The profile of deregulated miRNAs found in EVs of these cell lines was as follows: i. miR-122-5p, miR-451a, miR-30b-5p, 381-3p, 99a-5p were upregulated and miR-20a-5p, miR-17-5p, miR-93-5p, miR-335-5p, miR-378c were down-regulated in Caco-2 cell line produced EVs, ii. miR-215-5p was downregulated in HcoEpic cell line produced EVs, and; iii. miRNAs expression was not deregulated in HCT116 cell line EVs. After 48- and 72-hours LPS treatment miRNA expression was deregulated only in HCOepic cell line: miR-451a, miR-147b-3p, miR-146a-5p, but not in the EVs secreted by this cell line. This research shows that host cells hold a sorting mechanism that guides specific miRNAs into exosomes. It is important to mention that we noticed that certain miRNAs sequences were found only in EVs of cells, but not in the cells, and some of them (even the most overexpressed) had not been packaged into EVs.Conclusion: The miRNA profile of extracellular vesicles differs from miRNA profile of the host cells after stimulation with lipopolysaccharide.