Analysis of differentially expressed microRNA of TNF-α-stimulated mesenchymal stem cells and exosomes from their culture supernatant

Abstract

IntroductionTo analyze the microRNA expression of tumor necrosi factor α (TNF-α) stimulated mesenchymal stem cells (MSCs) and exosomes from their culture supernatant.Material and methodsTNF-α (20 ng/ml) was used to stimulate MSCs, which were then regarded as TNF-α cells (TC), while unstimulated cells were the normal control cells (NCC). MSCs and their culture supernatant were harvested after 48 h. Subsequently, exosomes were isolated from culture supernatants with ExoQuick-TC and were divided into two groups, TNF-α exosomes (TE) and normal control exosomes (NCE). Then, the microRNAs were measured by high-throughput sequencing and the results were differentially analyzed. Finally, the correlation of the target genes corresponding to differently expressed microRNAs was analyzed by gene ontology (GO) and KEGG pathway analysis.ResultsHigh-throughput sequencing showed that the cellular compartment (TC vs. NCC) had 280 microRNAs. miR-146a-5p was a uniquely up-regulated microRNA (p < 0.001) and the most significantly down-regulated microRNA among the 279 microRNAs included was miR-150-5p (p < 0.001). There were 180 differentially expressed microRNAs in the exosome compartment (TE vs. NCE), where miR-146-5p (p < 0.001) was one of 176 upregulated microRNAs and miR-203b-5p (p < 0.001) was one of 4 downregulated microRNAs. Coincidentally, bioinformatics analysis showed that IRAK1 was a critical target gene of miR-146-5p related to the Toll-like receptor (TLR) signaling pathway.ConclusionsIn contrast with the control group, there were significantly differentially expressed microRNAs in both MSCs and exosomes. Interestingly, miR-146a-5p was up-regulated in both comparative groups, and its target gene IRAK1 plays a crucial part in the TLR signaling pathway. These investigations demonstrate a new direction for subsequent inflammation mechanistic studies.

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