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

Introduction: To analyze the microRNA expression of tumor necrosi factor α (TNF-α) stimulated mesenchymal stem cells (MSCs) and exosomes from their culture supernatant.

Material and methods: TNF-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.

Results: High-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.

Conclusions: In 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.