Next generation-sequencing of extracellular RNAs identified extracellular full-length mature miRNAs which are not associated with extracellular vesicles

Extracellular RNAs (exRNAs) have been proposed to play a role in cell-to-cell communication and regulate gene expression when they are imported inside cells. While some exRNAs are found within extracellular vesicles, some other exRNAs such as miRNAs have been reported not to be associated with extracellular vesicles. However, properties of non-vesicular exRNAs, such as length, species, sequences and stability have remained unclear. In this study, by using next-generation sequencing, we analyzed non-vesicular exRNAs from cell culture media, in comparison with vesicular exRNAs and intracellular RNAs. In order to assess whether non-vesicular exRNAs contain intact RNAs, we analyzed RNAs under ~150 bases. We found that non-vesicular exRNAs contained 366 full-length mature miRNAs. In contrast, full-length tRNAs were not detected in non-vesicular exRNAs. We also found that ~40% of non-vesicular miRNAs in serum can exist stable during incubation for 24 hours at 37 °C. Moreover, we showed that non-vesicular artificial miRNA which was added to cell culture media without any transfection regents can enter cultured cells and reduce target mRNA expression. Collectively, our results indicate that extracellular miRNA outside of extracellular vesicles can exist intact and enter cells to affect target gene expression.