Abstract
A recent study demonstrated that intracellular small/microRNAs are released from cells, and some of these extracellular RNAs are embedded in vesicles, such as ceramide-rich exosomes, on lipid-bilayer membranes. In the present study, we examined the effects of sphingomyelin phosphodiesterase 3 (SMPD3), which generates ceramide from sphingomyelin, on the release of small/microRNAs from intracellular to extracellular spaces. In these experiments, SW480 human colorectal and HuH-7 human hepatocellular cancer cells were cultured for 48 h in serum-free media. Culture supernatants were then collected, and floating cells and debris were removed by centrifugation and filtration through a 0.22-µm filter. Extracellular small RNAs in purified culture supernatants were stable for 4 weeks at room temperature, after 20 freeze-thaw cycles and exposure to pH 2.0, and were resistant to ribonuclease A degradation. Amino acid sequence analyses of SMPD3 showed high homology between mammals, indicating evolutionary conservation. Therefore, to investigate the mechanisms of cellular small/microRNA export, SW480 and HuH-7 cells were treated with the SMPD3 inhibitor GW4869 in serum-free media. Culture supernatants were collected for microarray and/or reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments. The number of microRNAs in culture supernatants was decreased following treatment with GW4869. Among these, extracellular and intracellular miR-638 were dose-dependently decreased and increased, respectively. These data suggest that SMPD3 plays an important role in the release of microRNAs into extracellular spaces.