Secretion of small/microRNAs including miR-638 into extracellular spaces by sphingomyelin phosphodiesterase 3.
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.
Department of Medical Technology, Hirosaki University School of Health Sciences, Hirosaki, Aomori 036-8564, Japan.
Shiori
S
Kubota
Department of Biomedical Sciences, Division of Medical Life Sciences, Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori 036-8564, Japan.
Mitsuru
M
Chiba
Department of Medical Technology, Hirosaki University School of Health Sciences, Hirosaki, Aomori 036-8564, Japan.
Miki
M
Watanabe
Department of Medical Technology, Hirosaki University School of Health Sciences, Hirosaki, Aomori 036-8564, Japan.
Maki
M
Sakamoto
Department of Medical Technology, Hirosaki University School of Health Sciences, Hirosaki, Aomori 036-8564, Japan.
Narumi
N
Watanabe
D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001597:Benzylidene Compounds / Q000494:pharmacology; D045744:Cell Line, Tumor; D017124:Conserved Sequence; D005110:Extracellular Space; D015870:Gene Expression; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000502:physiology*
D000814:Aniline Compounds; D001597:Benzylidene Compounds; C468773:GW 4869; C572154:MIRN638 microRNA, human; D035683:MicroRNAs; C491484:SMPD3 protein, human; D013108:Sphingomyelin Phosphodiesterase