Secretion of small/microRNAs including miR-638 into extracellular spaces by sphingomyelin phosphodiesterase 3

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.

Figure 1

Extracellular small RNAs in cell culture media are stable against several external conditions. (A–D) Stability of extracellular small RNAs from HuH-7 cells, which were seeded at 1×10⁵ cells/well in 12-well plates. After 24 h, cells were washed three times in serum-free media. Serum-free (1 ml) media were then added, and the cells were incubated at 37°C for 48 h. Culture supernatants were then collected and purified by centrifugation and filtration. Culture supernatants of HuH-7 cells were (A) treated with a final concentration of 4 μg/ml of ribonuclease A (RNase A) at 37°C for 30 min, (B) incubated for 4 weeks at room temperature, (C) subjected to 20 freeze-thaw cycles, and (D) were subjected to a pH decrease to 2.0. Small RNAs were extracted from 200 μl aliquots and were detected using an Agilent bioanalyzer.

Figure 2

Evolutionary conservation of sphingomyelin phosphodiesterase 3 (SMPD3) in mammals. Amino acid sequence alignment analyses of SMPD3; two hydrophobic segments, two palmitoylation sites, and a catalytic domain were highly conserved in Homo sapiens, Pan troglodytes, Mus musculus, and Bos taurus. Asterisks indicate matched amino acids in all 4 sequences, while dots indicate matched amino acids in 3 of 4 mammals.

Figure 3

SMPD3 is involved in the secretion of small/microRNAs from cells. (A) Comparison of SMPD3 mRNA expression in SW480 and HuH-7 cells by electrophoresis of reverse transcription polymerase chain reaction (RT-PCR) products and RT quantitative PCR (RT-qPCR) using the primer pairs described in Table I. GAPDH was used as an internal control. Expression levels were determined using the comparative Ct method and were normalized to those of SW480 cells. (B) Effects on the quantities of small RNAs released from SW480 or HuH-7 cells after treatment with the SMPD3 inhibitor GW4869. SW480 and HuH-7 cells were seeded at 1×10⁵ cells/well in 12-well plates. After 24 h, the cells were washed three times in serum-free media and were incubated in 1 ml of serum-free media at 37°C for 48 h. Culture supernatants were collected and purified by centrifugation and filtration and extracellular RNAs were isolated from 400-μl aliquots. Quantitative analysis of small RNAs in culture supernatants of SW480 or HuH-7 cells treated with GW4869 using an Agilent bioanalyzer. The quantities of small RNAs in culture supernatants decreased following treatment with 10.0 μM GW4869. (C) Scatter plot of microRNA expression in culture supernatants from HuH-7 cells after treatment with 0 and 10.0 μM GW4869. Analyses were performed using a Toray microRNA microarray system. (D and E) Expression levels of extracellular and intracellular miR-638 in SW480 and HuH-7 cells treated with GW4869. Samples of cell pellets and supernatants from (D) HuH-7 cultures (each n=8) and (E) SW480 (each n=6) were prepared, RNA was extracted, and RT-qPCR analyses were performed. Cel-miR-39 and U6 small nuclear RNA (snRNA) were used as internal controls. Bars indicate the mean ± standard error of the mean (SEM) values. Multiple comparisons were made using one-way ANOVA followed by Dunnett’s multiple comparisons post hoc test. Double asterisks indicate significant differences (P<0.01 vs. 0 μM); n.s., not significant.