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

Kubota,Shiori; Chiba,Mitsuru; Watanabe,Miki; Sakamoto,Maki; Watanabe,Narumi

doi:10.3892/or.2014.3605

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

Authors:
- Shiori Kubota
- Mitsuru Chiba
- Miki Watanabe
- Maki Sakamoto
- Narumi Watanabe
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Affiliations: Department of Medical Technology, Hirosaki University School of Health Sciences, Hirosaki, Aomori 036-8564, Japan, Department of Biomedical Sciences, Division of Medical Life Sciences, Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori 036-8564, Japan
Published online on: November 13, 2014 https://doi.org/10.3892/or.2014.3605
Pages: 67-73
Copyright: © Kubota et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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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.

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1	Valadi H, Ekström K, Bossios A, Sjostrand M, Lee JJ and Lötvall JO: Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 9:654–659. 2007. View Article : Google Scholar : PubMed/NCBI
2	Hu G, Drescher KM and Chen XM: Exosomal miRNAs: biological properties and therapeutic potential. Front Genet. 3:562012. View Article : Google Scholar : PubMed/NCBI
3	Arroyo JD, Chevillet JR, Kroh EM, et al: Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci USA. 108:5003–5008. 2011. View Article : Google Scholar : PubMed/NCBI
4	Turchinovich A, Weiz L, Langheinz A and Burwinkel B: Characterization of extracellular circulating microRNA. Nucleic Acids Res. 39:7223–7233. 2011. View Article : Google Scholar : PubMed/NCBI
5	Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD and Remaley AT: MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol. 13:423–433. 2011. View Article : Google Scholar : PubMed/NCBI
6	Wang K, Zhang S, Weber J, Baxter D and Galas DJ: Export of microRNAs and microRNA-protective protein by mammalian cells. Nucleic Acids Res. 38:7248–7259. 2010. View Article : Google Scholar : PubMed/NCBI
7	Brase JC, Wuttig D, Kuner R and Sultmann H: Serum microRNAs as non-invasive biomarkers for cancer. Mol Cancer. 9:3062010. View Article : Google Scholar : PubMed/NCBI
8	Huang Z, Huang D, Ni S, Peng Z, Sheng W and Du X: Plasma microRNAs are promising novel biomarkers for early detection of colorectal cancer. Int J Cancer. 127:118–126. 2010. View Article : Google Scholar
9	Moon PG, Lee JE, You S, et al: Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy. Proteomics. 11:2459–2475. 2011. View Article : Google Scholar : PubMed/NCBI
10	Michael A, Bajracharya SD, Yuen PS, et al: Exosomes from human saliva as a source of microRNA biomarkers. Oral Dis. 16:34–38. 2010. View Article : Google Scholar :
11	Lässer C, Alikhani VS, Ekström K, et al: Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med. 9:92011. View Article : Google Scholar : PubMed/NCBI
12	Taylor DD and Gercel-Taylor C: MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol. 110:13–21. 2008. View Article : Google Scholar : PubMed/NCBI
13	Kosaka N, Izumi H, Sekine K and Ochiya T: microRNA as a new immune-regulatory agent in breast milk. Silence. 1:72010. View Article : Google Scholar : PubMed/NCBI
14	Ge Q, Zhou Y, Lu J, Bai Y, Xie X and Lu Z: miRNA in plasma exosome is stable under different storage conditions. Molecules. 19:1568–1575. 2014. View Article : Google Scholar : PubMed/NCBI
15	Karlsson M, Lundin S, Dahlgren U, Kahu H, Pettersson I and Telemo E: ‘Tolerosomes’ are produced by intestinal epithelial cells. Eur J Immunol. 31:2892–2900. 2001. View Article : Google Scholar : PubMed/NCBI
16	Muturi HT, Dreesen JD, Nilewski E, et al: Tumor and endothelial cell-derived microvesicles carry distinct CEACAMs and influence T-cell behavior. PLoS One. 8:e746542013. View Article : Google Scholar : PubMed/NCBI
17	King HW, Michael MZ and Gleadle JM: Hypoxic enhancement of exosome release by breast cancer cells. BMC Cancer. 12:4212012. View Article : Google Scholar : PubMed/NCBI
18	Montecalvo A, Larregina AT, Shufesky WJ, et al: Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood. 119:756–766. 2012. View Article : Google Scholar :
19	Lee JK, Park SR, Jung BK, et al: Exosomes derived from mesenchymal stem cells suppress angiogenesis by down-regulating VEGF expression in breast cancer cells. PLoS One. 8:e842562013. View Article : Google Scholar
20	Escola JM, Kleijmeer MJ, Stoorvogel W, Griffith JM, Yoshie O and Geuze HJ: Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J Biol Chem. 273:20121–20127. 1998. View Article : Google Scholar : PubMed/NCBI
21	Bobrie A, Colombo M, Raposo G and Thery C: Exosome secretion: molecular mechanisms and roles in immune responses. Traffic. 12:1659–1668. 2011. View Article : Google Scholar : PubMed/NCBI
22	Hanson PI and Cashikar A: Multivesicular body morphogenesis. Annu Rev Cell Dev Biol. 28:337–362. 2012. View Article : Google Scholar : PubMed/NCBI
23	Raiborg C and Stenmark H: The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature. 458:445–452. 2009. View Article : Google Scholar : PubMed/NCBI
24	Trajkovic K, Hsu C, Chiantia S, et al: Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science. 319:1244–1247. 2008. View Article : Google Scholar : PubMed/NCBI
25	Marchesini N, Luberto C and Hannun YA: Biochemical properties of mammalian neutral sphingomyelinase 2 and its role in sphingolipid metabolism. J Biol Chem. 278:13775–13783. 2003. View Article : Google Scholar : PubMed/NCBI
26	Kosaka N, Iguchi H, Yoshioka Y, Takeshita F, Matsuki Y and Ochiya T: Secretory mechanisms and intercellular transfer of microRNAs in living cells. J Biol Chem. 285:17442–17452. 2010. View Article : Google Scholar : PubMed/NCBI
27	Lee DH, Kim SH, Ahn KH, et al: Identification and evaluation of neutral sphingomyelinase 2 inhibitors. Arch Pharm Res. 34:229–236. 2011. View Article : Google Scholar : PubMed/NCBI
28	Rodriguez-Boulan E, Kreitzer G and Müsch A: Organization of vesicular trafficking in epithelia. Nat Rev Mol Cell Biol. 6:233–247. 2005. View Article : Google Scholar : PubMed/NCBI
29	Jahn R and Fasshauer D: Molecular machines governing exocytosis of synaptic vesicles. Nature. 490:201–207. 2012. View Article : Google Scholar : PubMed/NCBI
30	Savina A, Vidal M and Colombo MI: The exosome pathway in K562 cells is regulated by Rab11. J Cell Sci. 115:2505–2515. 2002.PubMed/NCBI
31	Ostrowski M, Carmo NB, Krumeich S, et al: Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat Cell Biol. 12:19–30. 2010. View Article : Google Scholar
32	Söllner T, Whiteheart SW, Brunner M, et al: SNAP receptors implicated in vesicle targeting and fusion. Nature. 362:318–324. 1993. View Article : Google Scholar : PubMed/NCBI