Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells

Abstract

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumorsuppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.It has been well known that extracellular RNAs circulate in the blood of healthy people and diseased patients with sufficient integrity, although ribonuclease is present in both plasma and serum (1). The spectrum of RNAs whose presence was demonstrated in plasma and other body fluids, such as urine and breast milk, extends from housekeeping genes to fetal genes detected in pregnant women and genes overexpressed in a variety of different tumors (2). To explain the stability of circulating RNAs, it was suggested that extracellular RNAs are included within lipoprotein vesicles. Indeed, exogenous RNAs added to plasma or blood are immediately degraded, whereas endogenous plasma RNAs are stable for hours under the same conditions (3). Moreover, the treatment of some detergents results in immediate degradation of plasma extracellular RNAs, apparently due to disruption of the lipid vesicles. These findings clearly indicate that extracellular RNAs are packaged in some kinds of secretory particles including apoptotic bodies and exosomes, and thus, they are protected from dominantly existing ribonucleases.Apoptotic bodies are small membranous particles released during programmed cell death (4), and exosomes are small intraluminal vesicles (50 -100 nm in diameter) of multivesicular bodies (MVB) 3 released on exocytic fusion of MVB with plasma membranes (5). Currently, accumulating evidence suggests that these secretory vesicles can function as intercellular transmitters to convey their contents, in particular, microRNA (miRNA) (6 -8). Recent studies reported that extracellular exosomal miRNAs were transferred into other cells and that apoptotic bodies delivered miR-126 into endothelial cells (9). Despite these advances, however, the underlying mechanism of the secretory process and the biological function of circulating miRNAs are not yet...

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