Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells

Takahiro Ochiya,Yusuke Yoshioka,Fumitaka Takeshita,Nobuyoshi Kosaka,Haruhisa Iguchi,Yasushi Matsuki

doi:10.1074/jbc.m110.107821

Takahiro Ochiya, Yusuke Yoshioka + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.107821

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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 tumor-suppressive 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.

Highlights

Nous plasma RNAs are stable for hours under the same conditions [3]
We have shown that secretion of miRNAs is controlled by neutral sphingomyelinase 2, which is known as a rate-limiting enzyme of ceramide biosynthesis
Overexpression of miRNA in Cells Leads to an Increased Secretion—To reveal the physiological roles of extracellular miRNAs, we investigated the mechanism of their secretion and whether or not secretory miRNAs can function in cells beyond their own cell origin

Summary

EXPERIMENTAL PROCEDURES

Reagents—Rabbit polyclonal anti-nSMase (H-195) (sc67305), goat polyclonal anti-Alix (Q-19) (sc-49268), and donkey anti-goat IgG (horseradish peroxidase) (sc-2020) were purchased from Santa Cruz Biotechnology. Preparation of Conditioned Medium and Exosome—Prior to collection of culture medium, HEK293 and COS-7 cells were washed three times with Advanced RPMI containing antibiotic-antimycotic and 2 mM L-glutamine (medium A), and the medium was switched to fresh medium A. HEK293 cells were transfected with 0.5 ␮g of pLucNeo vector or pri-miR-146a expression vector at 90% confluency in 24-well dishes using a Lipofectamine LTX reagent in accordance with the manufacturer’s instructions. Luciferase Reporter Assay—HEK293 and COS-7 cells were cultured at a density of 5 ϫ 104 and 1 ϫ 104 cells/well, respectively, in 96-well tissue culture plates overnight, and miRNA transfections or the addition of conditioned medium were performed as described under the legend for Figs. The following day, the cells were transfected with mature miRNAs or were incubated with a conditioned medium. The following day, the cells were transfected with nSMase expression vector or were treated with 10 ␮M cisplatin. After a 12-h incubation, the fluorescence of each well was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using Envision (Wallac)

RESULTS

PCR analysis showed that the blocked

DISCUSSION

Matsuki and Takahiro Ochiya