Secretory mechanisms and intercellular transfer of microRNAs in living cells

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.

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)(7)(8). Recent papers reported that extracellular exosomal miRNAs were transferred into other cells, and that apoptotic bodies delivered miR-126 into endothelial cells (9). In spite of these advances, however, the underlying mechanism of the secretory process and the biological function of circulating miRNAs are not yet fully understood.
MiRNAs, small 20-22 nucleotide-long members of the non-protein-coding RNA family, are expressed in the vast majority of eukaryotes, including humans (10).
Not only do they inhibit translation of their target genes, they also degrade the target mRNAs through recognition of imperfect complementary sites, usually located in the 3' untranslated regions (3'-UTR) of the target mRNAs, endowing miRNAs with the capacity to regulate numerous biological processes. Over the past several years, it was evident that dysregulations of many kinds of miRNAs have been linked to the initiation and progression of human cancer (11).
Interestingly, the amounts of secretory miRNAs are up-regulated in the plasma of patients bearing tumors, including B cell lymphoma, prostate cancer, lung cancer and ovarian cancer (12)(13)(14)(15). Thus, detection and monitoring of tumors are now becoming possible by the evaluation of tumor-derived secretory miRNAs.
In this study, we have shown that secretion of miRNAs is controlled by neutral sphingomyelinase 2 (nSMase2), which is known as a rate-limiting enzyme of ceramide biosynthesis. Furthermore, we provide evidences that miRNAs secreted from donor cells can be taken up and function in recipient cell. These findings propose a novel mechanism of intercellular communication mediated by secretory miRNAs. RNAs were purified using mirVana miRNA isolation kit (Ambion) as described above.

Quantitative Real Time PCR (QRT-PCR)---
The method for QRT-PCR has been previously described (13). PCR was carried out in 96-well plates using 7300 Real Time PCR System (Applied Biosystems). All

Establishment of stable cell lines---Stable HEK293
cell lines expressing firefly luciferase or miR-146a were generated by selection with 300 µg/ml Geneticin.  To exactly estimate what percentages of the miRNAs were excreted, we re-evaluated cellular amounts of the miRNAs by the same normalization method as the extracellular miRNAs (Supplemental Fig. 1B  To address the question as to whether miRNA secretion is regulated by the ceramide dependent pathway or ESCRT pathway, we first treated HEK293 cells with a nSMase inhibitor, GW4869, which is known to inhibit ceramide biosynthesis (18). where the amount of β-actin was not affected (Fig. 7C).

Overexpression of miRNA in cells leads
To confirm that the cell growth inhibition resulted from the transferred exosomal miR-146a, we examined whether the suppressive effect can be cancelled by a reduced exosome secretion.
Conditioned medium from GW4869-treated donor cells had no inhibitory activity on cell proliferation after a 4-day incubation (Fig. 7D; compare lanes 1 and 2). We also did not observe any direct effect of GW4869 on the restoration of miR-146a-suppressed cell growth (Fig. 7D; compare lanes 3 and 4). These findings suggest that itinerant exosomal miRNAs can induce some phenotypic changes in the recipient cells.   represents not significant.