MicroRNAs (miRNA) are short non-coding RNA molecules that modulate the activity of specific mRNA targets in normal development and disease, typically by compromising messenger RNA (mRNA) stability. MiRNAs are released by cells in a variety of vesicles or associated in complexes with proteins [reviewed in (1
)]. Exosomes were the first extracellular vesicles shown to contain miRNA (2–12
). Exosomes originate from multivesicular bodies (MVBs) of the endosomal compartment and may contain miRNA as a consequence of loading into the RNA-induced silencing complex and unloading at the MVBs (13
The association of miRNA with exosomes is significant, in that exosomes can transfer cancer-specific molecules to other cells (15
). Through this transfer of material, exosomes have been shown to contribute to tumor progression (Duelli et al
., 2005), modification of the microenvironment (17
) and the host (18
), through induction of angiogenesis, production of niches for metastasis (19
) and immune modulation (21–25
). The packaging and export of miRNAs from cells and delivery of them to other cells may be important processes for extracellular miRNA-signaling pathways, although these mechanisms are not well understood.
Exosomes are perhaps the best studied of extracellular vesicles, although some cells, in particular tumor cells, actually release a heterogeneous population of microvesicles of uncertain subcellular origin (reviewed in (1
)). Recent work suggests that the profile of microvesicles released from cells may be indicative of the cell type or cell state and may play an important role in cell–cell transfer and communication. For example, solid tumor cells, including breast cancer cells, release heterogeneous microvesicles that contain a variety of molecules (27
) that can be transferred to recipient cells and influence signaling pathways (29
). Although miRNA has been established as cargo of exosomes, the miRNA distribution in cells that release multiple vesicles is not known.
We recently demonstrated that miRNAs are released from breast cancer cells in a selective manner. We have identified three categories of released miRNA in mammary epithelial cells, which were classified based on the ratio between the amount of miRNA released from the cells and the amount retained in the cell () (30
). The first group is selectively released miRNAs, or ‘s-miRNA’. These miRNAs are characterized by being released excessively from breast cancer cells with relatively low concentrations of miRNA remaining in the cell. Alternatively, normal cells release nearly none of these miRNAs (30
). Another group of miRNAs is in equal abundance within the cell and extracellularly, i.e. neutrally released miRNA, ‘n-miRNAs’ (30
). N-miRNAs include putative biomarkers miR-16, miR-21 and other miRNAs for which the abundance in microvesicles reflects the increased abundance in the malignant cells of origin.
Table 1. MiRNA that are differentially retained and exported in benign and malignant mammary epithelial cells (30)
The selectivity of release of individual miRNAs, and thereby their categorical grouping, differs depending on the cell type (6
). In particular, selection is affected by malignant transformation. For example, malignant mammary epithelia release >99% of miR-451 and miR-1246 produced by the cells, while in benign epithelial cells these miRNAs are mostly retained (30
). Both of these s-miRNAs are linked to cancer. MiR-451 is a tumor suppressor (32
), affecting proliferation (33
) and cell polarity (35
), by deregulating several oncogenic pathways (36–39
). MiR-451 also induces sensitivity to chemotherapeutic drugs (40
), allows adaptation to metabolic stress (42
) and can induce endocrine resistance in breast cancer cells (44
). MiR-1246 induces p53-dependent apoptosis triggered by DNA damage (45
). The changes in the release of cancer-related miRNAs may suggest a role for selective miRNA export in malignant transformation, and it may provide a cancer signature within the exported, circulating miRNA population.
N-miRNAs include miR-16 and miR-720 (30
). MiR-16 is one of the first miRNAs identified to have tumor-suppressor activity (46
), and circulating miR-16 is currently being evaluated as a serum marker for diagnosis or prognosis of several cancers (48
) and pre-malignant syndromes (50
). MiR-16 regulates chemosensitivity (51
), apoptosis (52
) and the cell cycle (53
), while its biogenesis is induced by radiation (54
) and is modulated by p53 (55
). MiR-720 is the most abundantly released miRNA from malignant mammary epithelial cells (30
). MiR-720 is up-regulated in pre-neoplastic syndromes (56
) and has roles in p63 regulation and cell differentiation (57–59
). The importance and roles of these vesicular miRNAs in cancer and in the extracellular environment are beginning to emerge. However, much remains to be determined regarding the nature of the vesicles that shuttle these miRNAs outside of the cell.
The observations that cells can selectively release miRNAs and also release a heterogeneous population of vesicles raise the possibility that the differential release of miRNAs is associated with different microvesicles. We thus analyzed the nature of the microvesicles that are associated with s-miRNAs. We find that s-miRNAs and n-miRNAs are released from cells in different types of particles. The identification and purification of sub-populations of extracellular microvesicles that carry distinct miRNA cargo allows for the possible assignment of the subcellular origin, extracellular function and cellular targets of these vesicles, as well as for the development of cancer diagnosis and prognosis based on the association of specific miRNA with specific extracellular vesicles.