Each miRNA binds to its target mRNA with incomplete homology, generally recognizes the 3ʹUTR of the target gene, and destabilizes the target mRNA or represses its translation by translation inhibition (Macfarlane and Murphy 2010).
Transcriptional repression mediated by miRNAs is known to play an important role in a wide variety of biological processes such as normal development, teratogenicity, cell proliferation and differentiation, apoptosis, and cancer formation (Alberti and Cochella 2017; Gueta et al. 2010; Mor et al. 2014; Macfarlane and Murphy 2010).
The standard miRNA production process consists of the following five steps: (1) transcriptional synthesis of primary miRNA (pri-miRNA); (2) cleavage of the pri-miRNA in the nucleus, to produce precursor miRNA (pre-miRNA); (3) nuclear export of the pre-miRNA; (4) cleavage of the pre-miRNA in the cytoplasm to produce miRNA duplexes; and (5) maturation of the miRNA.
These processes are collectively referred to as “miRNA processing” (see Figure 3.3).
1 Transcriptional synthesis of primary miRNA (pri-miRNA). The first miRNA precursor transcripts are produced in the nucleus through the transcription of the miRNA genes, primarily by RNA polymerase II and III, the same polymerases that transcribe those genes that are translated into proteins and polIII-driven repeat sequences (Lee et al. 2004; Borchert et al. 2006). Primary transcripts containing 60–70 nt RNA hairpin structures are called pri-miRNAs (Han et al. 2006). Most mammalian miRNAs are located within intergenic regions or within the introns of protein-encoding genes and non-coding RNAs. Less commonly, miRNA precursors may be located in exons of transcripts and in antisense transcripts.
2 Cleavage of pri-miRNA in the nucleus to produce precursor miRNA (pre-miRNA). The ribonuclease III endonuclease (RNase III), Drosha, along with DGCR8 and multiple cofactors, cleaves the hairpin base of the pri-miRNA in the nucleus to produce a 60–70-base pre-miRNA with a hairpin structure, which is an intermediate (Denli et al. 2004; Gregory et al. 2004; Han et al. 2004; Lee et al. 2003; Lee et al. 2002).
3 Nuclear export of pre-miRNA. Subsequent pre-miRNA processing occurs in the cytoplasm. The pre-miRNA is mainly transported from the nucleus to the cytoplasm by nucleocytoplasmic transporter containing exportin-5 (XPO5) and Ran-GTP, which prevents its degradation in the nucleus and facilitates its translocation into the cytoplasm (Bohnsack Czaplinski, Gorlich 2004; Okada et al. 2009; Yi et al. 2003; Zeng and Cullen 2004).
4 Cleavage of pre-miRNA in the cytoplasm to produce miRNA duplexes. The pre-miRNA undergoes a second cleavage event, whichis mediated by another RNase III endonuclease, Dicer, with its cofactor TRBP. The resulting small RNA is a 21-to-24-base miRNA duplex (miRNA-5p/miRNA-3p) (Hutvagner et al. 2001; Chendrimada et al. 2005).
5 Maturation of miRNA. A double-stranded miRNA duplex is incorporated into the Ago protein, and only one side of the RNA strand (the miRNA strand, guide strand, or mature miRNA) finally forms a stable complex with the Ago protein, forming an RNA-induced silencing complex (RISC) (Kwak and Tomari 2012; Iwasaki et al. 2010; Hammond et al. 2000). Finally, this single-stranded mature miRNA acts as a guide for gene expression regulation. RISC binds to its target mRNA, which has a sequence that is partially complementary to the miRNA incorporated into the RISC, and generally suppresses the output of the target mRNA to the protein (Jonas and Izaurralde 2015).
Figure 3.3 Canonical miRNA biogenesis pathway.
To date, there have been several reports on the mechanism of EV capture of miRNAs. Those miRNAs whose expression levels increase in T cells upon activation are not significantly increased in exosomes (Villarroya-Beltri et al. 2013). A similar trend was observed for mRNAs, which suggests that miRNA and mRNA loading into exosomes is not a passive process. Several miRNAs were more highly represented in EVs than in cells, for example miR-575, miR-451, miR-125a-3p, miR-198, miR-601 and miR-887. Analysis of these expression profiles revealed that miRNAs that are preferentially sorted to EVs contain specific short motifs, called “EXO motifs.” The sumoylated heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) controls the loading of specific miRNAs into EVs by binding to these motifs. It was also demonstrated that HnRNPA2B1 could bind to an RNA trafficking sequence (RTS) of myelin basic protein (MBP) mRNA with a length of 21 nt and regulate mRNA trafficking to axons in neural cells (Munro et al. 1999). Sumoylation of HnRNPA2B1 is also required for the binding of RNA to HnRNPA2B1. Thus at least HnRNPA2B1 contributes to the loading of miRNAs into EVs (Villarroya-Beltri et al. 2013).
Proteins
After EVs are released from cells, EV-associated miRNAs will be delivered to recipient cells through the body fluids. Surface proteins on EVs help EVs to first adhere to target cells, which is a fundamental step for EV-recipient cell communication. Tetraspanins are thought to have roles in adhesion, motility, signal transduction, and cell activation; and they are highly abundant on the exosome surface (Figure 3.4). These tetraspanins include CD9, CD53, CD63, CD81, and CD82; they may contribute to the spatial assembly of factors for antigen recognition and may partially dictate the signal induced by EVs (Simons and Raposo 2009; Mathivanan et al. 2010). In fact, the treatment of recipient cells with antibodies against CD81 or CD9 can reduce the uptake of EVs by recipient cells, which suggests that tetraspanins have a role in EV uptake. Owing to their endosomal origin, exosomes are commonly enriched in endosome-associated proteins. Some of these proteins, for example ALIX and TSG101, are involved in MVB biosynthesis and are normally used as exosome markers along with the tetraspanins CD63, CD81, and CD9. Integrins are also enriched in the exosome membrane and are related to exosome tropism. The exosomal integrins α6β4 and α6β1 are associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. In fact, targeting the integrins α6β4 and αvβ5 decreased respectively exosome uptake as well as lung and liver metastasis.
Figure 3.4 Schematic representation of the major components of exosomes. Common exosome markers include tetraspanins (CD9, CD63, and CD81), integrins, TSG101, and Alix. Exosomes also contain other proteins, different species of RNA, and DNA.
It is biologically important to determine the organ where metastasis might be facilitated by such a mechanism. Accumulating evidence suggests that exosome-mediated activities play an important role in various diseases, especially cancer.
Physiological and Cellular Functions of EVs
Angiogenesis is the physiological process through which new blood vessels are formed from pre-existing vessels (Birbrair et al. 2014; Birbrair et al. 2015). Angiogenesis is regulated by angiogenic factors, extracellular matrix components, and endothelial cells (ECs) (Carmeliet and Jain 2000).
Angiogenesis is also promoted by the uptake of cancer cell-derived EVs that are rich in matrix metalloproteinases, especially MMP-2, MMP-9 and MMP-13. These proteins have been found in glioblastoma-, melanoma-, myeloma- and nasopharyngeal carcinoma-derived exosomes (Skog et al. 2008; Ekstrom et al. 2014; You et al. 2015; Wang et al. 2016; Giusti et al. 2016; Chan et al. 2015). It has been recently reported that metastatic breast cancer-derived EVs, which express high levels of the proangiogenic protein annexin II (Anx II), promote angiogenesis and that head and neck squamous cell carcinoma-derived EVs regulate angiogenesis through ephrin-B reverse signaling (Sato et al. 2019).
EV-associated miRNAs are also known to promote angiogenesis. EV-associated miR-23a, derived from hypoxic lung cancer cells, promotes angiogenesis by targeting prolyl hydroxylase and the tight junction protein ZO-1 (Hsu et al. 2017). It
