Unlocking the
Mysteries of
Extracellular RNA
Communication

Once thought to exist only inside cells, RNA is
known to travel outside of cells and play a role in newly
discovered mechanisms of cell-to-cell communication.

Extracellular vesicles (EVs) play an important role in cell-to-cell communication. Recently, EVs have been shown to be involved in immune modulation, tumor biology, and tissue regeneration. The mechanisms of action of EVs are associated with their ability to stimulate target cells directly and to transfer proteins, biologically active lipids, and nucleic acids to the target cells. In fact, mRNAs, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs) can be compartmentalized into EVs, escape enzymatic degradation, and be delivered to target cells. This horizontal transfer of extracellular RNAs carried by EVs can induce epigenetic alterations in recipient cells. The result is a change of phenotype or even a genetic and functional reprogramming of the recipient cells. Furthermore, EVs carry a selection of miRNAs different from the miRNAs most expressed in the cells of origin. However, little is known about the mechanisms of miRNA enrichment in EVs.

Argonaute2

Argonaute2


 
Alix

Alix

We hypothesized a possible interaction between the Alix and Argonaute 2 (Ago2) proteins. The resulting complex may have a role in miRNA transport into EVs. Ago2 is a reasonable candidate to play a role in miRNA packaging within multivesicular bodies during EV biogenesis because of its central role in miRNA maturation. We observed that Ago2, as well as several other ribonucloproteins involved in RNA storage and stability, is expressed in EVs derived from adult human liver stem-like cells (HLSCs). Cells which express mesenchymal and embryonic markers.
 

Alix Figure 1

Alix is a multifunctional protein commonly used as a marker of EVs. It is an accessory protein of the Endosomal Sorting Complex Required for Transport (ESCRT), and several studies indicate that ESCRT is involved in the biogenesis of EVs.

We observed that HLSC-derived EVs express both Alix and Ago2. Co-immunoprecipitation (Co-IP) experiments with Alix or Ago2 antibody showed that the two proteins are associated. We also found that the miRNAs enriched in HLSC-EVs precipitate with the Alix – Ago2 complex. After the incubation of HLSC-EVs with human endothelial cells, we observed that miRNAs from HLSC-EVs are transferred to these cells.
 

Alix Figure 4

After the silencing of Alix expression in HLSCs, we observed the absence of both Alix and Ago2 proteins in EVs derived from the knockdown HLSCs and a strong reduction in the number of miRNAs normally enriched in HLSC-EVs. On the other hand, EV size, surface expression of CD63 and Tsg101, and the number of released EVs were not affected. After incubation with endothelial cells, EVs derived from Alix-knockdown HLSC do not transfer miRNAs to cells.

Alix is known to be involved in endocytic membrane trafficking and cytoskeletal remodeling. It is also associated with the ESCRT machinery, which participates in processes of vesiculation and cargo sorting, including multivesicular body biogenesis. Our data suggest that Alix binds Ago2 and drives it into EVs together with the associated miRNAs.
 

Alix Figure 5

This might be a general mechanism of miRNA transport into EVs, common to other cell types. Enrichment of a selected set of miRNAs might also depend on the affinity of miRNAs for carrier proteins such as Ago2.

Source: Iavello A, Frech VS, Gai C, Deregibus MC, Quesenberry PJ, Camussi G. Role of Alix in miRNA packaging during extracellular vesicle biogenesis. Int J Mol Med. 2016 37:958-966. doi: 10.3892/ijmm.2016.2488. PMID: 26935291.

Members of the Extracellular RNA Communication Consortium have recently elucidated a mechanism through which hypoxia leads to increased tumor aggressiveness and metastasis. Anil Sood and his group at University of Texas MD Anderson Cancer Center have identified a miRNA that downregulates the very pathway responsible for miRNA biogenesis, a finding that should generate excitement due to the identification of a potential new way of treating cancers: through miRNA or RNA interference-based gene targeting.

The development of tumors is a complex process that involves the cooperation of cancer cells with non-cancer cells, together making up the tumor microenvironment that is vital for tumor survival. As tumors grow and become dense, they begin signaling for the formation of new blood vessels to bring oxygen and nutrients to their core, a process called angiogenesis. Inhibiting angiogenesis is thus a highly attractive therapeutic avenue and is typically accomplished through the targeting of vascular endothelial growth factor (VEGF) and the subsequent induction of hypoxia, or a condition of low oxygen, in the tumor. Hypoxia, however, comes with its own set of problems.

The recent study suggests that hypoxia itself can lead to an increase in the aggressiveness and metastatic potential of a tumor (Rupaimoole et al, 2016). Previous studies have found that hypoxic conditions lead to the downregulation of Drosha and Dicer, two components of the miRNA biogenesis pathway, and that this decrease in expression is associated with poor clinical outcomes through a decrease in the pool of miRNA present in the tumor. In investigating the root cause of Dicer downregulation, Sood and his team identified a miRNA, miR-630, that is upregulated during hypoxia and that targets the 3’ UTR of Dicer. The study validated this targeting by monitoring Dicer mRNA and protein levels both in cells and in vivo in mouse models. Mice that had miR-630 delivered to them via nanoliposomes developed larger tumors and metastases in more places than control mice.

Of particular importance, the researchers treated mice with a combination of anti-miR-630 or anti-VEGF therapy (bevacizumab). Mice that were treated with both bevacizumab and anti-miR-630 developed smaller tumors and fewer metastatic nodules compared with mice treated with bevacizumab alone; Dicer expression was rescued upon treatment with anti-miR-630.

Apart from furthering our understanding of Dicer regulation during cancer, this research demonstrates the potential of treating cancers with anti-miRNA therapies, which would be particularly useful in situations where antibodies or chemical agents are not able to reach a target.

The potential of extracellular vesicle (EV) RNA as biomarkers of disease is increasingly being recognized. Circulating extracellular RNAs can potentially indicate the presence of disease without the need for an invasive biopsy of diseased tissue. A recent study by Yuan et al (Scientific Reports, Jan 2016) performed a systematic analysis of circulating exRNA in plasma obtained from 50 healthy individuals and 142 cancer patients. The authors conducted the largest RNA sequencing study reported to date for profiling circulating extracellular RNA (exRNA) species in order to provide useful insights into their baseline expression level. High-throughput multivariate statistical analysis identified a set of RNA candidates that were associated with age, sex, and cancer type.

The study directly addressed a major challenge in the field of exRNA, namely the lack of a baseline reference to accurately determine RNA abundance. Currently qRT-PCR is the most common method used to quantitate gene expression, but it relies on well-established reference controls for normalization. However, information on reference controls has not been established for exRNA. Control RNAs used for normalization in qRT-PCR experiments of cellular RNA cannot be reliably used for exRNA. Exogenous spike-in RNAs such as those derived from species like C. elegans cannot be used to normalize across biological or pathological states. Here the authors surveyed a pool of almost 200 exRNA profiles to identify a few potential reference candidates for exRNA quantification. Most candidate were miRNAs like miR-99a-5p. The study also provided convincing evidence for the presence in plasma of species of RNA other than miRNA, such as piwiRNA (see figure).

The dataset generated by this study is available in the exRNA Atlas for other researchers to explore. Future studies will be needed to validate these findings; however, with the current study we have taken a big leap towards the goal of determining the biomarker potential of exRNA for human diseases.
 
 

RNA species detected in the plasma of 142 cancer patients and 50 healthy controls

RNA species detected in the plasma of 142 cancer patients and 50 healthy controls
Source: Scientific Reports / CC BY 4.0