Exosomal non-coding RNAs (Exo-ncRNAs) in cardiovascular health

Abstract

Extracellular vesicles (EVs) play a role in the pathophysiological processes and in different diseases, including cardiovascular disease. Out of several categories of EVs, exosomes (smallest – 30 to 150 nm) are gaining most of the focus as the next generation of biomarkers and in therapeutic strategies. This is because exosomes can be differentiated from other types of EVs based on the expression of tetraspanin molecules on the surface. More importantly, exosomes can be traced back to the cell of origin by identifying the unique cellular marker(s) on the exosomal surface. Recently, several researchs have demonstrated an important and underappreciated mechanism of paracrine cell-cell communication involving exosomal transfer, and its subsequent functional impact on recipient cells. Exosomes are enriched in proteins, mRNAs, miRNAs, and other non-coding RNAs, which can potentially alter myocardial function. Additionally, different stages of tissue damage can also be identified by measuring these bioactive molecules in the circulation. There are several aspects of this new concept still unknown. Therefore, in this review, we have summarized the knowledge we have so far and highlighted the potential of this novel concept of next generation biomarkers and therapeutic intervention.

Introduction

Far from being completely understood, it is now known that genomic sequence can be transcribed into a wide variety of protein-coding RNAs (mRNAs) and non-coding RNAs (ncRNAs) [1]. While 80–95% of the prokaryotic genome is mapped to protein-coding sequences, only about 2% of the human genome codes for known proteins. However, at least 60–70% of the mammalian genome is transcribed on one or both strands [2]. Recently, it has been identified that ncRNAs can have different biological functions, such as involvement in chromosome dynamics, splicing, RNA editing, translational inhibition and mRNA degradation [2].

ncRNAs are classified by length into long ncRNAs (lncRNAs; > 200 nucleotides long), and small ncRNAs (< 200 nucleotides long). The small ncRNAs are divided into microRNAs (miRNAs), small interfering RNAs (siRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and P-element-induced wimpy testis (piwi)-interacting RNAs (piwiRNAs) [3].

Long ncRNAs are more heterogeneous and difficult to classify and characterize compared to small ncRNAs. The 200 nucleotide long cut-off was selected based on RNA-purification protocols [4]. Considering their reduced abundance, lncRNAs were initially thought to be transcriptional noise resulting from low RNA polymerase fidelity [5]. LncRNAs predominantly localize into the nucleus and can function both in cis- or trans- forms at the transcription site [6]. In mammalian cells, relative lncRNA expression changes occur in a disease-, tissue and developmental stage-specific manner [7]. Unlike miRNAs or proteins, a specific lncRNA function can’t be easily inferred from its sequence or structure. These functions include epigenetic, transcriptional and post-transcriptional regulation [5]. LncRNAs are also classified by their structure as linear and circular lncRNAs (circRNAs) [8]. In the heart, lncRNAs have been implicated in the development of several diseases [[9], [10], [11]] as well as in normal tissue development [[10], [11], [12], [13]].

Circular RNAs (circRNAs) are the most recently discovered class of ncRNAs that form a covalently closed circle structure [14]. As a general characteristic, circRNAs are widespread exhibiting cell type-specific expression. CircRNAs are abundant and in some cases, the expression is higher than their related linear isoforms [15,16]. CircRNAs are involved in several physiological and pathological processes, mainly to down-regulate miRNAs by working as “sponges”. [8]. Considering the circular structure, circRNAs have significantly longer half-lives than linear RNA molecules. Mammalian cells can secret circRNAs. CircRNAs can be detected in plasma, making them candidates for disease biomarkers [14].

Competing endogenous RNAs (ceRNAs) contain miRNA response elements (MREs) and can communicate with and regulate each other by competing for shared binding sites for the same miRNAs [[17], [18], [19]]. All types of RNAs may compete with each other for microRNAs, acting as “miRNAs sponges”. It’s been shown that ceRNAs can be non-coding RNAs, mRNAs and/or pseudogenes [[17], [18], [19]]. Although there is still some discussion about the feasibility of ceRNA function in vivo [20], several lncRNAs have been postulated as ceRNAs of different miRNAs involved in the development of cardiac hypertrophy [11,[21], [22], [23], [24], [25]].

Among the small ncRNAs, miRNAs and siRNAs are the most studied molecules. miRNAs and siRNAs are short RNA fragments that are approximately 19–25 nucleotides long, derived either from hairpin or double-stranded RNA precursors (dsRNA), respectively. siRNAs are derived from dsRNA of hundreds or thousands of base pairs in length that are converted into 21–25 nt length dsRNAs. These dsRNAs associate with Argonaute 2 (Ago2) to bind to complementary mRNA targets [26]. On the other hand, miRNAs are derived from primitive forms of primary RNAs (pri-miRNAs) that are either encoded within introns of protein-coding genes or autonomous miRNA genes. Pri-miRNAs, contain multiple hairpin structures of which each hairpin structure is about 70 nucleotides long. Inside the nuclear component, Drosha (dsRNA-specific endonuclease) cuts these hairpin structures from pri-miRNA to form a pre-mature miRNAs (pre-miRNA). Pre-miRNAs are then exported to the cytosol by a nuclear protein called Exportin. Pre-miRNAs are spliced into 19–25 nucleotide long single strand RNAs (ssRNA) by another dsRNA-specific endonuclease, Dicer [[27], [28], [29]]. miRNAs form the RNA-induced silencing complex (RISC) with several other proteins, including Ago2 and Dicer [[27], [28], [29]]. RISC is the functional unit of both miRNA and siRNA. A seed sequence of 6–8 nucleotides in the 5’-end of the miRNAs and siRNAs binds to a complementary sequence on the 3’-untranslated region (UTR) of the target mRNA. Depending on whether the pairing of the seed sequence to the target mRNA is complete, or partial, the interaction results in mRNA degradation or translation inhibition, respectively [26]. Normally, siRNAs lead to mRNA degradation, and miRNAs inhibit the translational process. However, recently there were some ncRNAs, such as miR369-3 and Let-7, that were shown to increase mRNA translation by interacting with the UTRs of target mRNAs [[30], [31], [32]]. In addition, it’s been shown that one miRNA can silence several genes, and multiple miRNAs can regulate the same gene. In fact, an entire cellular pathway can be regulated by an individual miRNA or miRNA clusters, and the binding of several miRNAs to the same mRNA (in different MREs) can function in a combinatorial manner [27]. Interestingly, miRNAs have also been shown to target the 5′-UTR and the coding sequence of mRNAs as well as pseudogenes and lncRNAs [[33], [34], [35]]. Several miRNAs with known and even unknown functions have been observed in plasma, and are seen as promising tools for disease biomarkers [36,37].

There are also other types of small ncRNAs with known physiological and pathological roles. Particularly, Y RNAs are a class of small ncRNAs from approximately 100 nucleotides long with a stem-loop secondary structure, involved in RNA processing and quality control and also, in DNA replication [38]. Fragments of Y RNAs have been found in body fluids and are gaining interest as possible disease biomarkers.

Cellular release of molecules within membrane vesicles has been recognized for some time. Initially believed to be cell debris, it is now clear that cells release vesicles of varying sizes both through the endosomal pathway and by budding from the plasma membrane. These vesicles are collectively termed extracellular vesicles (EVs) [39]. EVs are small vesicles (30 nm–5  μm) released from the cell surface by membrane fusion of many different cell types into different body fluids that contain proteins, lipids, and RNA representative of the host cell [40]. EVs can be classified into apoptotic bodies (800–5000 nm diameter) that are released by cells undergoing programmed cell death; microvesicles or ectosomes (50–1000 nm diameter) that are produced by budding from the plasma membrane, and exosomes (30–150 nm diameter) from an endocytic origin [40]. EVs are a mechanism of horizontal transfer of intercellular information between cells mainly in the form of proteins and RNA molecules [41]. Both mRNAs and ncRNAs are present within EVs and can be transferred into recipient cells with functional consequences [39]. A single cell can release EVs of different sizes, with a different biogenesis mechanism and cargo. These can mediate autocrine, paracrine, and endocrine functions. EVs have different physiological roles, such as cellular migration and invasion, immunity, development, and nervous system communication. EVs have also been implicated in the pathogenesis of multiple diseases such as cancer and neurodegenerative diseases, among others (reviewed in [39]) and can be used as markers of disease and prognosis [3,27,42].

Exosomes are the best-characterized class of vesicles and represent a specific subset due to the intracellular origin from the endosome. Exosomes express certain molecular signatures, such as tetraspanins, heat shock proteins, GTPases, and endosomal proteins and markers, which also differentiate exosomes from other EVs [41,42]. Most importantly, exosomes express specific proteins from their parental cell type [41,43] allowing for the identification of the exosomal source for clinical implications. Different populations of exosomes released from the apical and basolateral cell surface not only demonstrate the heterogeneity of vesicles but also strongly suggest the existence of very specialized mechanisms to control the selective sorting of cargo into these vesicles [44].

The inward budding of the late endosomal membrane of multivesicular bodies (MVB) results in the formation of intraluminal vesicles. These vesicles are the origin of exosomes and are released by the cells through the fusion of the MVB with the cell membrane. All eukaryotic cell types secrete exosomes [42]. Based on their 30–150 nm size distribution, exosomes are separated at higher sedimentation speeds (100,000 ×g) compared to other EVs. Electron microscopy is one of the most robust methods that allows for the visualization of exosomes [41]. It has been shown that the mRNAs carried by exosomes can be translated into proteins in the target cell, while miRNAs can be functionally transferred to recipient cells, and subsequently silence gene expression, indicating that exosomes can act as a vector of genetic information [41,45].

Although cargo transfer from exosomes into the recipient cells is not completely understood, the general consensus is that exosomal content is internalized into recipient cells via endocytosis or into an endocytic compartment [39]. In this manner, several groups have been studying different proteins that could be involved in specifically direct exosomes to specific tissues or cell types [[46], [47], [48]]. Particularly, at least two recent papers proved that Cardiac Homing Peptide expressed in exosomes could increase exosome transfer to cardiac tissue, with beneficial effects on heart function [49,50]. Taking all this into account, exosomes are increasingly recognized as one of the major players in both local and distant cell-cell communication [44,45].

Exosomes have been shown to carry different classes of ncRNAs. The lipid bilayer of exosomes protects ncRNAs from degradation in the extracellular space and body fluids [51]. lncRNAs have been detected in exosomes from body fluids of cancer patients [52] and are capable of modulating angiogenesis and metastasis [52]. Other exosomal ncRNAs, such as miRNAs and piwiRNAs, can be used as potential cancer biomarkers [3]. Other types of small ncRNAs as transfer RNA, ribosomal RNA, small nuclear RNA, small nucleolar RNA and fragments of YRNAs and mitochondrial RNAs have been also detected in human exosomes [53]. Exosomal miRNAs modulate inflammatory responses and circRNAs can be used as biomarkers for cancer diagnosis [54] (reviewed in [3]).