The emerging roles of exosomal circRNAs in diseases

Exosomes, 30–150 nm in size, originate from the membrane vesicles of the endosomes and are secreted by almost all cells from different organisms, and are considered to be key roles in biological processes under normal and pathological conditions [1, 2]. Exosomes contain a variety of functional molecules, including various growth factors, proteins, metabolites, DNA, lipids, transfer RNA (tRNA), messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), long non‑coding RNA (lncRNA), circRNA and so on [3]. Numerous studies have demonstrated that exosomes are crucial mediators of intercellular communication. After exosomes are released into the tissue fluid, through a series of movements, exosomes arrive at the recipient cells and begin to deliver their internal active substances (such as circRNAs), thus initiating functional responses and inducing subsequent phenotypic changes [4‐6].

Exosomes not only target the target cells to trigger downstream signals but also transfer genetic material to target cells, exerting anti-inflammatory, antiapoptotic and immune-suppressive effects, promoting tissue repair and improving cytokine levels [7, 8]. In addition, exosomes are involved in the development of many pathological processes such as in cancer, diseases of the nervous system and endocrine system [9‐11]. Exosomes accumulate and circulate freely in different body fluids, and also contain a variety of functional molecular carriers. Therefore, exosomes may become a very promising non-invasive liquid biomarker, which will play an important role in future clinical diagnosis and treatment [12, 13]. CircRNAs are new endogenous non-coding RNAs, which are produced by splicing events or back-splicing events via exons or (and) introns circularization of the original transcription [14]. With the classical linear RNA with 5′caps and 3′poly(A) tails, circRNAs have a covalent closed-loop structure to avoid being degraded by RNA external enzymes or RNase R, so the number of circRNAs is more stable than linear mRNA, which makes them more rich than the typical linear transcription products of the same gene [15]. Due to the application of RNA sequencing (RNA-seq) technology coupled with novel bioinformatics approaches, circRNA has been found to occur widely and stably in human, animal and plant cells, even in mammalian tissues [16]. CircRNAs could regulate gene expression at transcriptional, posttranscriptional and translational levels. They participate in many pathological processes such as Alzheimer’s disease, diabetes, atherosclerosis and myogenesis through regulating alternative splicing, sponging miRNAs, sequestering functional proteins or even encoding proteins [17‐20]. Particularly, circRNAs play an important role in cancer growth, metastasis, recurrence and therapy resistance [21]. Surprisingly, recent studies have shown that large and stable expression of circRNA can be detected in exosomes extracted from human circulation and urine [22]. For instance, it has been reported that tumor-derived exosomes are involved in the development, invasion and metastasis of gastric cancer, and circRNA can be transmitted to recipient cells through exosomes to promote the malignant process of gastric cancer [23, 24]. Moreover, exosomal circRNAs were also found in pancreatic ductal adenocarcinoma (PDAR) [25], hepatocellular carcinoma [26], platelet-derived extracellular vesicles [27]. As can be seen from the above studies, exosome circRNAs may participate in a variety of life activities and pathological processes in the human body, and the combination of the two may further increase the potential clinical applications of these molecules as diagnostic and prognostic markers. In the past, exosomal lncRNAs and miRNAs have been widely reported, but relatively little attention has been devoted to exosomal circRNAs. In this review, we briefly address the biogenesis and biological functions of exosome and circRNAs, the significance and role of exosomal circRNAs in various diseases. Besides, we propose exosomal circRNAs can as a promising diagnostic marker and therapeutic punctuation for diseases, especially in cancer.

Exosomes are a type of extracellular vesicle consisting of a phospholipid bimolecular cell membrane, which are found in various body fluids such as blood, saliva, urine, breast milk and so on [28]. Early endosomes were formed by intracavity vesicles on the plasma membrane, which were further folded by the phospholipid bilayer to produce multiple intracavity vesicles, thus forming multi-vesicular body (MVB). It could not only fuse with the plasma membrane to release its contents, namely exosomes, but also transport to lysosomes to degrade the contents of vesicles [29, 30]. In addition, the membranes of exosomes are rich in a variety of proteins and lipids, such as cholesterol and sphingolipids. There are other proteins on the surface of exosomes, including four major transmembrane proteins (CD9, CD63, CD81 and CD82), which are enriched on their membranes and considered as ideal markers for exosome characterization [31], as shown in Fig. 1. It is noteworthy that exosomes play an important role in regulating normal physiological processes, such as repair ischemic injury [32], healthy pregnancy [33] and tissue repair [34]. In addition, exosomes also participate in the development of various diseases in the human body, playing a role that cannot be ignored. Studies have shown that CD317 protein and epidermal growth factor receptor (EGFR) are highly concentrated on the surface of exosomes in serum of non-small cell lung cancer (NSCLC), which is therefore considered as an important marker for the diagnosis of NSCLC [35]. Exosomes released by cancer cells can mediate phenotypical changes in the cells of tumor microenvironment (TME) to promote tumor growth and therapy resistance, for example, fibroblast- and macrophages-induced differentiation [36]. Therefore, exosomes are biomarkers with diagnostic and prognostic significance, and we will further study their potential in clinical application.

Early endosomes were formed by intracavity vesicles on the plasma membrane, which were further folded by the phospholipid bilayer to produce multiple intracavity vesicles, thus forming MVB. It can fuse with the plasma membrane to release its contents, namely exosomes, also transport to lysosomes to degrade the contents of vesicles.

The structure of exosomes include, tetraspanins (CD9, CD63, CD81, CD82). Lipid rafts, such as sphingolipids, cholesterol. Immunoregulatory molecules include major histocompatibility complex I, II (MHC I, MHC II), CD86. The receptors on the exosome membrane are different depending on the host cell, eg. EGFR. Exosome contents have proteins, nucleic acids such as DNA, tRNA, mRNA, rRNA, miRNA, lncRNA, circRNA and so on.

Exosomes have great potential in clinical diagnosis, biomarker detection, target recognition of molecular therapy and personalized medical application. However, there are still some obstacles such as super-sensitive detection technology, efficient isolation method and quantitative analysis. In the clinical environment, a fast and reliable test standard is urgently needed. A large number of approaches have been used for exosome and extracellular vesicle (EV) isolation, such as ultracentrifugation, density-gradient centrifugation, size exclusion chromatography, microfiltration, immunomagnetic beads, and several polymer-based precipitation techniques [37‐41]. These separation techniques rely either on the size and density of the EVs or the existing specific surface proteins on the exosomes such as, CD9, CD63, CD81, TSG101 and HSP70 [41]. Fortunately, with the in-depth research, different approaches with recognition element were reported, including nanoparticles tracking analysis (NTA), tunable resistive pulse sensing (RPS), dynamic light scattering (DLS), flow cytometry, cryo-EM, a platform combining surface plasmon resonance (SPR) with atomic Force Microscope (AFM), or by other techniques with similar capabilities have been developed [42‐44]. Besides, Western Blotting can be utilized to verify the presence of biomarker proteins in EVs. Traditional flow cytometry is suitable for identification of large vesicles, but not nanoscale ones. High-sensitivity flow cytometer (HSFCM) has been found that can increase the detection limit from about 500 nm to as low as 40 nm, making it possible to detect other large and small vesicle populations, such as exosomes [45]. Although these techniques and methods are fast-changing, there are still many limitations and shortcomings in their clinical application. First, many of methods require state-of-the-art instruments. Second, impure recoveries with regard to remnant matrix species (e.g., proteins, genetic material) and are performed on clinically irrelevant time and volume scales. Third, the selectivity for some specific exosomes is deficient. Fourth, the accuracy is often influenced by unreliable nanoparticle counting, which is from large proteins and lipoprotein particles, and even some exosomes less than a certain size being omitted [46]. We expect that in the future a technology will be developed to break down these barriers and translate exon detection of disease sources into clinical applications for early screening in primary care settings.

Numerous studies have shown that exosomes contain various nucleic acid substances, such as mRNA, miRNA, lncRNA, rRNA, circRNA, etc., when they transferred to the recipient cells, causing a series of biological reactions and play an important role in regulating the physiological process of cells [73‐75]. Moreover, the rich and stable expression of circRNAs in extracellular vesicles has been identified, providing new clues and support for the functional research of circRNAs. It was shown that compared with parental cells, circRNAs in exosomes were more abundant, larger in number and more stable in expression than linear mRNA subtypes [22, 76]. Possible mechanisms for circRNAs to be more abundant in exosomes than in its producing cells include, first, the relatively long half-life of circRNAs, as they are covalently closed-loop structures without a poly A tail or a 5′–3′ end, thereby avoiding degradation by endonuclease [62]. Studies have shown that most circRNAs have a half-life of more than 48 h, while linear RNAs have a half-life of less than 20 h. Therefore, compared with linear transcripts in cells, circRNAs are more stable and enriched [15]. Second, the extracellular vesicles release circRNAs, which are then exported to the extracellular microenvironment to form a mechanism for circRNA clearance, so circRNAs are more enriched in the extracellular space [77]. Finally, due to the protective effect or certain characteristics of the exosome envelope, it can safely prevent further removal of contents or degradation by RNA enzyme, so exosomal circRNAs are highly stable and abundant [78]. Exosomes contain different types of active substances whose sources reflect the state and type of cells; however, exosomes from different or identical sources may contain the same or different molecules, indicating that the molecular subsets in exosomes are tissue-specific and uniquely species-specific. Reverse transcriptional quantitative PCR and high-throughput analysis were used to detect exosome-derived circRNA, and the results revealed the difference of circRNA types in serum and exosomes [22], which also indicated that circRNA entered exosomes actively, including selectivity in the sorting process [79]. In addition, studies have shown that RNA-binding complexes endosoma-sorting complex required for transport II (ESCRT‑II) play an important role in the development of MVB and may help EV to selectively incorporate RNA [1]. Other studies have shown that intracellular miRNA may regulate the process of circRNA entering exosomes [22].

In summary, we need to further investigate and elucidate the complex mechanisms by which RNAs selectively and specifically enters exosomes, and we speculate that multiple types of sorting mechanisms may be at work simultaneously. These findings will greatly expand the potential of circRNAs as a prognostic and diagnostic biomarker.

Increasing evidences show that exosomal circRNAs play a crucial role in a variety of cancers and participate in many important biological processes, promoting or inhibiting cancer. It could provide us with further insights into the functional mechanism of exosomal circRNAs. Table 1 illustrates the role of some exosomal circRNAs in different cancers.

Not only do exosomal circRNAs play a vital role in tumors, but also play an important role in diseases other than tumors, such as nervous system diseases and cardiovascular diseases, as summarized in Table 3.

Human platelets are particularly rich in circRNA compared to other hematopoietic cell types, and since platelets are essential for central physiological processes such as hemostasis, wound healing, inflammation, and cancer metastasis, these findings could greatly expand the potential of circRNA as a prognostic and diagnostic biomarker [27, 111, 112]. Exosomes from the extracellular space of the brain, where differentially-expressed circRNAs might be related to the growth and repair of neurons, the development of the nervous system, and the transmission of nerve signals. The most highly correlated pathways that identified were involved primarily with glutamatergic synapse and the cyclic guanosine monophosphate-protein kinase G signaling pathway [113]. CircHIPK3 in hypoxic exosomes (HPC-exos) plays a role in cardiac microvascular endothelial cells (CMVECs) under oxidative conditions through miR-29a-mediated IGF-1 expression, leading to a decrease in oxidative stress-induced CMVECs dysfunction. These data suggest that the exosomal circRNA in CMs is a potential target to control CMVECs dysfunction under oxidative conditions [107].

To explored the role of smooth muscle SIRT1 in endothelial angiogenesis after ischemia and the underlying mechanisms, we performed a femoral artery ligation model using VSMC specific human SIRT1 transgenic (−Tg) and knockout (KO) mice. −Tg VSMCs inhibited endothelial angiogenic activity induced by hypoxia via the exosome cZFP609. The cZFP609 was delivered into ECs, and detained HIF1α in the cytoplasm via its interaction with HIF1α, thereby inhibiting VEGFA expression and endothelial angiogenic functions [108].

As an important carrier of exosomes, circRNAs play an important regulatory role in various physiological and pathological biological processes, such as inflammation, tissue repair, cell proliferation, migration, invasion, metastasis, apoptosis, and chemotherapy & radiotherapy resistance. Due to their unique features and high specificity, their ability to mediate cell communication, what is more, exosomal circRNAs is abundant, stable, and present in different extracellular fluids, including saliva, blood, and urine [28], making it an ideal diagnostic marker and therapeutic target as well as potential vectors for drug delivery. However, compared with other ncRNAs, such as miRNA and lncRNA, the studies of circRNAs in exosomes are still incomplete. Therefore, we need to further explore its functional mechanism.

At present, the study of the structure of endogenous circRNAs sponges may contribute to the design and development of effective artificial sponges to regulate disease progression. As a stable and effective miRNA inhibitor, the artificial miRNA "sponge" technique may be a new strategy for RNA gene therapy in the future. It can simultaneously inhibit the expression of other miRNAs and produce a more long-lasting inhibiting effect. Therefore, the use of nanoparticles to deliver circRNAs to generate specific types of exosomes provides an option for selective targeted cancer cell elimination [117‐120].

However, the specificity of exosomal circRNAs structure and the insufficiency of technology limit the discussion of its function. There are still many challenges and difficulties in clinical application. First, a standardized method for collecting, processing and isolating exosome samples has not yet been established [28, 121]. Ultracentrifugation method is the gold standard for exosome separation, but it is time and labor-consuming, requires a large amount of raw materials and expensive equipment, and is inefficient for high-throughput measurement [122], which cannot be effectively applied to clinical work. Second, in the clinical collection of patient samples, blood or other body fluids contain many impurities, such as protein complexes, nucleic acid lysates, etc., which are challenging to extract correct exosomes. Third, circRNA and its corresponding linear mRNA sequences are partially overlapped, so circRNA expression and function cannot be accurately assessed. Fourth, due to the low abundance of circRNAs, it is difficult to detect them in exosomes using accurate methods. Therefore, it is important to develop and use appropriate methods and techniques to elucidate the molecular mechanisms and regulatory networks of exosomal circRNAs.