The transcriptome holds a wide array of information to actively regulate the cellular system. This information comes in various forms such as mRNAs, microRNAs, long non-coding RNAs, and piwi-interacting RNAs (Palazzo and Lee, 2015). There is a wide gap of information that we have yet to retrieve from non-coding RNAs, but based on our current understanding, they are reported to be involved in various processes such as gene expression, transcription, protein expression, and scaffolding (Palazzo and Lee, 2015). One group of recently rediscovered non-coding RNAs includes circular RNAs. Circular RNAs were once thought to be a tangential splicing event and were regarded as an error (Chen et al., 2016). There are already several known circular RNAs derived from viroids or other viruses (Rezaian, 1999). However, only recently the interest in circular RNAs in humans has increased (Hentze and Preiss, 2013; Li et al., 2015a; Chen et al., 2016). Advances in molecular techniques such as next generation sequencing and bioinformatics analysis have provided key insights into the features of circular RNAs such as abundance, stability, conservation, and tissue-specific expression. In this mini-review, we will attempt to illuminate the potential use of circular RNAs as biomarkers.

In the non-coding RNA population, there are multiple species of RNA; therefore, there must be a robust way to specifically detect circular RNAs. More importantly, the experimental design and the bioinformatics analysis platform used should be validated and optimized. To detect circular RNAs, there are several major pipelines that have been developed and validated, which are find_circ, CIRI (Gao et al., 2015), MapSplice (Burd et al., 2010; Jeck et al., 2013), CIRCexplorer (Zhang et al., 2014), circRNA_finder, and deepBase (Zheng L.-L. et al., 2016). There are also multiple repositories for circular RNAs available such as circBase (Glažar et al., 2014), CircNet (Liu et al., 2016b), circInteractome (Dudekula et al., 2016), and circ2traits (Ghosal et al., 2013). Circular RNAs are characterized by a covalently continuous loop from the 5′ to 3′ ends (Lasda and Parker, 2014). There are different types of circular RNAs such as exonic circular RNA, intronic circular RNA, and intergenic circular RNA (Lasda and Parker, 2014). The most common type of circular RNA is the exonic circular RNA and is always referred to as circRNA (Valdmanis and Kay, 2013; Lasda and Parker, 2014). Circular RNAs are formed by a “back-splicing” process which is an event mediated by a spliceosome that splices a downstream 5′ splice site (splice donor) and joins it to an upstream 3′ splice site (splice acceptor; Lasda and Parker, 2014; Wilusz, 2015). Circular RNAs have been recently discovered and have not been fully characterized yet. The diversity of circular RNAs is wide; circular RNAs can be derived from numerous genes and can have different levels of expression. Moreover, circular RNAs can also have different sizes ranging from 100 nucleotides to 4 kb (Lasda and Parker, 2014; Ebbesen et al., 2016). Additionally, circular RNAs may contain different numbers of exons with different sized introns flanking the back-splice site (Lasda and Parker, 2014; Ebbesen et al., 2016). It is postulated that circular RNAs are conserved between human and the mouse (Jeck et al., 2013). For instance, Jeck et al. found that there are 69 mice circular RNAs that have high homology to its respective human circular RNAs (Jeck et al., 2013). However, there is evidence that the percentage of the conserved region may not be as high as previously thought (Guo et al., 2014). Furthermore, the ratio between the circular RNA and the corresponding linear RNA varies between different cell types and conditions. Some studies report that the presence of circular RNAs is less than 10% of the linear RNA, however, other studies report that certain circular RNAs are more enriched than the linear RNAs depending on different circumstances (Lasda and Parker, 2014). Thus, there is a wide variation of circular RNAs but more research should be done to discover if the variations are biologically relevant to develop circular RNAs into biomarkers.

Infectious circular RNAs are known since long to be present in multiple living organisms, including plants and animals. In plants, infectious circular RNAs endowed of autonomous replication (viroids) or depending on a helper virus (satellites) have been reported (Rao and Kalantidis, 2015). In humans, hepatitis delta virus, a well characterized infectious circular RNA sharing structural properties with viroids (Flores et al., 2016), is found in patients co-infected with hepatitis B virus. However, other non-infectious endogenous circular RNAs have been reported in animals and their role(s) remains sometimes inconclusive (Wang et al., 2014). Some circular RNAs are reported to be regulators of transcription in cis (Lasda and Parker, 2014), while others are proposed to function as micro RNA (miRNA) sponges (Chen et al., 2016). To date, there are several circular RNAs that were reported to interact with miRNAs (Burd et al., 2010; Hansen et al., 2013a,b; Jeck and Sharpless, 2014). MiRNAs are known to have specific binding sites for “sponges” such as mRNAs or in this case, circular RNAs. If changes occur in the binding regions of these “sites,” a downstream dysregulation of the entire network will bound to occur and will lead to major events such as cancer progression, neurological diseases, and cardiovascular diseases. One of the most reported and extensively studied circular RNA is the ciRS-7 (Hansen et al., 2013a,b; Liu et al., 2014). Bioinformatics analysis revealed that there are approximately seventy miR-7 binding sites present on ciRS-7 (Hansen et al., 2013b; Jeck and Sharpless, 2014). A study by Memczak et al. using zebrafish proved that there is an interaction between miR-7 and ciRS-7. The findings demonstrated that ciRS-7 managed to reduce the level of miR-7 activity by providing binding sites for miR-7 (“sponging”; Hansen et al., 2013b; Memczak et al., 2013). Additionally, ciRS-7 has also been implicated in neurological diseases such as prion disease and neuropathy (Satoh et al., 2009; Liu et al., 2014). In Alzheimer's disease (AD) and Parkinson's disease (PD) for example, ciRS-7 was found to be inactivated which in turns, increased the level of miR-7 and eventually down regulated AD- and PD- related targets (Lukiw, 2013; Memczak et al., 2013).

Another well-known circular RNA is the SRY circular RNA that is expressed in the testis of mouse (Capel et al., 1993). This circular RNA is known to be a sponge for miR-138 (Capel et al., 1993; Ebert et al., 2007; Hansen et al., 2013a). Recently, another circular RNA has been identified, circHIPK3, that was reported to contribute to cancer progression. This particular circular RNA can be derived from the exon of the HIPK3 gene and was found to be abundant in cancer cells. Zheng et al. silenced the circHIPK3 circular RNA and found that it affected the growth of cancer cells. Moreover, Zheng et al. also discovered multiple miRNA binding sites on circHIPK3, and found that miR-124 exhibited the most prominent binding effect (Zheng Q. et al., 2016). Additionally, another circular RNA, circ-Foxo3, was found to be involved in cell cycle progression and is highly expressed in non-cancer cells (Du et al., 2016). It is suggested that circ-Foxo3 forms a ternary complex with p21 and CDK2 to regulate the cell cycle process (Du et al., 2016). It has also been reported that another circular RNA, cZNF292, is regulated by hypoxia and displays pro-angiogenic activities in endothelial cells (Boeckel et al., 2015). Nevertheless, it is also suggested that cZNF292 does not act as a miRNA sponge, thus providing insight that different circular RNAs have different features and contributes to the regulatory network distinctively from one another (Boeckel et al., 2015).

It has been proposed that circular RNAs might be involved in protein expression since it has been shown that circular RNAs have IRES (Hentze and Preiss, 2013; Jeck and Sharpless, 2014). However, Guo et al. rebutted this concept because they did not find any evidence that could explain whether circular RNAs could be translated into proteins (Guo et al., 2014; Jeck and Sharpless, 2014). Therefore, the validity of this theory remains obscure and more research should be conducted to confirm this notion. Additionally, the theory of circular RNAs interacting with RNA-binding proteins remains plausible as circular RNAs have been shown to bind to argonaute proteins and pol II (Hentze and Preiss, 2013; Jeck and Sharpless, 2014). Recently, it has been shown by Conn et al. (2015) that the RNA-binding protein Quaking regulates the biogenesis of several circular RNAs in response to epithelial-mesencyhmal transition (EMT) process (Conn et al., 2015). Conn et al. findings suggest that a wide number of circular RNAs are involved in EMT-related functions such as invasion, migration, and adhesion (Conn et al., 2015).

Biomarkers are defined as biological entities that can be found in blood, bodily fluids, or tissues that can be a sign of a normal or abnormal process, or of a condition or disease (Henry and Hayes, 2012). The use of biomarkers has emerged as a means of early detection and diagnosing different diseases as well as measuring responses to certain treatments (Mayeux, 2004; Henry and Hayes, 2012). There are several features of a biomarker that renders it suitable for the use at the clinical setting. These features include stability, sensitivity and specificity (Mayeux, 2004; Henry and Hayes, 2012; Drucker and Krapfenbauer, 2013).

The use of circular RNA as biomarkers is a promising approach due to several reasons; (1) Circular RNAs are stable as they are not as susceptible to nucleases as linear RNA (Jeck and Sharpless, 2014). Moreover, circular RNAs have longer half-lives as compared to their linear counterparts (Enuka et al., 2016). (2) Circular RNAs are relatively abundant, especially in the blood (Jeck et al., 2013). The development and utilization of blood-based biomarkers are increasing because the analysis is fast, reliable, and cost-effective. Moreover, minimally-invasive procedures are deemed to be more favorable. (3) The level of circular RNAs is different between healthy and diseased subjects which increases the sensitivity and specificity of the target. The popularity of circular RNAs has steadily increased over the past years, thus understanding the functional mechanism of circular RNAs would be of great value. Hopefully in the future, the elucidation of the role of circular RNAs will be established and the use of circular RNAs will become routine in the clinical practice.

NA, RJ conceived the idea and wrote the manuscript.

This manuscript was funded by the UKM Medical Molecular Biology Institute (UMBI), Malaysia.