Circular RNAs in cell cycle regulation: Mechanisms to clinical significance

Abstract Circular RNAs (circRNAs), a type of non‐coding RNA, are single‐stranded circularized molecules characterized by high abundance, evolutionary conservation and cell development‐ and tissue‐specific expression. A large body of studies has found that circRNAs exert a wide variety of functions in diverse biological processes, including cell cycle. The cell cycle is controlled by the coordinated activation and deactivation of cell cycle regulators. CircRNAs exert mutifunctional roles by regulating gene expression via various mechanisms. However, the functional relevance of circRNAs and cell cycle regulation largely remains to be elucidated. Herein, we briefly describe the biogenesis and mechanistic models of circRNAs and summarize their functions and mechanisms in the regulation of critical cell cycle modulators, including cyclins, cyclin‐dependent kinases and cyclin‐dependent kinase inhibitors. Moreover, we highlight the participation of circRNAs in cell cycle‐related signalling pathways and the clinical value of circRNAs as promising biomarkers or therapeutic targets in diseases related to cell cycle disorder.


| INTRODUC TI ON
Different RNA species, such as protein-coding messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs), including circular RNAs (circRNAs), pseudogenes and long non-coding RNAs (lncRNAs), display complex crosstalk. These RNA transcripts function as natural microRNA (miRNA) sponges or competing endogenous RNAs (ceR-NAs) that communicate with and co-regulate each other by competing or interacting with shared miRNAs. Deeper understanding of this novel RNA crosstalk will help shed light on gene regulatory networks and broaden the horizon on the study of circRNAs in human development and disease. 1 CircRNAs are a covalently closed loop molecules without 5′ caps and 3′ poly (A) tails that confers resistance to RNase R, making them exceptionally more stable than linear RNAs. 2 CircRNAs are highly enriched in eukaryotes, and several of these are evolutionary conserved and expressed in a cell type-, developmental stage-and tissue-specific pattern. 3 Emerging studies have shed new light on how circRNAs execute diverse biological roles with cell type-or tissue-specific expression in various organisms. 4 Recently, independent studies have shown that the dysregulation of circRNAs is involved in a series of phenotypic changes, including proliferation and apoptosis. 4,5 The mammalian cell cycle is precisely regulated by diverse cyclins and cyclin-dependent kinases (CDKs) as well as RB and p53-related pathways. 6,7 Recently, several circRNAs have been implicated in the modulation of critical cell cycle regulators, including p53, pRB, CDKs, CDK inhibitors (CKIs) and cyclins. Therefore, dysregulation of the cell cycle-associated circRNAs may be involved in pathophysiological process, including disease aetiology, and may serve as potential novel molecular targets for diagnosis, prognosis and treatment of diseases such as cancer. 8,9 However, the detailed functions and mechanisms of cell cycle regulation by circRNAs remain largely obscure. Recent studies, however, have revealed the diverse roles of miRNA and lncRNAs in cell cycle regulation. 10,11 To date, crosstalk between circRNAs and cell cycle has not been systematically elucidated. This review summarizes current research on the emerging landscape of circRNAs in the cell cycle regulation. We highlight the multifaceted functions of circRNAs and their underlying molecular mechanisms in cell cycle. A deeper understanding of circRNA functions in cell cycle may provide promising candidates for diagnosis and treatment of cell proliferation-related diseases.

| Characteristics of circRNAs
The widespread use of sequencing technologies and bioinformatics has facilitated the detection of circRNAs in diverse species, including humans, fungi, plants and other organisms. Emerging evidence demonstrates that circRNAs are widely expressed in different cells and tissues. 12 Based on the diversity of source sequences, circRNAs are grouped into four major categories: exonic circRNAs (ecircRNAs), intronic circRNAs (ciRNAs), exonic-intronic circRNAs (EIciRNAs) and circRNAs generated from tRNAs (tricRNAs). 13,14 Currently, different circRNAs are being constantly discovered and recognized as hotspots in diverse diseases, such as cancer and Alzheimer's disease. 15 Accumulating evidence indicates that circRNAs manipulate different physiological and pathological processes via a diverse range of mechanisms, including transcription, miRNA sponge, protein sponge/decoy and translation. 5 (Figure 1).
Although circRNAs are generated in the nucleus, most are predominantly located in the cytoplasm, 16,17 suggesting specific rules for circRNA transportation or localization. 18 On the one hand, most of circRNAs are localized in the cytoplasm, suggesting their roles in transcriptional and post-transcriptional regulation; for example, certain circRNAs orchestrate gene expression via diverse mechanisms of action, including modulation of transcription, pre-mRNA splicing and mRNA stability. On the other hand, circRNAs interact with a broad repertoire of biomacromolecules, thereby contributing to RNA or protein modifications, influencing functional peptide encoding. Furthermore, circRNAs exhibit developmental stage-and tissue-specific expression. 19 Moreover, unlike linear RNAs, circRNAs with loop structure are much more stable and resistant to RNases. Therefore, circRNA-mediated gene regulation is a complex biological process that exists in a series of diseases including cancers, which provides avenues for prospective therapeutic interventions.

| CircRNAs as mutifunctional players in cell cycle
CircRNAs play a diverse range of roles in multiple phenotypic aspects, including cell proliferation, apoptosis, invasion and metastasis. 20 Therefore, it is worthwhile to further explore the interactive mechanisms between circRNAs and diseases. The elucidation of circRNA functions as effective molecular biomarkers or potential therapeutic targets will provide promising prospects for application, including early diseases diagnosis, treatment evaluation, prognosis prediction and even disease-specific gene treatment. 21 Clinically, cir-cRNAs are differentially expressed in several of diseases, including cancers, indicating their regulatory role. 22 CircRNAs have emerged as important modulators in a wide spectrum of pathophysiological processes as well as cell cycle regulation and proliferation. 23,24 Cyclins, CDKs and CKIs reportedly orchestrate diverse cellular processes involved in cell cycle regulation. The kinase activity of CDK/cyclin complexes is closely modulated by interplay between a serine/threonine-specific catalytic core and regulatory subunits, termed as cyclins, which control phase transitions during the cell cycle. Under adverse conditions, CKIs act as brakes to impede cell cycle progression. [25][26][27] The regulation of cell proliferation is a complex process driven by a large number of signals that lead to cell division and growth. [28][29][30][31] Thus, disorders affecting cell cycle regulation play an important role in excessive cell proliferation. 27 Recent studies revealed that dysregulation of circRNAs exert accelerative and suppressive roles in human diseases, including cancer.
Herein, we summarize the multiple regulatory functions of cir-cRNAs in cell proliferation and apoptosis via diverse mechanisms ( Figure 2). (i) miRNA sponges: Different sponge types can act as competing endogenous RNAs (ceRNA) to sequester miRNAs and dampen the gene expression of miRNA targets. 1 Interestingly, cir-cRNAs, such as circTP63 and circAGFG1, exert both promotive and suppressive roles to regulate the cell cycle by sponging miRNAs (e.g. miR-873-3p, miR-195-5p). 9,32 (ii) Binding proteins: CircRNAs affect cell cycle progression by binding to RNA-binding proteins (RBPs), thereby affecting RNA processing and mRNA stability. 33,34 For example, ectopic expression of circ-Foxo3 suppresses cell cycle progression by interacting with the cell cycle proteins CDK2 and p21, forming a ternary complex to block cell cycle progression -G 1 /S transition. CDK2 binds to cyclin A and cyclin E to promote cell cycle entry. 35 Another important role of circRNAs is their influence on protein interactions. For example, circNfix enhanced the interaction between Y-box binding protein 1 (Ybx1) and Nedd4l (an Together, these reports indicate that circRNAs play a diverse range of roles for regulating cell cycle progression by different molecular mechanisms (Table 1). Therefore, understanding the mechanisms of circRNA-mediated cell cycle may provide potential novel and promising therapeutic options suitable for clinical application.

| CircRNAs regulate cyclins and CDKs
Cyclins and CDKs play versatile roles as regulators in G 1 /S phase and G 2 /M phase transitions. The current classification of cyclins and CDKs is based on functional relevance and evolutionary conservation. 42,43 In the 'classical' model of the mammalian cell cycle, specific CDK-cyclin complexes drive the numerous events that occur during the interphase in a sequential and orderly manner. 27 At the  Another study identified circPUM1 was significantly up-regulated in lung adenocarcinoma and increased cyclin D1 and Bcl-2 expression by sponging miR-138-5p, thereby facilitating cell proliferation, migration and invasion of lung adenocarcinoma. 50 9 Xie et al. 65 proved that the elevated expression of  Recent studies showed that circRNAs orchestrate cell cycle progression by mediating cell cycle inhibitors, such as p21 and p27, in mRNA, promoting p21 mRNA stability and elevating its production.
Silencing circPCNX increased AUF1-p21 mRNA binding, thus reducing p21 production and promoting cell division. 79 Furthermore, circRNAs regulate CKIs through other mechanisms. CircRNAs target miRNAs that in turn target tumour suppressor genes. This axis is used to regulate cell cycle progression and considered an indicator of healing. For example, circ_0021977 was found to suppress CRC proliferation, migration and invasion by modulating the miR-10b-5p/p21 and p53 axis. Low circ_0021977 expression in CRC patients was correlated with higher TNM stage and poorer prognosis. 80 Additionally, circYAP1 acts as a tumour suppressor to restrain cell growth and invasion via targeting the miR-367-5p/ p27 Kip1 axis, which may offer a promising prognostic indicator of survival in patients with gastric cancer. 81

| CircRNAs mediate other pathways
In addition to the p53 pathway, many other signalling pathways are regulated by a range of circRNAs in the cell cycle. Here, we discuss two circRNA-mediated signalling pathways involved in cell cycle regulation: RB-E2F pathway and PI3K/AKT pathway.

| RB-E2F Pathway
The transition from G 1 to S phase is regulated by the interplay between CDKs and retinoblastoma protein (Rb) phosphorylation, thereby releasing E2F transcription factors to facilitate the expression of S phase genes. It is widely recognized that pRB targets members of the E2F family. 82 As subgroups of E2F family, E2F1, E2F2 and E2F3 are the 'activating' E2Fs that function as transcriptional activators to induce the G 1 /S transition. 83,84 The pRB-E2F pathway it promoted RCC proliferation and invasion by competitively binding miR-127-3p to up-regulate the downstream gene, CDKN3 and the E2F1 pathway. 86 Similarly, circ-Foxo3 was highly expressed in ageing cardiac tissues and facilitated cellular senescence by interacting with the anti-senescence proteins E2F1 and ID1 to influence impacted their subcellular translocation. Moreover, overexpression of E2F1 in miR-205-expressing cells could partly reverse the senescent phenotype. 87 Another study reported that circRNA CDR1 promotes E2F3 expression by binding miR-7-5p, thereby facilitating nasopharyngeal carcinoma growth and glucose metabolism. 88 Highly expressed hsa_ circ_0008039 exerted oncogenic effects in breast cancer, while its depletion markedly inhibited cell cycle progression and proliferation via sponging miR-432-5p and elevating E2F3 expression. 89

| PI3K/AKT Pathway
The phosphoinositide 3-kinase (PI3K) pathway plays an integral role in many cellular processes and is frequently altered in cancer, contributing to tumour growth and survival. 90,91 Small molecule inhibitors have been developed that target the three major nodes of this pathway: PI3K, AKT and mammalian target of rapamycin (mTOR). [92][93][94][95] CircRNAs have also been shown to target this axis. For example, a study identified that silencing of circ-ZNF609 specifically blocks the G 1 /S transition via inhibiting phosphorylated Rb:Rb ratio.
By contrast, circ-ZNF609 expression promotes cell proliferation in rhabdomyosarcoma, highlighting circ-ZNF609 as a new regulator of cell proliferation-related pathways that abrogates p-Akt proteasomedependent degradation. 96 Similarly, circ-IGF1R was significantly up-regulated, acting as an oncogene in HCC. siRNA-mediated knockdown of circ-IGF1R inhibited cell proliferation and triggered cell apoptosis. Further analysis indicated that circ-IGF1R activates PI3K/AKT signalling pathway, contributing to cell cycle. 97 Another study confirmed that exosomal circNRIP1 could be transported between gastric cancer cells and promoted the proliferation, migration and invasion by sponging miR-149-5p to activate the AKT1/mTOR signalling pathway. 98 Despite being the most well-studied signalling pathway implicated in cancer molecular mechanisms, the physiological functions of these kinases in cells and organisms are far more complex than previously assumed. 99 Therefore, a comprehensive investigation of underlying mechanism may also provide novel insights into the crosstalk in PI3K/AKT pathway and cell cycle regulation.

| P OTENTIAL D IAG NOS TI C AND THER APEUTI C APPLI C ATI ON S OF CIRCRNA S
Emerging studies have shown that certain circRNAs act as promising biomarkers and therapeutic targets in various diseases. The expression patterns and characteristics of circRNAs make them perfect candidates as valuable biomarkers to improve diagnostic efficacy.
Furthermore, circRNAs are abundant in human body fluids, such as blood and saliva, which makes them easily accessible and easy to detect, and thus, viable biomarkers for cancer detection, particularly via liquid biopsies. Moreover, a series of circRNAs, such as F-circEA, are more suitable for developing biomarkers than the linear RNA transcripts. 100,101 Thus, circRNAs may function to be prospective diagnostic biomarkers or novel therapeutic targets for disease treatment. In the following sections, we discuss circRNAs as prospective tools in the clinic for diagnosis and treatment of different disease types ( Figure 4).

| CircRNAs as promising biomarkers
Considering that circRNAs exert key roles in disease progression and cell cycle as well as feature abundance, conservation stability and prevalence, 102-104 they are appropriate candidates for biomarkers.
High-throughput sequencing has identified a number of circRNAs that may be prospective non-invasive biomarkers for diagnosis and

| CircRNAs as therapeutic targets
Several studies have focused on the clinical utility of circRNAs as promising therapeutic targets in many diseases. 54 119 To date, prominent advances in many cancer models could pave the way for their utilization in cell cycle regulation.
Currently, several specific CDK4/6 inhibitors targeting CDK-Rb-E2F pathway are either approved or in clinical trials for the treatment of different cancer types. 120 For example, a clinical phase II trial reported that the combination of CDK4/6 inhibitors and conventional chemotherapy (carboplatin and gemcitabine) increases progression-free survival in TNBC patients. 121 Similarly, a combination of palbociclib and letrozole to target CDK4/6 was effective in metastatic HER2-negative ER-positive breast cancer in postmenopausal women. 122 Ribociclib, the pyridopyrimidine palbociclib, and its isosteres ribociclib and abemaciclib are highly selective for CDK4 and CDK6, and induce G 1 /S arrest exclusively. 123 However, clinical applications of targeting circRNAs is still in its infancy, extensive studies need urgently to develop progressive techniques and efficient agents for clinical applications. Therefore, the combination of CKIs with specific circRNAs is a promising treatment strategy for several diseases. Moreover, advances in cancer models in vivo can pave the way for their utilization in cell cycle-related diseases.
However, more clinical trials are needed to drive the development of ncRNA-based diagnostic tests and therapeutic interventions for cancer patients.

| CON CLUS I ON AND FUTURE PRE S PEC TIVE S
In this review, we emphasized the expanding functions of circRNAs Despite these recent advances, the exact mechanisms of action of circRNAs remain largely unknown, certain challenges and limitations in this field need to be overcome. Therefore, the following key questions should be answered: (i) As sequence conservation was identified in most cyclin/CDK families, are circRNAs that regulate these cyclins/CDKs also conserved? (ii) Considering that cyclins are periodic, are the circRNAs that regulate these cyclins also periodic?