circ‐ZNF609: A potent circRNA in human cancers

Abstract Circular RNAs (circRNAs) are a novel group of endogenous RNAs with a circular structure. Growing evidence indicates that circRNAs are involved in a variety of human diseases including malignancies. CircRNA ZNF609 (circ‐ZNF609), derived from the ZNF609 gene sequence, has been demonstrated to be involved in the development and progression of many diseases. circ‐ZNF609 is thought to be a viable diagnostic and prognostic biomarker for several diseases and might be a new therapeutic target, but further research is needed to accelerate clinical application. Here, we review the biogenesis and function of circRNAs and the functional roles and molecular mechanism related to circ‐ZNF609 in neoplasms and other diseases.

species. 9 circ-ZNF609 was initially reported in the central nervous system. 10 Recently, it has been reported that abnormal expression of circ-ZNF609 is associated with tumours and other diseases, in- F I G U R E 1 Biogenesis and conservation of circ-ZNF609. The smaller boxes represented the 5′ and 3′ UTRs while the larger boxes indicated the Coding sequences (CDS). Start codons are shown in green while stop codons are shown in red. circ-ZNF609 (right) originates from ZNF609 mRNA (above), ZNF609 pre-mRNA (centre) and ZNF609 s exon (below)

F I G U R E 2 Biogenesis of circRNAs.
CircRNAs are generated from lariatdriven circulation or back-splicing events. (Three main mechanisms: intron pairing-driven circularization, RBPsmediated circularization and lariat-driven circularization). RBPs, RNA-binding proteins F I G U R E 3 Functions of circRNAs. CircRNAs can act as biomarkers (A), miRNAs sponges (B), be combined with RBPs (C), Act as templates for protein translation (D). Abbreviations: circRNAs, circular RNAs. miRNAs, microRNAs. RBPs, RNA-binding proteins In this review, we aim to describe the biological roles and molecular mechanism of circ-ZNF609 in tumour and other diseases and further explore its diagnostic, prognostic and therapeutic value.

| B I OG ENE S ISOFCIRCRNA S
We summarize three potential models of circRNA biogenesis according to recent studies ( Figure 2). In the first model, the pre-mRNA partially folds during transcription, prompting the 3′ splice site of the downstream intron to be attacked by the 5′ splice site of the upstream intron. Reverse splicing of the folded region produces circR-NAs, and the remaining sequence forms linear RNA. Most circRNAs are produced by this mechanism. 11 The circRNAs produced by the second model can be divided into two types: circRNAs with intron sequences retained and coexisting with exons (EIcircRNAs) and cir-cRNAs with intron sequences removed (EcircRNAs). The circRNAs in the EcircRNA group are produced by reverse splicing via reverse complementary sequences at introns. 11 The third model requires a common sequence in which a 7-nt GU-rich element is near the 5′ splice site and an 11-nt C-rich element is near the branchpoint site. 12 3 | FUN C TI ON SOFCIRCRNA S

| Biomarkers
Currently, mounting evidence indicates that circRNAs are ideal biomarkers for the diagnosis and prognosis of many diseases, especially malignancies ( Figure 3A). In pancreatic ductal adenocarcinoma (PDAC), it has been reported that the expression levels of circ-PDE8A and circ-IARS are correlated with tumour progression and postoperative survival time, which might be ideal biomarkers for PDAC diagnosis or progression. 13,14 In addition, exosomal circRNAs were found to be significantly upregulated in DKs-8 cells compared with DLD-1 and DKO-1 cells, suggesting that exosomal circRNAs may be promising biomarkers in CRC. 15 Lu et al. 16 found that circ-RanGAP1 expression was positively correlated with an advanced TNM stage and shorter survival time. Moreover, Li and colleagues 17 found that hsa_circ_0057762 and hsa_circ_0003090 play an important role in differentiating systemic lupus erythematosus (SLE) children from healthy controls.

| FunctionasmiRNAsponges
One of the most widely studied function of circRNAs is serving as miRNA sponges in the cytoplasm ( Figure 3B). 18 CircRNAs have been proven to be involved in various diseases as competitive endogenous RNAs (ceRNAs). 19,20 In DR, circular RNA ZNF532 promoted the viability, proliferation, and differentiation of pericytes and elevated the recruitment of pericytes towards endothelial cells by acting as a miR-29a-3p sponge and inducing increased expression of NG2, LOXL2 and CDK2. 21 In osteoarthritis, circ-SERPINE2 could act as a sponge of miR-1271-5p and functioned in human chondrocytes (HCs) through targeting miR-1271-5p and ERG. 22 Yu et al. 23 found that circ-BIRC6 was enriched in the AGO2 complex and directly interacts with miR-34a-3p and miR-145-3p to attenuate the downregulation of these target genes and suppressed human embryonic stem cells differentiation. Besides, Zheng et al. 24 generated ribosomal-depleted RNA sequencing data from normal and cancer tissues, and detected at least 27,000 circRNA candidates. They demonstrated that circ-HIPK3 was able to sponge 9 miRNAs with 18 potential binding sites, which might have a regulatory role in cancers.

| Interactionwithproteins
CircRNAs can form circRNA-protein complexes (circRNPs) by interacting with different proteins, thereby regulating the mode of action of proteins or the expression of target genes ( Figure 3C). For example, circ-MBL can interact with mannose-binding lectin (MBL) and modulate MBL and circ-MBL via negative feedback. 25 circ-PABPN1 can bind to HuR, which inhibits the binding of HuR to PABPN1 mRNA, thus reducing the translation of PABPN1. 26 Moreover, circular RNA DNA methyltransferase 1 (circ-DNMT1) binds to p53 and AU binding factor 1 (AUF1), thereby accelerating breast cancer progression. 27

| CircRNAsastemplatesforproteintranslation
In the past, circRNAs were thought as lack of protein translation potential. However, many recent reports have shown that circRNAs with ORFs and an internal ribosome entry site (IRES) can be efficiently translated into peptides or proteins ( Figure 3D). The IRES, which initiates protein translation independently of the 5′-cap structure, is a specific nucleotide sequence in endogenous circRNAs. 28 For example, circ-FAM188B encodes a novel protein called circ-FAM188B-103aa, which was suggested to promote proliferation but inhibit differentiation of chicken skeletal muscle satellite cells. 29 Gprc5a is an essential protein in bladder cancer stem cells. However, in the absence of a protein encoded by circ-Gprac5a, Gprc5a did not function, which indicated that the circ-Gprc5a-peptide-Gprc5a pathway was closely associated with bladder cancer progression. 30 In addition, Yang et al. 31 demonstrated that N 6 -methyladenosine can promote circRNAs to effectively initiate protein translation in the response of cells to environmental stress.

| CircRNAsaffectparentalgeneexpression
Emerging evidence has demonstrated that some circRNAs retained in the cell nucleus could affect their parental gene expression ( Figure 3E). Li et al. 32 found that circ-EIF3J and circ-PAIP2 could interact with RNA Pol II and promote their parental gene transcription in interaction with U1 snRNP in the nucleus of cells. Another study indicated that circ-ANKRD52 could specifically combine with the elongation RNA Pol II complex and directly promote ANKRD52 transcription. 12 Moreover, overexpression of circ-Yap could suppress the interaction of PABP with eIF4G and consequently inhibit the translation initiation of Yap mRNA. 33 circ-PABPN1 might repress HuR binding to PABPN1 mRNA and hinder the translation of PABPN1. 26 These studies verified that circRNAs might affect their parental gene expression at both the transcriptional and translational levels.

| circ-ZNF609incancersandotherdiseases
Recently, emerging evidence has demonstrated that circ-ZNF609 is

| circ-ZNF609incancers
Mounting evidence has demonstrated that circ-ZNF609 could exert vital functions in a variety of human malignancies. circ-ZNF609 can act as ceRNA to regulate several target genes via sponging different miRNAs, so as to participate in the initiation and development of cancers ( Figure 4).

| Renal cell carcinoma (RCC)
RCC is the third most common malignant tumour of the genitourinary system. 34 Although numerous studies have focused on identifying molecular biomarkers of RCC, few of these markers have been proven useful in determining disease prognosis and applied in clinical practice. 35 Therefore, it is urgent to find new biomarkers and therapeutic targets for RCC.
Xiong et al. 36 found that circ-ZNF609 expression was significantly increased in RCC tissues and cell lines. In vitro experiments showed that silencing circ-ZNF609 had a negative effect on cell proliferation and invasion. Further molecular assays demonstrated that the circ-ZNF609/miR-138-5p/FOXP4 regulatory axis was involved.
In summary, the preliminary study concluded that circ-ZNF609 exerts an important role in the pathogenesis of RCC by regulating FOXP4 expression through sponging of miR-138-5p.

| Colorectal cancer (CRC)
CRC is a common malignant tumour worldwide that has a high death rate and molecular heterogeneity. The incidence and mortality of CRC rank at near the top, and the incidence of CRC in younger TA B L E 1 Functional characterization of circ-ZNF609 in various malignancies patients appears to be increasing. 37 Although multiple treatments for CRC are available, the prognosis of CRC patients remains poor. 38 Therefore, exploration of potential mechanism and early diagnostic biomarker are key and urgent.
Wu et al. 39 found that circ-ZNF609 and Gli1 were highly expressed in CRC tissues. Knockdown of circ-ZNF609 had a negative impact on the migration of HCT116 cells. Further study showed that circ-ZNF609 could regulate Gli1 expression through miR-150-5p, thus affecting cancer metastasis. circ-ZNF609 might be a novel target for subsequent CRC screening and treatment, while the downstream target genes for this pathway remains unexplored.

| Rhabdomyosarcoma (RMS)
RMS is one of the most common soft tissue sarcomas in children, accounting for 4.5% of all paediatric malignant tumours. 40 Although a combination of surgery, radiotherapy and chemotherapy is widely

| Nasopharyngeal carcinoma (NPC)
NPC is one of the most frequently diagnosed head and neck cancer types. Radiotherapy is the first-line treatment for NPC. 43 In recent years, with the progress in radiotherapy technology and the improvement in comprehensive treatments combining radiotherapy and chemotherapy, the overall survival (OS) rate and local control rate for NPC patients have improved to some extent, but the distant metastasis rate remains high. 44,45 Therefore, finding specific markers that can predict the occurrence and development of NPC is urgent.
To date, three studies have found that circ-ZNF609 was highly expressed in NPC tissues, but their mechanisms of action are different. Zhu et al. 46 found that high expression levels of circ-ZNF609 and Sp1 could promote the proliferation, migration and invasion of NPC cells, while high levels of miR-150-5p exerted the opposite effects.

TA B L E 2 Functional characterization of circ-ZNF609 in non-cancer diseases
The researchers further found that circ-ZNF609 could elevate the expression of the oncogene Sp1 by binding to miR-150-5p, thus accelerating the progression of NPC. Liu et al. 47 reported high circ-ZNF609 expression contributed to a poor prognosis and circ-ZNF609 regulated HRAS through miR-338-3p to promote the progression of NPC. Wang et al. 48 found that silencing circ-ZNF609 inhibited proliferation, migration and angiogenesis of NPC cells, while miR-145-5p inhibition reversed these effects. Furthermore, STMN1, the downstream target of miR-145-5p and circ-ZNF609, promotes angiogenesis in NPC. Therefore, circ-ZNF609 might have the potential to act as a diagnostic marker and therapeutic target in NPC.

| Gastric cancer (GC)
GC is a malignant neoplasm with high heterogeneity. Its pathogenesis is mainly related to genetic variations, Helicobacter pylori and Epstein-Barr virus infection, and epigenetic changes. 49 At present, surgical resection with chemotherapy is one of the conventional treatment approaches for GC patients. A series of chemotherapeutic and molecular-targeted drugs, including 5-fluorouracil or oxaliplatin followed by Herceptin, are routinely recommended. 50 However, due to widespread toxicity/side effects, the prognosis remains far from optimistic. 51 Therefore, it is necessary to identify novel molecular therapeutic targets for GC and explore their correlation with the prognosis of GC to further optimize precision therapy for GC patients.

| Lung cancer (LC)
In 2020, with 2.2 million new LC cases and 1.8 million deaths reported worldwide, LC is the second most commonly diagnosed cancer and the leading cause of cancer death. 53 LC is pathologically classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), 54 while NSCLC can be further divided into three subtypes: large cell lung carcinoma, lung squamous cell carcinoma and lung adenocarcinoma (LUAD). 55 Recently, despite significant advances in the diagnosis and treatment of LC have been achieved, there are few valuable molecular biomarkers, and the prognosis of LC patients remains dismal, with a 5-year OS rate of only 16.6%. 56 Therefore, it is of great value to explore the mechanism and novel targets for different LC types.

| Hepatocellular carcinoma (HCC)
HCC is a malignant tumour that seriously threatens human health.
Approximately 750,000 new cases are diagnosed every year, and the 5-year OS rate is 5%~9%. 60 The effect of surgery and chemoradiotherapy is not satisfactory. With the development of molecular biology techniques and the application of targeted therapy, it is imperative to study the molecular mechanism underlying the occurrence and development of HCC and find the corresponding effective therapeutic targets. 61,62 Liao and colleagues 63 first found that circ-ZNF609 was overexpressed in HCC tissues and positively correlated with poor prognosis.
In vitro experiments showed that knockout of circ-ZNF609 effectively suppressed the activity, migration and invasion of cancer cells.
Simultaneously, in vivo experiments showed that knockout of circ-ZNF609 inhibited tumour growth and metastasis in mice. It was further found that the effect of circ-ZNF609 on tumour cells was mainly realized through the circ-ZNF609/miR-342-3p/RAP2C regulatory axis.

| Prostate cancer
Prostate cancer is one of the most common malignant tumours of the urinary and male reproductive systems in middle-aged and elderly men. Prostate cancer is usually asymptomatic in its early stage, and most patients are diagnosed in the advanced stages. Endocrine therapy is the preferred treatment for advanced prostate cancer.
However, for castration-resistant prostate cancer, endocrine therapy is less effective. 64 At present, the molecular mechanism of prostate cancer remains elusive. Therefore, in-depth study of the key genes related to prostate cancer is of great significance for the early diagnosis and targeted therapy.
Du and colleagues 65 confirmed that circ-ZNF609 is abnormally upregulated in prostate cancer tissues and cell lines. Silencing of circ-ZNF609 inhibited cell survival, metastasis, radiation tolerance and apoptosis by inhibiting glycolysis. An important finding in prostate cancer is that silencing circ-ZNF609 increases the radiosensitivity of prostate cancer cells in vivo. In terms of the mechanism, miR-501-3p was a direct target of circ-ZNF609, and HK2 was an indirect target of circ-ZNF609. HK2 was regulated by the circ-ZNF609/miR-501-3p axis in prostate cancer cells, which affected the survival, metastasis, radiation resistance and apoptosis of prostate cancer cells. Study of circ-ZNF609 in prostate cancer will provide a novel potential target for the treatment of prostate cancer, although further exploration is needed.

| Cervical cancer (CC)
CC is the most common gynaecological malignant tumour and the fourth leading cause of cancer death in women. 66 It is estimated that there were 604,000 new cases of CC and 342,000 deaths worldwide in 2020. 53 In present, studies have confirmed that high-risk human papillomavirus (HPV) infection is the main cause of CC, about 95% of CC is related to HPV infection, leading to precancerous lesions and invasion of CC. 67 Recently, NF-YA and EphA2 were identified as key regulators in the pathogenesis of CC, without detailed exploration in the clinical setting. 68,69 Therefore, it is of great clinical significance to deeply explore the pathogenesis of CC and find more potential molecular markers of CC.

| Glioma
Glioma is the most common primary intracranial tumour. Glioma can be classified into low-grade and high-grade subtypes. High-grade glioma is characterized by rapid invasive growth, vascular dysplasia and destruction of brain tissue, which make it difficult to treat and have a poor prognosis. 71 Currently, surgical resection with radiotherapy and chemotherapy is the standard of treatment for glioma patients, but the OS is not satisfactory. Therefore, it remains an urgent priority to identify the potential pathogenesis and treatment of glioma.
Du et al. 72 observed that circ-ZNF609 was upregulated in glioma. Elevated circ-ZNF609 expression led to increased migration and invasion of glioma cells both in vitro and in vivo whereas silencing circ-ZNF609 decreased migration and invasion of glioma cells.
Mechanistically, circ-ZNF609 could directly bind miR-1224-3p and further promote the expression of target gene PLK1. However, this paper relied only on the use of luciferase assays in the presence of mimics to explore the interacting miRNAs of circ-ZNF609, and more experiments such as pull-down assays can be added in future to increase the credibility.

| Hirschsprung's disease (HSCR)
HSCR is caused by the abnormal development of intestinal neurons.
The incidence of HSCR in males is four times higher than that in females, and HSCR is a common digestive tract malformation in paediatrics. At present, the absence of ganglion cells is generally believed to be related to a halt in the development of the intestinal nervous system, which is caused by disordered migration of neural crest cells, resulting in loss or abnormal development of ganglion cells from the intermuscular plexus of the intestinal wall and the submucosal nerve. 73 To date, fourteen HSCR-related genes have been identified, but most cases still cannot be explained at the mechanistic level. Thus, the molecular mechanism of HSCR remains to be further explored. 74 Peng et al. 75

| Vascular endothelial dysfunction
Vascular endothelial dysfunction plays a key role in the progression of various malignant, inflammatory, ischaemic, infectious and immunological diseases. 76,77 These diseases are associated with vascular endothelial barrier destruction, vascular leakage, inflammation response and angiogenesis. 78 Therefore, studying the mechanism of vascular endothelial dysfunction is of great value in the prevention and treatment of vascular complications.
Liu et al. 79 found that circ-ZNF609 was significantly upregulated in high-glucose and low-oxygen environment in vivo and in vitro.
Silencing of circ-ZNF609 reduced retinal vascular loss and pathological angiogenesis in vivo. Mechanistically, circ-ZNF609 might act as an endogenous miR-615-5p sponge to increase the expression of the downstream target gene MEF2A. Importantly, the authors detected a high level of circ-ZNF609 expression in clinical samples from patients with diabetes, hypertension and coronary artery disease, suggesting that targeted regulation of circ-ZNF609 expression is a potential approach for the treatment of vascular dysfunction.

| Corneal neovascularization (CNV)
Under normal circumstances, the cornea is in an avascular state, with peripheral blood vessels terminating at the limbus of the cornea and forming a vascular network. The avascular state of the cornea is the key to maintaining corneal transparency and ensuring good vision.
In the pathological state, new capillaries invade the cornea from the limbus, leading to the formation of CNV. According to statistics, 4.14% of ophthalmic patients in the United States exhibit CNV. 80 The formation and eventual blindness related to CNV caused by various ophthalmic diseases have become important problems in the ophthalmic field. 81 Therefore, explorations of the mechanism underlying CNV and identification of therapeutic targets are urgently needed.
Wu et al. 82 found that circ-ZNF609 and miR-184 intervention might be promising therapeutic measures for pathologic CNV.
They found that circ-ZNF609 is highly expressed in CNV tissues while miR-184 is lowly expressed. High expression of miR-184 can maintain the proliferation and migration of human corneal epithelial keratinocytes cells and inhibit the formation of blood vessels in vivo, while circ-ZNF609 exerted the opposite effect. In terms of the mechanism, circ-ZNF609 could interacted with miR-184 to regulate the downstream AKT and VEGF signalling pathways.

| Glaucoma
Glaucoma is the most irreversible blindness-inducing disease in the world and seriously affects the visual health of humans. It is estimated that the number of cases of glaucoma will increase to 112 million in 2040. 83 This is a disease characterized by progressive loss

| Neuropathic pain
Neuropathic pain is initiated and maintained by mediators released by neurons and glial cells that cause neuronal sensitization in the peripheral and central nervous systems. 87 Currently, the main treatment for neuropathic pain is pain relief with drugs. Approximately 50% of patients fail to achieve the expected effects, and many adverse events occur. 88 Therefore, it is of urgency to understand the pathogenesis of neuropathic pain and identify alternative therapeutic targets.
Li et al. 89 found that miR-22-3p and its downstream target ENO1 contribute to neuropathic pain by regulating inflammatory factors, such as TNFα, IL-1 and IL-6. Moreover, downregulation of miR-22-3p can promote the progression of neuropathic pain.
After further investigation, it was found that the upstream regulator of miR-22-3p was circ-ZNF609, and it was further found that circ-ZNF609 could promote the progression of neuropathic pain.
The discovery of the circ-ZNF609/miR-22-3p/ENO1 axis provides a new perspective for the study of neuropathic pain. However, the study was limited in a rat model without further exploration in human beings.

| Other non-cancerous diseases
With the development of high-throughput screening technology, high expression of circ-ZNF609 has been found in some other benign diseases. Christine Voellenkle et al. 90 found that four transcripts (CDYL, HIPK3, RTN4_03 and circ-ZNF609) were increased in biopsies of skeletal muscle from DM1 patients. Moreover, the expression of circ-ZNF609 was also increased in differentiated myogenic cell lines from DM1 patients. Zhang et al. 91 used circRNA microarray to detect the circRNA expression profiles in diabetic and nondiabetic human retinas, found that circ-ZNF609 was upregulated in DR. Luan et al. 92 reported the high expression of circ-ZNF609 in LN tissues compared to normal kidney tissues, which was significantly correlated with clinical parameters of LN disease activity. However, the above-mentioned studies only identified circ-ZNF609 expression patterns in these diseases, and the biological functions and mechanisms remain to be studied.

| CON CLUS I ON SANDFUTURE PER S PEC TIVE S
There are still some unsolved questions about circRNA biology. At present, we know little about the reasons why circRNAs are dysregulated in diseases. It has been speculated that the abnormal expression of circRNAs may be explained in part by genetic and/or epigenetic changes, such as mutations in spliceosomal genes, but evidence is lacking. 93 The second issue is the sensitivity of circRNAs For ceRNA mechanistic studies, the circRNAs should be quantified using a circRNA-specific method, such as RNase R and Actinomycin D assays. Then, Nuclear and cytoplasmic extraction assay and RNA FISH assay should be conducted to verify the subcellular localization of circRNA. As is known to all, circRNAs perform the function of ceRNA mainly in the cytoplasm. Besides, bioinformatics prediction, luciferase reporter assay, RNA immunoprecipitation and RNA pull-down should be conducted to explore circRNA-miRNA interactions.
However, the results presented in many original papers involved in this review always lack very important controls. Legnini et al. 8 demonstrated that circ-ZNF609 is related to heavy polysomes, and it could be translated into a protein in a splicing-dependent and capindependent manner, while most of the studies did not explore the protein-coding potential of circ-ZNF609. Besides, FISH assay could more intuitively explore the localization of circ-ZNF609 and miR-NAs, while the results of nucleus-cytoplasmic separation assay were not convincing. Moreover, most of the studies found the potential miRNAs which might bind to circ-ZNF609 via bioinformatics prediction and verified the two RNA molecules interact to each other by luciferase reporter and RNA Immunoprecipitation assay. However, pull-down assay should be performed to analyse the interacting miRNAs of circ-ZNF609, which is more convincing.
In several studies, the increased expression of circ-ZNF609 is accompanied by the decreased expression of miRNAs. The corresponding upregulation of downstream genes is difficult to be explained whether the sponge binding effect of circ-ZNF609 or the reduced degradation effect of miRNAs. Furthermore, the circ-ZNF609 mutant type should be constructed to be transfected with cell and explore its potential effects on cell function and downstream genes.
Recent studies have shown that circRNA is enriched in exosomes and plays an important role in intercellular communication. 95 Exosomal circRNAs are stable in blood. Serum exosomal circRNA has been well documented to distinguish cancer patients from healthy controls and can be used as a biomarker for liquid biopsy. 96 Shang et al. 97 revealed that the cancer-derived exosome circ-PACRGL plays an oncogenic role in the progression of CRC. Another study reported exosomal circ-SHKBP1 could promote GC progression and serve as a promising circulating biomarker for GC diagnosis. 98 However, the role of circ-ZNF609 in exosomes remains to be explored.
Taken together, these studies highlight the molecular mechanisms and biological functions of circ-ZNF609 in various diseases.
However, there are some limitations in these studies, such as a lack of rescue tests and limited sample size, and thus, the findings need to be further verified by independent cohort studies and multicentre studies. Besides, most of involved studies did not provide strong theoretical support to the sponge hypothesis and the experiments provided to support such models are based on inconclusive data. In addition, future studies should focus on its translational application in clinical diagnosis and prognosis assessments.

CO N FLI C TO FI NTE R E S T
The authors have declared that no competing interest exists.

DATAAVA I L A B I L I T YS TAT E M E N T
Data sharing is not applicable to this review article as no new data were generated or analysed in this study.