Translation role of circRNAs in cancers

Abstract Circular RNAs (circRNAs) constitute a class of covalently closed RNA molecules. With the continuous advancement of high‐throughput sequencing technology and bioinformatics tools, many circRNAs have been identified in various human tissues and cell lines. Notably, recent studies have indicated that some circRNAs have translational functions. Internal ribosome entry sites and the N6‐methyladenosine modification mediate cap‐independent translation. This review describes these two translation mechanisms and verification methods at the molecular level. Databases (including ORF Finder, Pfam, BLASTp, CircRNADb, CircBase, CircPro, CircCode, IRESite, IRESbase) were used to analyze whether circRNAs have the structural characteristic of translation. CircRNA minigene reporter system containing green fluorescent protein (GFP) confirmed the translation potential of circRNAs. Also, we briefly summarize the roles of proteins/peptides encoded by circRNAs (circFBXW7, circFNDC3B, circLgr4, circPPP1R12A, circMAPK1, circβ‐catenin, circGprc5a, circ‐SHPRH, circPINTexon2, circAKT3) that have been verified thus far in human cancers (triple‐negative breast cancer, colon cancer, gastric cancer, hepatocellular carcinoma, bladder cancer, glioblastoma). Those findings suggest circRNAs have a great implication in translation of the human genome.

regulating the translation function of circRNAs. [10][11][12] One involves internal ribosome entry sites (IRESs), 13 and the other involves the N 6 -methyladenosine (m 6 A) modification 14 ; both are potential mechanisms for cap-independent translation of circRNAs. In other words, cap-independent translation of circRNAs greatly expands our understanding of the biological functions of circRNAs and provides new perspectives for cancer treatment.

| Verification of circRNAs translation
In general, traditional translation can be initiated not only by the initiation codon (AUG), a suitable translation sequence and open reading frames (ORFs), but also by a dissociative 5′ end. 10 Therefore, novel elements are essential for activating cap-independent translation. To easily understand the requirements for cap-independent translation, several databases are listed for reference (Table 1). First, the ORFs with coding potential in circRNAs must be identified. ORF Finder 15 is a graphical sequence analysis tool that can search for all possible ORFs and deduce the translated amino acid sequence.
This amino acid sequence is entered into BLASTp or Pfam tools 16 to confirm that the original search result was successful. Second, a comprehensive circRNA database that can better evaluate the coding potential of circRNAs is accessed. CircRNADb 17 includes circRNA genome sequence, IRES, and ORF information. CircBase 18 is constructed through the collection and integration of published circRNA data, which can quickly return circRNA information and supporting evidence. CircPro 19 can also be used to predict and identify circRNAs with coding potential. CircCode 20 recognizes translatable circRNAs in ribose sequence reads (ribo-seq). Finally, IRES is identified. IRESite 21 contains many published and verified IRESs and provides experimental evidence of these IRESs. IRESbase 22 includes IRESs related to circRNAs and long noncoding RNAs (lncRNAs), and the function of an IRES has been experimentally verified.
In addition to using bioinformatics tools, Yang Y et al used the circRNA minigene reporter system to identify functional translation mediated by IRES and m 6 A. 23

| Translation mediated by IRESs
Internal ribosome entry site-mediated cap-independent translation is a relatively mature mechanism currently studied. CircRNAs with ORFs and IRESs upstream can be effectively translated by this mechanism in various human cancers ( Figure 2).

A circRNA-translated protein in triple-negative breast cancer (TNBC)
Breast cancer is a common type of malignant tumor and the second leading cause of cancer mortality among women worldwide; TNBC is known as the subtype with the worst prognosis. 27 Therefore, it is necessary to explore possible mechanisms underlying the progression of TNBC. A study found that circFBXW7 blocks miR-197-3p and that the spanning junction ORF of circFBXW7 encodes FBXW7-185aa, 28 mediated by IRES, regulates the expression of FBXW7 in TNBC, and exhibits a tumor suppression effect. Therefore, circF-BXW7 and FBXW7-185aa may be potential therapeutic targets.

CircRNA-translated proteins/peptides in colon cancer (CC)
Colon cancer is the second leading cause of cancer-related death worldwide, and it is a matter of great urgency for researchers to develop more effective molecular targets for CC therapy. 29 A study reported that circFNDC3B can encode a novel protein, circFNDC3B-218aa. 30

A circRNA-translated protein in hepatocellular carcinoma (HCC)
Hepatocellular carcinoma (HCC) has a high mortality rate, which is attributed to a lack of efficient diagnostic and therapeutic tools. 34 The Wnt/β-catenin pathway extensively participates in tumor growth, but the reason it is overactivated in HCC is still unknown. 35 A recent study showed that circ-0004194 (circβ-catenin) encodes a new protein, β-catenin-370aa, which is a subtype of β-catenin. 36 Additionally, circβ-catenin regulates the expression of β-catenin at the protein level, not at the transcription level. β-catenin-370aa acts as bait for GSK3β and binds to it, thereby reducing the ubiquitination

CircRNA-translated proteins/peptides in glioblastoma
Glioblastoma is one of the most lethal human cancers that may occur at any age and exhibits an extremely poor response to approved therapies. 39,40 Obviously, new targets/treatments urgently need to be explored. The FBXW7-185aa protein encoded by circFBXW7 plays a role not only in the aforementioned TNBC, 28 as mentioned above, but also in glioblastoma. 41 C-Myc is a key regulator of tumorigenesis. FBXW7-185aa competitively interacts with USP28 to prevent the binding of USP28 and FBXW7α and then promotes c-Myc ubiquitination and degradation. Therefore, FBXW7-185aa inhibits the proliferation and delays cell cycle progression of glioma cells.
Circ-SHPRH and its encoded protein SHPRH-146aa are highly expressed in normal human brains. 42

| Translation mediated by m 6 A modification
CircRNAs modified by m 6 A can also undergo cap-independent translation. 23  proteins. 46,50 The protein containing the YTH domain was the first reader to be recognized. YTHDC1 has been proven to regulate the reverse splicing and export of circRNAs. 51,52 YTHDF1 was found to increase translation efficiency through the binding of m 6 A and YTHDF3. 53,54 Then, YTHDF3 and eIF4G 2 physically associate with endogenous circRNAs to promote the coding of proteins and control cell proliferation. Furthermore, translation from circRNAs is weakened when m 6 A demethylates fat mass and obesity-associated protein (FTO). 23,55 A study reported that oncogenic human papillomavirus 16 (HPV16) generates circE7, which translated to produce E7 oncoprotein in CaSki cervical carcinoma cells. 56 The expression level of E7 oncoprotein affects cancer cell growth both in vitro and tumor xenografts, but the exact carcinogenic mechanism of E7 peptide remains uncertain.
In summary, in addition to the IRES-dependent pathway (Table 2), the m 6 A residues in circRNAs can serve as m 6 A-induced internal ribosome-binding sites (MIRES), thereby promoting capindependent translation (Figure 3). 14

| PER S PEC TIVE S
The biological functions of the proteins/peptides encoded by circR-NAs have begun to emerge. We summarized the coding functions of circRNAs in human tumors that have been confirmed thus far and the tumor-promoting or tumor-inhibiting effects of their products.
The idea that circZNF609 57 in myogenesis and circMBL 58  Therefore, circRNAs have great potential for use in disease treatment.
In the near future, more circRNA-encoding functions will be verified, and other possible encoding mechanisms will even be discovered.
Although the future of circRNA translation function is very impressive, there are still some problems that need to be considered in depth. For example, the current understanding of the cap-independent translation mechanism of circRNAs is still unsatisfactory. Are other coding mechanisms possible? The proteins/ peptides encoded by circRNAs may serve as new drugs for the treatment of cancers due to their high specificity, low toxicity, and clear biological function. However, their poor stability and short half-life also greatly limit their clinical application. Therefore, these problems require further research to be solved.

ACK N OWLED G M ENTS
This work was supported by grants from Zhejiang Natural Science

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data presented in this study can be found in online repositories.
The name of repositories and reference number can be found in the review.