Diagnostic and prognostic value of circular RNAs in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the sixth most common malignant tumour, which has posed a heavy health and financial burden worldwide. Due to limited symptoms at the early stage and the limitation in current biomarkers, HCC patients are usually diagnosed at the advanced stage with a pessimistic overall survival rate. Circular RNAs (circRNAs) are a subclass of single‐stranded RNAs characterized by a covalently closed loop structure without 3’‐ or 5’‐end. With advances in high‐throughput sequencing technology and bioinformatics, accumulating studies have demonstrated the promotor or suppressor roles of circRNAs in the carcinogenesis, progression, and metastasis of HCC. Moreover, circRNAs are characteristic of higher abundance, stability and conservation compared with linear RNAs. Therefore, circRNAs have emerged as one of the most promising diagnostic and prognostic biomarkers for HCC with reliable accuracy, sensitivity and specificity. In this review, we briefly introduce the characteristics of circRNAs and summarize the roles of circRNAs in the biological procedures of HCC. Furthermore, we provide an overview on the potential diagnostic and prognostic value of circRNAs as biomarkers for patients with HCC. Finally, we discuss future perspectives of circRNAs in cancer research.

Circular RNAs (circRNAs) are a subclass of single-stranded RNAs characterized by a covalently closed loop structure. Base on whether they can be translated, circRNAs can be divided into non-coding circRNAs and coding circRNAs. 6 With the advances in high-throughput sequencing technology and bioinformatics, a variety of circRNAs have been demonstrated to play essential roles in regulating gene expression at transcriptional or post-transcriptional levels [7][8][9][10] and participate in multiple biological processes of HCC. 11 Moreover, owing to the single-stranded closed circular structure, circRNAs show higher abundance, stability and conservation compared with linear RNAs. These advantages make cir-cRNAs one of the most promising biomarkers in the diagnosis and prognosis of HCC. 12,13 In this review, we briefly introduce the characteristics of cir-cRNAs and summarize their roles in the biological processes of HCC.
Furthermore, we provide an overview on the potential diagnostic and prognostic value of circRNAs as biomarkers. Finally, we give an insight into future perspectives of circRNAs in cancer research.

| CHAR AC TERIS TI C S OF CIRCRNA S
CircRNAs were initially discovered as aberrant by-products or abnormally spliced transcripts in human cells, while their specific roles remained vague due to the limitation of traditional RNA sequencing technologies. 14,15 Owing to the improvement of specialized computational pipelines in the last decade, accumulating information has been gathered in this area, and the roles of circRNAs in various diseases are becoming increasingly evident. [16][17][18][19] CircRNAs are single-stranded transcripts arisen from exons (ecircRNAs), 20 introns (ciRNAs) 21 or both (EIciRNAs) 18,22 (Figure 1).
CircRNAs are extensively expressed in eukaryotic cells and characterized by high abundance, stability, conservation, as well as a tissueor developmental-specific expression pattern. 11,[23][24][25] The covalently closed circular structure makes circRNAs more stable and resistant to exonuclease-induced degradation compared with linear RNAs. 26 Moreover, circRNAs display evolutionary conservation across multiple species and the expression profiles are tissue-or developmental stage-specific, suggesting the broad participation in various physiological and pathophysiological processes. 23,[27][28][29] Accumulating evidence has revealed the function of circRNAs in regulating gene transcription and expression, and the miRNA sponge activity is the hallmark function of circRNAs. 30 CircRNAs have a significant number of binding sites for miRNAs and can act as sponges of miRNAs, which would subsequently influence the stability and translation of target RNAs. 31 Apart from miRNA sponge activity, circRNAs can also interact with RNA binding proteins, 32 function as transcriptional or translational regulators, 22 influence splicing of pre-mRNAs 33 and participate in protein translation. 34,35

| ROLE S OF CIRCRNA S IN H CC
Recently, the knowledge of circRNAs is accumulating at an increasing pace, and plenty of studies have revealed the association between circRNAs and various biological processes in cancers, including cell proliferation, apoptosis, invasion, as well as metastasis. In HCC, numerous circRNAs are dysregulated in tumour tissues and may play oncogenic or suppressor roles in cancer development and progression. [36][37][38][39][40][41][42][43] Generally, most of the up-regulated circRNAs are positively associated with HCC progression, whereas down-regulated circRNAs usually act as suppressors preventing the development of HCC. Table 1 summarizes recent studies on the regulating effect of circRNAs in HCC. [44][45][46][47][48][49][50][51][52][53][54] Cdr1as (hsa_circ_0001946), a sponge of miR-7, is one of the classic circRNAs acting as a promotor in the progression of HCC.
Cdr1as was reported to be significantly up-regulated in HCC cell lines and HCC tissues compared with the non-tumour ones, and the expression level of Cdr1as was positively associated with hepatic microvascular invasion as well as deterioration. 55,56 As a target of Cdr1as, miR-7 targets several oncogenes directly, and the up-regulation of miR-7 inhibits the proliferation and invasion of HCC cells. 57 Moreover, knockdown of Cdr1as would suppress the HCC cell proliferation and invasion, 56 while overexpression of Cdr1as could lead to the opposite effect. 58 Importantly, exosomes acquired from HCC cells overexpressing Cdr1as could also accelerate the proliferation and migration of surrounding normal cells. 58 Guan et al 59

| D IAG NOS TI C VALUE OF CIRCRNA S IN H CC
Due to limited symptoms at the early stage and the limitation in current biomarkers, HCC patients are usually diagnosed at the advanced stage with a pessimistic overall survival rate. 65 Moreover, current biomarkers, such as α-fetoprotein (AFP) and AFP-L3, show modest diagnostic performance for HCC. 65 Since circRNAs are closely as-

| PROG NOS TI C VALUE OF CIRCRNA S IN H CC
Since circRNAs are involved in multiple biological processes in HCC, the prognostic value of circRNAs also attracts wide attention. hazards among hsa_circ_000520 in patients with HCC. And the Cox multivariate analysis indicated the expression level of hsa_ circ_000520 as an independent prognostic factor of RFS. 71 Similarly, low expression of circTRIM33-12 was closely correlated with poor prognosis and was demonstrated as an independent predictor for OS (P = .001 in univariate analysis; P = .007 in multivariate analysis) as well as post-operative recurrence (P = .001 in univariate analysis; P = .005 in multivariate analysis). 64 In addition, circASAP1, a competing endogenous RNA for miR-326 and miR-532-5p, was closely associated with pulmonary metastasis of HCC after curative resection. 74

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
HCC is the sixth most common malignant tumour with a pessimistic survival, 76 which accounts for approximately 90% of primary liver cancers. Due to limited symptoms at the early stage and the lack of satisfying biomarkers, more than 75% of HCC patients are diagnosed at the advanced stage with a reduced overall survival rate. 65 Despite the advances in treatment strategies against HCC, such limitation makes HCC remain one of the most lethal cancers, which accounts for at least 700,000 deaths worldwide annually. 77

ACK N OWLED G EM ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.