An update on the roles of circular RNAs in osteosarcoma

Abstract Osteosarcoma is the most common primary bone malignancy and is a neoplasm thought to be derived from the bone‐forming mesenchymal stem cells. Aberrant activation of oncogenes and inactivation of tumour suppressor genes by somatic mutations and epigenetic mechanisms play a pivotal pathogenic role in osteosarcoma. Aside from alterations in these protein‐coding genes, it has now been realized that dysregulation of non‐coding RNAs (ncRNAs), including microRNAs (miRNAs), long non‐coding RNAs (lncRNAs) and the recently discovered circular RNAs (circRNAs), is crucial to the initiation and progression of osteosarcoma. CircRNAs are single‐stranded RNAs that form covalently closed loops and function as an important regulatory element of the genome through multiple machineries. Recently, an increasing number of studies suggested that circRNAs also played critical roles in osteosarcoma. This review summarizes recent development and progression in circRNA transcriptome analysis and their functions in the modulation of osteosarcoma progression.


| INTRODUC TI ON
Osteosarcoma, a neoplasm thought to be derived from the bone-forming mesenchymal stem cells, is the most common primary bone malignancy, predominantly involving metaphyseal regions of the long bones (eg, proximal end of tibia or humerus and distal end of femur) which are the most rapidly growing parts of the skeleton in children and adolescents. [1][2][3][4][5] The incidence of osteosarcoma follows a bimodal age distribution with ages between 10 and 30 primarily affected. [6][7][8][9][10] The second peak appears in the elderly in which osteosarcoma very often comes as a later cancer secondary to radiation exposure or is associated with Paget disease (a disorder of bone remodelling resulting in structural weakening). [11][12][13][14][15][16] With the advent of neoadjuvant and adjuvant chemotherapy with cisplatin, doxorubicin and high-dose methotrexate and the advances in surgery, the 5-year survival rate of patients with osteosarcoma has improved from ~20% before the 1980s to currently ~ 70%. [17][18][19][20][21][22] Nevertheless, half of the patients still do not survive for longer than 10 year. [23][24][25] Thus, it is important to identify new therapeutic targets for tackling this devastating and potentially fatal disease ( Figure 1).
Previously, Zhang et al 68 elegantly summarized the roles of circRNAs in osteosarcoma. As circRNA research is a rapidly evolving field, over a dozen of original articles related to circRNAs in osteosarcoma have been published since then. It is therefore timely to update the recent progress.
In this review, we first summarize circRNA profiling studies on osteosarcoma so as to provide a comprehensive curation of available datasets which may facilitate subsequent studies, followed by in-depth discussion of functions and mechanisms of action of newly discovered osteosarcoma-related circRNAs not covered by the review of Zhang et al. 68 The potential diagnostic, prognostic and therapeutic utilities of circRNAs in the clinical management of osteosarcoma are also addressed.

| circRNA E XPRE SS I ON PROFILING AND INTEG R ATIVE ANALYS IS IN OS TEOSARCOMA
Profiling circRNA expression using microarray or whole-transcriptome sequencing followed by validation with reverse transcriptionquantitative PCR (RT-qPCR) is the most frequent method to identify and confirm deregulated expression of circRNAs in specific disease states. [69][70][71][72] In this connection, attempts have been made to profile circRNAs specific to osteosarcoma or related to its resistance to chemotherapy.
Liu et al 65  Pathway analyses of the differentially expressed circRNAs and their parental genes revealed biological processes pertinent to chemoresistance, including glycolysis/gluconeogenesis, ABC transporters, and vascular endothelial growth factor signalling ( Table 1).

| CircTCF25
circTCF25 was first identified as an oncogenic circRNA in bladder carcinoma. 77 Wang et al reported that the expression of circTCF25 was significantly higher in osteosarcoma tissues as compared with morphologically normal bone tissues from the same patients.

| CircPVT1
CircPVT1 is a circRNA derived from the genomic region that also encodes the oncogenic lncRNA PVT1. 79 Liu et al 80

| Circ_001621
Circ_001621 was identified as the top upregulated circRNA in osteosarcoma by PCR array, and its high expression was associated with more advanced TNM staging and shortened overall survival. 74 In this regard, miR-578 was found to be the target of circ_001621

| Circ_0001658
Circ_0001658 was found to be upregulated in osteosarcoma tissues as compared with normal bone tissues. Functional experiments suggested that circ_0001658 promoted the proliferation, migration, and invasion of osteosarcoma cells and impeded apoptosis. These pro-tumorigenic effects were mediated by sponging miR-382-5p and the subsequent depression of YB-1. Taken together, circ_0001658 is an oncogenic circRNA via modulating the miR-382-5p/YB-1 axis. 86

| Circ_0102049
The expression of circ_0102049 was found to be higher in osteosarcoma tissue samples as compared with their paired non-cancerous tissues. 87

| CircEPSTI1
Tan et al 88 reported that circEPSTI1 was significantly upregulated in osteosarcoma where knockdown of this circRNA suppressed cell proliferation and migration, suggesting an oncogenic role.
Mechanistically, circEPSTI1 upregulated the expression of MCL1 (myeloid cell leukaemia 1) via reducing the availability of miR-892b. 88 These data suggested an important role for circEPSTI1-miR-892b-MCL1 axis in the progression of osteosarcoma.

| Circ-0060428
Cao and Liu reported that Circ-0060428 was expressed at significantly higher levels in osteosarcoma cell lines (U2OS, 143B, SAOS-2, HOS) as compared with the human normal osteoblast hFOB1. 19. 89 Functionally, knockdown of circ-0060428 induced apoptosis of U2OS and HOS osteosarcoma cells, suggesting this circRNA functioned as an oncogenic circRNA. Mechanistic studies found that circ-0060428 bound to miR-375 and thereby depressing RBPJ (a transcription factor positively regulating Notch signalling). In this connection, miR-375 inhibitor nullified the pro-apoptotic effect of circ-0060428 knockdown. 89 These findings suggest that upregulation of circ-0060428 could confer apoptosis resistance to osteosarcoma cells by sponging miR-375. Nevertheless, whether RBPJ is functionally involved in the oncogenic action of circ-0060428 remains unclear. Moreover, the upregulation of circ-0060428 needs to be confirmed in human osteosarcoma tissues.

| CircMYO10
Chen et al 91 illustrated that miR-370-3p was decreased and circ-MYO10 was overexpressed in osteosarcoma samples and cells.
Inhibition expression of circMYO10 suppressed EMT development and growth through regulating miR-370-3p/RUVBL1/β-catenin/ LEF1 axis. These results revealed that circMYO10 played as one oncogene role in progression of osteosarcoma.

| CircLRP6
Previous study noted that circLRP6 level was overexpressed in osteosarcoma samples and was correlated with poor overall survival and shorter disease-free survival. 92 Knockdown of circLRP6 inhibited cell migration, invasion and growth and induced cell cycle arrested and apoptosis. The interaction between EZH2 and LSD1 with circLRP6 regulates their binding to promoter regions of APC and KLF2. Inhibition expression of circLRP6 decreased binding abilities of EZH2, LSD1 to APC, KLF2. Furthermore, the oncogene effect of circLRP6 on osteosarcoma can be reversed with APC. These data indicated that circLRP6 served as one oncogene through binding to EZH2 and LSD1 to suppress APC and KLF2 expression.

| CircSAMD4A
Zhao et al 67 noted that circSAMD4 was upregulated in osteosarcoma specimens compared with adjacent non-cancerous specimens.
Ectopic expression of circSAMD4A induced osteosarcoma cell growth in vitro and in vivo and increased cells stemness characteristics. Moreover, they illustrated that circSAMD4A sponged miR-1244 and identified MDM2 was one direct target gene of miR-1244. Their data illustrated that miR-1244/MDM2/ circSAMD4A modulator loop may be one treatment target for osteosarcoma.

| Circ-0001785
Previous reference noted that circ-0001785 was overexpressed in cell lines of osteosarcoma. 93 Downregulation expression of circ-0001785 inhibited cell growth and enhanced osteosarcoma cell apoptosis. In addition, they showed that circ-0001785 can sponge miR-1200 expression and suppress HOXB2 expression, which is one target gene of miR-1200. They showed that circ-0001785 modulated Bcl-2 pathway and Akt/PI3K signalling in the osteosarcoma. These results suggested that circ-0001785 modulates osteosarcoma pathogenesis via regulating miR-1200 to enhance HOXB2 expression.

| Circ_0000885
Zhu et al 97 indicated that circ_0000885 expression was upregulated in serum samples and tissue from osteosarcoma patients and higher expression of circ_0000885 was correlated with Enneking stage.
Cases with high tumour and serum levels of the circ_0000885 had the lower rates of overall survival and disease-free survival. These

| Circ0021347
The expression of circ0021347 was lower in human osteosarcoma tissues and cell lines than their normal counterparts. In patients with osteosarcoma, the low expression of circ0021347 was associated with more advanced tumour-node-metastasis (TNM) staging and shortened overall survival, suggesting circ0021347 functions as a tumour suppressor. 98 Nevertheless, the mechanism of action of circ0021347 is presently unclear.

| Circ_0000190
Circ_0000190 has been shown to inhibit the progression of multiple myeloma through sponging miR-767-5p. Li

| Circ-LARP4
Hu et al 100 showed that circ-LARP4 level was decreased in osteosarcoma tissues compared to non-tumour samples and circ-LARP4 was associated with Enneking stage. Patients with high circ-LARP4 level indicated enhanced tumour cell necrosis rates to chemotherapy after resection. The higher expression of circ-LARP4 was associated with prolonged overall survival and disease-free survival.
Ectopic expression of circ-LARP4 enhanced MG63 cells chemosensitivity to doxorubicin and cisplatin but not the methotrexate.
Their data suggested that high expression of circ-LARP4 was associated with prolonged survival profiles and decreased Enneking stage and overexpression of circ-LARP4 enhanced chemosensitivity to doxorubicin and cisplatin through sponging miR-424 in development of osteosarcoma.

| Circ-ITCH, a circRNA with an ambiguous role
Circ-ITCH was found to act as a tumour-suppressing circRNA in oesophageal squamous cell carcinoma, 101 colorectal cancer 102 and lung cancer. 103 In contrast with its inhibitory role in these cancer types, Li et al 104  of cir-ITCH. 104 Ren et al 105 also found that circ-ITCH played asa tumour suppressor in progression of osteosarcoma by modulating miR-22 (Tables 2 and 3; Figures 2 and 3).

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
Osteosarcoma is one multi-factor and multi-step comprehensive disease, pathogenesis mechanism is still unclear, and more efforts are in urgent need. CircRNAs function as an important regulatory element of the genome via several machineries, including transcription regulation, modulation of alternative splicing, sponging of miRNAs and direct interactions with RNA-binding proteins. CircRNAs may play as tumour suppressors or activators in tumour and have been applied in modulating cancer metastasis and chemoresistance. As summarized in our review, circRNAs are acting critical roles in many biological processes in the procedures in the progression and development of osteosarcoma including cell apoptosis, invasion, growth, differentiation, migration, drug resistance and cachexia. As one crucial biomarker for prognosis and diagnosis of osteosarcoma, circRNA is one potential target for therapy of osteosarcoma. This use of cir-cRNAs as therapeutics has vast tremendously and may supply a new method to treat osteosarcoma.
Here are some future suggestions for circRNAs study. Firstly, more exact mechanisms about how circRNAs participated in progression of osteosarcoma need to further research, potentially via using online databases. Secondly, further study on circRNAs in osteosarcoma needs to use non-invasive specimens such as saliva, urine and blood. Thirdly, since the purpose of circRNA study is to F I G U R E 3 The representative diagram of circRNA mediated ceRNA network and oncogene signalling pathways. The current study in osteosarcoma highlights the regulatory relationship of the circRNA-miRNA-mRNA network for different signalling pathways. Multiple identified circRNAs function as miRNA sponge and subsequently upregulate relevant target genes expression level use circRNAs for clinical application for osteosarcoma, generous clinical markers experiments and cell function mechanism studies are required in future.

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