Recent Advances of Circular RNAs as Biomarkers for Osteosarcoma

Introduction

Osteosarcoma is the most common primary malignant bone tumor in young adult, which is prone to early metastasis and poor prognosis. Because the early diagnosis rate is still low, clinical diagnosis and treatment delay seriously affects patients’ diagnosis, treatment, and prognosis. Compared with invasive procedures such as tissue biopsy, liquid biopsy is portable and straightforward and can be used in early screening and prognosis analysis. Therefore, it is of great significance to find biomarkers for the early diagnosis of osteosarcoma. Circular RNA (circRNA) is a covalently blocked noncoding RNA. There are differences in expression profiles between osteosarcoma patients and healthy people. It is closely related to the occurrence, development, metastasis, and drug resistance of osteosarcoma. It suggests that target regulation of circRNA may become a breakthrough in the treatment of osteosarcoma. Liquid biopsy, circRNA-based prognosis analysis, circRNA-based targeted therapy, etc will be a breakthrough in the individualized diagnosis and treatment of osteosarcoma. In this paper, we reviewed the mechanism of circRNA in the development of osteosarcoma and its clinical application in recent years.

Definition of Circular RNA

Circular RNA (circRNA) is a covalently blocked endogenous non-coding RNA. It was first found in plant viroids,¹ yeast mitochondrial RNA,² and δ Hepatitis B virus.³ Later, circRNAs were accidentally found in exons or introns, which were considered by-products of splice body mediated splicing errors. With the further researches, their functions are gradually discovered. A new type of endogenous non-coding RNA molecule is circRNA, which widely and stably exists in eukaryotic cells. It does not have a 5’ terminal cap and a 3’ terminal poly (a) tail and forms a closed-loop structure with covalent bonds. Genome-wide annotation shows that circRNA is widely expressed in different cell lines and species,^3,4 with different expression levels, and is widely involved in the occurrence and development of various diseases such as cancer.

Formation of Circular RNA

Different from the standard splicing of linear RNA connecting upstream (5 ‘) splice donor site with downstream (3’) splice acceptor site, circRNA is produced by reverse splicing of precursor mRNA (connecting downstream splice donor site with upstream splice acceptor site) or exon jumping.⁵ CircRNA is cyclized into a continuous closed loop structure, lacking poly(a) tail, and a 5 ‘cap. According to its composition, circRNA can be divided into four categories: exonic circRNA (ecircRNA), exon-intron circRNA (EIciRNA), circular intronic RNA (ciRNA), and tRNA intronic circRNA (tricRNA). At present, three mechanisms of circRNA biogenesis have been widely accepted: RNA binding protein (RBP)-mediated cyclization, intron pairing driven cyclization, and lasso protein driven cyclization. RBP mediated and intron pairing driven cyclization of circRNA occurs through direct reverse splicing. RBP plays an essential role in promoting circRNA biogenesis by regulating adjacent splice sites. In addition, cyclization can be achieved by including reverse complementary sequences. Finally, we promote lasso-driven cyclization by exon jumping events. Internal splicing helps remove the flanking intron sequence and produce ecircRNA; If these flanking sequences are retained, the construct is called EIciRNAs. In addition, ciRNA is generated by lasso-driven cyclization. TricRNA is produced by combining released intron ends produced from spliced pre-tRNA by tRNA splicing endonuclease complex.⁶

Molecular Characteristics of Circular RNA

CircRNAs were first identified in RNA viruses in 1976. They were once considered as “splicing noise” in organisms. With the development of high-throughput sequencing technology and bioinformatics, they have increasingly become a research hotspot. Unlike linear RNA, due to the lack of poly (a) tail, circRNA is stable and resistant to exonuclease (including RNAse R). Its half-life is longer than its corresponding linear RNA, and it can stably exist in various tissues and organs.⁷ Its covalent closed loop structure makes it stable in plasma and exosomes. It can be used as a diagnostic marker of tumors and other diseases.⁸

Identification of Circular RNA

In recent years, a new method, RNAse R processing, has been developed to enrich high purity circRNA by polyadenylation and poly (a) + RNA depletion (RPAD). In this method, RNAse R is first used to treat total RNA to deplete linear RNA. The remaining 3 ‘- OH terminal RNA was polyadenylated, consumed by poly (a) + RNA, and passed through oligomeric (DT) beads. RPAD method eliminates the interference of linear RNA and dramatically improves data reliability.⁹ The presence of circRNA could not be guaranteed by the circRNA ligation detected by qPCR. If two consecutive exons have very similar sequences or a given RNA is a transcribed product that leads to exon replication or reorganization, these atypical splicing points may originate from linear RNA.¹⁰

Moreover, template conversion activity from reverse transcriptase can also lead to repetitive sequences in cDNA. Therefore, circRNA validation is a key step in any circRNA research. Strict circRNA verification usually requires 3 ‘exonuclease RNAse R and Northern blotting. Compared with RT qPCR, this is a more accurate method to fully express the RNA types in a given exon.¹¹ Although RT qPCR is a better quantitative method, it can not identify the true circRNA. It is essential that, since RNase R processing results in a significant change in the concentration and composition of RNA mixtures to be RT-qPCR, adequate internal controls (ie, incorporation of known amounts of foreign RNA) must be used to evaluate RNase R sensitivity of specific circRNA candidates. Another essential step in circRNA validation is to confirm the existence of trans-splicing by Sanger sequencing.

Function of Circular RNA

Circular RNA has many functions in cells^12,13 (Figure 1). (A) CircRNA acts as a miRNA sponge with multiple miRNA binding sites that indirectly control gene expression and play a competitive endogenous RNA role. (B) CircRNA acts as a transcriptional regulator by binding to RNA polymerase II. (C) CircRNA interacts with proteins. In humans and Drosophila, RBP (MBL) binds to circRNA, which plays a role in gene regulation by competing with linear splicing.¹⁴ In mice, cell cycle-related proteins bind to circRNA to enhance p21/CDK2 interaction and block cell cycle progression.¹⁵ (D) CircRNA containing open reading frame (ORF) and stop codon in the frame is translated into protein in a splicing dependent and cap-dependent manner. (E) Other functions: regulating intercellular signaling pathways, affecting cell differentiation, forming pseudogenes, and other functions.^16–18

Figure 1 Function of circular RNA; potential application of circular RNA in the diagnosis and treatment of osteosarcoma.

The Role and Regulatory Mechanism of Circular RNA in the Occurrence and Development of Osteosarcoma

Osteosarcoma (OS) is one of the most common malignant bone tumors. It originates from bone mesenchymal cells. The production of a bone-like matrix characterizes it and occurs in adolescents aged 10–20 years old. The most common sites are the distal femur, proximal tibia, and proximal humerus metaphysis. It is highly malignant and prone to local recurrence and metastasis. The prognosis of patients is poor. Somatic mutation and epigenetic mechanism play an important role in abnormal activation of oncogenes (such as cps3, PIK3CA, etc.) and inactivation of tumor suppressor genes (such as TP53, BRCA1/2, etc.).¹⁹ In addition to the changes in these protein-coding genes, the role and regulatory mechanism of circRNA in the occurrence and development of osteosarcoma have become a hot spot in recent research (Table 1).

Table 1 Expression and Association of CircRNAs in Osteosarcoma

Carcinogenic Circular RNA

CircTCF25

The first discovery of circTCF25 is the carcinogenic cyclic RNA in bladder cancer.²⁰ Functionally, the higher expression of circTCF25 promotes the proliferation, migration, and invasion of osteosarcoma cells, accompanied by the corresponding changes of phenotype-related proteins (up-regulation of CyclinD1 and Cdk6 to increase cell proliferation; up-regulation of CyclinD1 and Cdk6 to increase cell proliferation; up-regulation of CyclinD1 and Cdk6 to increase cell proliferation); Up-regulation of MMP2, MMP9, and vimentin, down-regulation of TIMI-1 enhance cell migration and invasion. At the same time, overexpression of circTCF25 can reduce the level of miR-206 in osteosarcoma cells. In this regard, MEK/ERK and AKT/mTOR pathways were identified as downstream pathways inhibited by miR-206.²¹

CircMMP9

Studies have shown that circMMP9 promotes the occurrence and development of glioblastoma.²² Pan et al²³ found that the expression level of circMMP9 in osteosarcoma was high, which may lead to the rapid development of tumor and is related to TNM staging. TNM (tumor–node–metastasis) staging system codes the extent of the primary tumor (T), regional lymph nodes (N), and distant metastases (M) and provides a “stage grouping” based on T, N, and M.²⁴ The overall survival rate of osteosarcoma patients with high expression of circMMP9 was also lower than that of patients with low expression of circMMP9. Functionally, knockout of circMMP9 decreased the viability, colony-forming ability, migration, and invasion ability of osteosarcoma cells, suggesting that circMMP9 has a carcinogenic effect. It was found that the carcinogenesis of circMMP9 was mediated by sponging miR-1265 and decreased subsequent inhibition of CHI3L1 (chitinase-3-like protein 1). MiR-1265 was directly bound to the 3ʹ UTR of CHI3L1 mRNA and inhibited its expression. CHI3L1 levels were regulated by circMMP9/miR-1265 axis.

Circ001621

Circ001621 was identified as an upregulated circRNA in osteosarcoma by PCR array. Its high expression was associated with a higher TNM stage and shorter overall survival.²⁵ In this regard, miR-578 was the target of circ001621 in osteosarcoma, and their expression showed a strong negative correlation. Functional experiments and mechanism studies showed that circ001621 promoted the proliferation and migration of osteosarcoma cells in vitro by inhibiting miR-578 mediated VEGF inhibition and CDK4 and MMP9 expression. Significantly, circ001621 enhances the metastatic ability of osteosarcoma to the lung and liver and upregulates VEGF, CDK4, and MMP9.²⁵ In conclusion, circ001621 is a carcinogenic circular RNA that promotes osteosarcoma metastasis by regulating the miR-578/VEGF pathway.

Circ0001658

Circ0001658 was found to be upregulated in osteosarcoma compared with normal bone tissue. Functional experiments showed that circ0001658 promoted the proliferation, migration, and invasion of osteosarcoma cells and hindered apoptosis. These tumor-promoting effects are mediated by sponging miR-382-5p and increasing the expression of YB-1. There was a negative expression correlation between circ0001658 and miR-382-5p as well as miR-382-5p and YB-1. The expression of circ0001658 was positively correlated with YB-1. In conclusion, circ0001658 is a circular RNA that can promote cancer by regulating the miR-382-5p /YB-1 axis.²⁶

CircEPSTI1

Tan et al²⁷ reported that circEPSTI1 was significantly upregulated in osteosarcoma. Knockout of circRNA inhibits cell proliferation and migration, suggesting its carcinogenic effect. CircEPSTI1 upregulates the expression of MCL1 by reducing the availability of miR892b. These data suggest that the circEPSTI1-miR-892b- MCL1 axis plays a vital role in osteosarcoma progression.

CircANKIB1

Du et al²⁸ showed that circANKIB1 increased miR-19b expression in osteosarcoma cells, enhanced the expression of circANKIB1-miR-19b, and inhibited the expression of the downstream target gene SOCS3. After knockout of circANKIB1-miR-19b, the body inhibits cell invasion and growth by regulating the STAT3 Pathway, thus inducing tumor cell apoptosis. These data suggest that circANKIB1 is an oncogene and a potential therapeutic target for osteosarcoma.

Carcinostatic Circular RNA

CircLARP4

Hu et al²⁹ showed that the level of circLARP4 in osteosarcoma tissue was lower than that in non-tumor tissue, and circLARP4 was related to the Enneking stage. In patients with high circLARP4 levels, the necrosis rate of tumor cells increased after resection and chemotherapy. High expression of circLARP4 was associated with prolonged overall survival and disease-free survival. Overexpression of circLARP4 enhanced the chemosensitivity of MG63 cells to adriamycin and cisplatin but did not affect the chemosensitivity of MG63 cells to methionine. In addition, overexpression of miR-424 can reduce the expression of circLARP4, thus affecting the chemosensitivity and therapeutic effect of MG63 cells. Their data suggest that overexpression of circLARP4 shortens the Enneking phase by sponging miR-424, down-regulate the expression of miR-424, and overexpression of circLARP4 can enhance the chemosensitivity of cells to adriamycin and cisplatin, thereby interfering with the occurrence and development of osteosarcoma.

Circ0002052

Wu et al³⁰ showed that overexpression of Hsa_circ_0002052 significantly inhibited the proliferation, migration and invasion of OS cells, and promoted apoptosis in vitro. Hsa_circ_0002052 inhibited Mir-1205, which targeted APC2, a negative regulator of Wnt/b-catenin signaling pathway. In this study, hsa_circ_0002052 was identified as a mir-1205 sponge to facilitate APC2 expression and consequently inactivate Wnt/b-catenin signaling pathway, leading to reduced OS progression. The Hsa_circ_0002052/ Mir-1205/APC2/Wnt/b-catenin axis may be a potential target for OS treatment.

CircROCK1-E3/E4

The study of Liu et al³¹ showed that circROCK1-E3/E4, produced from exons 3 and 4 of ROCK1 precursor mRNA, was downregulated in patients with osteosarcoma and associated with poor prognosis. Downregulation of circROCK1-E3/E4 promoted proliferation and migration in U2OS and HOS cells. In contrast, overexpression of circROCK1-E3/E4 suppressed the proliferation and migration ability. In U2OS and HOS cells, CircROCK1-E3/E4 sponges miR-532-5p which promotes proliferation and migration by targeting PTEN. Up-and downregulation of miR-532-5p inversely regulated PTEN expression. It was revealed that miR-532-5p was upregulated in 20 osteosarcoma plasma samples by using 15 healthy controls.

CircNRIP1

Hei et al³² showed that circNRIP1 was down-regulated in OS cell lines. CircNRIP1 expression could significantly suppress OS cell proliferation, migration and invasion by sponging miR-1200 in vitro and in vivo. To investigate the in vivo function of circNRIP1 in OS cell xenograft growth, they established and evaluated a xenograft model of OS cells in nude mice. Increased MIA2 expression was observed in tissues from mice overexpressed circNRIP1. CircNRIP1 acts as a miRNA sponge by interacting with miR-1200 and its target gene MIA2, leading to upregulation of MIA2 expression and ultimately inhibition of osteosarcoma progression. CircNRIP1 inhibits OS progression through the miR-1200 /MIA2 axis.

CircITCH, a Multifunctional CircRNA

In esophageal squamous cell carcinoma, circITCH was found as a tumor suppressor RNA, and it was also found in³³ colorectal cancer³⁴ and lung cancer,³⁵ but its inhibitory effect in these cancer types was different. Li et al³⁶ reported that the expression level of circITCH in osteosarcoma cell lines was significantly higher than in hfob1.19 normal osteoblasts. Notably, the higher expression of circITCH in osteosarcoma promotes the proliferation, migration, and invasion of osteosarcoma cells, while the knockout of circITCH has the opposite effect. Current studies on osteosarcoma emphasize the regulation of different signaling pathways by the circRNA network. Therefore, erlotinib (an EGFR tyrosine kinase inhibitor) can eliminate the role of circITCH in promoting tumor growth and enhancing tumor invasion.³⁶ Ren et al³⁷ also found that circITCH can inhibit osteosarcoma progression by down-regulating miR-22. circITCH may function through PTEN/PI3K/AKT and SP-1pathways. PTEN negatively regulates PI3K/AKT pathway. PTEN expression was significantly escalated when overexpressing circ-ITCH and declined via upregulating miR-22.

Application of circRNA in the Diagnosis and Treatment of Osteosarcoma

With the development of science and medical technology, many tumors are no longer incurable, and the survival time of patients and quality of life have been significantly improved. For the treatment of osteosarcoma, surgery and neoadjuvant chemotherapy are mainly used, but the 5-year survival rate of patients still needs to be improved. Xu et al³⁸ found no survival advantage between preoperative chemotherapy and immediate surgery for a specific group of non-metastatic high-grade pelvic osteosarcoma patients with an immense tumor burden and an inadequate response to chemotherapy. At 5 years, the overall survival rate was 43% in the neoadjuvant group and 40% in the immediate surgery group, with no significant difference. It is necessary to seek new therapeutic methods. Recent studies have shown that circRNA can be used as a target for tumor target therapy, and the expression of some circRNAs is significantly correlated with the grade of osteosarcoma and the survival time of patients³⁹ (Figure 1).

Diagnosis and Prognosis Analysis

CircRNA can stably exist in large quantities in various body fluids, including plasma, serum, exosomes, and urine.⁴⁰ The expression of circRNA was significantly different between tumor patients and healthy controls, which indicated that circRNA in body fluid could be used as a new biomarker for monitoring tumor development.⁴¹ Zhang et al⁴² showed that circRNA UBAP2 was overexpressed in OS, and its expression level was significantly correlated with the differentiation degree and prognosis of OS. The detection of circRNA UBAP2 content in serum exosomes can help the early diagnosis of osteosarcoma. The study of Long et al⁴³ showed that compared with normal adjacent tissues, circ001569, circCDRLAS, circHIPK3 and circfoxo3 were significantly down-regulated in osteosarcoma, among which circHIPK3 was significantly correlated with Enneking stage, lung cancer metastasis, and survival time, suggesting that the lower the expression of circHIPK3, the shorter the survival time and the worse the prognosis. Many studies have shown that the expression level of circRNAs can be used for early diagnosis, classification, staging, and prognosis analysis of osteosarcoma. Compared with invasive tests such as needle biopsy, the development of tumor markers diagnostic kit for circRNAs in body fluid is expected to make tumor diagnosis safer and more portable, which is also of great significance for early tumor screening (Figure 1).

Drug Resistance

Zhu et al⁴⁴ showed that circRNA PVT1 was screened to determine its carcinogenic effect on OS. circPVT1 was significantly upregulated in OS tissues, serum, and chemoresistant cell lines, which was related to the poor prognosis of OS patients. ROC curve showed that circPVT1 might be a better biomarker than alkaline phosphatase (ALP), with higher sensitivity and specificity. In addition, functional analysis showed that siRNA knockdown of circPVT1 could reduce the resistance of OS cells to adriamycin and cisplatin by reducing the expression of ABCB1. This may provide new insights into the role of circpvt1 as a biomarker for the diagnosis and treatment of OS. Gu et al⁴⁵ showed that has_circ_0010220 increased DOX-resistance in OS by miR-574-3p/IL-6 axis regulation. Reducing DOX resistance in vitro was achieved by silencing Has_circ_0010220. It suggested potency of circRNA as a promising biomarker for treating OS.

RNA Vaccine

Researchers from Tsinghua University show that⁴⁶ circRNA vaccine platform provides a novel prospect for the development of cancer RNA vaccines in a wide range of hard-to-treat malignancies. CircRNA can elongate the production of tumor antigen, thus prolonging the antigen presentation of antigen-presenting cells. The purified circRNA has low immunogenicity and cannot provide an inflammatory microenvironment for the activation of cytotoxic T cells. To solve this problem, they cooperated with a new type of ionizable lipid, which can induce the release of proinflammatory cytokines into LNP carrier. This binding exerts the long-term protein translation ability of circRNA, and provides an inflammatory immune environment suitable for cytotoxic T cell activation.

CircRNA-Based Drug Delivery Systems

Nanoparticles can carry drugs and deliver them to therapeutic targets. Lipid-based nanoparticles (LNPs) can be used to target specific cells using endogenous or exogenous ligands by encapsulating circRNA.⁴⁷ Gold nanoparticle (AuNP), a well-studied non-viral vector with high stability, purity, and easy surface modification, has been used to deliver circRNAs plasmids.⁴⁸ Research showed AuNP delivery of circFoxo3 plasmid can promote tumor cell apoptosis and inhibits tumor growth.⁴⁹

Nucleic Acid Aptamer

RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and regulate cellular function.⁵⁰ Because aptamers can bind specific domains and conformations of proteins,⁵¹ they can either inhibit proteins or modulate their function. Circular RNA can be used as RNA aptamer. Comparable to the levels of proteins and the most abundant endogenous small RNAs, circular RNAs are highly stable and achieve significantly high expression levels in cells.⁵²

The study of Al-Sudani et al⁵⁰ proved that there are two types of aptamer selection, circular and linear. AC3, a circular aptamer, stood up as the optimal aptamer for inducing SIRT1 activity and exerting anticancer action. The biophysical interactions between AC3 and SIRT1 were described using a modified library. Confirmed by co-localisation, AC3 was recruited to intracellular SIRT1. AC3 has shown anticancer activity against several cancer models including lung cancer, liver cancer and osteosarcoma. AC3 was non-toxic on the non-cancerous cell-line Beas2B, implying it might be safe for non-cancerous tissues.

Circular RNAs in Exosomes

The expression profiles of circRNA in the body fluids between osteosarcoma patients and healthy people are different,⁵³ such as circRNAs in exosomes. Exosomal circRNAs can be taken up by neighboring or distant cells and influence physiological and pathological status of the recipient cells.⁵⁴ The study of Pan et al⁵⁵ showed that hsa_circ_103801 was highly enriched in CDDP-resistant OS cell-derived exosomes. CDDP-resistant cell-derived exosomes could transfer hsa_circ_103801 to sensitive cells and enhance the resistance of OS cells. The survival time of OS patients with high expression of hsa_circRNA_103801 was shorter. These findings suggest that reducing hsa_circ_103801 expression in CDDP-resistant cell-derived exosomes may be a potential strategy to overcome chemotherapy resistance to OS. The study of Dou et al⁵⁶ showed that OS patients had higher circ_0056285 levels in serum exosomes than those in healthy volunteers. ROC curve showed that the level of exosomal hsa_circ_0056285 had high diagnostic value for OS. Circ_0056285 promoted OS progression by sponging miR-1244 and increasing TRIM44 expression. Functional experiments showed that circ_0056285 silencing inhibited the progression of OS cells by inhibiting cell proliferation and glycolysis and inducing cell apoptosis. Circ_0056285 knockdown blocked tumor growth in nude mice. It provided a promising therapeutic target for OS.

Exosomes represent a new way of intercellular communication, rich in circRNAs.⁵⁷ Many studies have shown that exosomal circRNA is involved in cell–cell communication in the tumor microenvironment and regulates many characteristics of tumors, such as proliferation, invasion and metastasis, angiogenesis, immune escape, and drug resistance.⁵⁸ In addition, exosomal circRNA carries the biological information related to the primary tumor, easily crosses various biological barriers, and exists in a large number of body fluids from different sources, which brings great hope for the diagnosis and treatment of osteosarcoma and can be used as a biomarker for early diagnosis and prognosis evaluation.⁵⁹ The isolation and detection of circRNA from tumor-associated exosomes need cutting-edge technology, so it is necessary to develop more accurate and straightforward detection methods to monitor and find new therapeutic targets.

Tumor-derived exosomes of nanometer size are easy to pass through biological barriers such as the blood–brain barrier.⁶⁰ They have natural homing characteristics, stability of bilayer lipid structure, and high biocompatibility. Therefore, tumor exosomes are becoming an ideal carrier for tumor target therapy; especially the engineered exosomes show the excellent effect of tumor-targeted intervention therapy. The application prospect of tumor exosomes in circRNA-related target therapy is worth looking forward to and further exploring.

Conclusion

CircRNA plays a vital role in various diseases, participates in the physiological and pathological regulation process, and can be used as a promising biomarker in disease detection and treatment.^61,62 But at present, as a cancer biomarker, specific circRNA is required to be differentially expressed after transformation or in the early stage of specific cancer and needs the support of related quantitative technology. This technology still needs to be studied for clinical application. It will be fruitful work to study the role of circRNA in cancer. Using circRNA as a cancer biomarker can be used for early cancer screening and increase the survival rate.

Understanding the role of circRNA will change and supplement the traditional understanding of the occurrence and development of osteosarcoma and help understand the molecular mechanism related to the occurrence and development of osteosarcoma. CircRNA is widely involved in gene expression regulation and protein translation⁶³ and plays an indispensable role in the occurrence and development of osteosarcoma.⁶⁴ Liquid biopsy, circRNA-based prognosis analysis, RNA vaccine, circRNA-based drug delivery systems, reduction of circRNA-related drug resistance, application of nuclear acid aptamer, application of engineered exosome, etc. will benefit the individualized diagnosis and treatment of osteosarcoma in the future.