Circular RNA hsa_circ_0000658 inhibits osteosarcoma cell proliferation and migration via the miR‐1227/IRF2 axis

Abstract Osteosarcoma (OS) is the most frequently occurring bone cancer. Circular RNAs (circRNAs) have been shown to exert pivotal impact in modulation of gene expression, but their roles in OS are still not fully understood. In this study, we analysed the role of circ‐0000658 in OS. Thereafter, molecular techniques such as Western blot, qRT‐PCR, RNA‐binding protein immunoprecipitation and Dual‐Luciferase reporter assays were implemented to investigate the role of circ‐0000658/miR‐1227/interferon regulatory factor‐2 (IRF2) axis in OS. Eventually, the impact of circ‐0000658 on tumour growth and metastasis was observed in a xenograft mouse model. The results of this study revealed that circ‐0000658 exhibits low levels in OS tissues and cell lines. Moreover, circ‐0000658 repression promoted cell cycle, proliferation, invasion and migration but inhibited the apoptosis of OS cells. Researches have previously shown that circ‐0000658 contains a binding site for miR‐1227 and thus can abundantly sponge miR‐1227 to up‐regulate the expression of its target gene IRF2. Moreover, both inhibition of miR‐1227 and overexpression of IRF2 reversed cell proliferation and invasion, which was triggered by circ‐0000658 repression. Conclusively, circ‐0000658 modulates biological function of OS cells through the miR‐1227/IRF2 axis. Therefore, circ‐0000658 might act as a possible novel therapeutic target for the treatment of OS.

methods for OS. Therefore, new effective treatment regimen is urgently required for OS.

Circular RNAs (circRNAs) are a class of non-coding RNAs (ncRNAs)
with limited ability to encode proteins and usually form a closed circular structure covalently joined by the 3′ and 5′ ends. 8,9 Accumulating evidences about circRNAs have demonstrated that they can impact diverse biological processes and are also implicated in tumour generation and development. [10][11][12] The most investigated function of cir-cRNAs is as master regulators of gene expression that act to sequester or 'sponge' other gene expression regulators, such as miRNAs. 13,14 For instance, recent studies have indicated that circIFT80 functions as a competing endogenous RNA (ceRNA) of miR-1236-3p to promote the development of colorectal cancer. 15 circFBLIM1 has been shown to act as a ceRNA that promotes the development of hepatocellular cancer by acting as a sponge of miR-346. 16 Other studies have confirmed that a variety of circRNAs, such as circRNA_100876, 17 circRNA_UBAP2, 18 circ_HIPK3 19 and circRNA_100269, 20 participate in the pathophysiological processes of OS by exhibiting competitive binding to miRNAs.
However, the function of circ-0000658 in OS remains unknown.
In this study, we verified that the expression of circ-0000658 is low in OS cell lines and tissues. Circ-0000658, as a ceRNA, modulated OS pathogenesis by sponging miR-1227, thereby promoting the expression of IRF2 and continuously modulating the behaviour of OS cells. Therefore, circ-0000658 might act as a possible novel therapeutic target for the treatment of OS.

| Collection of OS samples and culture of OS cells
Sixty pairs of OS samples were collected from the patients at the  Gibco, Carlsbad, CA, USA) in an incubator (Temp: 37°C; CO 2 : 5%).

| Real-time PCR assay, transfection of cells, as well as production and transduction of lentiviruses
TRIzol reagent (Invitrogen) was used for total RNA extraction from OS cells as per the manufacturer's protocol. Thereafter, the extracted total RNA was subjected to reverse transcription to generate cDNA by using the reverse transcription kit from Takara. qPCR was performed using the SYBR Green PCR kit (Takara, Dalian, China). U6 was used for normalization of the miRNA whereas GAPDH was used for the normalization of mRNA or circRNA. The primers used are listed in Table S1.
Lentiviral particles carrying scrambled circ-0000658 shRNA and circ-0000658-expressing vectors were generated through HEK293T cells. OS cells were then infected with recombinant lentiviruses, followed by selection with 2 μg/mL puromycin.

| Transwell, cell cycle and apoptosis assays
Cells were inoculated into the transwell chambers that were sub- Beijing, China PR) was used assess cell apoptosis. specific primary antibodies were applied to the membrane, and then, the membrane was incubated overnight at 4°C. Secondary antibodies conjugated with horseradish peroxidase were then used, and the membrane was incubated at room temperature for 1 hour. Finally, the BioSpectrum 600 Imaging System (UVP, CA, USA) was used to obtain the images.

| RNase R digestion
The total RNAs (5 μg) were incubated at 37°C for 15 minutes with RNase R (Epicentre Biotechnology, Shanghai, China) that was used to remove the linear RNAs at a concentration of 3 units/1 μg. After RNase R treatment, the expression of circ-0000658 was detected via qRT-PCR analysis.

| RNA-binding protein immunoprecipitation experiment
The RNA-binding protein immunoprecipitation (RIP) assay was performed by using the EZ-Magna RIP Kit (Millipore, Billerica, MA, USA). After the transfection of miR-1227 or circ-0000658 into the cells, Ago2-RIP assays were performed. First, the cells were lysed using the RIP lysis buffer along with RNase (Millipore) and proteinase inhibitors (Millipore). Second, the RIP lysates were placed in RIP buffer containing magnetic beads conjugated with human anti-Ago2 antibody or non-specific mouse IgG antibody (Millipore). Next, the immunoprecipitates were digested with proteinase K, and the precipitates were examined for circ-0000658 expression by RT-PCR analysis and gel staining. Finally, the RNA concentration was measure by using the NanoDrop spectrophotometer, and qRT-PCR analysis was conducted by using the purified RNA.

| Dual-Luciferase reporter assay
Following the amplification of 3′-UTRs of IRF2 and circ-0000658, they were independently cloned into the firefly luciferase gene

| Tumour formation in vivo
Five-week-old BALB/c (nu/nu) mice were subcutaneous seeded with Each mouse was inoculated with HOS cells (1 × 10 7 ) that were stably injected in the tail vein, so as to establish an advanced-stage pulmonary metastasis model. Four weeks later, the mice were sacrificed, followed by lung removal, and haematoxylin and eosin (HE) staining.

| Statistical analysis
Data are presented as mean ± SD. Differences in categorical variables were determined by using Fisher's exact test, and comparison between the groups was performed by two-tailed Student's t test or one-way ANOVA. Correlation analysis was performed by assessing

Pearson's correlation coefficient. The log-rank test and Kaplan-
Meier's method were used to assess the survival rates. Differences with P < 0.05 were considered as statistically significant.

| Circ-0000658 expression level is decreased in OS cells and tissues
The microarray GSE96964 from platform GPL19978, containing seven human OS cell lines (U2OS, MTX300, HOS, MG63, X143B, ZOS and ZOSM) and the human osteoblast hFOB1.19, was used to perform the expression analysis. Through this analysis, circ-0000658, which is the most significantly down-regulated circRNA in OS cell lines, was selected as the study subject ( Figure 1A). As circ-000658 is resistant to RNase R digestion, the circRNA characteristics of circ-000658 were corroborated ( Figure 1B). qRT-PCR was performed to examine circ-0000658 expression level in the pairs of primary OS tissues and non-tumour tissues. The results revealed that the non-tumour tissues expressed circ-0000658 at a higher level than the OS tissues ( Figure 1C). Similarly, OS cells expressed circ-0000658 at a notably lower level than HFOB1.19 cells ( Figure 1D). in high-expression group exhibit higher overall survival rate than in low-expression group ( Figure 1E).

| Circ-0000658 restricts OS proliferation and invasion in vitro
First, circ-0000658 expression level in HOS cells was increased, while it was decreased in MG63 cells after transfection (Figure 2A).
Subsequently, it was found that an increase in circ-0000658 expression markedly suppressed the cell proliferation and colony formation abilities of cells, as shown in the results of CCK-8, EdU and colony formation assays ( Figure 2B-D). Moreover, flow cytometry analysis revealed that the S phase in circ-0000658 group was lower than in Lv-NC group ( Figure 2E). Later, whether circ-0000658 could exert an impact on apoptosis was examined by performing the apoptosis assay. The results revealed that circ-0000658 promotes OS cell apoptosis ( Figure 3A). Finally, transwell invasion assay was performed to determine the impact of circ-0000658 on OS cell invasion.
It was found that circ-0000658 inhibits OS cell invasion ( Figure 3B).

| Mutual inhibition between circ-0000658 and miR-1227 expression
MiRNAs with complementary base matching circ-0000658 were identified using the CircInteractome (https://circi ntera ctome.nia. nih.gov/); as a result, miR-1227 was identified, which was previously found to be increased in many cancer cells. 23,24 qRT-PCR analysis revealed that miR-1227 expression was increased in OS tissues than in non-tumour tissues ( Figure 4A) and that there was a negative correlation between the expression levels of circ-0000658 and miR-1227 in OS tissues ( Figure 4B). The biding sites of miR-1227 on circ-0000658 are depicted in Figure 4C. The speculated miR-1227 binding site on circ-0000658 (circ-0000658 WT) and a mutant miR-1227 binding site on circ-0000658 (circ-0000658 MUT) were cloned into reporter plasmids. Co-transfection with miR-1227 and circ-0000658 WT was shown to markedly weaken the luciferase activity, whereas co-transfection with miR-1227 and circ-0000658 MUT exerted no such impact on the luciferase activity ( Figure 4C). Furthermore, RIP assays verified that circ-0000658 and miR-1227 were gathered in Ago2 immunoprecipitates ( Figure 4D). Finally, in both MG63 and HOS cells, shcirc-0000658 increased and circ-0000658 decreased miR-1227 levels significantly ( Figure 4E). Collectively, the above data imply that circ-0000658 is able to directly bind to miR-1227 and thus inversely regulates miR-1227 expression.

| IRF2 is a direct target of miR-1227
IRF2 was selected from the list of putative target genes of miR-1227 for future research. The binding sites between miR-1227 and IRF2 are presented in Figure 5A. Dual-Luciferase reporter assay revealed that HEK293T cells co-transfected with miR-1227 and IRF2 WT exhibited reduced luciferase activity relative to other groups ( Figure 5B). Then, IRF2 expression level in OS tissues was evaluated ( Figure 5C), and we found that there was a negative correlation between the expression levels of IRF2 and miR-1227 in OS, but a positive correlation between the expression levels of IRF2 and circ-0000658 ( Figure 5D,E). Moreover, qRT-PCR and Western blot analyses validated that miR-1227 inhibited IRF2 expression in both MG63 and HOS cells ( Figure 5F,G). Additionally, the level of IRF2 was found to be increased upon circ-0000658 overexpression, while co-transfection with miR-1227 reversed this effect ( Figure 5H,I). Altogether, these results suggest that IRF2 is a downstream target gene of miR-1227 that can be modulated by circ-0000658.

| Increase in circ-0000658 expression impedes tumour growth and metastasis
Whether an increase in circ-0000658 expression impedes tumour growth in the body was investigated further. It was found that the growth of xenograft tumours was reduced upon circ-0000658 overexpression ( Figure 6A). Moreover, the volume and average weight of xenograft tumours in circ-0000658 group were less than those in Lv-NC group ( Figure 6B,C). Thereafter, the expression of circ-0000658 in the resected tumour tissues was examined. In addition, upon staining the tumour sections to detect IRF2 expression, the results revealed that the IRF2 expression level was also higher in circ-0000658 group than in Lv-NC group ( Figure 6E).
Further, to investigate whether the increase in circ-0000658 expression impedes tumour metastasis, a lung metastasis model was established in vivo. Overexpression of circ-0000658 notably reduced lung metastases ( Figure 6F). Finally, tunnel staining revealed that the overexpression of circ-0000658 notably induced cell apoptosis ( Figure 6G).  Functional assays revealed that circ-0000658 restricted the cell proliferation and invasion and protected cells from undergoing apoptosis to some extent. Moreover, circ-0000658 repression was found to promote the G1 to S phase transition of cell cycle. Further, elevated circ-0000658 expression group exhibited longer overall survival of OS patients than low circ-0000658 expression group.

| D ISCUSS I ON
The above-mentioned findings imply that circ-0000658 is a possible biomarker for the prognosis of OS patients and that it inhibits the progression of OS.
The IRF protein family is a pivotal adaptive immune factor and is known to modulate cellular responses implicated in tumour generation. 28,29 Interferon regulatory factor-2 (IRF2) of the IRF family is able to exert anti-oncogenic effects. IRF2 is down-regulated in many primary human cancers, including gastric cancer and hepatocellular carcinoma. 28,30,31 In this study, IRF2 was found to be expressed in OS tissues at a lower level as compared to that in non-tumour tissues.
Conversely, up-regulated expression of IRF2 reversed the promotion of cell proliferation and invasion, which were induced by circ-0000658 repression.
Conclusively, the results of this study demonstrate that circ-0000658 is notably decreased in OS tissues and can successfully combine with miR-1227 to regulate IRF2 expression. Moreover, circ-0000658 overexpression inhibits cell proliferation and invasion by targeting the miR-1227/IRF2 axis in OS cells. Therefore, circ-0000658 may act as a novel therapeutic target for OS treatment and also as a potential biomarker for its prognosis.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflicts of interest in this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed in the present study can be provided by the corresponding author on reasonable request.