Circ‐CTNNB1 drives aerobic glycolysis and osteosarcoma progression via m6A modification through interacting with RBM15

Abstract Objectives Circular RNAs (circRNAs) are a subclass of noncoding RNAs, playing essential roles in tumorigenesis and aggressiveness. Recent studies have revealed the pivotal functions of circ‐CTNNB1 (a circular RNA derived from CTNNB1) in cancer progression. However, little is known about the role of circ‐CTNNB1 in osteosarcoma (OS), a highly malignant bone tumour in children and adolescents. Methods Circ‐CTNNB1 was analysed by qRT‐PCR, and the results were confirmed by Sanger sequencing. The interaction and effects between circ‐CTNNB1 and RNA binding motif protein 15 (RBM15) were analysed through biotin‐labelled RNA pull‐down and mass spectrometry, in vitro binding, and RNA electrophoretic mobility shift assays. In vitro and in vivo experiments were performed to evaluate the biological functions and underlying mechanisms of circ‐CTNNB1 and RBM15 in OS cells. Results Circ‐CTNNB1 was highly expressed in OS tissues and predominantly detected in the nucleus of OS cells. Ectopic expression of circ‐CTNNB1 promoted the growth, invasion, and metastasis of OS cells in vitro and in vivo. Mechanistically, circ‐CTNNB1 interacted with RBM15 and subsequently promoted the expression of hexokinase 2 (HK2), glucose‐6‐phosphate isomerase (GPI) and phosphoglycerate kinase 1 (PGK1) through N6‐methyladenosine (m6A) modification to facilitate the glycolysis process and activate OS progression. Conclusions Circ‐CTNNB1 drives aerobic glycolysis and OS progression by facilitating RBM15‐mediated m6A modification.

development of metastases. [1][2][3] With the application of chemotherapy and surgery over the last 30 years, the prognosis of OS remains poor for high rate of metastasis and rapid progression. 4 Therefore, revealing the molecular mechanisms underlying OS development and progression is critical for developing specific therapies.
CircRNAs, a group of transcripts with closed continuous loops generated from a process called back-splicing, are described as key regulators of gene expression, [5][6][7] and dysregulated circRNAs have been identified in almost all types of cancer. 8 Previous studies have reported various circRNA functions and their underlying mechanisms in cellular physiology including sponging miRNA, binding with proteins, and translating into peptides and proteins, exerting transcriptional or translational regulation. [9][10][11] Recent studies have shown the vital functions of circRNAs in the control of gene expression via physical interaction with proteins. For example, circ-Amotl1 promotes tumorigenesis through binding to c-myc and increasing the retention of nuclear c-myc in breast cancer. 12 CircAGO2 binds and facilitates the recruitment of HuR protein in the 3 0 -untranslated region of the target gene, and promotes cancer progression. 13 In a previous study, we have identified that circ-CTNNB1 interacts with protein and drives tumour progression through the activation of the Wnt/β-catenin signalling pathway in gastric cancer. 14 However, the specific functions and underlying mechanistic involvement of circ-CTNNB1 in OS have not yet been explored.
N6-methyladenosine (m6A), which results from the methylation of adenosine at the N6 position of almost every type of RNA molecule, has been reported to play vital roles in cancer biology through modulating RNA maturation, localization, translation and metabolism. [15][16][17] Aberrant level of global m6A abundance has been reported recently in cancers, and the dysregulation could be associated with malignant progression and clinical outcome. 18 The biological effects of m6A are mainly regulated by three kinds of methylation modulators, namely methylation transferases (Writers), demethylases (Erasers) and methylated readers (Readers), based on which the m6A modification is involved in regulating gene expression of cancer. 19,20 The 'writers' are proteins involved in catalysing the m6A modification of adenosine on target RNA. Accumulating evidence has demonstrated that some methylation modulators are regulated by circRNAs. For example, circKIAA1429 maintains the expression of Zeb1 in a YTHDF3 (a m6A reader protein)-dependent manner to accelerate the progression of liver cancer, 21 circMAP2K4 promoted hepatocellular carcinoma cell proliferation by expediting YTHDF1 expression through m6A RNA methylation. 22 In this study, we detected high expression of circ-CTNNB1 in OS tissues and cell lines, which was associated with lung metastasis in OS patients and malignant progression of OS cells in vitro and in vivo.
Mechanistically, circ-CTNNB1 interacts with m6A regulator RBM15, which facilitates the ability of the latter to elevate the m6A levels at the 3 0 -UTR of the key aerobic glycolysis genes hexokinase 2 (HK2), glucose-6-phosphate isomerase (GPI) and phosphoglycerate kinase 1 (PGK1), ensuring a more stable activity and elevated expression of the target genes. Therefore, OS cells are able to obtain metabolic survival advantage from aerobic glycolysis via the upregulation of circ-CTNNB1.

| Real-time PCR and quantitative realtime PCR
The detection of circRNA, mRNA samples were described before, 14 and primers are shown in Table S1.

| Northern blot
The junction probe for circ-CTNNB1 was synthesized and labelled with digoxigenin, as described in our previous study. 14
2.6 | RNA fluorescence in situ hybridization (RNA-FISH) A biotin-labelled antisense probe for the circ-CTNNB1 junction sequence and probes for GAPDH and U1 were synthesized as we previously described. 14 The probes were hybridized using the Fluorescent In Situ Hybridization kit (RiboBio) following the manufacturer's instructions. The nuclei of OS cells were counterstained with 4 0 ,6-diamidino-2-phenylindole (DAPI), and the images were analysed using a Nikon A1Si Laser Scanning Confocal Microscope (Nikon, Japan).

| Dual-luciferase reporter assay
The TOP-FLASH and FOP-FLASH reporters for the activity of the canonical Wnt pathway were obtained from Millipore (Temecula, CA, USA). The promoter fragments of human HK2 (À1813/+424), GPI (À1854/+247), PGK1 (À882/+246) and 3 0 -UTR of target genes amplified from genomic DNA (Table S2)  System (Invitrogen). The dual-luciferase assay was performed according to the manufacturer's instructions (Promega). The luciferase signal in the promoter activity assay was normalized to the firefly/Renilla ratio, while the activity of the 3 0 -UTR reporter was measured by the Renilla/firefly ratio.

| Biotin-labelled RNA pull down and mass spectrometry analysis
The biotin-labelled RNA probe for circ-CTNNB1 was in vitro transcribed using the Biotin RNA Labeling Mix kit (Roche) and T7 RNA polymerase, as described in our previous study. 23 RNA pull-down assay was performed at room temperature, and the biotinylated proteins were detected by mass spectrometry (MS) at the Wuhan Institute of Biotechnology (Wuhan, China). 2.10 | Aerobic glycolysis and seahorse extracellular flux assays Cellular Aerobic glycolysis activity and glucose uptake, lactate production and adenosine triphosphate (ATP) levels were detected as previously described. 23 Extracellular acidification rate and oxygen consumption rate (ECAR, OCR) were measured in response to glucose  (Table S1).

| In vitro binding assay
Five truncates of RBM15 were cloned with primers (Table S1) into vectors with flag tags as we described previously. 13 The Flag-RBM15 and circ-CTNNB1 complexes were pulled down using Flag beads (Sigma, USA). Circ-CTNNB1 was measured by RT-PCR with divergent primers (Table S1), and protein was validated by western blot.

RNA EMSA was conducted according to the instructions of LightShift
Chemiluminescent RNA EMSA Kit (Thermo Fisher Scientific, Inc.)

| In vitro cell viability, growth, and invasion assays
The in vitro viability, growth and invasion capabilities of OS cells were detected by MTT colorimetry, colony formation and matrigel invasion assays, as described previously. 24

| Statistical analysis
All data are presented as the mean ± standard error of the mean (SEM) processed by GraphPad Prism 5.0 (La Jolla, USA). Student's t-test or one-way analysis of variance (one-way ANOVA) was used to evaluate differences between groups. All statistical tests were two sided. A value of p < 0.05 was considered statistically significant.

| Circ-CTNNB1 is upregulated in human OS tissues and cells
In a previous study, we identified that circ-CTNNB1 drives cancer growth, invasion, and metastasis through the activation of β-catenin in cancer, 14 while no further studies have been conducted on this cir-cRNA in OS. The generation of circ-CTNNB1 from CTNNB1 was analysed by RT-PCR with divergent primers, and Sanger sequencing confirmed the predicted back-splicing junction in OS cells ( Figure 1A). Furthermore, using divergent primers of circ-CTNNB1, PCR products could only be amplified from cDNA but not from genomic DNA in 143B and MG-63 cells ( Figure 1B). Circ-CTNNB1 was resistant to RNase R digestion, while the linear RNA of CTNNB1 was significantly reduced after RNase R treatment in 143B and MG-63 cells ( Figure 1C). Higher endogenous expression levels of circ-CTNNB1 were observed in OS cells than those of hFOB 1.19 cells in qRT-PCR assay ( Figure 1D) and Northern blot assay ( Figure 1E). Moreover, we detected the expression profiles in samples of clinical patients with OS tissues, revealing that circ-CTNNB1 was upregulated and associated with lung metastasis in OS patients ( Figure 1F

| Circ-CTNNB1 directly interacts with m6A regulator RBM15
Previous studies have shown that circRNA can regulate cancer progression through binding with RNA binding proteins. Given the nuclear location of circ-CTNNB1, we hypothesized that circ-CTNNB1 may regulate OS progression via potential protein partners. To evidence our hypothesis, we performed a proteomic analysis of circ-CTNNB1-associated proteins in 143B cells by the RNA pull-down  20,26 The MS assay revealed 89 proteins of circ-CTNNB1 pull-down, and overlapped it with m6A regulators and differentially expressed genes in GSE87624 to search critical gene aberrantly expressed in OS that binding with circ-CTNNB1, indicating two potential protein ( Figure 3A, Table S3). Furthermore, RIP assay validated the interaction of circ-CTNNB1 with the m6A writer RNA binding motif protein 15 (RBM15) but not with insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in OS cells ( Figure 3B).
Notably, the expression levels of RBM15 were upregulated in OS ( Figure S3A-C), and circ-CTNNB1 has no regulatory effect on RBM15 in OS cells ( Figure S3D,E). Moreover, transfection of circ-CTNNB1 increased its enrichment in RNA co-precipitated by RBM15 antibody in 143B cells ( Figure 3C). Dual RNA-FISH and immunofluorescence assays confirmed the nuclear colocalization of circ-CTNNB1 and RBM15 in 143B and MG-63 cells ( Figure 3D). Consistently, the RNA electrophoretic mobility shift assay (EMSA) showed that circ-CTNNB1 interacted strongly with endogenous RBM15 in nuclear extracts ( Figure 3E). We further investigated the interaction domain between circ-CTNNB1 and RBM15. The in vitro binding assay indicated that the RRM1 domain, but not other domains of FLAG-tagged RBM15 protein, was crucial for the interaction of RBM15 with circ-CTNNB1 ( Figure 3F). These results indicated that circ-CTNNB1 interacted with RBM15.

| Circ-CTNNB1 promotes aerobic glycolysis in OS
Aerobic glycolysis is a hallmark of metabolic reprogramming in various cancers. However, the mechanisms regulating the glycolytic activity remain elusive in OS. It was reported that circRNA could regulate aerobic glycolysis and cancer progression in an m6A-dependent manner. 27 Given the nuclear interaction described above, we investigated the potential effects of circ-CTNNB1 binding with RBM15 in the aerobic glycolysis in OS. RBM15 was shown to promote the glycolytic process in OS ( Figure S4A-E). Five crucial glycolytic genes were identified as targets of the circ-CTNNB1/RBM15 axis by the comprehensive analysis of RBM15 CLIP-seq and glycolytic genes ( Figure 4F, Table S3).
Notably, ectopic expression or knockdown of RBM15 and circ-CTNNB1 increased and decreased, respectively, the transcripts and protein levels of GPI, HK2 and PGK1, but not of fructosebisphosphate A (ALDOA) or enolase 1 (ENO1), in OS cells ( Figure 4G-I). These findings indicated that circ-CTNNB1 promotes aerobic glycolysis in OS cells.

| Circ-CTNNB1 facilitates RBM15-mediated gene activation via m6A regulation
We further investigated the effects of the interplay and the underlying mechanisms between circ-CTNNB1 and RBM15 on the regulation of target genes (GPI, HK2 and PGK1) and cancer progression in OS cells. As m6A modification is involved in the post-transcriptional control of gene expression, the mRNA-stabilizing function was tested.
We interfered the process of transcription by the RNA polymerase II inhibitor actinomycin D to observe the degradation rate of mRNA.  Figure S5C,D). Next, the RNA methylation quantification assay was used to analyse whether circ-CTNNB1 and RBM15 regulate target gene expression in a m6A-dependent manner. As expected, the MeRIP-qPCR assay showed that the 3 0 -UTR of GPI, HK2 and PGK1 was effectively enriched by m6A-specific antibody, and m6A level was remarkably increased in circ-CTNNB1-overexpression cells, which were prevented by knockdown of RBM15 ( Figure 5D). Importantly, RBM15 wild type, but not ΔRRM1 truncation of RBM15, abol- In the dual-luciferase assay with a reporter containing the 3 0 -UTR of GPI, HK2 and PGK1, ectopic expression or interference of circ-CTNNB1 and RBM15 promoted and attenuated the 3 0 -UTR activity in OS cells, respectively ( Figure 5E,F). Importantly, stable overexpression of circ-CTNNB1 increased the wild-type 3 0 -UTR activity, which was reduced by stable knockdown of RBM15, while the luciferase activity of mutated 3 0 -UTR was not affected in OS cells ( Figure 5G). These results showed that circ-CTNNB1 facilitated RBM5-mediated GPI, HK2, and PGK1 activation via m6A modification.

| Circ-CTNNB1 promotes aerobic glycolysis and OS progression by interaction with RBM15
Next, the effects of the interplay of circ-CTNNB1 and RBM15 in OS cells were analysed. The ectopic expression of circ-CTNNB1 promoted the ECAR and reduced the OCR in OS cells, which were prevented by knockdown of RBM15 ( Figure 6A,B). Accordingly, stable interference of RBM15 abolished the increase of the glucose uptake, lactate production and ATP levels induced by circ-CTNNB1 overexpression in 143B and MG-63 cells ( Figure 6C-E). Notably, stable circ-CTNNB1 overexpression was facilitated, and MTT, colony formation, and matrigel invasion assays showed the viability, growth and invasiveness of 143B and MG-63 cells, which were abolished by the knockdown of RBM15, respectively ( Figure 6F-H). Taken together, these results showed that circRNA facilitated the aerobic glycolysis process and OS progression through the circ-CTNNB1/RBM15/m6A axis (Figure 7).

| DISCUSSION
OS is the most prevalent primary bone cancer and ranks as the top malignancy among adolescents. 1 The 5-year survival rate of patients with OS has increased in the past 30 years; however, the prognosis in drug-resistant or metastatic OS is still not satisfactory. 29 To provide insights into the pathogenesis and metastasis of OS, we identified the novel role of circular RNA circ-CTNNB1 in regulating OS progression.
In this study, we confirmed that upregulated circ-CTNNB1 exerted an oncogenic role in OS tumour progression in a RBM15-dependent manner. Mechanistically, circ-CTNNB1 binds RBM15 to perform the m6A modification of the key aerobic glycolysis genes HK2, GPI and PGK1 and thus stabilize the mRNA levels of the target genes. Consistently, we observed a dynamic aerobic glycolysis process in OS cells (i.e., decrease and increase of the glucose uptake, lactate production, and ATP levels of OS cells for knockdown or overexpression of circ-CTNNB1, respectively). Then, the process of aerobic glycolysis ensures a survival advantage of tumour cells in the development and progression of cancer.
Recently, numerous studies demonstrate that circRNAs have a crucial role in cancer. 30 CircRNAs could recruit or sponge proteins to the specific regions of target genes to regulate gene expression. 31,32 Emerging evidence indicates that circRNAs are aberrant expressed and involved in miRNA inhibition, endothelial-mesenchymal transition F I G U R E 7 Mechanisms underlying circ-CTNNB1-promoted OS progression (EMT), initiation and progression of OS, and thus could be potential therapeutic targets for OS. 33,34 For example, circTADA2A promotes OS progression and metastasis via sponging miR-203a-3p and facilitating CREB3 expression, 35 circTCF25 drives carcinogenesis in OS cells by suppressing miR-206 expression. Our previous study demonstrated the essential roles of the circ-CTNNB1/DDX3/YY1 axis in cancer progression. 14 In this study, to identify specific functions and underlying mechanistic involvement of circ-CTNNB1 in OS, we demonstrate that circ-CTNNB1 drives aerobic glycolysis and OS progression via m6A modification through interacting with RBM15.
RBM15 is a member of the SPEN (split-end) family of proteins, which interacts with RNA by binding with spliceosome components, 36 playing vital roles in the mechanism of mRNA methylation as a m6A methyltransferase 'writer', 37 and exerting oncogenic role in cancer. 38 RBM15 promoted the invasion, migration and metastasis of OS with a high correlation with metastasis and the decreased survival rate, 39 and m6A regulators were confirmed to play vital roles in regulating glycolysis of cancer cells. 18,40 Additionally, studies have revealed that circRNA could regulate aerobic glycolysis and cancer progression in an m6A-dependent manner. 27,28 In the current study, gain-and lossof-function studies show that RBM15 facilitates aerobic glycolysis and malignant progression of OS cells, suggesting the oncogenic roles of RBM15 in OS. Additionally, our results indicate that RRM1 domain is essential for the interaction between circ-CTNNB1 and RBM15.
The excessive demand of tumours for nutrients is also associated with severe metabolic challenges. Through metabolic remodelling, tumours have evolved a unique metabolic regulation system. 41,42 Aerobic glycolysis, which is known as the 'Warburg effect', 43 is the first discovered and most important event in the metabolic reprogramming process. This metabolic reprogramming not only provides ATP for tumour cells but also essential macromolecules for their protein and nucleotide biosynthesis. 44 The metabolic reprogramming process could be a promising treatment target, and our research provides a potential choice for OS therapy from the metabolic point of view.
The circ-CTNNB1/RBM15/aerobic glycolysis pathway could be intervened in different ways. CircRNAs are typically knocked down by RNA interference (RNAi)-based strategies. However, this is accompanied by many limitations, including their instability, lack of cell specificity, low intracellular entry, immune system activation and other offtarget effects. 45 Using nanoparticles or exosomes as delivery systems can partly improve their efficacy. 46 Disturbance of the interaction between circ-CTNNB1 and RBM15 with dominant-negative mutants or small molecular inhibitors are also relatively specific strategies. In our previous study, the growth and aggressiveness of various other cancer cells were efficiently suppressed by a cell-penetrating inhibitory peptide, which blocks the interaction of circ-CTNNB1 and that of the partner protein DDX3. 14 Therefore, we will further study a way to target this pathway and provide transformation value.
In summary, oncogenic circ-CTNNB1 is upregulated in OS tissues and cells. High circ-CTNNB1 expression was associated with increased aggression of OS cells. Circ-CTNNB1 interacted with RBM15 to facilitate m6A modification of its aerobic glycolysis genes, resulting in more stable mRNA and activation of target genes. Moreover, the aerobic glycolysis level was elevated, which increased the survival advantage of OS cells. Our study provides a potential target for the treatment of OS.