Circuitous Ways of EWS::FLI1 Using Circular RNA ZNF609 to Evade Translational Repression by miR-145 in Ewing’s Sarcoma

Abstract

Background: Ewing’s sarcoma (EwS) is a pediatric bone and soft tissue cancer driven by the oncogenic fusion protein EWS::FLI1. Currently, EwS lacks targeted therapies, necessitating the identification of novel regulatory mechanisms. While the role of microRNAs and long non-coding RNAs has been explored in EwS, the presence and functional significance of circular RNAs (circRNAs) in EwS is not reported. This is the first study to report the presence and role of oncogenic circRNA, circZNF609 in EwS tumor progression. Methods: Expression of circZNF609 was validated in 5 different EwS cell lines using qPCR. Cellular localization of circZNF609 was identified using circFISH. Functional assays for proliferation, migration and apoptosis were performed in wild type and circZNF609 knocked down (KD) cell lines to confirm its oncogenic role. The impact of circZNF609 on EWS::FLI1 protein levels was confirmed using western blots, immunofluorescence, and polysome fractionation. Mechanistic insights were gained utilizing bioinformatic, dual-luciferase reporter assays, rescue experiments, and microscopy to identify and validate the circRNA-miRNA-mRNA regulatory axis. Results: We report the first identification of circZNF609 in EwS, demonstrating that its expression is EWS::FLI1-dependent. Functional analysis reveals that circZNF609 promotes cell proliferation and metastasis while inhibiting apoptosis. Mechanistically, circZNF609 acts as a molecular sponge for miR-145-5p. By sequestering this miRNA, circZNF609 prevents the translational repression of EWS::FLI1, thereby sustaining oncogenic signaling. Conclusions: These findings identify circZNF609 as a novel post-transcriptional regulator of EWS::FLI1 and establish its critical role in EwS pathogenesis. Our results suggest that targeting the circZNF609/miR-145-5p/EWS::FLI1 axis may offer a promising therapeutic strategy for EwS.

1. Introduction

Ewing’s sarcoma (EwS) is a bone and soft tissue cancer in children, with an incidence of approximately 2–4 cases per million, and it is associated with a very high mortality rate upon metastasis [1]. The molecular cause of EwS is a chromosomal rearrangement between the EWSR1 gene on chromosome 22 and members of the ETS gene family (most commonly FLI1, in 90% of cases) on chromosome 11, resulting in the formation of the aberrant oncogenic fusion protein EWS::FLI1. EWS::FLI1 is known to function as an enhancer, transcription factor, and splicing mediator to initiate tumorigenic changes; however, the precise mechanisms by which this single, highly disordered oncogenic protein drives tumor progression remain incompletely understood [2,3]. The lack of structural information and the unstable nature of the fusion protein have made it challenging to develop drugs targeting EWS::FLI1, which would otherwise be an ideal therapeutic target. Although the EWSR1-FLI1 fusion has been recognized as the molecular cause of EwS for over 40 years, no targeted therapies are currently available. As a result, alternative approaches have focused on understanding the interacting partners and downstream effectors of EWS::FLI1, with most work to date emphasizing chromatin regulation [4], epigenetics [5], and protein partners [6]. However, the contribution of non-coding RNAs (ncRNAs) to Ewing’s sarcoma pathogenesis remains poorly understood. Given that EwS cells have a relatively stable genome [1], it is important to explore non-genetic regulatory factors that may contribute to EWS::FLI1-mediated transformation in order to identify novel therapeutic targets.

The majority of the human genome is transcribed into ncRNAs, whose regulatory roles are only beginning to be appreciated [7]. Among these, a newly recognized class of ncRNAs, circular RNAs (circRNAs), has been shown to play regulatory roles in various cancers, and their mechanisms of action are an emerging area of research [8,9]. CircRNAs are covalently closed RNA molecules with no free ends, typically formed by base pairing between repeat regions of long introns flanking exons, leading to back-splicing and the release of a circular molecule. While circRNAs can be derived from exons, introns, or a combination of both, exonic circRNAs are considered the most clinically relevant [10,11]. Although circRNAs were discovered about 40 years ago, they were long considered splicing errors until the last decade, when their roles in various physiological processes began to be elucidated [8]. The most studied function of circRNAs is their ability to act as microRNA (miRNA) sponges [12]. A growing body of literature has identified functional roles for circRNAs in the progression of many cancers [8,9]. Reducing their expression or blocking their interactions with targets has led to dramatic changes in cancer progression, suggesting that circRNAs may be promising therapeutic targets [9,13]. Furthermore, due to their resistance to exonucleases and their enrichment in extracellular vesicles (EVs), circRNAs are promising biomarkers for non-invasive and early diagnosis of many solid tumors [9]. Despite the recognition of multiple critical roles for circRNAs in various cancers, including osteosarcoma [13], there have been limited studies characterizing circRNAs in EwS cells or in EVs derived from EwS cells. While the roles of miRNAs and long ncRNAs in EwS oncogenesis have been previously studied [14,15,16], the expression and function of circRNAs in EwS remain unexplored. In this study, we focus on circZNF609, a known oncogenic circRNA previously reported in several cancers, including breast cancer and hepatocellular carcinoma [17,18]. CircZNF609 has also been reported to act as a sponge for miR-145, which has been extensively studied in EwS, as well as other miRNAs in several cancers [18,19]. Here, we report that circZNF609 is expressed in an EWS::FLI1-dependent manner in EwS and acts as a miR-145-5p sponge, thereby preventing miR-145-5p from binding to the 3′ UTR of EWS::FLI1. This relieves miR-145-5p-mediated suppression of EWS::FLI1 and contributes to EwS oncogenesis.

2. Materials and Methods

Cell lines and cloning: HEK 293T (ATCC, Manassas, VA, USA, CRL-3216) cell line was cultured in DMEM (Millipore Sigma, St. Louis, MO, USA, D6429) supplemented with 10% FBS (Millipore Sigma, St. Louis, MO, USA, F2442), penicillin/streptomycin (Millipore Sigma, St. Louis, MO, USA, P4333), and L-glutamine (Millipore Sigma, St. Louis, MO, USA, G7513). A673 inducible cell lines were a gift from Dr. Olivier Delattre, Institut Curie, and all other EwS cell lines were a gift from Dr. James Wells, Harvard Medical School. A673 inducible cells lines and 6647 cells were cultured in the above mentioned DMEM, further supplemented with HEPES (Millipore Sigma, St. Louis, MO, USA, H0887) and non-essential amino acids (Millipore Sigma, St. Louis, MO, USA, M7145). TC32 and A4573 cell lines were cultured in RPMI media (Millipore Sigma, St. Louis, MO, USA, R8758) supplemented with 10% FBS, penicillin/streptomycin, and HEPES. SKES-1 cells were cultured in McCoy’s 5A media (Millipore Sigma, St. Louis, MO, USA, M9309) supplemented with 10% FBS and penicillin/streptomycin. The human immortalized myoblast AB678 cell line was a gift from Dr. Myriam Gorospe, laboratory of Genetics and Genomics at National Institute of Aging at NIH. AB678 were cultured in 1:1 HAMS F-10 (Cytiva, Marlborough, MA, USA, SH30025.01) and Promocell skeletal muscle cell growth medium (PromoCell Inc., Heidelberg, Germany, C-23060) supplemented with 20% FBS and 1% penicillin/streptomycin. For cloning, shRNA targeting the BSJ of circZNF609 was cloned into the pLKO.1-TRC cloning vector (Addgene Plasmid 10878) and transfected into 293T EBNA cells for virus production. The shRNA sequences used are provided in Table S1. The scrambled shRNA sequence was cloned into pLKO.1-TRC cloning vector and was used as a negative control. A673 inducible and TC32 cells were transduced with the virus, and stable cell lines were selected via puromycin (Gibco, Waltham, MA, USA, A11138-03). All cells were cultured at 37 °C with 5% CO₂. For imaging studies, all cells were grown on 0.1% gelatin (Bio-Rad, Hercules, CA, USA, 170-6537)-coated coverslips in 100 cm² Petri dishes until 70–80% confluent before fixing in 4% PFA (Millipore Sigma, St. Louis, MO, USA, F8775) and permeabilizing in 70% ethanol for hybridization.

Probe synthesis and purification: Sets of 45–50 linear oligonucleotide probes, each 20 nucleotides in length, were designed to be complementary to specific regions of ZNF609 and circZNF609 RNA molecules, along with an amino group on their 3′ ends (Biosearch Technologies, Novato, CA, USA) to generate PC and PL probes for the ZNF609. The probes were pooled in equimolar concentrations and conjugated with Texas Red [TR] (Invitrogen, Waltham, MA, USA, T6134) or Cy5 (GE Healthcare, Chicago, IL, USA, PA25001) fluorophores and purified by high-pressure liquid chromatography as previously described in [20]. The list of probe sequences used is provided in Supplementary Table S2.

In situ circFISH Hybridization: Coverslips with fixed adherent cells were washed with 2× saline sodium citrate solution (Ambion, Austin, TX, USA, AM9763) containing 20% formamide (Ambion, Austin, TX, USA, AM9342/44) and 2 mM ribonucleoside–vanadyl complex (New England Biolabs, Ipswich, MA, USA, S1402S), then cells were hybridized with probe sets in hybridization buffer containing 25 ng/μL of each probe set. Hybridization was performed overnight at 37 °C in a moist chamber. The next day, the coverslips were washed three times and stained with DAPI (Sigma, St. Louis, MO, USA, D9542) and mounted on glass slides for imaging. For circRNA-miRNA imaging, circFISH and miRNA detection probes (Qiagen, Hilden, Germany, 339350) were used to visualize the interaction between circZNF609 and miR-145-5p, following the protocol adapted by [21].

Fluorescence imaging and analysis: All images were captured with a 100× oil objective using a Nikon TiE Inverted epi Fluorescence microscope equipped with a pixis 1024b camera (Princeton Instruments, Trenton, NJ, USA). The images were obtained using Metamorph imaging software, version 7.8.13.0 (Molecular Devices, San Jose, CA, USA). Z-stack images were captured for each fluorescent wavelength at 2 sec exposures, for a total of 16 stacks, 0.2 mm apart. The compiled z-stack images were analyzed using an in-house-designed algorithm with MATLAB software, version 23.2.0.2365128 (R2023b) (MathWorks, Natick, MA, USA) that identifies spot-like signals in each image and determines their three-dimensional coordinates, then identifies spots that have a counterpart within a 250 nm distance in the other channel. Spots meeting this criterion are classified as co-localized. Each imaging experiment was performed to obtain at least 100 cell counts. The numbers present average molecules with errors indicating 95% confidence interval. The p-values were obtained using Student’s t-test.

qRT-PCR: Total RNA was extracted from cells lysed in Trizol (Sigma, St. Louis, MO, USA, T9424), and equal concentrations of RNA from different cells were used for cDNA synthesis using iScript Reverse Transcription Supermix (Bio-RAD, Hercules, CA, USA, 1708841), and gene expression was analyzed using iTaq Universal SYBR Green Supermix (Bio-RAD, Hercules, CA, USA, 1725124) according to the manufacturer’s protocol. List of primers is provided in Supplementary Table S3.

Immunofluorescence: For immunofluorescence, the permeabilized A673 and TC32 cells were incubated with blocking buffer followed by overnight incubation at 4 °C with primary antibodies. The subsequent day, another blocking step was performed, followed by incubation with a secondary antibody for 1 h at room temperature, and then followed by four to five washes with PBS. For imaging, the cells were mounted with DAPI containing mounting medium and imaged using a 100× oil objective in a Nikon TiE inverted fluorescence microscope equipped with a CCD Princeton Pixis 1024b camera. The images were acquired using Metamorph software. The corrected total cell fluorescence (CTCF) was calculated using ImageJ 1.54d (National Institutes of Health, Bethesda, MD, USA) [22], and the following formula was applied: CTCF = integrated density − (area of selected cell × mean fluorescence of background readings), as per [23]. A list of antibodies is provided in Supplementary Table S4.

Proliferation assay: Cell proliferation was performed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) Cell Proliferation kit (Roche, Basel, Switzerland, 11465007001). After harvesting the cells, they were reseeded in 96-well plates with approximately 5000 cells/well with media and maintained at 37 °C in the CO₂ incubator for 96 h. Cells were treated based on the manufacturer’s protocol at 0, 24, 48, 72, and 96 h timepoints using a plate reader.

Annexin V assay: Annexin V/Propidium Iodide staining was performed using Annexin V Alexa Fluor 488 and Propidium Iodide kit (Invitrogen, Waltham, MA, V13241). In brief, EwS cells were washed with PBS and harvested. Cells were then resuspended in Annexin V binding buffer and diluted to approximately 1 × 10⁶ cells. Later, based on the manufacturer’s protocol, the assay was performed in a BD Fusion Aria flow-cytometer and analyzed using FlowJo software, version 10.10.0.

Soft agar colony assay: EwS cells with and without circZNF609-KD were passaged and seeded in a 12-well plate (5000 cells/well) in a soft agar assembly. The bottom agar layer was 0.5% agar mixed with media, and the top layer constituted 0.3% agar mixed with cells and media, and the soft agar assembly was further topped with media to prevent drying of agar. Cells were cultured at 37 °C for 2 weeks. The wells were then stained with 0.01% crystal violet for 30 min and destained with dH20. The colonies were counted under a dissection microscope using a general protocol [24].

Polysome fractionation: Ribosomal subunits and polysomes were resolved from A673 WT, A673 EWS::FlI1, and A673 circZNF609-KD cell extracts by size-exclusion chromatography according to a published protocol [25]. Briefly, cells were grown to 80% confluency before performing lysis. The size-exclusion column (SEC) employed for polysome fractionation was Bio SEC-5, 5 μm particles, 2000 Å, and 7.8 × 300 mm (Agilent Technologies, Santa Clara, CA, USA, 5190-2541), using a Dionex Ultimate 3000 uHPLC system (Thermo Fisher Scientific, Waltham, MA, USA). Later, total RNA was extracted from each of the fractions using Trizol extraction. Equal concentrations of RNA from all the cell lines were used to make cDNA using iScript Reverse Transcription Supermix. The obtained cDNA was used as a template to analyze expression of EWS::FLI1 as well as reference genes (GAPDH and B-Actin) using iTaq Universal SYBR Green Supermix (Bio-RAD, Hercules, CA, USA, 1725124).

Western blotting: Cells were lysed in cold RIPA buffer containing protease inhibitor cocktail for 30 min. Lysate was centrifuged @ 14,000× g/4 °C for 15 min. A total of 10 ug of total protein was boiled with 2× loading dye, and denatured proteins were run on 12% SDS-PAGE gel, following which an overnight wet transfer assembly system was used to transfer the proteins on the gel to a PVDF membrane. The following day, the membrane was washed, blocked, and then treated with the corresponding antibody overnight at 4 °C. The next day, the membrane was washed thrice with 1× TBST and incubated with secondary antibody dissolved in 5% blocking buffer for 1 h at RT. Membrane was later washed with 1× TBST 3–5 times before imaging with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA, 34580), as per manufacturer’s protocol.

Luciferase assay: Cells were cultured in a 12-well plate at around 40–50% confluency, after which they were transfected with either psiCHECK2-FLI1 3′ UTR F1, psiCHECK2-FLI1 3′ UTR F2, or psiCHECK2-empty vector as a control using Bio-T transfection reagent (Bioland Scientific LLC, Paramount, CA, USA), as per the manufacturer’s protocol. After 24–48 h, lysates were harvested using passive lysis buffer. Later, Firefly and Renilla luciferase readings were taken in a plate reader using the Dual-Luciferase Reagent Assay System (Promega, Madison, WI, USA, E1910), as per the manufacturer’s protocol. Firefly luciferase (expressed constitutively) obtained after transfection was used to normalize bioluminescence values attained for Renilla luciferase, which was expressed in fusion with the 3′ UTR of FLI1. The corrected values were then sequentially normalized to the levels obtained in mock and psiCHECK2-empty vector transfections. Each experiment was repeated in triplicate, and Student’s t-test was performed to obtain p-values.

Bioinformatic Prediction and Analyses: Functionally relevant miRNAs targeting FLI1 3′ UTR were predicted using TargetScan and miRDB databases. MiRNAs interacting with circZNF609 and FLI1 3′ UTR were predicted using miRanda v3.3a, circInteractome/TargetScan, and miRDB [26,27,28]. Further comparison of the common miRNAs targeting FLI1 3′ UTR and circZNF609 with miRTarBase v8 identified high-confidence miRNAs potentially involved in the circZNF609/EWS::FLI1/miRNA regulatory axis.

3. Results

Characterizing CircZNF609 in EwS: CircZNF609 (hsa_circ_0000615) is generated from exon 2 of the ZNF609 pre-mRNA and is 874 nucleotides in length (Figure 1A) [29]. We assessed circZNF609 expression in EwS using RT-qPCR and found it to be constitutively expressed at higher levels in the five different EwS cell lines compared to a control non-Ewing sarcoma cell line (immortalized human myoblast AB678 cells) (Figure 1B). Further validation using divergent primers amplified the circular isoform. Finally, RNase R treatment of total RNA led to a reduction in linear ZNF609 levels while circZNF609 expression persisted, confirming its circular nature; GAPDH was used as a control to demonstrate the effectiveness of RNase R on linear transcripts (Figure 1C). This is expected because RNase R is an exoribonuclease that selectively degrades linear RNA, whereas circRNAs are resistant to RNase R digestion [30,31]. Finally, the PCR product was validated by Sanger sequencing to confirm the presence of the back-splice junction (Figure 1D).

Conventional techniques for analyzing non-coding RNAs, including circRNAs, do not provide information about their cellular localization, which is important for understanding their function. We used our recently developed circFISH method [32] to determine the cellular localization of circZNF609 in EwS cell lines at single-molecule resolution (Figure 2A). This method allows simultaneous visualization of both linear and circular isoforms via colocalization analysis of signals from a combination of two probe sets (Figure 2B). Upon quantification, both linear ZNF609 and circZNF609 were found to be predominantly localized in the cytoplasm compared to the nucleus in both A673 and TC32 cells (Figure 2C and Figure S1). The expression levels of circZNF609 were approximately half that of linear ZNF609 (Figure 2D). To confirm whether the signal corresponding to circZNF609 is indeed from the circular isoform and not from any splice variants of linear ZNF609, we performed in situ RNase R treatment [32]. Indeed, we found a significant loss of linear RNA upon treatment with RNaseR (labeled as T; the not-treated panel is labeled as NT), but almost no reduction in circRNA after RNase R treatment, confirming that we are visualizing the circular isoform (Figure 2D). These findings suggest that, being mainly cytoplasmic in localization, circZNF609 may regulate EwS oncogenesis post-transcriptionally, potentially via miRNA or RNA-binding protein (RBP) sponging or via being translated into a functional peptide.

3.1. Downregulation of CircZNF609 Impairs EwS Cell Proliferation, Tumorigenicity, and Viability

To understand the underlying molecular mechanism circZNF609 plays in regulating EwS, we performed a shRNA-mediated knockdown of circZNF609 by targeting the BSJ of circZNF609 (Figure 3A). We found a significant reduction in circZNF609 RNA levels in A673 and TC32 cells, while there was no change in the level of linear ZNF609 expression in either of these cell lines as measured by RT-qPCR (Figure 3B and Figure S2). These results confirmed the specificity and effectiveness of the knockdown.

To understand if circZNF609 has an oncogenic role in EwS, we evaluated the functional effect of circZNF609 knockdown on the proliferative capacity of EwS cells. Oncogenic circular RNAs are known to result in reduced apoptosis and higher proliferation. We used Annexin V and propidium Iodide (PI) staining to compare cells in early and late apoptosis stages from the WT and circZNF609KD cells. A scrambled KD, ZSCR, was used as a negative control (Figure 4A and Figure S3A). The circZNF609 knockdown in both TC32 and A673 cells resulted in an increase in the number of apoptotic cells as seen in the late apoptotic (LA) and early apoptotic (EA) quadrants for Annexin V and PI double-positive cells, indicating that circZNF609 knockdown induces apoptosis (Figure 4B and Figure S3B). We then analyzed the impact of circZNF609 knockdown on proliferation rate by performing MTT assays and observed a significant reduction in proliferation rate after 72 h in TC32 cells and after 96 h in A673 cells compared to wild-type (WT) cells (Figure 4C and Figure S3C). Furthermore, soft agar colony formation assays showed a significant reduction in the number of colonies formed by circZNF609-KD cells compared to WT cells (Figure 4D,E), indicating reduced tumorigenicity. In summary, these functional assays confirmed that circZNF609 knockdown impairs cell proliferation and tumor growth and induces apoptosis. These results are consistent with existing data reporting that circZNF609 functions as an oncogenic circRNA in other cancers [19,33,34,35]. Our results validated the functional oncogenic role of circZNF609 in EwS for the first time.

3.2. Downregulation of CircZNF609 Reduced the Level of EWS::FLI1 Protein in EwS Cells

To investigate the mechanism by which circZNF609 exerts its functional effects, we examined whether it influences the levels of the oncogenic driver fusion protein EWS::FLI1. We first assessed EWS::FLI1 mRNA levels and found no significant difference between WT and circZNF609 knockdown cells, suggesting that circZNF609 does not affect EWS::FLI1 transcription (Figure S4). Given that circRNAs often regulate gene expression post-transcriptionally, we next evaluated EWS::FLI1 protein levels and observed a reduction in EWS::FLI1 protein in circZNF609 knockdown cell lines, as determined by both immunofluorescence (Figure 5A,B) and Western blot analysis (Figure 5C,D). Further, we also performed polysome fractionation and found a decreased association of EWS::FLI1 mRNA with polysomes in circZNF609 knockdown cells compared to WT cells (Figure 5E,F). An inducible EWS::FLI1 knockdown (EFKD) in A673 cells was used as a positive control that also showed reduced EWS::FLI1 protein, as expected. To confirm that this is not a cell line-specific phenomenon, we repeated the Western blot and immunofluorescence analysis in an additional EwS cell line, TC32, and found similar results (Supplementary Figure S5). These findings further indicate that circZNF609 affects the translation of EWS::FLI1 mRNA.

Mechanism of EWS::FLI1 downregulation by circZNF609: Since the most well-studied function of circRNAs is mediated by sponging of miRNAs [11,12,36], we investigated whether a similar regulatory mechanism occurs in EwS cells. Several miRNAs have been reported in the literature to be differentially regulated by EWS::FLI1, although not all directly inhibit EWS::FLI1 expression [37]. Likewise, many miRNAs have been reported to be sponged by circZNF609 in various cancers [18,19,35,38,39,40]. Given the observed reduction in EWS::FLI1 protein in circZNF609 knockdown cells, we aimed to identify miRNAs that both bind to EWS::FLI1 and have binding sites on circZNF609. Using TargetScan and miRDB, we identified 152 miRNAs predicted to bind the 3′ UTR of EWS::FLI1 and found them in both databases (Figure 6A), which we further narrowed down to 46 that are functionally relevant miRNAs using miRDB (Figure 6B). We then identified all the miRNAs predicted to have a binding site for circZNF609 and compared them to all miRNAs predicted to bind FLI1 3′ UTR. Finally, we compared this analysis to the miRTarBase v8 hg to find which of these common miRNAs are reported to be expressed in human cells, and we identified seven common miRNAs (Figure 6C). All seven were tested by qRT-PCR to determine their levels in EwS cells, and we found that miR-145-5p showed the most significant EWS::FLI1-dependent expression (Figure 6D). Numerous studies have reported that circZNF609 acts by sponging miR-145-5p, as seen in glioblastoma [18,39,41]. Furthermore, miR-145-5p is a key miRNA in EwS, and its reduction has been reported to promote oncogenesis in EwS via a yet unknown mechanism [14,42]. Finally, miR145-5p has been shown to regulate the expression of FLI1 in osteosarcoma [43].

Direct visualization of circZNF609 and miR145-5p interaction: To directly visualize the interaction between circZNF609 and miR-145-5p, we combined our circFISH assay with miRNA imaging to assess their co-localization. CircZNF609 was detected using probes labeled with Cy5, and after hybridization with circFISH probes, cells were further stained with miRNA probes requiring signal amplification. We observed that a subset of circZNF609 RNA molecules co-localized with the miR-145-5p signal. Although the signals were somewhat diminished due to multiple rounds of washing, staining, and signal processing, the data clearly demonstrate that circZNF609 has a binding site for miR-145-5p and that this interaction occurs in A673 cells (Figure 7).

Functional validation of the role of circZNF609 sponging of miR145-5p in EwS: It is well established that miR-145-5p is significantly repressed by EWS::FLI1 and functions in a feedback regulatory loop in EwS, although the mechanism of miR-145-5p suppression is not understood [14,44,45]. Interestingly, we observed a reduction in circZNF609 levels in EWS::FLI1 knockdown (EFKD) cells, indicating that its expression is positively correlated with EWS::FLI1 (Figure 8A). Consistent with the existing literature, miR-145-5p levels showed a significant increase in EFKD cells (Figure 8B), but there was no reduction in miR-145-5p levels in circZNF609 knockdown cells, suggesting that circZNF609 does not regulate the expression of miR-145-5p (Figure 8C) [14]. This is not unexpected, as circRNAs typically do not affect the expression but rather the function of their target miRNAs [46]. To assess the impact of circZNF609 on miR-145-5p function, we performed a luciferase assay by cloning two fragments of the FLI1 3′ UTR into the psiCHECK2 vector downstream of the Renilla luciferase coding region. Fragment 1 contains the binding site for miR-145-5p, while fragment 2 does not (Figure 8D). TC32 cells (WT, ZSCR, and circZNF609-KD) were transfected with these constructs, along with empty vector controls. We observed a change in luciferase expression only with FLI1 3′ UTR fragment 1, with reduced luciferase activity in circZNF609-KD cells, while fragment 2 remained unchanged (Figure 8E), suggesting that circZNF609 sponges miR-145-5p. Finally, to show that the effect is due to the sponging of miR-145 by circZNF609, we added a mimic of miR145-5p to WT cells that should overexpress the miRNA and thus reverse the sponging effect of circZNF609. Indeed, addition of a miR-145-5p mimic showed a dose-dependent response reduction in luciferase activity in TC32 WT cells expressing fragment 1 of FLI1 3′ UTR (Figure 8F), with a marked effect seen at 5 nM concentration of the mimic. To further confirm the specificity of this effect, we repeated this experiment using 5 nM of a negative control mimic, which did not affect luciferase activity (Figure 8G). These results confirmed that the effect is specific to miR-145 binding to the 3′ UTR, which is also a target of circZNF609. Together, the direct visualization of the circRNA:miRNA interaction and the functional luciferase assay validated and provided the first evidence for the functional circZNF609–miR-145–EWS::FLI1 regulatory axis in EwS.

circZNF609 functions as a regulatory RNA in EwS: Although circRNAs are generally classified as non-coding RNAs, a few have been shown to be translated into short peptides via cap-independent mechanisms, such as internal ribosome entry sites [47]. Notably, circZNF609 is among the few circRNAs with demonstrated coding potential, and its peptide has been shown to have functional roles in myogenesis and acute kidney disease [29,48]. This prompted us to investigate whether the peptide form of circZNF609 plays a role in EwS. We performed polysome fractionation and found no significant changes in the association of circZNF609 RNA with polysome fractions among WT, EFKD, and circZNF609KD cells, suggesting that if the peptide is produced, it does not play a functional role in EwS. These results confirm that circZNF609 functions as a regulatory RNA in EwS (Supplementary Figure S6).

4. Discussion

Over the past decade, ample evidence has demonstrated the impact of circRNAs on the development and progression of various diseases, particularly human cancers such as lung, prostate, colon, breast, and hepatocellular carcinomas, as well as osteosarcoma [13,19,35,49,50,51,52,53]. CircRNAs are highly stable due to their resistance to exonucleases and have been detected in extracellular vesicles and bodily fluids, making them promising candidates for biomarkers and therapeutics [9,13,54,55].

CircZNF609 is a very well-studied oncogenic circRNA [35]. Existing research has demonstrated the importance of circZNF609 in regulating cell proliferation, migration, and metastasis in several human malignancies, including hepatocellular carcinoma, breast cancer, lung adenocarcinoma, gastric cancer, and glioma [17,18,35,56,57]. However, the expression and function of circZNF609 have not previously been reported in EwS. Here, we present the first characterization of a functional circular RNA in EwS. We validated the expression, role, and mechanism of action of circZNF609 in EwS and identified the circZNF609:miR-145-5p:FLI1 regulatory axis. Our data show that circZNF609 is expressed in an EWS::FLI1-dependent manner, and it is well established that miR-145-5p is a critical miRNA capable of suppressing EWS::FLI1 expression. Furthermore, FLI1 is not expressed in normal cells, which is why EWS::FLI1 is often detected and studied using antibodies against the 3′ end of FLI1 [58]. Integrating our new functional data with existing studies, we propose a model in which, under normal conditions, cells express miR-145-5p, which binds to the 3′ UTR of FLI1 and keeps EWS::FLI1 translationally inhibited after the translocation occurs. However, as EWS::FLI1 expression increases, its concentration likely surpasses the regulatory capacity of miR-145-5p, allowing EWS::FLI1 protein to accumulate. Early expression of EWS::FLI1 induces circZNF609 and other effectors that repress miR-145-5p expression by yet unknown mechanisms. The expression of circZNF609 leads to sponging of miR-145-5p, thereby blocking its function and enabling higher levels of EWS::FLI1 protein, which, in turn, promotes aberrant gene expression and increases proliferation, invasiveness, and growth in EwS (Figure 9). This positive feedback loop ensures that EWS::FLI1 protein concentrations remain high in the cells, representing a novel strategy by which EWS::FLI1 evades repression by miRNAs.

This is the first report describing the role of circular RNAs in EwS. A recent work by Chen et al. has reported another circRNA called circCAMSAP1 to sponge miR-145 to de-repress FLI1 expression in osteosarcoma in both cell lines and animal models, providing a comparative perspective on circRNA functions across different sarcomas as well as the biological relevance of reducing miR145-FLI1 interaction [43]; however, this circRNA has not been identified in EwS cell lines. Through genome profiling, we identified more circRNAs, of which several, including circZNF609, are upregulated, and some are downregulated in EwS, indicating diverse functional roles for these regulatory RNAs that remain to be explored (unpublished data). We acknowledge that a limitation of this study is the lack of analysis of circZNF609 levels in patient samples. Future studies are planned to test the expression of circZNF609 in patient samples, as well as to functionally characterize the role of other circRNAs. A recent study showed that EWS::FLI1 is not just a “rogue” transcription factor (regulating gene expression at the DNA level), but it also functions independently as an mRNA decay factor (regulating the lifespan of genetic messages) [59]. By identifying and characterizing the mechanism of action of the first circular RNA in EwS, our study provides another possible function of EWS::FLI1 as an “miRNA silencer” and will set the foundation for future research aimed at developing better diagnostic markers and therapeutic targets for EwS.