Exosomal circVANGL1 Enhances Cisplatin Resistance in Osteosarcoma Cells by Regulating the miR-145-5p/E2F3 Axis

Exosomal circVANGL1 Enhances Cisplatin Resistance in Osteosarcoma Cells by Regulating the miR-145-5p/E2F3 Axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exosomal circVANGL1 Enhances Cisplatin Resistance in Osteosarcoma Cells by Regulating the miR-145-5p/E2F3 Axis Fan Mo, Hao Wang, Qi Xie, Dahong Huang, Yuan Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6734085/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background and Objectives: osteosarcoma (OS) is a malignant bone tumor originating directly from bone tissue, predominantly affecting adolescents. Cisplatin (DDP)-based chemotherapy is commonly used in the treatment of OS, but the emergence of DDP resistance poses a significant challenge to effective management. The study aimed to investigate the role of circRNA VANGL1 (circVANGL1) in mediating DDP resistance in OS. Materials and Methods the OS cell lines U2OS and MG-63 were used, and DDP-resistant cell models (U2OS-DR and MG-63-DR) were established. Transfection with circVANGL1 siRNA was performed to silence circVANGL1 expression, and its impact on cell proliferation, apoptosis, DDP resistance, and miR-145-5p level was studied. Fluorescence in situ hybridization was employed to localize circVANGL1. TargetScan prediction was utilized to identify the interaction between circVANGL1 and miR-145-5p. Co-transfection experiments of si-circVANGL1 with anti-miR-145-5p or E2F3 overexpression vector (E2F3-oe) were conducted to assess their effects on DDP-resistant cells. Results circVANGL1 expression was greatly elevated in U2OS-DR and MG-63-DR cells. Transfection with si-circVANGL1 effectively suppressed cell proliferation and DDP resistance while promoting apoptosis in DDP-resistant cells. circVANGL1 is transferred via exosomes and primarily localized in the cytoplasm of U2OS and MG-63 cells. TargetScan prediction indicated a target relationship between circVANGL1 and miR-145-5p. Co-transfection of si-circVANGL1 with anti-miR-145-5p or E2F3-oe counteracted the changes in proliferation and apoptosis observed with si-circVANGL1 transfection alone in DDP-resistant cells. Conclusion Exosomal circVANGL1 contributes to DDP resistance in OS cells via modulation of miR-145-5p/E2F3 axis. osteosarcoma cisplatin Drug resistance circRNA circVANGL1 miR-145-5p E2F3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Osteosarcoma (OS) is a malignant tumor originating from bone tissue, commonly found in children and adolescents, characterized by high metastatic potential and mortality rates (1). It is estimated that approximately 12,000 new cases of OS are diagnosed worldwide each year, with most patients presenting with micrometastases at the time of initial diagnosis (2). Although the combination of surgical resection and adjuvant chemotherapy has significantly improved survival rates, OS remains one of the most common malignant bone tumors in children and adolescents, and the prognosis remains poor (3). Chemotherapy is one of the primary treatment modalities for OS. Cisplatin (DDP), a standard chemotherapy drug, is widely used in the treatment of OS. However, the emergence of DDP resistance significantly limits the effectiveness of chemotherapy and poses a major challenge in OS treatment (4,5). Therefore, investigating the molecular mechanisms underlying DDP resistance and identifying novel therapeutic targets is crucial for improving survival rates and enhancing prognosis for OS patients. Circular RNAs (circRNAs) are a type of non-coding RNA. Initially, circRNAs were thought to be rare and even misinterpreted as transcriptional noise and artifacts (6), but in recent years, they have been studied many times in tumor biology (7). Some circRNAs were identified, which were closely correlated with OS (8). By interacting with the RNA-binding protein RBM15, circ-CTNNB1 regulates the expression of key glycolysis-related genes, such as HK2, GPI, and PGK1, through m6A modification, thereby promoting aerobic glycolysis and driving the progression and metastasis of OS (9). Huang et al. (2020) (10) reported that increased expression of circRNAs is associated with adverse clinical features, with oncogenic circRNAs negatively impacting overall survival, while upregulation of tumor-suppressive circRNAs extends survival. CircVANGL1 is derived from two exons of the Van Gogh-like 1 (VANGL1) gene. Studies have shown that circ-VANGL1 downregulates RUNX2 expression by binding to miRNA-217, thereby promoting the development of osteoporosis (11). Research indicated that circVANGL1 is upregulated in various tumors and is closely related to tumorigenesis and progression (12,13). Additionally, it has been found that the deletion of circVANGL1 can inhibit the viability of bladder cancer cells in vitro, induce apoptosis, and reduce resistance to doxorubicin (14). However, the specific role of circVANGL1 in DDP resistance in OS and its underlying molecular mechanisms remain unclear. This study aimed to investigate the function of circVANGL1 in DDP resistance in OS and its regulatory mechanisms. By establishing a DDP-resistant OS cell model, this study examined changes in circVANGL1 expression and its effects on cell proliferation, apoptosis, and DDP resistance. Furthermore, this study explored whether circVANGL1 contributes to DDP resistance in OS by regulating the miR-145-5p/E2F3 axis. The goal was to elucidate the role and mechanisms of circVANGL1 in DDP resistance in OS, fill the current gap in circRNA research in this field, and provide a theoretical foundation for future clinical applications. Materials and methodologies Cell transfection OS cells (U2OS and MG-63) (provided by Manassas, USA) were cultured in DMEM (Invitrogen, USA) +FBS (HyClone, USA), 100U/mL penicillin, and 100μg/mL streptomycin at 37°C in an incubator with 5% CO 2 . DDP-resistant OS cells were exposed to parental cells to increased concentrations (0.5–25 µM) of cis-diamineplatinum(II) dichloride (cisplatin; Sigma-Aldrich, USA) for 3 months. si-circVANGL1, siRNA for negative control (NC) (si-NC), miR-145-5p mimics (miR-145-5p), mimics for NC (miR-NC), anti-miR-145-5p, and inhibitor for NC (anti-miR-NC) were supplied by Guangzhou RiboBio Co., Ltd. (China). To construct E2F3 overexpression plasmid, E2F3 cDNA sequence was prepared and cloned into pcDNA3.1 vector (Invitrogen). An empty vector was undertaken as the NC. After seeding in 6-well plates, the cells were cultured to 70-80% confluence. Transient transfection was performed using Lipofectamine 3000 (Invitrogen). After two days, RT-qPCR was employed to analyze the transfection efficacy. Establishment and screening of DDP-resistant cell model DDP-resistant cell lines, U2OS-DR and MG-63-DR, were developed by gradually increasing the DDP concentration. The initial concentration was 0.1 μM, which was progressively raised to 1.0 μM. After each increase in DDP concentration, cells were subcultured 2-3 times and stabilized in drug-free medium for one week to ensure adaptation to the new drug concentration. During the entire culture process, cells were periodically cryopreserved to prevent experimental interruptions. After 7 months of continuous culture, the drug-resistant cell lines were successfully established and able to survive at a DDP concentration of 1.0 μM. Prior to functional assays, the resistant cell lines were maintained in drug-free complete medium for at least 2 weeks to restore normal growth conditions. For cell passaging, cells were centrifuged at 1000 rpm for 5 minutes to separate the cells and remove the spent culture medium. Isolation of exosomes The samples were first centrifuged at 300, 2,000, and 10,000×g for 10, 15, and 30 minutes to remove the cells and cell debris. Next, supernatant was further ultracentrifuged at 120,000×g for 2 h, and the exosome pellet was resuspended in 100 μL PBS. Transmission electron microscopy (TEM) observation Exosomes were negatively stained by 2% phosphotungstic acid for around 2 minutes on a copper grid carbon-coated. Then, the grid with stained exosomes was dried at the ambient temperature, and examined using a JEM-1200 EX microscope (JEOL, Japan). RT-qPCR Here, TRIzol reagent (Invitrogen) extracted total RNA, and miRNeasy Serum/Plasma kit (Qiagen, USA) was used to extract total RNA from exosomes. The target gene expression was computed via 2 -ΔΔCt (15). The expression levels of circVANGL1 were analyzed using GAPDH as the internal reference gene, and the expression levels of miR-145-5p were analyzed using U6 as the internal reference gene. The primers used for the analysis were as follows: circVANGL1 forward primer: 5’-GTCCGCTCCACCGATGGCGA-3’, reverse primer: 5’-CTGAACTTCCTCTGTCCGAGT-3’; GAPDH forward primer: 5’-CCGGGAAACTGTGGCGTGATGG-3’, reverse primer: 5’-AGGTGGAGGAGTGGGTGTCGCTGTT-3’; miR-145-5p forward primer: 5’-CAGTCTTGTCCAGTTTTCCCAG-3’, reverse primer: 5’-TATGCTTGTTCTCGTCTCTGTGTC-3’; U6 forward primer: 5’-CTCGCTTCGGCAGCACA-3’, reverse primer: 5’-AACGCTTCACGAATTTGCGT-3’.” MTT assay MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to assess the impact of DDP treatment on the viability of U2OS and MG-63 cells. The detailed procedure is as follows: U2OS, MG-63, U2OS-DR, and MG-63-DR cells were seeded into 96-well plates at a density of 5×10 3 cells per well. After cell attachment, the cells were treated with different concentrations of DDP (1.5, 3, 6, 12, and 24 μM). A well without DDP was set as the control group. After 2 days of treatment, 20 μL of MTT solution (5 mg/mL; Sigma-Aldrich) was added to each well, and the cells were incubated for 4 hours at 37°C in a 5% CO 2 incubator. Following incubation, 150 μL of DMSO was added to each well to dissolve the purple formazan crystals formed. After shaking the plate for 10 minutes in the dark at room temperature, the absorbance (OD value) at 570 nm was measured using a microplate reader. The cell viability was evaluated by calculating the OD ratio of each treatment group compared to the control group. All experiments were repeated three times to ensure the reliability and reproducibility of the data. Cell apoptosis After DDP treatment, cells were harvested, resuspended in 1×binding buffer, and labeled with 5µL Annexin V-FITC and 10µL PI utilizing Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, USA) in the dark. After 15 minutes, cell apoptosis was evaluated via a flow cytometry (BD Biosciences). Western blot (WB) The RIPA lysis buffer (Solarbio, China) was employed for extraction of the total protein. Equal amounts of protein were separated via SDS-PAGE, transferred to PVDF membranes (Merck Millipore). 5% FBS was employed to block the membranes, which were then mixed with primary antibodies for incubation at 4°C overnight and with HRP-conjugated secondary antibody for 60 minutes. Protein bands were visualized by using GAPDH and β-actin as a control. Fluorescence in situ hybridization (FISH) Cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized in PBS with 0.5% Triton X-100 for 5 minutes, and hybridized with specific Cy3-labeled circVANGL1 probe (Guangzhou RiboBio Co., Ltd., China) at 37 °C overnight. DAPI was applied for nuclear complex staining. Slides were photographed with a Leica SP5 confocal microscope (Leica Microsystems, Germany). Dual-luciferase reporter assay (DLRA) Sequence of circVANGL1 or E2F3 mRNA was amplified and subcloned into psiCHECK-2 vector (Promega, USA). circVANGL1-wt/mut or E2F3-wt/mut were co-transfected with miR-145-5p or miR-NC into cells. After two days of incubation, luciferase activity (LA) was detected by the DLRA system (Promega). Statistical analysis All experiment procedures were implemented and repeated three times, based on which the average values of each indicator were calculated. Results were denoted in mean value ± standard deviation (SD). At the same time, Student’s t -test/one-way ANOVA assessed statistical significance. Correlation was assessed by Pearson coefficient. P <0.05 meant statistically significant. Results circVANGL1 is elevated in OS cells with property of DDP resistance U2OS and MG-63 cells were exposed to incremental doses of DDP to construct DDP-resistant U2OS-DR cell and MG-63-DR cell, respectively, for further analysis. MTT assay verified that the U2OS-DR cell and MG-63-DR cell upon DDP treatment showed remarkably increased viability compared to that of parental cells (Fig. 1 A). In addition, U2OS-DR cell and MG-63-DR cell had lower apoptosis rates after 48 h of DDP (1.5 µM) treatment (Fig. 1 B). The RT-qPCR results in Fig. 1 C show that circVANGL1 expression was elevated in U2OS-DR and MG-63-DR cells. circVANGL1 knockdown reduces DDP resistance of OS cells To investigate the functions of circVANGL1, we carried out loss-of-function experiments. siRNA targeting circVANGL1 was synthesized and incorporated into DDP-resistant OS cells. In addition, the levels of circVANGL1 in U2OS-DR cell and MG-63-DR cell were notably decreased after transfection. Data was not shown. Figure 2 demonstrated the DDP resistance of OS cells reduced by the circVANGL1 knockdown. MTT assay further showed that circVANGL1 knockdown markedly reduced the viability of both U2OS-DR cell and MG-63-DR cell upon DDP treatment (Fig. 2 A). Besides, the apoptosis rates of both U2OS-DR cell and MG-63-DR cell cells after 48 h of DDP (1.5 µM) treatment were remarkably increased by circVANGL1 knockdown (Fig. 2 B). ExosomalcircVANGL1 spreads DDP resistance in OS cells Figure 3 illustrates the spread of DDP resistance promoted by the exosomal circVANGL1. The morphology of collected exosomes demonstrated a cup-shaped morphology with a median diameter of ~ 100 nm (Fig. 3 A). WB detection revealed positive expression of TSG101, CD63, and CD81 in the isolated exosomal fractions, but the case was not same in the cell extracts (Fig. 3 B). We further observed that circVANGL1 expression was markedly increased in the exosomes from U2OS-DR cell and MG-63-DR cell compared with their parental cells (Fig. 3 C). Moreover, as shown in Fig. 3 D and Fig. 3 E, co-culture with exosomes of DDP-resistant OS cells for 48 h obviously induced DDP resistance in their parental cells. circVANGL1 functions in miR-145-5p in OS circVANGL1 functions in miR-145-5p in OS were illustrated in Fig. 4 . It was verified that circVANGL1 was expressed in cytoplasm of U2OS and MG-63 cells (Fig. 4 A), suggesting circVANGL1may function at the post-transcriptional level. miR-145-5p harbor a complementary binding sequence of circVANGL1 (Fig. 4 B). What’s more, the interaction was further validated by DLRA. The results shown in Fig. 4 C suggested miR-145-5p considerably reduced LA of circVANGL1-wt in U2OS and MG-63 cells, but the inhibitory was abolished after maturity of binding sites. In addition, miR-145-5p decline in both U2OS-DR cell and MG-63-DR cell was notably reversed by circVANGL1 knockdown (Fig. 4 D). Meanwhile, we noted miR-145-5p in exosomes from both cells was markedly decreased (U2OS-DR cell and MG-63-DR cell) (Fig. 4 E). miR-145-5p inhibition abates role of circVANGL1 knockdown on DDP-resistant OS cells After analysis on TargetScan ( http://www.targetscan.org/vert_71/ ), we noted a putative region in 3′-UTR of E2F3 mRNA that may bind to miR-145-5p (Fig. 5 A). As demonstrated in Fig. 5 B, LA of E2F3-wt in U2OS cells was notably reduced, and it was the same case in the MG-63 cells. The elevated expressed E2F3 in U2OS-DR cell and MG-63-DR cell cell was blocked by circVANGL1 knockdown, and miR-145-5p inhibition changed the effects reversely (Fig. 5 C). miR-145-5p inhibition or E2F3 overexpression damaged effects of circVANGL1 knockdown on the two cells (Fig. 5 D-E ) . Discussion circRNAs is identified as potential functional regulators in cancer progression. circVANGL1 is generally reported as an oncogene, and increased circRNAs level is associated with malignant phenotypes in various kinds of human cancers ( 16 , 17 ). Some circRNAs were identified, which were closely correlated with OS. Chemotherapy is a common therapeutic option used against cancer, and DDP is a standard drug for OS therapy; however, DDP resistance can hinder the OS management greatly. The development of DDP resistance is a multifactorial and multistep process involving mechanisms such as cell proliferation, apoptosis, DNA repair, and drug efflux ( 18 ). This study found that the expression of circVANGL1 was significantly upregulated in DDP-resistant OS cells, suggesting that circVANGL1 may be involved in the regulation of DDP resistance. By knocking down circVANGL1, we observed a significant reduction in the proliferative capacity of DDP-resistant cells, suppression of DDP resistance, and a notable increase in cell apoptosis. These results indicated that circVANGL1 plays a crucial role in maintaining DDP resistance, likely through the regulation of cell proliferation and apoptosis-related signaling pathways. Exosomes are secreted by most eukaryotic cells and are naturally present in body fluids. They are important messengers in the intricate intercellular communication for progression of related cancers ( 19 , 20 ). circRNAs are enriched in exosomes ( 21 ). Our research proved that circVANGL1 could be incorporated into exosomes and transmitted to DDP-sensitive OS cells, thus disseminating DDP resistance. Detection of exosomal circRNAs in blood samples can provide additional evidence for conventional diagnostic methods ( 22 ), and the clinical values of circulating exosomal circVANGL1 in OS need further work. Many miRNA binding elements exist in circRNAs and can exert the role of ceRNAs to indirectly regulate the target protein of miRNAs ( 23 ). CeRNAs are frequently involved in many cancers’ pathogenesis, and deciphering ceRNA interplay may provide opportunities for cancer therapy exploration ( 24 ). According to previous records, miR-145-5p is a tumor suppressor in OS ( 25 ), and circVANGL1 interacts with miR-145-5p in OS directly. circVANGL1 knockdown also caused increased miR-145-5p in DDP-resistant OS cells. E2F3 is oncogenic in tumorigenesis, and it can be activated/inhibited by numerous miRNAs ( 26 ). The results indicated that circVANGL1 regulates the expression of E2F3 through direct interaction with miR-145-5p, thereby affecting the proliferation, apoptosis, and chemoresistance of DDP-resistant OS cells. MiR-145-5p is a tumor-suppressive miRNA, and its expression is downregulated in various cancers, potentially contributing to drug resistance. As a ‘sponge’ for miR-145-5p, circVANGL1 may sequester miR-145-5p, inhibiting its regulation of target genes, thereby promoting cell proliferation and suppressing apoptosis, leading to the development of DDP resistance. Overexpression of E2F3 partially reversed the effects of circVANGL1 knockdown on cell proliferation and apoptosis in DDP-resistant cells, suggesting that circVANGL1 may influence DDP resistance through the regulation of E2F3 expression. The cytoplasm is a key site for circRNA function, and circVANGL1, located in the cytoplasm, may mediate intercellular signaling via exosome transport, potentially interacting with cytoplasmic RNA-binding proteins or miRNAs to influence drug resistance regulation in the tumor microenvironment. This mechanism warrants further investigation in the context of tumor drug resistance. These findings suggest that circVANGL1 is a crucial regulatory factor in DDP-resistant OS cells, and targeting circVANGL1 may provide a novel therapeutic strategy to overcome DDP resistance. By regulating miR-145-5p and E2F3, circVANGL1 could be combined with other targeted therapies or immunotherapies to further enhance therapeutic efficacy. To conclude, in this research, we firstly demonstrated that exosomal circVANGL1 enhances resistance of OS cells against DDP partly through regulating miR-145-5p/E2F3 axis. This study explored the mechanism by which circVANGL1 regulates DDP resistance through the miR-145-5p/E2F3 axis. However, there are certain limitations to this study. The upstream transcription factors, epigenetic regulatory factors, and RNA-binding proteins that modulate circVANGL1 were not thoroughly investigated. Additionally, whether other molecules participate in regulating this network remains to be further explored. Furthermore, the silencing effect of circVANGL1 in vivo and its safety still need to be validated. Future work will focus on further investigating the upstream regulatory mechanisms of circVANGL1, as well as exploring other potential miRNAs or pathways involved. Additionally, the delivery system for circVANGL1 will be optimized, and its safety and efficacy in in vivo applications will be assessed, with the goal of gaining a more comprehensive understanding of its role in OS development and treatment. In conclusion, this work provides a new mechanism for understanding DDP resistance in OS. Declarations Acknowledgment: This work was supported by Self-funded scientific research project of Guangxi Health Commission (project No. Z-A20221074). Data Availability The data used to support the findings of this study are available from the corresponding author upon request. Conflicts of Interest The authors declare no conflicts of interest. Clinical trial number: Not applicable Consent to Publish declaration: Not applicable Consent to Participate declaration: Not applicable Ethics declaration: Not applicable Conflicts of Interest ： The authors declare no conflicts of interest. References Isakoff MS, Bielack SS, Meltzer P, Gorlick R. Osteosarcoma: Current Treatment and a Collaborative Pathway to Success. J Clin oncology: official J Am Soc Clin Oncol. 2015;33(27):3029–35. Cole S, Gianferante DM, Zhu B, Mirabello L, Osteosarcoma. 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Supplementary Files CD63.tif CD81.tif TSG101.tif Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Oct, 2025 Reviews received at journal 20 Oct, 2025 Reviews received at journal 20 Oct, 2025 Reviewers agreed at journal 05 Oct, 2025 Reviews received at journal 04 Oct, 2025 Reviewers agreed at journal 02 Oct, 2025 Reviewers agreed at journal 30 Sep, 2025 Reviewers invited by journal 29 Sep, 2025 Editor assigned by journal 21 Aug, 2025 Submission checks completed at journal 31 Jul, 2025 First submitted to journal 31 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":64390,"visible":true,"origin":"","legend":"\u003cp\u003ecircVANGL1 in DDP-resistant OS cells. Figure A: viability of OS cells upon DDP treatment. Figure B: apoptosis of OS cells upon DDP treatment. Figure C: expression levels of circVANGL1 in OS cells. \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 was marked by *.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6734085/v1/3c1e12f80bbac55697d8a9fb.png"},{"id":93234257,"identity":"c29c9abd-a004-46ae-8fa5-76b90c2ec189","added_by":"auto","created_at":"2025-10-10 13:41:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60094,"visible":true,"origin":"","legend":"\u003cp\u003ecircVANGL1 knockdown reduces DDP resistance of OS cells. Figure A: MTT assay showing the DDP response of DDP-resistant OS cells after circVANGL1 knockdown. Figure B: Flow cytometric analysis showing the DDP-induced apoptosis of DDP-resistant OS cells after circVANGL1 knockdown. \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 was marked by *.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6734085/v1/dc4eef2c094eb995310a821f.png"},{"id":93233205,"identity":"bae6115e-46f3-42db-9a3d-e3997be3e4f8","added_by":"auto","created_at":"2025-10-10 13:33:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":199754,"visible":true,"origin":"","legend":"\u003cp\u003eExosomal circVANGL1 spreads DDP resistance in OS cells. (A) Themorphology of collected exosomes under TEM. (B) Expression levels of exosomal protein markers (TSG101, CD9, CD63) in purified exosomes and cell extracts detected by WB. (C) Expression levels of circVANGL1 in the exosomes of OS cells. (D) DDP response of parental OS cells after exosomes treatment. (E) The DDP-induced apoptosis of parental OS cells after exosomes treatment measured by Flow cytometric analysis. \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 was marked by *.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6734085/v1/5336235fcfc25006badb1584.png"},{"id":93233207,"identity":"903c55d0-2ae6-4006-9e9c-c84d5bfb3e93","added_by":"auto","created_at":"2025-10-10 13:33:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":121469,"visible":true,"origin":"","legend":"\u003cp\u003ecircVANGL1 for miR-145-5p in OS. Figure A: FISH analysis showing the subcellular distribution of circVANGL1. Figure B: miR-145-5p-predicted binding site. C: DLRA verifying binding between circVANGL1 and miR-145-5p. D: levels of miR-145-5p in OS cells with property of DDP resistance after circVANGL1 knockdown. Figure E: miR-145-5p levels in exosomes of OS cells. \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 was marked by * in the above figure.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6734085/v1/0a6275abbb1e394371320280.png"},{"id":93234261,"identity":"97cfba7f-a987-4c13-8fcd-4b656d5bec4b","added_by":"auto","created_at":"2025-10-10 13:41:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":120193,"visible":true,"origin":"","legend":"\u003cp\u003emiR-145-5p inhibition abates circVANGL1 knockdown on DDP-resistant OS cells. (A) miR-145-5p binding site within E2F3 mRNA. Figure B: DLRA verifying the binding between E2F3 mRNA and miR-145-5p s. Figure C: WB analysis showing the expression of E2F3 protein in DDP-resistant OS cells after circVANGL1 knockdown/miR-145-5p inhibition. (D) MTT assay showing the DDP response of OS cells with property of DDP resistance after miR-145-5p inhibition/E2F3 overexpression. (E) Flow cytometric analysis showing the DDP-induced apoptosis of OS cells with property of DDP resistance after miR-145-5p inhibition/E2F3 overexpression. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6734085/v1/9e21d515616bda49ee4f0ed4.png"},{"id":93234608,"identity":"b829b952-a41e-411f-9d33-707258eb2998","added_by":"auto","created_at":"2025-10-10 13:49:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1165902,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6734085/v1/e28b8aab-a48c-4a16-9e37-568cc5c54a8b.pdf"},{"id":93233203,"identity":"2386296d-60a5-469d-ba4f-66ec8ff06ccd","added_by":"auto","created_at":"2025-10-10 13:33:36","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":671830,"visible":true,"origin":"","legend":"","description":"","filename":"CD63.tif","url":"https://assets-eu.researchsquare.com/files/rs-6734085/v1/cdbbc1b7669bb96e5693397b.tif"},{"id":93234260,"identity":"687681db-4e7f-4003-bdcb-38503b773b93","added_by":"auto","created_at":"2025-10-10 13:41:36","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":718450,"visible":true,"origin":"","legend":"","description":"","filename":"CD81.tif","url":"https://assets-eu.researchsquare.com/files/rs-6734085/v1/48084c31d6b644bc162ecc1a.tif"},{"id":93234264,"identity":"a18424fb-c35b-4c53-bfd1-841e7e52585d","added_by":"auto","created_at":"2025-10-10 13:41:37","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":649052,"visible":true,"origin":"","legend":"","description":"","filename":"TSG101.tif","url":"https://assets-eu.researchsquare.com/files/rs-6734085/v1/43455b21e7e97a787a1f5f0a.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exosomal circVANGL1 Enhances Cisplatin Resistance in Osteosarcoma Cells by Regulating the miR-145-5p/E2F3 Axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteosarcoma (OS) is a malignant tumor originating from bone tissue, commonly found in children and adolescents, characterized by high metastatic potential and mortality rates (1). It is estimated that approximately 12,000 new cases of OS are diagnosed worldwide each year, with most patients presenting with micrometastases at the time of initial diagnosis (2). Although the combination of surgical resection and adjuvant chemotherapy has significantly improved survival rates, OS remains one of the most common malignant bone tumors in children and adolescents, and the prognosis remains poor (3). Chemotherapy is one of the primary treatment modalities for OS. Cisplatin (DDP), a standard chemotherapy drug, is widely used in the treatment of OS. However, the emergence of DDP resistance significantly limits the effectiveness of chemotherapy and poses a major challenge in OS treatment (4,5). Therefore, investigating the molecular mechanisms underlying DDP resistance and identifying novel therapeutic targets is crucial for improving survival rates and enhancing prognosis for OS patients.\u003c/p\u003e\n\u003cp\u003eCircular RNAs (circRNAs) are a type of non-coding RNA. Initially, circRNAs were thought to be rare and even misinterpreted as transcriptional noise and artifacts\u0026nbsp;(6), but in recent years, they have been studied many times in tumor biology (7). Some circRNAs were identified, which were closely correlated with OS (8). By interacting with the RNA-binding protein RBM15, circ-CTNNB1 regulates the expression of key glycolysis-related genes, such as HK2, GPI, and PGK1, through m6A modification, thereby promoting aerobic glycolysis and driving the progression and metastasis of OS (9). Huang et al. (2020) (10) reported that increased expression of circRNAs is associated with adverse clinical features, with oncogenic circRNAs negatively impacting overall survival, while upregulation of tumor-suppressive circRNAs extends survival. CircVANGL1 is derived from two exons of the Van Gogh-like 1 (VANGL1) gene. Studies have shown that circ-VANGL1 downregulates RUNX2 expression by binding to miRNA-217, thereby promoting the development of osteoporosis (11). Research indicated that circVANGL1 is upregulated in various tumors and is closely related to tumorigenesis and progression (12,13). Additionally, it has been found that the deletion of circVANGL1 can inhibit the viability of bladder cancer cells in vitro, induce apoptosis, and reduce resistance to doxorubicin (14).\u003c/p\u003e\n\u003cp\u003eHowever, the specific role of circVANGL1 in DDP resistance in OS and its underlying molecular mechanisms remain unclear. This study aimed to investigate the function of circVANGL1 in DDP resistance in OS and its regulatory mechanisms. By establishing a DDP-resistant OS cell model, this study examined changes in circVANGL1 expression and its effects on cell proliferation, apoptosis, and DDP resistance. Furthermore, this study explored whether circVANGL1 contributes to DDP resistance in OS by regulating the miR-145-5p/E2F3 axis. The goal was to elucidate the role and mechanisms of circVANGL1 in DDP resistance in OS, fill the current gap in circRNA research in this field, and provide a theoretical foundation for future clinical applications.\u003c/p\u003e"},{"header":"Materials and methodologies","content":"\u003cp\u003e\u003cstrong\u003eCell transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOS cells (U2OS and MG-63) (provided by Manassas, USA) were cultured in DMEM (Invitrogen, USA) +FBS (HyClone, USA), 100U/mL penicillin, and 100μg/mL streptomycin at 37°C in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eDDP-resistant OS cells were exposed to parental cells to increased concentrations (0.5–25 µM) of cis-diamineplatinum(II) dichloride (cisplatin; Sigma-Aldrich, USA) for 3 months.\u003c/p\u003e\n\u003cp\u003esi-circVANGL1, siRNA for negative control (NC) (si-NC), miR-145-5p mimics (miR-145-5p), mimics for NC (miR-NC), anti-miR-145-5p, and inhibitor for NC (anti-miR-NC) were supplied by Guangzhou RiboBio Co., Ltd. (China). To construct E2F3 overexpression plasmid, E2F3 cDNA sequence was prepared and cloned into pcDNA3.1 vector (Invitrogen). An empty vector was undertaken as the NC. After seeding in 6-well plates, the cells were cultured to 70-80% confluence.\u0026nbsp;Transient transfection was performed using Lipofectamine 3000 (Invitrogen). After two days, RT-qPCR was employed to analyze the transfection efficacy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment and screening of DDP-resistant cell model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDDP-resistant cell lines, U2OS-DR and MG-63-DR, were developed by gradually increasing the DDP concentration. The initial concentration was 0.1 μM, which was progressively raised to 1.0 μM. After each increase in DDP concentration, cells were subcultured 2-3 times and stabilized in drug-free medium for one week to ensure adaptation to the new drug concentration. During the entire culture process, cells were periodically cryopreserved to prevent experimental interruptions. After 7 months of continuous culture, the drug-resistant cell lines were successfully established and able to survive at a DDP concentration of 1.0 μM. Prior to functional assays, the resistant cell lines were maintained in drug-free complete medium for at least 2 weeks to restore normal growth conditions. For cell passaging, cells were centrifuged at 1000 rpm for 5 minutes to separate the cells and remove the spent culture medium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of exosomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples were first centrifuged at 300, 2,000, and 10,000×g for 10, 15, and 30 minutes to remove the cells and cell debris. Next, supernatant was further ultracentrifuged at 120,000×g for 2 h, and the exosome pellet was resuspended in 100 μL PBS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy (TEM) observation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExosomes were negatively stained by 2% phosphotungstic acid for around 2 minutes on a copper grid carbon-coated. Then, the grid with stained exosomes was dried at the ambient temperature, and examined using a JEM-1200 EX microscope (JEOL, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHere, TRIzol reagent (Invitrogen) extracted total RNA, and miRNeasy Serum/Plasma kit (Qiagen, USA) was used to extract total RNA from exosomes. The target gene expression was computed via 2\u003csup\u003e-ΔΔCt\u0026nbsp;\u003c/sup\u003e(15). The expression levels of circVANGL1 were analyzed using GAPDH as the internal reference gene, and the expression levels of miR-145-5p were analyzed using U6 as the internal reference gene. The primers used for the analysis were as follows: circVANGL1 forward primer: 5’-GTCCGCTCCACCGATGGCGA-3’, reverse primer: 5’-CTGAACTTCCTCTGTCCGAGT-3’; GAPDH forward primer: 5’-CCGGGAAACTGTGGCGTGATGG-3’, reverse primer: 5’-AGGTGGAGGAGTGGGTGTCGCTGTT-3’; miR-145-5p forward primer: 5’-CAGTCTTGTCCAGTTTTCCCAG-3’, reverse primer: 5’-TATGCTTGTTCTCGTCTCTGTGTC-3’; U6 forward primer: 5’-CTCGCTTCGGCAGCACA-3’, reverse primer: 5’-AACGCTTCACGAATTTGCGT-3’.”\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMTT assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to assess the impact of DDP treatment on the viability of U2OS and MG-63 cells. The detailed procedure is as follows: U2OS, MG-63, U2OS-DR, and MG-63-DR cells were seeded into 96-well plates at a density of 5×10\u003csup\u003e3\u003c/sup\u003e cells per well. After cell attachment, the cells were treated with different concentrations of DDP (1.5, 3, 6, 12, and 24 μM). A well without DDP was set as the control group. After 2 days of treatment, 20 μL of MTT solution (5 mg/mL; Sigma-Aldrich) was added to each well, and the cells were incubated for 4 hours at 37°C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Following incubation, 150 μL of DMSO was added to each well to dissolve the purple formazan crystals formed. After shaking the plate for 10 minutes in the dark at room temperature, the absorbance (OD value) at 570 nm was measured using a microplate reader. The cell viability was evaluated by calculating the OD ratio of each treatment group compared to the control group. All experiments were repeated three times to ensure the reliability and reproducibility of the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell apoptosis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter DDP treatment, cells were harvested, resuspended in 1×binding buffer, and labeled with 5µL Annexin V-FITC and 10µL PI utilizing Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, USA) in the dark.\u0026nbsp;After 15 minutes, cell apoptosis was evaluated via a flow cytometry (BD Biosciences).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot (WB)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RIPA lysis buffer (Solarbio, China) was employed for extraction of the total protein. Equal amounts of protein were separated via SDS-PAGE, transferred to PVDF membranes (Merck Millipore). 5% FBS was employed to block the membranes, which were then mixed with primary antibodies for incubation at 4°C overnight and with HRP-conjugated secondary antibody for 60 minutes. Protein bands were visualized by using GAPDH and β-actin as a control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence in situ hybridization (FISH)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were fixed with 4% paraformaldehyde\u0026nbsp;for 10 minutes, permeabilized in PBS with 0.5% Triton X-100 for 5 minutes, and hybridized with specific Cy3-labeled circVANGL1 probe (Guangzhou RiboBio Co., Ltd., China) at 37 °C overnight. DAPI was applied for nuclear complex staining. Slides were photographed with a Leica SP5 confocal microscope (Leica Microsystems, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual-luciferase reporter assay (DLRA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence of circVANGL1 or E2F3 mRNA was amplified and subcloned into psiCHECK-2 vector (Promega, USA). circVANGL1-wt/mut or E2F3-wt/mut were co-transfected with miR-145-5p or miR-NC into cells. After two days of incubation, luciferase activity (LA) was detected by the DLRA system (Promega).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiment procedures were implemented and repeated three times, based on which the average values of each indicator were calculated. Results were denoted in mean value ± standard deviation (SD). At the same time, Student’s \u003cem\u003et\u003c/em\u003e-test/one-way ANOVA assessed statistical significance. Correlation was assessed by Pearson coefficient. \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 meant statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ecircVANGL1 is elevated in OS cells with property of DDP resistance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eU2OS and MG-63 cells were exposed to incremental doses of DDP to construct DDP-resistant U2OS-DR cell and MG-63-DR cell, respectively, for further analysis. MTT assay verified that the U2OS-DR cell and MG-63-DR cell upon DDP treatment showed remarkably increased viability compared to that of parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In addition, U2OS-DR cell and MG-63-DR cell had lower apoptosis rates after 48 h of DDP (1.5 \u0026micro;M) treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The RT-qPCR results in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC show that circVANGL1 expression was elevated in U2OS-DR and MG-63-DR cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ecircVANGL1 knockdown reduces DDP resistance of OS cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the functions of circVANGL1, we carried out loss-of-function experiments. siRNA targeting circVANGL1 was synthesized and incorporated into DDP-resistant OS cells. In addition, the levels of circVANGL1 in U2OS-DR cell and MG-63-DR cell were notably decreased after transfection. Data was not shown. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e demonstrated the DDP resistance of OS cells reduced by the circVANGL1 knockdown. MTT assay further showed that circVANGL1 knockdown markedly reduced the viability of both U2OS-DR cell and MG-63-DR cell upon DDP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Besides, the apoptosis rates of both U2OS-DR cell and MG-63-DR cell cells after 48 h of DDP (1.5 \u0026micro;M) treatment were remarkably increased by circVANGL1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eExosomalcircVANGL1 spreads DDP resistance in OS cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the spread of DDP resistance promoted by the exosomal circVANGL1. The morphology of collected exosomes demonstrated a cup-shaped morphology with a median diameter of ~\u0026thinsp;100 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). WB detection revealed positive expression of TSG101, CD63, and CD81 in the isolated exosomal fractions, but the case was not same in the cell extracts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). We further observed that circVANGL1 expression was markedly increased in the exosomes from U2OS-DR cell and MG-63-DR cell compared with their parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Moreover, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, co-culture with exosomes of DDP-resistant OS cells for 48 h obviously induced DDP resistance in their parental cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ecircVANGL1 functions in miR-145-5p in OS\u003c/b\u003e\u003c/p\u003e\u003cp\u003ecircVANGL1 functions in miR-145-5p in OS were illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. It was verified that circVANGL1 was expressed in cytoplasm of U2OS and MG-63 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), suggesting circVANGL1may function at the post-transcriptional level. miR-145-5p harbor a complementary binding sequence of circVANGL1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). What\u0026rsquo;s more, the interaction was further validated by DLRA. The results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC suggested miR-145-5p considerably reduced LA of circVANGL1-wt in U2OS and MG-63 cells, but the inhibitory was abolished after maturity of binding sites. In addition, miR-145-5p decline in both U2OS-DR cell and MG-63-DR cell was notably reversed by circVANGL1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Meanwhile, we noted miR-145-5p in exosomes from both cells was markedly decreased (U2OS-DR cell and MG-63-DR cell) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003emiR-145-5p inhibition abates role of circVANGL1 knockdown on DDP-resistant OS cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter analysis on TargetScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.targetscan.org/vert_71/\u003c/span\u003e\u003cspan address=\"http://www.targetscan.org/vert_71/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), we noted a putative region in 3\u0026prime;-UTR of E2F3 mRNA that may bind to miR-145-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, LA of E2F3-wt in U2OS cells was notably reduced, and it was the same case in the MG-63 cells. The elevated expressed E2F3 in U2OS-DR cell and MG-63-DR cell cell was blocked by circVANGL1 knockdown, and miR-145-5p inhibition changed the effects reversely (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). miR-145-5p inhibition or E2F3 overexpression damaged effects of circVANGL1 knockdown on the two cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ecircRNAs is identified as potential functional regulators in cancer progression. circVANGL1 is generally reported as an oncogene, and increased circRNAs level is associated with malignant phenotypes in various kinds of human cancers (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Some circRNAs were identified, which were closely correlated with OS. Chemotherapy is a common therapeutic option used against cancer, and DDP is a standard drug for OS therapy; however, DDP resistance can hinder the OS management greatly. The development of DDP resistance is a multifactorial and multistep process involving mechanisms such as cell proliferation, apoptosis, DNA repair, and drug efflux (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). This study found that the expression of circVANGL1 was significantly upregulated in DDP-resistant OS cells, suggesting that circVANGL1 may be involved in the regulation of DDP resistance. By knocking down circVANGL1, we observed a significant reduction in the proliferative capacity of DDP-resistant cells, suppression of DDP resistance, and a notable increase in cell apoptosis. These results indicated that circVANGL1 plays a crucial role in maintaining DDP resistance, likely through the regulation of cell proliferation and apoptosis-related signaling pathways.\u003c/p\u003e\u003cp\u003eExosomes are secreted by most eukaryotic cells and are naturally present in body fluids. They are important messengers in the intricate intercellular communication for progression of related cancers (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). circRNAs are enriched in exosomes (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Our research proved that circVANGL1 could be incorporated into exosomes and transmitted to DDP-sensitive OS cells, thus disseminating DDP resistance. Detection of exosomal circRNAs in blood samples can provide additional evidence for conventional diagnostic methods (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), and the clinical values of circulating exosomal circVANGL1 in OS need further work.\u003c/p\u003e\u003cp\u003eMany miRNA binding elements exist in circRNAs and can exert the role of ceRNAs to indirectly regulate the target protein of miRNAs (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). CeRNAs are frequently involved in many cancers\u0026rsquo; pathogenesis, and deciphering ceRNA interplay may provide opportunities for cancer therapy exploration (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). According to previous records, miR-145-5p is a tumor suppressor in OS (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), and circVANGL1 interacts with miR-145-5p in OS directly. circVANGL1 knockdown also caused increased miR-145-5p in DDP-resistant OS cells. E2F3 is oncogenic in tumorigenesis, and it can be activated/inhibited by numerous miRNAs (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The results indicated that circVANGL1 regulates the expression of E2F3 through direct interaction with miR-145-5p, thereby affecting the proliferation, apoptosis, and chemoresistance of DDP-resistant OS cells. MiR-145-5p is a tumor-suppressive miRNA, and its expression is downregulated in various cancers, potentially contributing to drug resistance. As a \u0026lsquo;sponge\u0026rsquo; for miR-145-5p, circVANGL1 may sequester miR-145-5p, inhibiting its regulation of target genes, thereby promoting cell proliferation and suppressing apoptosis, leading to the development of DDP resistance. Overexpression of E2F3 partially reversed the effects of circVANGL1 knockdown on cell proliferation and apoptosis in DDP-resistant cells, suggesting that circVANGL1 may influence DDP resistance through the regulation of E2F3 expression. The cytoplasm is a key site for circRNA function, and circVANGL1, located in the cytoplasm, may mediate intercellular signaling via exosome transport, potentially interacting with cytoplasmic RNA-binding proteins or miRNAs to influence drug resistance regulation in the tumor microenvironment. This mechanism warrants further investigation in the context of tumor drug resistance. These findings suggest that circVANGL1 is a crucial regulatory factor in DDP-resistant OS cells, and targeting circVANGL1 may provide a novel therapeutic strategy to overcome DDP resistance. By regulating miR-145-5p and E2F3, circVANGL1 could be combined with other targeted therapies or immunotherapies to further enhance therapeutic efficacy.\u003c/p\u003e\u003cp\u003eTo conclude, in this research, we firstly demonstrated that exosomal circVANGL1 enhances resistance of OS cells against DDP partly through regulating miR-145-5p/E2F3 axis. This study explored the mechanism by which circVANGL1 regulates DDP resistance through the miR-145-5p/E2F3 axis. However, there are certain limitations to this study. The upstream transcription factors, epigenetic regulatory factors, and RNA-binding proteins that modulate circVANGL1 were not thoroughly investigated. Additionally, whether other molecules participate in regulating this network remains to be further explored. Furthermore, the silencing effect of circVANGL1 in vivo and its safety still need to be validated. Future work will focus on further investigating the upstream regulatory mechanisms of circVANGL1, as well as exploring other potential miRNAs or pathways involved. Additionally, the delivery system for circVANGL1 will be optimized, and its safety and efficacy in in vivo applications will be assessed, with the goal of gaining a more comprehensive understanding of its role in OS development and treatment. In conclusion, this work provides a new mechanism for understanding DDP resistance in OS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u0026nbsp;\u003c/strong\u003eThis work was supported by Self-funded scientific research project of Guangxi Health Commission (project No. Z-A20221074).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate declaration:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eIsakoff MS, Bielack SS, Meltzer P, Gorlick R. 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Tumour biology: J Int Soc Oncodevelopmental Biology Med. 2015;36(2):479\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi H, Pan R, Lu Q, Ren C, Sun J, Wu H, et al. MicroRNA1455p inhibits osteosarcoma cell proliferation by targeting E2F transcription factor 3. Int J Mol Med. 2020;45(5):1317\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao Y, Feng B, Lu L, Han S, Chu X, Chen L, et al. MiRNAs and E2F3: a complex network of reciprocal regulations in human cancers. Oncotarget. 2017;8(36):60624\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu C, Li B, Wang J, et al. miR-145-5p Modulates Gefitinib Resistance by Targeting NRAS and MEST in Non-Small Cell Lung Cancer. Ann Clin Lab Sci. 2021;51(5):625\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen CK, Cheng R, Demeter J, et al. Structured elements drive extensive circular RNA translation. Mol Cell. 2021;81(20):4300\u0026ndash;e431813.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"osteosarcoma, cisplatin, Drug resistance, circRNA, circVANGL1, miR-145-5p, E2F3","lastPublishedDoi":"10.21203/rs.3.rs-6734085/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6734085/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and Objectives:\u003c/h2\u003e\u003cp\u003eosteosarcoma (OS) is a malignant bone tumor originating directly from bone tissue, predominantly affecting adolescents. Cisplatin (DDP)-based chemotherapy is commonly used in the treatment of OS, but the emergence of DDP resistance poses a significant challenge to effective management. The study aimed to investigate the role of circRNA VANGL1 (circVANGL1) in mediating DDP resistance in OS.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e\u003cp\u003ethe OS cell lines U2OS and MG-63 were used, and DDP-resistant cell models (U2OS-DR and MG-63-DR) were established. Transfection with circVANGL1 siRNA was performed to silence circVANGL1 expression, and its impact on cell proliferation, apoptosis, DDP resistance, and miR-145-5p level was studied. Fluorescence in situ hybridization was employed to localize circVANGL1. TargetScan prediction was utilized to identify the interaction between circVANGL1 and miR-145-5p. Co-transfection experiments of si-circVANGL1 with anti-miR-145-5p or E2F3 overexpression vector (E2F3-oe) were conducted to assess their effects on DDP-resistant cells.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003ecircVANGL1 expression was greatly elevated in U2OS-DR and MG-63-DR cells. Transfection with si-circVANGL1 effectively suppressed cell proliferation and DDP resistance while promoting apoptosis in DDP-resistant cells. circVANGL1 is transferred via exosomes and primarily localized in the cytoplasm of U2OS and MG-63 cells. TargetScan prediction indicated a target relationship between circVANGL1 and miR-145-5p. Co-transfection of si-circVANGL1 with anti-miR-145-5p or E2F3-oe counteracted the changes in proliferation and apoptosis observed with si-circVANGL1 transfection alone in DDP-resistant cells.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eExosomal circVANGL1 contributes to DDP resistance in OS cells via modulation of miR-145-5p/E2F3 axis.\u003c/p\u003e","manuscriptTitle":"Exosomal circVANGL1 Enhances Cisplatin Resistance in Osteosarcoma Cells by Regulating the miR-145-5p/E2F3 Axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-10 13:33:31","doi":"10.21203/rs.3.rs-6734085/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-27T05:57:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T20:36:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T20:27:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337407320328966858554848060298818460384","date":"2025-10-05T13:00:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-04T22:43:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153781258780881005234320136796770043253","date":"2025-10-02T07:36:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159433782160495489965645685516892178248","date":"2025-10-01T01:52:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-29T10:37:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-22T03:40:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-31T05:33:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Oncology","date":"2025-07-31T05:31:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"26f857e5-7266-42d6-8f2d-6dd64c75ca17","owner":[],"postedDate":"October 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-01T11:24:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-10 13:33:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6734085","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6734085","identity":"rs-6734085","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}