circCOL3A1 Facilitates Esophageal Squamous Cell Carcinoma Progression by Stabilizing YBX1 through Enhanced USP10-Mediated Deubiquitination

circCOL3A1 Facilitates Esophageal Squamous Cell Carcinoma Progression by Stabilizing YBX1 through Enhanced USP10-Mediated Deubiquitination | 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 circCOL3A1 Facilitates Esophageal Squamous Cell Carcinoma Progression by Stabilizing YBX1 through Enhanced USP10-Mediated Deubiquitination Xueting Hu, Yuyan Cao, Xiaotian He, Ruihao Liang, Guifen Fan, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9378154/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Recent findings suggest that circular RNAs (circRNAs) play important roles in tumor growth and cancer advancement. However, the specific biological functions and mechanisms of circRNAs in esophageal squamous cell carcinoma remain largely unclear. Methods To identify differentially expressed circular RNAs (circRNAs), we analyzed GEO circRNA microarray datasets. circCOL3A1 expression was validated in ESCC cell lines and a clinical cohort comprising 100 paired tissue samples using quantitative reverse transcription PCR (qRT-PCR). The circular nature of circCOL3A1 was confirmed through RNase R digestion assays and divergent primer-based PCR amplification. Functional investigations, including in vitro and in vivo experiments, were performed to assess the role of circCOL3A1 in cellular proliferation, migration, and invasion. Mechanistic studies were conducted using Western blot analysis, RNA immunoprecipitation (RIP), co-immunoprecipitation (Co-IP), and ubiquitination assays to elucidate the molecular pathways involving circCOL3A1. Results CircCOL3A1 expression was markedly elevated in both ESCC tissues and cell lines, exhibiting a significant association with advanced TNM stages and reduced overall survival. Functionally, circCOL3A1 facilitated ESCC cell proliferation, migration, and invasion in vitro, while also promoting tumor growth in vivo. Mechanistic investigations demonstrated that circCOL3A1 directly bound to the transcription factor YBX1, acting as a protein scaffold to strengthen its interaction with the deubiquitinase USP10. Consequently, this interaction enhanced YBX1 deubiquitination, increased its protein stability, and led to YBX1 accumulation, thereby accelerating ESCC progression. Rescue experiments further validated YBX1 as a pivotal downstream mediator of circCOL3A1. Conclusion Our study elucidates that circCOL3A1 exacerbates ESCC malignancy by stabilizing YBX1 via USP10-mediated deubiquitination. These findings suggest that circCOL3A1 could serve as both a prognostic biomarker and a therapeutic target in ESCC. Circular RNA CircPPP2R5A Esophageal Squamous Cell Carcinoma Deubiquitination YBX1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Esophageal cancer represents a significant global health challenge, accounting for approximately 510,000 new cases and 450,000 deaths annually. As the seventh leading cause of cancer-related mortality worldwide, it contributes to 4.6% of all cancer deaths [ 1 , 2 ]. In China, esophageal squamous cell carcinoma (ESCC) constitutes the predominant histological subtype, representing approximately 85% of all esophageal cancer cases and exceeding 90% of cases within the Chinese population [ 3 , 4 ]. Despite advancements in surgical techniques and multimodal therapies, ESCC remains characterized by high rates of recurrence and metastasis, resulting in a five-year overall survival rate below 30% [ 5 – 7 ]. Consequently, elucidating the molecular mechanisms underlying ESCC pathogenesis and progression is imperative for developing early diagnostic approaches and identifying potential therapeutic targets. Circular RNAs, known as circRNAs, are non-coding RNA molecules that have a covalently closed loop, produced through the back-splicing of pre-mRNA [ 8 ]. Their unique circular structure confers greater stability than linear RNAs by resisting degradation by RNases. CircRNAs are widely expressed in eukaryotes, with many exhibiting tissue- or cell-specific expression patterns. The abundance of certain circRNAs can even exceed that of their linear counterparts [ 9 ]. Functionally, circRNAs operate through diverse mechanisms, including acting as molecular sponges for miRNAs, regulating gene transcription, interacting with RNA-binding proteins to modulate their functions, and, in some cases, serving as templates for translation. Among these, the competitive endogenous RNA (ceRNA) mechanism, involving the sequestration of miRNAs, is the most extensively studied [ 10 – 12 ]. Due to their stability, distinct expression patterns, and significant regulatory functions, circRNAs are crucial in the onset and advancement of human diseases, especially cancer, underscoring their significant potential for clinical application [ 13 ]. Previous studies in ESCC have identified a series of functional circRNAs, such as circTMEM45A, circNF1, and circPRKCA, which regulate malignant phenotypes through various pathways [ 14 – 16 ]. Nonetheless, the exact function of circCOL3A1 in ESCC has yet to be investigated. In recent years, ubiquitination, a key post-translational modification (PTM) involved in regulating intracellular protein degradation, has garnered increasing attention [ 17 ]. Emerging evidence indicates that circRNAs can modulate protein ubiquitination levels. For instance, circWSB1 interacts with USP10, attenuating USP10-mediated p53 stabilization and promoting breast cancer (BC) progression [ 18 ]. Exosome-derived hsa_circ_0007132 also contributes to lenvatinib resistance by inhibiting the ubiquitin-mediated degradation of NONO[ 19 ]. Nevertheless, the role of circRNAs in regulating protein deubiquitination in ESCC remains largely uninvestigated. In this analysis, we discovered that circCOL3A1 is upregulated in ESCC and is significantly related to an unfavorable outcome. Functional experiments conducted both in vitro and in vivo showed that overexpressing circCOL3A1 enhances the proliferation, invasion, and migration of ESCC cells. Mechanistically, circCOL3A1 facilitates the interaction between YBX1 and USP10, leading to enhanced YBX1 deubiquitination, increased protein stability, and consequently elevated YBX1 expression, which collectively drive ESCC progression. These findings highlight the potential of the circCOL3A1-USP10-YBX1 axis as both a diagnostic marker and therapeutic approach for ESCC. Material and Methods Patient Samples A cohort of 100 treatment-naïve patients with histologically confirmed esophageal squamous cell carcinoma (ESCC), who underwent surgical resection at Sun Yat-sen Memorial Hospital between January 2019 and October 2024, was included in this study. None of the participants had received prior radiotherapy or chemotherapy. Tumor tissues and matched adjacent normal tissues were collected, immediately flash-frozen in liquid nitrogen, and stored for subsequent molecular analyses. The study protocol was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and was approved by the Institutional Ethics Committee of Sun Yat-sen Memorial Hospital. Cell Culture The human esophageal epithelial cell line (HEEC) and ESCC cell lines (TE1, KYSE30, KYSE150, and KYSE180) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (Eallbio, Beijing) and 1% penicillin-streptomycin (NCM Biotech, Suzhou), and maintained at 37°C in a humidified 5% CO₂ incubator. RNase R Digestion and Actinomycin D Assay To verify the circular structure of COL3A1, total RNA (3 µg) extracted from TE1 and KYSE30 cells was treated with 20 U/µL RNase R (Beyotime, Shanghai) at 37°C for 15 min. The digested RNA was then analyzed by nucleic acid electrophoresis using primers specific to either the circular or linear form of COL3A1. For RNA stability assessment, TE1 cells were treated with the transcriptional inhibitor Actinomycin D (Sigma, Germany) and harvested at 0, 6, 12, 18, and 24 h post-treatment. RNA was isolated at each time point, and the relative expression levels of circular COL3A1 and its linear mRNA counterpart were quantified by reverse transcription-quantitative PCR (RT-qPCR). Nuclear and Cytoplasmic Fractionation Nuclear and cytoplasmic RNA were isolated from TE1 cells using the PARIS™ Kit (Invitrogen, USA) according to the manufacturer's protocol. Briefly, 1×10⁷ TE1 cells were fractionated with 500 µL of ice-cold Cell Fractionation Buffer for 10 min. After centrifugation at 4°C for 5 min, the supernatant (cytoplasmic fraction) was carefully transferred to a new tube. The nuclear pellet was resuspended in 500 µL of ice-cold Cell Disruption Buffer. The RNA fractions were then washed, eluted, and stored at -80°C for subsequent analysis. Quantitative Real-Time PCR (qRT-PCR) Total RNA was isolated from both tissue and cell samples using TRIzol reagent (Invitrogen, USA), followed by quantification and purity assessment through spectrophotometric analysis. Prior to quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, the extracted RNA was reverse transcribed into complementary DNA (cDNA) using a standard protocol. Cell Counting Kit-8 (CCK-8) Assay To assess cellular proliferation, the CCK-8 assay kit (ApexBio, USA) was utilized. TE1 and KYSE30 cell lines were seeded in 96-well plates at a density of 2×10³ cells per well, with each well containing 100 µL of complete growth medium. After the designated incubation period, 10 µL of CCK-8 solution was added to each well, followed by an additional two-hour incubation at 37°C. Absorbance was subsequently measured at 450 nm using a microplate reader to quantify cellular viability. Colony Formation Assay Following transfection, TE1 and KYSE30 cells were seeded into 6-well plates at a density of 1,000 cells per well and cultured for 14 days under standard conditions to facilitate colony formation. The resulting colonies were subsequently fixed, stained with 0.1% crystal violet solution, and quantified using an optical microscope (Olympus, Japan) for documentation and analysis. EdU Assay To evaluate cellular proliferation, the BeoClick EdU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime, Shanghai) was utilized in accordance with the manufacturer's protocol. Fluorescence imaging was performed using an Olympus IX73 inverted fluorescence microscope system (Olympus, Japan) for quantitative analysis. Wound Healing Assay ESCC cells were seeded in 6-well culture plates and cultured until reaching approximately 95% confluency. A uniform linear wound was created in the monolayer using a sterile 200 µL pipette tip with a standardized width of 0.5 mm. The initial wound area was immediately imaged using phase-contrast microscopy, with subsequent imaging performed at 12 hours post-wounding. Quantitative assessment of wound closure kinetics was conducted through automated image analysis using a customized ImageJ macro, enabling precise measurement of cell migration rates. Cell Migration and Invasion Assay To evaluate invasive capacity, Transwell inserts (8-µm pore size, 24-well format) were pre-coated with Matrigel (Invitrogen, USA). ESCC cells suspended in serum-free medium were seeded in the upper chambers, while complete growth medium was added to the lower chambers as a chemoattractant. After 24 hours of incubation, cells that had migrated through the Matrigel matrix were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and quantified using an optical microscope (Olympus, Japan). For assessment of cell migration, the same experimental procedure was employed without Matrigel coating. Western Blot Analysis Total cellular proteins were extracted using RIPA lysis buffer (Beyotime, Shanghai) supplemented with a protease inhibitor cocktail (Servicebio, Wuhan). Protein samples (20 µg per lane) were separated by electrophoresis on 10% SDS-polyacrylamide gels and subsequently transferred onto PVDF membranes via electroblotting. The membranes were incubated overnight at 4°C with primary antibodies specific to YBX1 and USP10. Following extensive washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 40 minutes at room temperature. Protein bands were visualized using a chemiluminescence detection system. RNA Pull-Down Assay RNA-protein interactions were isolated utilizing the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, USA) in accordance with the manufacturer's protocol. Specifically bound proteins to biotinylated RNA probes were subsequently eluted and subjected to comprehensive characterization through mass spectrometry and immunoblotting analyses. RNA Immunoprecipitation (RIP) The RNA immunoprecipitation (RIP) assay was performed using a commercially available RIP kit (Geneseed, Guangzhou). Briefly, cellular lysates were prepared from approximately 1×10⁷ cells in RIPA buffer supplemented with protease inhibitors. Immunoprecipitation was carried out overnight at 4°C using either anti-FLAG antibody or control IgG pre-conjugated to protein A/G magnetic beads. Following extensive washing steps, the co-precipitated RNA was isolated and purified. Subsequent analysis of circCOL3A1 enrichment was conducted through reverse transcription-PCR (RT-PCR) followed by agarose gel electrophoresis. Co-immunoprecipitation (Co-IP) Protein-protein interactions were analyzed through co-immunoprecipitation using the Pierce immunoprecipitation kit (Beyotime, Shanghai) following the manufacturer's protocol. Cellular lysates, prepared with IP lysis buffer, were incubated overnight at 4°C with either anti-Ybx1 antibody or control IgG immobilized on Protein A/G magnetic beads. Following extensive washing, the immunoprecipitated complexes were eluted and subsequently subjected to Western blot analysis for detection of the target proteins. Immunohistochemistry (IHC) The transplanted tumor tissue sections were prepared using standard procedures, including fixation, dehydration, paraffin embedding, and microtomy. Following antigen retrieval, endogenous peroxidase activity was quenched, and nonspecific binding sites were blocked using a commercial immunohistochemistry (IHC) detection kit (ZGB-Bio, Beijing). Primary antibody incubation was performed at 4°C overnight. Subsequently, sections were incubated with a secondary antibody and visualized using 3,3'-diaminobenzidine (DAB) chromogenic substrate. Finally, the stained sections were examined and imaged under a light microscope. Xenograft Tumor Model For xenograft establishment, a suspension of 1×10⁷ stably transfected ESCC cells in phosphate-buffered saline (PBS) was subcutaneously injected into 4-6-week-old male BALB/c nude mice. All experimental animals were humanely euthanized at 6 weeks post-inoculation. Excised tumor specimens were systematically documented, weighed, and volumetrically analyzed by an investigator blinded to experimental groups using the standard ellipsoid formula: Volume = (length × width²)/2. Tumor tissues were subsequently fixed in appropriate solutions for immunohistochemical (IHC) analysis. The complete animal experiment was conducted under specific pathogen-free (SPF) conditions and received formal approval from the Institutional Animal Care and Use Committee (IACUC) of Laidi Biotechnology Statistical Analysis Quantitative data are presented as mean ± standard deviation (SD). Intergroup comparisons were performed using Student's t-test for two groups and one-way analysis of variance (ANOVA) for multiple groups. Categorical variables were analyzed using Pearson's chi-square test or Fisher's exact test, as appropriate. All statistical analyses were conducted using GraphPad Prism software (Version 8.0). A probability (P) value of < 0.05 was considered statistically significant, with specific significance levels denoted as *P < 0.05, **P < 0.01, and ***P < 0.001. Non-significant results were indicated as "ns." Results Identification of an ESCC-Associated Circular RNA: circCOL3A1 To identify the circular Rnas (circrnas) associated with esophageal squamous cell carcinoma (ESCC), we analyzed two datasets (GSE150476 and GSE250413) from the GEO database. By comparing the circRNA expression profiles of ESCC tumor tissues and adjacent normal tissues, we identified differentially expressed circrnas (fold change ≥ 2, p value < 0.05) (Fig. 1 A, 1 B). The intersection of upregulated circrnas in both datasets revealed eight common candidate circrnas. Of these, hsa_circRNA_057362 showed the most significant upregulation (Fig. 1 C). CircCOL3A1 (hsa_circRNA_057362) is a 747 nucleotide transcript derived from exons 5 to 17 of the COL3A1 gene on chromosome 2. Validation in our cohort of 100 pairs of esophageal squamous cell carcinoma and normal tissue confirmed that circCOL3A1 expression was significantly higher in tumor tissues (Fig. 1 D). In addition, patients with high circCOL3A1 expression in their tumors had significantly worse overall survival, as shown by Kaplan-Meier analysis (Fig. 1 E). To determine its circular structure, we designed divergence and convergence primers spanning the postsplice junction. As expected, circCOL3A1 was amplified only with different primers, using cDNA instead of gDNA as a template (Fig. 1 F, 1 G). The stability of circCOL3A1 was detected by actinomycin D. circCOL3A1 had a significantly longer half-life than linear COL3A1 (Fig. 1 H). qRT-PCR after nucleoplasmic separation showed that circCOL3A1 was mainly localized in the cytoplasm (Fig. 1 I). Comparative analysis across cell lines showed that the expression of circCOL3A1 was significantly increased in ESCC cells (TE1, KYSE30, KYSE150, KYSE180) compared with the normal esophageal epithelial cell line HEEC, with the highest expression in TE1 and the lowest expression in KYSE30 (Fig. 1 J). Fluorescence in situ hybridization (FISH) further confirmed the predominant cytoplasmic localization of circCOL3A1 in TE1 and KYSE30 cells (Fig. 1 K, 1 L). circCOL3A1 Promotes Proliferation, Migration, and Invasion in ESCC To determine the functional contribution of circCOL3A1 to ESCC pathogenesis, gain-of-function and loss-of-function studies were performed. We established lentivirus-mediated overexpression in KYSE30 cells and knockdown in TE1 cells, and verified the efficiency by qPCR (FIG. S1 ). Subsequent functional analyses, including CCK-8, colony formation, and EdU incorporation assays, consistently showed that enhanced circCOL3A1 expression significantly increased the proliferation rate of KYSE30 cells. In contrast, silencing of circCOL3A1 in TE1 cells resulted in significant inhibition of proliferation (Fig. 2 A-C). In the cell migration assay, we observed parallel results: circCOL3A1 overexpression enhanced cell migration, while knockdown of circCOL3A1 was impaired by wound healing and Transwell assays (Fig. 2 D, 2 E). Taken together, these results confirmed that circCOL3A1 could enhance the proliferation, migration and invasion properties of ESCC cells. circCOL3A1 Binds to and Stabilizes YBX1 by Promoting Its Deubiquitination To explore the molecular mechanism, we isolated circCOL3A1 interacting proteins using RNA pull-down assays and mass spectrometry, which identified YBX1 as an important binding partner (Fig. 3 A). This interaction was specifically confirmed by western blot, showing enrichment of YBX1 antisense but not sense circCOL3A1 probes (Fig. 3 B). In turn, RNA immunoprecipitation (RIP) using an anti-YbX1 antibody significantly enriched circCOL3A1 (Fig. 3 C), confirming the direct RNA-protein interaction. Notably, manipulation of circCOL3A1 levels significantly altered YBX1 protein abundance without affecting its mRNA expression, implying post-transcriptional control (Fig. 3 D, 3 E). We hypothesized that circCOL3A1 may regulate YBX1 stability through the ubiquitin-proteasome system. In support of this notion, the decrease in YBX1 protein caused by circCOL3A1 knockdown was rescued by the proteasome inhibitor MG-132 (Fig. 3 F). Cycloheximide (CHX) chase experiments further showed that circCOL3A1 knockdown shortened YBX1 half-life, whereas its overexpression prolonged YBX1 half-life (Fig. 3 G). Importantly, immunoprecipitation analysis performed in ha-ub expressing cells showed that circCOL3A1 overexpression reduced YBX1 polyubiquitination, whereas its knockdown enhanced YBX1 polyubiquitination (Fig. 3 H). Immunofluorescence microscopy confirmed cytoplasmic colocalization of circCOL3A1 and YBX1 (Fig. 3 I). These data collectively suggest that circCOL3A1 binds to YBX1 and promotes its protein stability by inhibiting ubiquitin-dependent proteasomal degradation. Knockdown of YBX1 Reverses circCOL3A1-Induced Malignant Phenotypes YBX1 was knocked down in KYSE30 cells overexpressing circcol3a1 for rescue experiments. CCK-8, colony formation, and EdU assays showed that YBX1 silencing significantly attenuated the pro-proliferation effect of circCOL3A1 (Fig. 4 A-C). Similarly, wound healing and Transwell migration assays confirmed that YBX1 knockdown reversed circCOL3A1-driven enhanced migration and invasion (Fig. 4 D, 4 E). These results indicate that YBX1 is a key downstream effector mediating the oncogenic function of circCOL3A1. circCOL3A1 Facilitates the YBX1-USP10 Interaction Mass spectrometry from the circCOL3A1 pull-down also identified the deubiquitinase USP10 (Fig. 5 A). Western blot analysis confirmed that the antisense circCOL3A1 probe coprecipitated USP10 and YBX1 (Fig. 5 B). Co-immunoprecipitation (Co-IP) assays revealed an endogenous interaction between YBX1 and USP10, which was enhanced when circCOL3A1 was overexpressed (Fig. 5 C). To determine whether USP10 acts as a deubiquitinating enzyme for YBX1, we regulated USP10 expression. USP10 overexpression increased YBX1 protein levels, whereas USP10 knockdown decreased YBX1 protein levels without changing YBX1 mRNA levels (Fig. 5 D). Importantly, USP10 knockdown significantly reversed the reduced YBX1 ubiquitination induced by circCOL3A1 overexpression (Fig. 5 E). The mechanism diagram showed that circCOL3A1 stabilized YBX1 protein by promoting its deubiquitination by promoting the YBX1-USP10 interaction (Fig. 5 F). This suggests that circCOL3A1 recruits USP10 to promote YBX1 deubiquitination and stabilization. circCOL3A1 Promotes ESCC Tumor Growth In Vivo by Upregulating YBX1 A subcutaneous xenograft tumor model was established to evaluate the in vivo function of circCOL3A1. Mice injected with circCOL3A1-overexpressing KYSE30 cells developed significantly larger and heavier tumors than controls (Fig. 6 A-C). In contrast, tumors formed from circcol3a1 knockdown TE1 cells were significantly smaller. Histologic (he) and immunohistochemical (IHC) analyses of resected tumors showed that circCOL3A1 overexpression resulted in increased YBX1 protein levels and a higher Ki-67 positivity rate, whereas circCOL3A1 knockdown had the opposite effect (Fig. 6 D). These in vivo findings confirm our in vitro data suggesting that the circCOL3A1-USP10-YBX1 axis promotes ESCC genesis. Discussion Esophageal squamous cell carcinoma (ESCC), the predominant histological subtype of esophageal cancer in China, remains associated with poor clinical outcomes despite progress in early detection and therapeutic interventions in recent years [ 20 ]. Consequently, the identification of reliable biomarkers for early diagnosis is imperative to improve the overall survival (OS) of ESCC patients. In this study, circCOL3A1 was identified as a novel circRNA significantly associated with ESCC through comprehensive microarray data analysis. The existence of circCOL3A1 was subsequently validated, and its canonical circular RNA characteristics were confirmed in both ESCC cell lines and clinical tissue specimens. Clinical sample analysis demonstrated that circCOL3A1 is markedly upregulated in ESCC tissues, with its elevated expression exhibiting a strong correlation with advanced TNM stage and diminished overall survival. Moreover, in vitro and in vivo functional assays revealed that circCOL3A1 enhances the proliferative, invasive, and migratory capacities of ESCC cells, thereby facilitating malignant progression. These findings collectively suggest that circCOL3A1 may serve as a promising diagnostic and prognostic biomarker for ESCC. Circular RNAs (circRNAs) play critical regulatory roles in tumorigenesis and cancer progression, functioning either as oncogenic drivers or tumor suppressors [ 21 , 22 ]. Their biological effects are primarily mediated through three well-characterized mechanisms. The most prevalent involves acting as microRNA sponges; for example, circPFKP inhibits gastric cancer progression by sequestering miR-346 and modulating CAMD3 expression [ 23 ]. Alternatively, circRNAs can function as protein scaffolds to regulate target protein activity, as exemplified by circPHLPP2, which interacts with ILF3 to modulate IL36γ transcription in colorectal cancer, thereby promoting tumor growth and conferring resistance to anti-PD-1 therapy [ 24 ]. Although traditionally classified as non-coding RNAs, certain circRNAs have been shown to encode functional peptides or proteins. For instance, hsa_circ_0085121 encodes a novel protein that drives prostate cancer progression by activating the PI3K/Akt/mTOR pathway and promoting AR-V7 alternative splicing [ 25 ]. In this study, we elucidated a scaffolding function of circCOL3A1, wherein it facilitates the interaction between the target protein YBX1 and the deubiquitinase USP10. This interaction enhances YBX1 deubiquitination, stabilizes the YBX1 protein, leads to its accumulation, and ultimately accelerates ESCC progression. YBX1, a member of the cold shock domain (CSD) protein family, serves as a critical transcription factor with diverse biological functions. It orchestrates extensive transcriptional regulation by specifically binding to Y-box sequences within the enhancer and promoter regions of numerous target genes [ 26 ]. Elevated YBX1 expression has been documented in various solid tumors, including hepatocellular carcinoma, colorectal cancer, non-small cell lung cancer, and gastric adenocarcinoma, with its overexpression strongly correlating with adverse clinical outcomes [ 27 – 31 ]. Consequently, YBX1 represents a promising therapeutic target for multiple malignancies. Beyond its transcriptional regulatory role, YBX1 activity is modulated by various post-translational modifications, such as phosphorylation and ubiquitination. For instance, MASTL-mediated phosphorylation of YBX1 facilitates transcriptional activation of PAK4, thereby promoting lenvatinib resistance in hepatocellular carcinoma [ 32 ]. Conversely, TRIM29 attenuates the PI3K/AKT signaling pathway by ubiquitinating and degrading YBX1, which reverses lenvatinib resistance in hepatocellular carcinoma cells [ 33 ]. Our study provides novel evidence that YBX1 interacts with the deubiquitinase USP10 in esophageal squamous cell carcinoma (ESCC), resulting in its stabilization via deubiquitination. However, the precise deubiquitination sites on YBX1 remain to be elucidated. Given that circCOL3A1 stabilizes YBX1 expression through deubiquitination, therapeutic strategies targeting circCOL3A1 may offer a novel approach for ESCC treatment, warranting further investigation. Conclusion In summary, our study revealed that circCOL3A1 functions as a tumor promoter in ESCC. It regulates the proliferation, invasion and migration of ESCC cells by regulating the USP10-YBX1 axis. These findings not only provide insights into the molecular mechanisms driving ESCC, but also highlight circCOL3A1 as a potential prognostic biomarker and a promising therapeutic target. CRediT authorship contribution statement Xueting Hu: Writing - original draft, Methodology, Data curation. Yuyan Cao: Validation, Methodology, Investigation. Xiaotian He: Methodology, Formal analysis. Ruihao Liang: Investigation, Data curation. Guifen Fan: Funding acquisition, Formal analysis. Yaxu Hu: Formal analysis. Wenjian Wang: Methodology, Formal analysis, Conceptualization. Minghui Wang: Writing - original draft, Visualization, Software, Funding acquisition, Conceptualization. Declarations CRediT authorship contribution statement Xueting Hu: Writing - original draft, Methodology, Data curation. Yuyan Cao: Validation, Methodology, Investigation. Xiaotian He: Methodology, Formal analysis. Ruihao Liang: Investigation, Data curation. Guifen Fan: Funding acquisition, Formal analysis. Yaxu Hu: Formal analysis. Wenjian Wang: Methodology, Formal analysis, Conceptualization. Minghui Wang: Writing - original draft, Visualization, Software, Funding acquisition, Conceptualization. Ethics statement The human studies were approved by the Institutional Review Board of Sun Yat-sen Memorial Hospital, while the animal experiments received ethical clearance from the Animal Care and Use Committee of Laidi Biotechnology. All procedures strictly adhered to applicable regulatory guidelines and institutional ethical standards. Funding This work was supported by the National Natural Science Foundation of China (Grant No. 82203451), the Guangdong Medical Science and Technology Research Foundation (No. A2025166), the Guangdong Basic and Applied Basic Research Foundation (No.2025A1515010792), the Shanwei Science and Technology Project (No.2024C049). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors appreciate the supports of our team. Data availability The data used to support the findings of this study are included within the article and the supplementary information file. References Leiter A, Veluswamy RR, Wisnivesky JP. The global burden of lung cancer: current status and future trends. Nat Rev Clin Oncol. 2023;20(9):624–39. Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229–63. Morgan E, Soerjomataram I, Rumgay H, et al. The Global Landscape of Esophageal Squamous Cell Carcinoma and Esophageal Adenocarcinoma Incidence and Mortality in 2020 and Projections to 2040: New Estimates From GLOBOCAN 2020. Gastroenterology. 2022;163(3):649–58. Han B, Zheng R, Zeng H, et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent. 2024;4(1):47–53. Rogers JE, Sewastjanow-Silva M, Waters RE, Ajani JA. Esophageal cancer: emerging therapeutics. Expert Opin Ther Targets. 2022;26(2):107–17. He S, Xu J, Liu X, Zhen Y. Advances and challenges in the treatment of esophageal cancer. Acta Pharm Sin B. 2021;11(11):3379–92. Codipilly DC, Qin Y, Dawsey SM, et al. Screening for esophageal squamous cell carcinoma: recent advances. Gastrointest Endosc. 2018;88(3):413–26. Zhou WY, Cai ZR, Liu J, Wang DS, Ju HQ, Xu RH. Circular RNA: metabolism, functions and interactions with proteins. Mol Cancer. 2020;19(1):172. Kristensen LS, Andersen MS, Stagsted LVW, Ebbesen KK, Hansen TB, Kjems J. The biogenesis, biology and characterization of circular RNAs. Nat Rev Genet. 2019;20(11):675–91. Liu L, Gu M, Ma J, et al. CircGPR137B/miR-4739/FTO feedback loop suppresses tumorigenesis and metastasis of hepatocellular carcinoma. Mol Cancer. 2022;21(1):149. Published 2022 Jul 20. Zhao M, Wang Y, Tan F, et al. Circular RNA circCCNB1 inhibits the migration and invasion of nasopharyngeal carcinoma through binding and stabilizing TJP1 mRNA. Sci China Life Sci. 2022;65(11):2233–47. Peng Y, Xu Y, Zhang X, et al. A novel protein AXIN1-295aa encoded by circAXIN1 activates the Wnt/β-catenin signaling pathway to promote gastric cancer progression. Mol Cancer. 2021;20(1):158. Kristensen LS, Jakobsen T, Hager H, Kjems J. The emerging roles of circRNAs in cancer and oncology. Nat Rev Clin Oncol. 2022;19(3):188–206. Meng L, Wu H, Wu J, et al. Aberrant CircTMEM45A Facilitates Inflammatory Progression of Esophageal Squamous Cell Carcinoma through m5C-Mediated NLRP3 Activation. Cancer Res. 2025;85(14):2694–713. Wang C, Ju C, Du D, et al. CircNF1 modulates the progression and immune evasion of esophageal squamous cell carcinoma through dual regulation of PD-L1. Cell Mol Biol Lett. 2025;30(1):37. Wu L, Gu L, Zheng Y, et al. CircPRKCA facilitates esophageal squamous cell carcinoma metastasis via m5C-dependent CSF2 mRNA stabilization. J Transl Med. 2025;23(1):385. Liu F, Chen J, Li K, et al. Ubiquitination and deubiquitination in cancer: from mechanisms to novel therapeutic approaches. Mol Cancer. 2024;23(1):148. Yang R, Chen H, Xing L, et al. Hypoxia-induced circWSB1 promotes breast cancer progression through destabilizing p53 by interacting with USP10. Mol Cancer. 2022;21(1):88. Cao M, Li Y, Su X, et al. Exosome-derived hsa_circ_0007132 promotes lenvatinib resistance by inhibiting the ubiquitin-mediated degradation of NONO. Noncoding RNA Res. 2025;14:1–13. Reichenbach ZW, Murray MG, Saxena R, et al. Clinical and translational advances in esophageal squamous cell carcinoma. Adv Cancer Res. 2019;144:95–135. Huang G, Liang M, Liu H, et al. CircRNA hsa_circRNA_104348 promotes hepatocellular carcinoma progression through modulating miR-187-3p/RTKN2 axis and activating Wnt/β-catenin pathway. Cell Death Dis. 2020;11(12):1065. Chen S, Li K, Guo J, et al. circNEIL3 inhibits tumor metastasis through recruiting the E3 ubiquitin ligase Nedd4L to degrade YBX1. Proc Natl Acad Sci U S A. 2023;120(13):e2215132120. Song X, Zhang G, Niu J, et al. Circular RNA circPFKP suppress gastric cancer progression through targeting miR-346/CAMD3 axis. Exp Cell Res. 2025;445(1):114390. Hu Y, Cai ZR, Huang RZ, Wang DS, Ju HQ, Chen DL, Circular. RNA circPHLPP2 promotes tumor growth and anti-PD-1 resistance through binding ILF3 to regulate IL36γ transcription in colorectal cancer. Mol Cancer. 2024;23(1):272. Li J, Qiu H, Dong Q, et al. Androgen-targeted hsa_circ_0085121 encodes a novel protein and improves the development of prostate cancer through facilitating the activity of PI3K/Akt/mTOR pathway and enhancing AR-V7 alternative splicing. Cell Death Dis. 2024;15(11):848. Prabhu L, Hartley AV, Martin M, Warsame F, Sun E, Lu T. Role of post-translational modification of the Y box binding protein 1 in human cancers. Genes Dis. 2015;2(3):240–6. Kwabiah D, Nagati V, Tripathi MK. Transcription factor YBX1 orchestrates drug resistance and tumor progression in HCC. Drug Discov Today. 2025;30(9):104439. Park MS, Jeong SD, Shin CH, et al. LINC02257 regulates malignant phenotypes of colorectal cancer via interacting with miR-1273g-3p and YB1. Cell Death Dis. 2024;15(12):895. Wang Y, Wei J, Feng L, et al. Aberrant m5C hypermethylation mediates intrinsic resistance to gefitinib through NSUN2/YBX1/QSOX1 axis in EGFR-mutant non-small-cell lung cancer. Mol Cancer. 2023;22(1):81. Xu J, Ji L, Liang Y, et al. CircRNA-SORE mediates sorafenib resistance in hepatocellular carcinoma by stabilizing YBX1. Signal Transduct Target Ther. 2020;5(1):298. Qin S, Liu Y, Zhang X, et al. lncRNA FGD5-AS1 is required for gastric cancer proliferation by inhibiting cell senescence and ROS production via stabilizing YBX1. J Exp Clin Cancer Res. 2024;43(1):188. Liang BG, Zheng YM, Xu MH et al. The MASTL/YBX1/PAK4 axis regulated by stress-activated STK24 triggers lenvatinib resistance and tumor progression in HCC. Hepatology. Tang Y, Fan S, Peng R, et al. TRIM29 reverses lenvatinib resistance in liver cancer cells by ubiquitinating and degrading YBX1 to inhibit the PI3K/AKT pathway. Transl Oncol. 2025;53:102294. Additional Declarations No competing interests reported. Supplementary Files FigureS1.tif Figure S1. qPCR analysis of circCOL3A1 expression in ESCC cells with circCOL3A1 knockdown or overexpression. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9378154","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":621919727,"identity":"910d117e-19cb-4bf4-8856-1aab62f70a30","order_by":0,"name":"Xueting Hu","email":"","orcid":"","institution":"Sun Yat-sen Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xueting","middleName":"","lastName":"Hu","suffix":""},{"id":621919728,"identity":"858c03b9-9cd9-48e8-b073-270f3fa6d737","order_by":1,"name":"Yuyan Cao","email":"","orcid":"","institution":"Sun Yat-sen Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuyan","middleName":"","lastName":"Cao","suffix":""},{"id":621919729,"identity":"42761395-cbde-46b1-813c-b6749d1c0ae0","order_by":2,"name":"Xiaotian He","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Xiaotian","middleName":"","lastName":"He","suffix":""},{"id":621919730,"identity":"f78d13d4-0e73-413b-a306-d6030825b58a","order_by":3,"name":"Ruihao Liang","email":"","orcid":"","institution":"Sun Yat-sen Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ruihao","middleName":"","lastName":"Liang","suffix":""},{"id":621919731,"identity":"b58f113c-09c1-4746-8f3a-5b230c8dd6b2","order_by":4,"name":"Guifen Fan","email":"","orcid":"","institution":"Sun Yat-sen Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guifen","middleName":"","lastName":"Fan","suffix":""},{"id":621919732,"identity":"3201dc58-cf31-4a8f-aa4e-ed801ffb3ed0","order_by":5,"name":"Yaxu Hu","email":"","orcid":"","institution":"Jieshou People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yaxu","middleName":"","lastName":"Hu","suffix":""},{"id":621919733,"identity":"c6d0be12-58ca-47f2-98d7-20f22d912d96","order_by":6,"name":"Wenjian Wang","email":"","orcid":"","institution":"Sun Yat-sen Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenjian","middleName":"","lastName":"Wang","suffix":""},{"id":621919734,"identity":"7662593d-8a68-499b-811d-f93ee90a2b69","order_by":7,"name":"Minghui Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYFACxgYgsmFgYGZugAkZEKMlDaiFkWgtYF2HIXqJ0mJw7XDbw687zkfztzM2MP5sq0tsYG/eJsFQcwe3ltuJ7cayZ27nzjjM2MDM23Y4sYHnWJkEw7FnOLWY3U5sk5Zsu53bANLC2HYgsUEix0wC4lS8Ws7lzj8Mc5j8G8JaJD+2HcjdANTCwNvGDLSFB78We5AtjG3JuRuBWg7znDts3MaTVmyRcAy3FsnZ6c8kf7bZ5c47f/jgwx9ldbL97Ic33vhQg1sLCDDzQBkHGNkYGNhArAS8GoBx+APO/ENA6SgYBaNgFIxIAAAFNFs1xUq6sQAAAABJRU5ErkJggg==","orcid":"","institution":"Sun Yat-sen Memorial Hospital","correspondingAuthor":true,"prefix":"","firstName":"Minghui","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-04-10 10:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9378154/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9378154/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106858609,"identity":"ff49ec69-d099-44bb-86f9-3e9b9d33031e","added_by":"auto","created_at":"2026-04-14 07:56:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4415867,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of an ESCC-Associated Circular RNA: circCOL3A1. \u003c/strong\u003e(A) Volcano plot displaying differentially expressed circular RNAs in ESCC tissues versus adjacent normal tissues from the GSE150476 dataset. (B) Volcano plot displaying differentially expressed circular RNAs in ESCC tissues versus adjacent normal tissues from the GSE250413 dataset. (C) Venn diagram showing the overlap of differentially expressed circular RNAs between the GSE150476 and GSE250413 datasets. (D) qPCR analysis of circCOL3A1 expression in 100 paired tumor and adjacent normal tissues from ESCC patients. (E) Kaplan-Meier survival curve of ESCC patients stratified into high and low circCOL3A1 expression groups based on the median relative expression value across all samples. (F. G) Gel electrophoresis of PCR products using divergent and convergent primers on cDNA and gDNA, along with RNase R treatment, to verify the circular structure and stability of circCOL3A1 in TE1 and KYSE30 cell lines. (H) Actinomycin D assay to evaluate the stability of circCOL3A1. (I) Nuclear and cytoplasmic RNA fractionation assay to determine the subcellular localization of circCOL3A1 in ESCC cells. (J) qPCR analysis of circCOL3A1 expression levels in different ESCC cell lines. (K. L) Fluorescence in situ hybridization (FISH) to visualize the subcellular localization of circCOL3A1 in ESCC cells.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9378154/v1/e20c36088fef72d9ea16b0c4.png"},{"id":106858611,"identity":"6c8ef4af-9566-4beb-9f30-91f3a15ba9f7","added_by":"auto","created_at":"2026-04-14 07:56:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7681281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecircCOL3A1 Promotes Proliferation, Migration, and Invasion in ESCC. \u003c/strong\u003e(A) The proliferative capacity of differently transfected ESCC cells was measured by the CCK-8 assay. (B) The colony formation ability of differently transfected ESCC cells was determined by the cell colony formation assay. Representative results are shown. (C) The proliferative capacity of differently transfected ESCC cells was measured by the EDU assay. (D) A wound healing assay was used to evaluate the migratory capacity of transfected ESCC cells. (E) Transwell migration and Matrigel invasion assays were employed to assess the migration and invasion capabilities of ESCC cells transfected with circCOL3A1 overexpression plasmid or sh-circCOL3A1.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9378154/v1/ac1f62c8196af86493c439e3.png"},{"id":106858703,"identity":"f4b9481f-3ba1-49c9-a38c-782d4433ec23","added_by":"auto","created_at":"2026-04-14 07:57:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4637103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecircCOL3A1 Binds to and Stabilizes YBX1 by Promoting Its Deubiquitination. \u003c/strong\u003e(A) Secondary mass spectra of the YBX1 protein identified by LC-MS/MS. (B) Western blot analysis using an anti-YBX1 antibody to detect YBX1 levels in proteins pulled down by the circCOL3A1 probe, with simultaneous nucleic acid electrophoresis of RNA enriched by the circCOL3A1 probe. (C) Nucleic acid electrophoresis to detect circCOL3A1 abundance bound by the RIP assay using an anti-YBX1 antibody, and Western blot to detect YBX1 protein levels. (D) Western blot analysis of YBX1 protein expression changes in ESCC cells transfected with circCOL3A1 overexpression plasmid or sh-circCOL3A1. (E) qPCR analysis of YBX1 mRNA expression changes in ESCC cells transfected with circCOL3A1 overexpression plasmid or sh-circCOL3A1. (F) Western blot analysis of the effect of MG-132 treatment on YBX1 protein. (G) Western blot analysis of YBX1 stability changes under different transfection conditions. (H) Detection of ubiquitination level changes in YBX1 under different treatments using anti-YBX1 antibody pull-down assay. (I) Immunofluorescence showing the colocalization of YBX1 and circCOL3A1.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9378154/v1/78f379928a9e8c9038f250e1.png"},{"id":106858689,"identity":"e2330853-c888-41bb-8799-cc3f95515d94","added_by":"auto","created_at":"2026-04-14 07:57:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9210783,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of YBX1 Reverses circCOL3A1-Induced Malignant Phenotypes. \u003c/strong\u003e(A) The proliferative capacity of differently transfected KYSE30 cells was measured by the CCK-8 assay.\u003cstrong\u003e \u003c/strong\u003e(B) The colony formation ability of differently transfected KYSE30 cells was determined by the cell colony formation assay, with representative results shown. (C) The proliferative capacity of differently transfected KYSE30 cells was measured by the EDU assay. (D) A wound healing assay was used to evaluate the migratory capacity of YBX1-knockdown KYSE30 cells. (E) Transwell migration and Matrigel invasion assays were employed to assess the migration and invasion capabilities of YBX1-knockdown KYSE30 cells.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9378154/v1/1fd3cbde762b50a778896b3b.png"},{"id":106858607,"identity":"ee4d2143-7d1e-41a6-b1a8-d9ad468ab79e","added_by":"auto","created_at":"2026-04-14 07:56:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2884591,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecircCOL3A1 Facilitates the YBX1-USP10 Interaction. \u003c/strong\u003e(A) Secondary mass spectra of the USP10 protein identified by LC-MS/MS.\u003cstrong\u003e \u003c/strong\u003e(B) Western blot analysis of YBX1 and USP10 protein levels in circCOL3A1 probe pull-down complexes using anti-YBX1 and anti-USP10 antibodies.\u003cstrong\u003e \u003c/strong\u003e(C) Co-immunoprecipitation with anti-YBX1 antibody followed by Western blot detection of the pulled-down proteins using anti-USP10 antibody.\u003cstrong\u003e \u003c/strong\u003e(D) Western blot analysis of YBX1 expression changes upon USP10 overexpression or knockdown.\u003cstrong\u003e \u003c/strong\u003e(E) Western blot detection of ubiquitination levels of YBX1 following USP10 knockdown.\u003cstrong\u003e \u003c/strong\u003e(F) Schematic diagram illustrating the mechanism by which circCOL3A1 promotes YBX1 stabilization through enhanced deubiquitination via interaction with both YBX1 and USP10.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9378154/v1/0f0a519aff0fba8e2bbe848a.png"},{"id":106858622,"identity":"e1a60954-d3e8-420c-8af3-79fc62d2b3b9","added_by":"auto","created_at":"2026-04-14 07:56:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":23579289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecircCOL3A1 Promotes ESCC Tumor Growth In Vivo by Upregulating YBX1. \u003c/strong\u003e(A) Representative images of xenograft tumors from different experimental groups.\u003cstrong\u003e \u003c/strong\u003e(B) Tumor growth curves of subcutaneous xenograft models (n=5).\u003cstrong\u003e \u003c/strong\u003e(C) Tumor weight measurements of subcutaneous xenograft tumors (n=5).\u003cstrong\u003e \u003c/strong\u003e(D) Immunohistochemical detection of KI67 and YBX1 protein expression levels in different ESCC groups.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9378154/v1/0a50f42badce95c38e354810.png"},{"id":108976460,"identity":"6c1dba4e-645b-4b42-8046-a312f5051019","added_by":"auto","created_at":"2026-05-11 11:22:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":48623506,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9378154/v1/f7f03dcc-1790-4754-8429-3905ab8e1d1e.pdf"},{"id":106858623,"identity":"2870f82e-b04e-414b-994c-144d2df7c10b","added_by":"auto","created_at":"2026-04-14 07:56:44","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":379788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1. qPCR analysis of circCOL3A1 expression in ESCC cells with circCOL3A1 knockdown or overexpression.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-9378154/v1/ed81e7aac3a32f1474988f3e.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"circCOL3A1 Facilitates Esophageal Squamous Cell Carcinoma Progression by Stabilizing YBX1 through Enhanced USP10-Mediated Deubiquitination","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEsophageal cancer represents a significant global health challenge, accounting for approximately 510,000 new cases and 450,000 deaths annually. As the seventh leading cause of cancer-related mortality worldwide, it contributes to 4.6% of all cancer deaths [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In China, esophageal squamous cell carcinoma (ESCC) constitutes the predominant histological subtype, representing approximately 85% of all esophageal cancer cases and exceeding 90% of cases within the Chinese population [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite advancements in surgical techniques and multimodal therapies, ESCC remains characterized by high rates of recurrence and metastasis, resulting in a five-year overall survival rate below 30% [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Consequently, elucidating the molecular mechanisms underlying ESCC pathogenesis and progression is imperative for developing early diagnostic approaches and identifying potential therapeutic targets.\u003c/p\u003e \u003cp\u003eCircular RNAs, known as circRNAs, are non-coding RNA molecules that have a covalently closed loop, produced through the back-splicing of pre-mRNA [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Their unique circular structure confers greater stability than linear RNAs by resisting degradation by RNases. CircRNAs are widely expressed in eukaryotes, with many exhibiting tissue- or cell-specific expression patterns. The abundance of certain circRNAs can even exceed that of their linear counterparts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Functionally, circRNAs operate through diverse mechanisms, including acting as molecular sponges for miRNAs, regulating gene transcription, interacting with RNA-binding proteins to modulate their functions, and, in some cases, serving as templates for translation. Among these, the competitive endogenous RNA (ceRNA) mechanism, involving the sequestration of miRNAs, is the most extensively studied [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Due to their stability, distinct expression patterns, and significant regulatory functions, circRNAs are crucial in the onset and advancement of human diseases, especially cancer, underscoring their significant potential for clinical application [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Previous studies in ESCC have identified a series of functional circRNAs, such as circTMEM45A, circNF1, and circPRKCA, which regulate malignant phenotypes through various pathways [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Nonetheless, the exact function of circCOL3A1 in ESCC has yet to be investigated.\u003c/p\u003e \u003cp\u003eIn recent years, ubiquitination, a key post-translational modification (PTM) involved in regulating intracellular protein degradation, has garnered increasing attention [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Emerging evidence indicates that circRNAs can modulate protein ubiquitination levels. For instance, circWSB1 interacts with USP10, attenuating USP10-mediated p53 stabilization and promoting breast cancer (BC) progression [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Exosome-derived hsa_circ_0007132 also contributes to lenvatinib resistance by inhibiting the ubiquitin-mediated degradation of NONO[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Nevertheless, the role of circRNAs in regulating protein deubiquitination in ESCC remains largely uninvestigated.\u003c/p\u003e \u003cp\u003eIn this analysis, we discovered that circCOL3A1 is upregulated in ESCC and is significantly related to an unfavorable outcome. Functional experiments conducted both in vitro and in vivo showed that overexpressing circCOL3A1 enhances the proliferation, invasion, and migration of ESCC cells. Mechanistically, circCOL3A1 facilitates the interaction between YBX1 and USP10, leading to enhanced YBX1 deubiquitination, increased protein stability, and consequently elevated YBX1 expression, which collectively drive ESCC progression. These findings highlight the potential of the circCOL3A1-USP10-YBX1 axis as both a diagnostic marker and therapeutic approach for ESCC.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient Samples\u003c/h2\u003e \u003cp\u003eA cohort of 100 treatment-na\u0026iuml;ve patients with histologically confirmed esophageal squamous cell carcinoma (ESCC), who underwent surgical resection at Sun Yat-sen Memorial Hospital between January 2019 and October 2024, was included in this study. None of the participants had received prior radiotherapy or chemotherapy. Tumor tissues and matched adjacent normal tissues were collected, immediately flash-frozen in liquid nitrogen, and stored for subsequent molecular analyses. The study protocol was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and was approved by the Institutional Ethics Committee of Sun Yat-sen Memorial Hospital.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003e The human esophageal epithelial cell line (HEEC) and ESCC cell lines (TE1, KYSE30, KYSE150, and KYSE180) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (Eallbio, Beijing) and 1% penicillin-streptomycin (NCM Biotech, Suzhou), and maintained at 37\u0026deg;C in a humidified 5% CO₂ incubator.\u003c/p\u003e\n\u003ch3\u003eRNase R Digestion and Actinomycin D Assay\u003c/h3\u003e\n\u003cp\u003eTo verify the circular structure of COL3A1, total RNA (3 \u0026micro;g) extracted from TE1 and KYSE30 cells was treated with 20 U/\u0026micro;L RNase R (Beyotime, Shanghai) at 37\u0026deg;C for 15 min. The digested RNA was then analyzed by nucleic acid electrophoresis using primers specific to either the circular or linear form of COL3A1. For RNA stability assessment, TE1 cells were treated with the transcriptional inhibitor Actinomycin D (Sigma, Germany) and harvested at 0, 6, 12, 18, and 24 h post-treatment. RNA was isolated at each time point, and the relative expression levels of circular COL3A1 and its linear mRNA counterpart were quantified by reverse transcription-quantitative PCR (RT-qPCR).\u003c/p\u003e\n\u003ch3\u003eNuclear and Cytoplasmic Fractionation\u003c/h3\u003e\n\u003cp\u003eNuclear and cytoplasmic RNA were isolated from TE1 cells using the PARIS\u0026trade; Kit (Invitrogen, USA) according to the manufacturer's protocol. Briefly, 1\u0026times;10⁷ TE1 cells were fractionated with 500 \u0026micro;L of ice-cold Cell Fractionation Buffer for 10 min. After centrifugation at 4\u0026deg;C for 5 min, the supernatant (cytoplasmic fraction) was carefully transferred to a new tube. The nuclear pellet was resuspended in 500 \u0026micro;L of ice-cold Cell Disruption Buffer. The RNA fractions were then washed, eluted, and stored at -80\u0026deg;C for subsequent analysis.\u003c/p\u003e\n\u003ch3\u003eQuantitative Real-Time PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from both tissue and cell samples using TRIzol reagent (Invitrogen, USA), followed by quantification and purity assessment through spectrophotometric analysis. Prior to quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, the extracted RNA was reverse transcribed into complementary DNA (cDNA) using a standard protocol.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell Counting Kit-8 (CCK-8) Assay\u003c/h2\u003e \u003cp\u003eTo assess cellular proliferation, the CCK-8 assay kit (ApexBio, USA) was utilized. TE1 and KYSE30 cell lines were seeded in 96-well plates at a density of 2\u0026times;10\u0026sup3; cells per well, with each well containing 100 \u0026micro;L of complete growth medium. After the designated incubation period, 10 \u0026micro;L of CCK-8 solution was added to each well, followed by an additional two-hour incubation at 37\u0026deg;C. Absorbance was subsequently measured at 450 nm using a microplate reader to quantify cellular viability.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eColony Formation Assay\u003c/h3\u003e\n\u003cp\u003eFollowing transfection, TE1 and KYSE30 cells were seeded into 6-well plates at a density of 1,000 cells per well and cultured for 14 days under standard conditions to facilitate colony formation. The resulting colonies were subsequently fixed, stained with 0.1% crystal violet solution, and quantified using an optical microscope (Olympus, Japan) for documentation and analysis.\u003c/p\u003e\n\u003ch3\u003eEdU Assay\u003c/h3\u003e\n\u003cp\u003eTo evaluate cellular proliferation, the BeoClick EdU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime, Shanghai) was utilized in accordance with the manufacturer's protocol. Fluorescence imaging was performed using an Olympus IX73 inverted fluorescence microscope system (Olympus, Japan) for quantitative analysis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWound Healing Assay\u003c/h2\u003e \u003cp\u003eESCC cells were seeded in 6-well culture plates and cultured until reaching approximately 95% confluency. A uniform linear wound was created in the monolayer using a sterile 200 \u0026micro;L pipette tip with a standardized width of 0.5 mm. The initial wound area was immediately imaged using phase-contrast microscopy, with subsequent imaging performed at 12 hours post-wounding. Quantitative assessment of wound closure kinetics was conducted through automated image analysis using a customized ImageJ macro, enabling precise measurement of cell migration rates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell Migration and Invasion Assay\u003c/h2\u003e \u003cp\u003eTo evaluate invasive capacity, Transwell inserts (8-\u0026micro;m pore size, 24-well format) were pre-coated with Matrigel (Invitrogen, USA). ESCC cells suspended in serum-free medium were seeded in the upper chambers, while complete growth medium was added to the lower chambers as a chemoattractant. After 24 hours of incubation, cells that had migrated through the Matrigel matrix were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and quantified using an optical microscope (Olympus, Japan). For assessment of cell migration, the same experimental procedure was employed without Matrigel coating.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e \u003cp\u003eTotal cellular proteins were extracted using RIPA lysis buffer (Beyotime, Shanghai) supplemented with a protease inhibitor cocktail (Servicebio, Wuhan). Protein samples (20 \u0026micro;g per lane) were separated by electrophoresis on 10% SDS-polyacrylamide gels and subsequently transferred onto PVDF membranes via electroblotting. The membranes were incubated overnight at 4\u0026deg;C with primary antibodies specific to YBX1 and USP10. Following extensive washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 40 minutes at room temperature. Protein bands were visualized using a chemiluminescence detection system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA Pull-Down Assay\u003c/h2\u003e \u003cp\u003eRNA-protein interactions were isolated utilizing the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, USA) in accordance with the manufacturer's protocol. Specifically bound proteins to biotinylated RNA probes were subsequently eluted and subjected to comprehensive characterization through mass spectrometry and immunoblotting analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA Immunoprecipitation (RIP)\u003c/h2\u003e \u003cp\u003eThe RNA immunoprecipitation (RIP) assay was performed using a commercially available RIP kit (Geneseed, Guangzhou). Briefly, cellular lysates were prepared from approximately 1\u0026times;10⁷ cells in RIPA buffer supplemented with protease inhibitors. Immunoprecipitation was carried out overnight at 4\u0026deg;C using either anti-FLAG antibody or control IgG pre-conjugated to protein A/G magnetic beads. Following extensive washing steps, the co-precipitated RNA was isolated and purified. Subsequent analysis of circCOL3A1 enrichment was conducted through reverse transcription-PCR (RT-PCR) followed by agarose gel electrophoresis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eProtein-protein interactions were analyzed through co-immunoprecipitation using the Pierce immunoprecipitation kit (Beyotime, Shanghai) following the manufacturer's protocol. Cellular lysates, prepared with IP lysis buffer, were incubated overnight at 4\u0026deg;C with either anti-Ybx1 antibody or control IgG immobilized on Protein A/G magnetic beads. Following extensive washing, the immunoprecipitated complexes were eluted and subsequently subjected to Western blot analysis for detection of the target proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eThe transplanted tumor tissue sections were prepared using standard procedures, including fixation, dehydration, paraffin embedding, and microtomy. Following antigen retrieval, endogenous peroxidase activity was quenched, and nonspecific binding sites were blocked using a commercial immunohistochemistry (IHC) detection kit (ZGB-Bio, Beijing). Primary antibody incubation was performed at 4\u0026deg;C overnight. Subsequently, sections were incubated with a secondary antibody and visualized using 3,3'-diaminobenzidine (DAB) chromogenic substrate. Finally, the stained sections were examined and imaged under a light microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eXenograft Tumor Model\u003c/h2\u003e \u003cp\u003eFor xenograft establishment, a suspension of 1\u0026times;10⁷ stably transfected ESCC cells in phosphate-buffered saline (PBS) was subcutaneously injected into 4-6-week-old male BALB/c nude mice. All experimental animals were humanely euthanized at 6 weeks post-inoculation. Excised tumor specimens were systematically documented, weighed, and volumetrically analyzed by an investigator blinded to experimental groups using the standard ellipsoid formula: Volume = (length \u0026times; width\u0026sup2;)/2. Tumor tissues were subsequently fixed in appropriate solutions for immunohistochemical (IHC) analysis. The complete animal experiment was conducted under specific pathogen-free (SPF) conditions and received formal approval from the Institutional Animal Care and Use Committee (IACUC) of Laidi Biotechnology\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eQuantitative data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Intergroup comparisons were performed using Student's t-test for two groups and one-way analysis of variance (ANOVA) for multiple groups. Categorical variables were analyzed using Pearson's chi-square test or Fisher's exact test, as appropriate. All statistical analyses were conducted using GraphPad Prism software (Version 8.0). A probability (P) value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant, with specific significance levels denoted as *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Non-significant results were indicated as \"ns.\"\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of an ESCC-Associated Circular RNA: circCOL3A1\u003c/h2\u003e \u003cp\u003eTo identify the circular Rnas (circrnas) associated with esophageal squamous cell carcinoma (ESCC), we analyzed two datasets (GSE150476 and GSE250413) from the GEO database. By comparing the circRNA expression profiles of ESCC tumor tissues and adjacent normal tissues, we identified differentially expressed circrnas (fold change\u0026thinsp;\u0026ge;\u0026thinsp;2, p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The intersection of upregulated circrnas in both datasets revealed eight common candidate circrnas. Of these, hsa_circRNA_057362 showed the most significant upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). CircCOL3A1 (hsa_circRNA_057362) is a 747 nucleotide transcript derived from exons 5 to 17 of the COL3A1 gene on chromosome 2. Validation in our cohort of 100 pairs of esophageal squamous cell carcinoma and normal tissue confirmed that circCOL3A1 expression was significantly higher in tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In addition, patients with high circCOL3A1 expression in their tumors had significantly worse overall survival, as shown by Kaplan-Meier analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). To determine its circular structure, we designed divergence and convergence primers spanning the postsplice junction. As expected, circCOL3A1 was amplified only with different primers, using cDNA instead of gDNA as a template (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The stability of circCOL3A1 was detected by actinomycin D. circCOL3A1 had a significantly longer half-life than linear COL3A1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). qRT-PCR after nucleoplasmic separation showed that circCOL3A1 was mainly localized in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Comparative analysis across cell lines showed that the expression of circCOL3A1 was significantly increased in ESCC cells (TE1, KYSE30, KYSE150, KYSE180) compared with the normal esophageal epithelial cell line HEEC, with the highest expression in TE1 and the lowest expression in KYSE30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Fluorescence in situ hybridization (FISH) further confirmed the predominant cytoplasmic localization of circCOL3A1 in TE1 and KYSE30 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ecircCOL3A1 Promotes Proliferation, Migration, and Invasion in ESCC\u003c/h2\u003e \u003cp\u003eTo determine the functional contribution of circCOL3A1 to ESCC pathogenesis, gain-of-function and loss-of-function studies were performed. We established lentivirus-mediated overexpression in KYSE30 cells and knockdown in TE1 cells, and verified the efficiency by qPCR (FIG. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Subsequent functional analyses, including CCK-8, colony formation, and EdU incorporation assays, consistently showed that enhanced circCOL3A1 expression significantly increased the proliferation rate of KYSE30 cells. In contrast, silencing of circCOL3A1 in TE1 cells resulted in significant inhibition of proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). In the cell migration assay, we observed parallel results: circCOL3A1 overexpression enhanced cell migration, while knockdown of circCOL3A1 was impaired by wound healing and Transwell assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Taken together, these results confirmed that circCOL3A1 could enhance the proliferation, migration and invasion properties of ESCC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003ecircCOL3A1 Binds to and Stabilizes YBX1 by Promoting Its Deubiquitination\u003c/h2\u003e \u003cp\u003eTo explore the molecular mechanism, we isolated circCOL3A1 interacting proteins using RNA pull-down assays and mass spectrometry, which identified YBX1 as an important binding partner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This interaction was specifically confirmed by western blot, showing enrichment of YBX1 antisense but not sense circCOL3A1 probes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In turn, RNA immunoprecipitation (RIP) using an anti-YbX1 antibody significantly enriched circCOL3A1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), confirming the direct RNA-protein interaction. Notably, manipulation of circCOL3A1 levels significantly altered YBX1 protein abundance without affecting its mRNA expression, implying post-transcriptional control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). We hypothesized that circCOL3A1 may regulate YBX1 stability through the ubiquitin-proteasome system. In support of this notion, the decrease in YBX1 protein caused by circCOL3A1 knockdown was rescued by the proteasome inhibitor MG-132 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Cycloheximide (CHX) chase experiments further showed that circCOL3A1 knockdown shortened YBX1 half-life, whereas its overexpression prolonged YBX1 half-life (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Importantly, immunoprecipitation analysis performed in ha-ub expressing cells showed that circCOL3A1 overexpression reduced YBX1 polyubiquitination, whereas its knockdown enhanced YBX1 polyubiquitination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Immunofluorescence microscopy confirmed cytoplasmic colocalization of circCOL3A1 and YBX1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). These data collectively suggest that circCOL3A1 binds to YBX1 and promotes its protein stability by inhibiting ubiquitin-dependent proteasomal degradation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of YBX1 Reverses circCOL3A1-Induced Malignant Phenotypes\u003c/h2\u003e \u003cp\u003eYBX1 was knocked down in KYSE30 cells overexpressing circcol3a1 for rescue experiments. CCK-8, colony formation, and EdU assays showed that YBX1 silencing significantly attenuated the pro-proliferation effect of circCOL3A1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Similarly, wound healing and Transwell migration assays confirmed that YBX1 knockdown reversed circCOL3A1-driven enhanced migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These results indicate that YBX1 is a key downstream effector mediating the oncogenic function of circCOL3A1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003ecircCOL3A1 Facilitates the YBX1-USP10 Interaction\u003c/h2\u003e \u003cp\u003eMass spectrometry from the circCOL3A1 pull-down also identified the deubiquitinase USP10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Western blot analysis confirmed that the antisense circCOL3A1 probe coprecipitated USP10 and YBX1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Co-immunoprecipitation (Co-IP) assays revealed an endogenous interaction between YBX1 and USP10, which was enhanced when circCOL3A1 was overexpressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). To determine whether USP10 acts as a deubiquitinating enzyme for YBX1, we regulated USP10 expression. USP10 overexpression increased YBX1 protein levels, whereas USP10 knockdown decreased YBX1 protein levels without changing YBX1 mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Importantly, USP10 knockdown significantly reversed the reduced YBX1 ubiquitination induced by circCOL3A1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The mechanism diagram showed that circCOL3A1 stabilized YBX1 protein by promoting its deubiquitination by promoting the YBX1-USP10 interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). This suggests that circCOL3A1 recruits USP10 to promote YBX1 deubiquitination and stabilization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003ecircCOL3A1 Promotes ESCC Tumor Growth In Vivo by Upregulating YBX1\u003c/h2\u003e \u003cp\u003eA subcutaneous xenograft tumor model was established to evaluate the in vivo function of circCOL3A1. Mice injected with circCOL3A1-overexpressing KYSE30 cells developed significantly larger and heavier tumors than controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). In contrast, tumors formed from circcol3a1 knockdown TE1 cells were significantly smaller. Histologic (he) and immunohistochemical (IHC) analyses of resected tumors showed that circCOL3A1 overexpression resulted in increased YBX1 protein levels and a higher Ki-67 positivity rate, whereas circCOL3A1 knockdown had the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These in vivo findings confirm our in vitro data suggesting that the circCOL3A1-USP10-YBX1 axis promotes ESCC genesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEsophageal squamous cell carcinoma (ESCC), the predominant histological subtype of esophageal cancer in China, remains associated with poor clinical outcomes despite progress in early detection and therapeutic interventions in recent years [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Consequently, the identification of reliable biomarkers for early diagnosis is imperative to improve the overall survival (OS) of ESCC patients. In this study, circCOL3A1 was identified as a novel circRNA significantly associated with ESCC through comprehensive microarray data analysis. The existence of circCOL3A1 was subsequently validated, and its canonical circular RNA characteristics were confirmed in both ESCC cell lines and clinical tissue specimens. Clinical sample analysis demonstrated that circCOL3A1 is markedly upregulated in ESCC tissues, with its elevated expression exhibiting a strong correlation with advanced TNM stage and diminished overall survival. Moreover, in vitro and in vivo functional assays revealed that circCOL3A1 enhances the proliferative, invasive, and migratory capacities of ESCC cells, thereby facilitating malignant progression. These findings collectively suggest that circCOL3A1 may serve as a promising diagnostic and prognostic biomarker for ESCC.\u003c/p\u003e \u003cp\u003eCircular RNAs (circRNAs) play critical regulatory roles in tumorigenesis and cancer progression, functioning either as oncogenic drivers or tumor suppressors [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Their biological effects are primarily mediated through three well-characterized mechanisms. The most prevalent involves acting as microRNA sponges; for example, circPFKP inhibits gastric cancer progression by sequestering miR-346 and modulating CAMD3 expression [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Alternatively, circRNAs can function as protein scaffolds to regulate target protein activity, as exemplified by circPHLPP2, which interacts with ILF3 to modulate IL36γ transcription in colorectal cancer, thereby promoting tumor growth and conferring resistance to anti-PD-1 therapy [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Although traditionally classified as non-coding RNAs, certain circRNAs have been shown to encode functional peptides or proteins. For instance, hsa_circ_0085121 encodes a novel protein that drives prostate cancer progression by activating the PI3K/Akt/mTOR pathway and promoting AR-V7 alternative splicing [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this study, we elucidated a scaffolding function of circCOL3A1, wherein it facilitates the interaction between the target protein YBX1 and the deubiquitinase USP10. This interaction enhances YBX1 deubiquitination, stabilizes the YBX1 protein, leads to its accumulation, and ultimately accelerates ESCC progression.\u003c/p\u003e \u003cp\u003eYBX1, a member of the cold shock domain (CSD) protein family, serves as a critical transcription factor with diverse biological functions. It orchestrates extensive transcriptional regulation by specifically binding to Y-box sequences within the enhancer and promoter regions of numerous target genes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Elevated YBX1 expression has been documented in various solid tumors, including hepatocellular carcinoma, colorectal cancer, non-small cell lung cancer, and gastric adenocarcinoma, with its overexpression strongly correlating with adverse clinical outcomes [\u003cspan additionalcitationids=\"CR28 CR29 CR30\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Consequently, YBX1 represents a promising therapeutic target for multiple malignancies. Beyond its transcriptional regulatory role, YBX1 activity is modulated by various post-translational modifications, such as phosphorylation and ubiquitination. For instance, MASTL-mediated phosphorylation of YBX1 facilitates transcriptional activation of PAK4, thereby promoting lenvatinib resistance in hepatocellular carcinoma [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Conversely, TRIM29 attenuates the PI3K/AKT signaling pathway by ubiquitinating and degrading YBX1, which reverses lenvatinib resistance in hepatocellular carcinoma cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our study provides novel evidence that YBX1 interacts with the deubiquitinase USP10 in esophageal squamous cell carcinoma (ESCC), resulting in its stabilization via deubiquitination. However, the precise deubiquitination sites on YBX1 remain to be elucidated. Given that circCOL3A1 stabilizes YBX1 expression through deubiquitination, therapeutic strategies targeting circCOL3A1 may offer a novel approach for ESCC treatment, warranting further investigation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our study revealed that circCOL3A1 functions as a tumor promoter in ESCC. It regulates the proliferation, invasion and migration of ESCC cells by regulating the USP10-YBX1 axis. These findings not only provide insights into the molecular mechanisms driving ESCC, but also highlight circCOL3A1 as a potential prognostic biomarker and a promising therapeutic target.\u003c/p\u003e \u003cp\u003eCRediT authorship contribution statement\u003c/p\u003e \u003cp\u003eXueting Hu: Writing - original draft, Methodology, Data curation. Yuyan Cao: Validation, Methodology, Investigation. Xiaotian He: Methodology, Formal analysis. Ruihao Liang: Investigation, Data curation. Guifen Fan: Funding acquisition, Formal analysis. Yaxu Hu: Formal analysis. Wenjian Wang: Methodology, Formal analysis, Conceptualization. Minghui Wang: Writing - original draft, Visualization, Software, Funding acquisition, Conceptualization.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003eCRediT authorship contribution statement\u003c/p\u003e\n\u003cp\u003eXueting Hu: Writing - original draft, Methodology, Data curation. Yuyan Cao: Validation, Methodology, Investigation. Xiaotian He: Methodology, Formal analysis. Ruihao Liang: Investigation, Data curation. Guifen Fan: Funding acquisition, Formal analysis. Yaxu Hu: Formal analysis. Wenjian Wang: Methodology, Formal analysis, Conceptualization. Minghui Wang: Writing - original draft, Visualization, Software, Funding acquisition, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human studies were approved by the Institutional Review Board of Sun Yat-sen Memorial Hospital, while the animal experiments received ethical clearance from the Animal Care and Use Committee of Laidi Biotechnology. All procedures strictly adhered to applicable regulatory guidelines and institutional ethical standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 82203451), the Guangdong Medical Science and Technology Research Foundation (No. A2025166), the Guangdong Basic and Applied Basic Research Foundation (No.2025A1515010792), the Shanwei Science and Technology Project (No.2024C049).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors appreciate the supports of our team.\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 included within the article and the supplementary information file.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLeiter A, Veluswamy RR, Wisnivesky JP. The global burden of lung cancer: current status and future trends. Nat Rev Clin Oncol. 2023;20(9):624\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorgan E, Soerjomataram I, Rumgay H, et al. The Global Landscape of Esophageal Squamous Cell Carcinoma and Esophageal Adenocarcinoma Incidence and Mortality in 2020 and Projections to 2040: New Estimates From GLOBOCAN 2020. Gastroenterology. 2022;163(3):649\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan B, Zheng R, Zeng H, et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent. 2024;4(1):47\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRogers JE, Sewastjanow-Silva M, Waters RE, Ajani JA. Esophageal cancer: emerging therapeutics. Expert Opin Ther Targets. 2022;26(2):107\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe S, Xu J, Liu X, Zhen Y. Advances and challenges in the treatment of esophageal cancer. Acta Pharm Sin B. 2021;11(11):3379\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCodipilly DC, Qin Y, Dawsey SM, et al. Screening for esophageal squamous cell carcinoma: recent advances. Gastrointest Endosc. 2018;88(3):413\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou WY, Cai ZR, Liu J, Wang DS, Ju HQ, Xu RH. Circular RNA: metabolism, functions and interactions with proteins. Mol Cancer. 2020;19(1):172.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKristensen LS, Andersen MS, Stagsted LVW, Ebbesen KK, Hansen TB, Kjems J. The biogenesis, biology and characterization of circular RNAs. Nat Rev Genet. 2019;20(11):675\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu L, Gu M, Ma J, et al. CircGPR137B/miR-4739/FTO feedback loop suppresses tumorigenesis and metastasis of hepatocellular carcinoma. Mol Cancer. 2022;21(1):149. Published 2022 Jul 20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao M, Wang Y, Tan F, et al. Circular RNA circCCNB1 inhibits the migration and invasion of nasopharyngeal carcinoma through binding and stabilizing TJP1 mRNA. Sci China Life Sci. 2022;65(11):2233\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng Y, Xu Y, Zhang X, et al. A novel protein AXIN1-295aa encoded by circAXIN1 activates the Wnt/β-catenin signaling pathway to promote gastric cancer progression. Mol Cancer. 2021;20(1):158.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKristensen LS, Jakobsen T, Hager H, Kjems J. The emerging roles of circRNAs in cancer and oncology. Nat Rev Clin Oncol. 2022;19(3):188\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng L, Wu H, Wu J, et al. Aberrant CircTMEM45A Facilitates Inflammatory Progression of Esophageal Squamous Cell Carcinoma through m5C-Mediated NLRP3 Activation. Cancer Res. 2025;85(14):2694\u0026ndash;713.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang C, Ju C, Du D, et al. CircNF1 modulates the progression and immune evasion of esophageal squamous cell carcinoma through dual regulation of PD-L1. Cell Mol Biol Lett. 2025;30(1):37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu L, Gu L, Zheng Y, et al. CircPRKCA facilitates esophageal squamous cell carcinoma metastasis via m5C-dependent CSF2 mRNA stabilization. J Transl Med. 2025;23(1):385.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu F, Chen J, Li K, et al. Ubiquitination and deubiquitination in cancer: from mechanisms to novel therapeutic approaches. Mol Cancer. 2024;23(1):148.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang R, Chen H, Xing L, et al. Hypoxia-induced circWSB1 promotes breast cancer progression through destabilizing p53 by interacting with USP10. Mol Cancer. 2022;21(1):88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao M, Li Y, Su X, et al. Exosome-derived hsa_circ_0007132 promotes lenvatinib resistance by inhibiting the ubiquitin-mediated degradation of NONO. Noncoding RNA Res. 2025;14:1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReichenbach ZW, Murray MG, Saxena R, et al. Clinical and translational advances in esophageal squamous cell carcinoma. Adv Cancer Res. 2019;144:95\u0026ndash;135.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang G, Liang M, Liu H, et al. CircRNA hsa_circRNA_104348 promotes hepatocellular carcinoma progression through modulating miR-187-3p/RTKN2 axis and activating Wnt/β-catenin pathway. Cell Death Dis. 2020;11(12):1065.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Li K, Guo J, et al. circNEIL3 inhibits tumor metastasis through recruiting the E3 ubiquitin ligase Nedd4L to degrade YBX1. Proc Natl Acad Sci U S A. 2023;120(13):e2215132120.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong X, Zhang G, Niu J, et al. Circular RNA circPFKP suppress gastric cancer progression through targeting miR-346/CAMD3 axis. Exp Cell Res. 2025;445(1):114390.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu Y, Cai ZR, Huang RZ, Wang DS, Ju HQ, Chen DL, Circular. RNA circPHLPP2 promotes tumor growth and anti-PD-1 resistance through binding ILF3 to regulate IL36γ transcription in colorectal cancer. Mol Cancer. 2024;23(1):272.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Qiu H, Dong Q, et al. Androgen-targeted hsa_circ_0085121 encodes a novel protein and improves the development of prostate cancer through facilitating the activity of PI3K/Akt/mTOR pathway and enhancing AR-V7 alternative splicing. Cell Death Dis. 2024;15(11):848.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrabhu L, Hartley AV, Martin M, Warsame F, Sun E, Lu T. Role of post-translational modification of the Y box binding protein 1 in human cancers. Genes Dis. 2015;2(3):240\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwabiah D, Nagati V, Tripathi MK. Transcription factor YBX1 orchestrates drug resistance and tumor progression in HCC. Drug Discov Today. 2025;30(9):104439.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark MS, Jeong SD, Shin CH, et al. LINC02257 regulates malignant phenotypes of colorectal cancer via interacting with miR-1273g-3p and YB1. Cell Death Dis. 2024;15(12):895.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Wei J, Feng L, et al. Aberrant m5C hypermethylation mediates intrinsic resistance to gefitinib through NSUN2/YBX1/QSOX1 axis in EGFR-mutant non-small-cell lung cancer. Mol Cancer. 2023;22(1):81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu J, Ji L, Liang Y, et al. CircRNA-SORE mediates sorafenib resistance in hepatocellular carcinoma by stabilizing YBX1. Signal Transduct Target Ther. 2020;5(1):298.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin S, Liu Y, Zhang X, et al. lncRNA FGD5-AS1 is required for gastric cancer proliferation by inhibiting cell senescence and ROS production via stabilizing YBX1. J Exp Clin Cancer Res. 2024;43(1):188.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang BG, Zheng YM, Xu MH et al. The MASTL/YBX1/PAK4 axis regulated by stress-activated STK24 triggers lenvatinib resistance and tumor progression in HCC. Hepatology.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang Y, Fan S, Peng R, et al. TRIM29 reverses lenvatinib resistance in liver cancer cells by ubiquitinating and degrading YBX1 to inhibit the PI3K/AKT pathway. Transl Oncol. 2025;53:102294.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Circular RNA, CircPPP2R5A, Esophageal Squamous Cell Carcinoma, Deubiquitination, YBX1","lastPublishedDoi":"10.21203/rs.3.rs-9378154/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9378154/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRecent findings suggest that circular RNAs (circRNAs) play important roles in tumor growth and cancer advancement. However, the specific biological functions and mechanisms of circRNAs in esophageal squamous cell carcinoma remain largely unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo identify differentially expressed circular RNAs (circRNAs), we analyzed GEO circRNA microarray datasets. circCOL3A1 expression was validated in ESCC cell lines and a clinical cohort comprising 100 paired tissue samples using quantitative reverse transcription PCR (qRT-PCR). The circular nature of circCOL3A1 was confirmed through RNase R digestion assays and divergent primer-based PCR amplification. Functional investigations, including in vitro and in vivo experiments, were performed to assess the role of circCOL3A1 in cellular proliferation, migration, and invasion. Mechanistic studies were conducted using Western blot analysis, RNA immunoprecipitation (RIP), co-immunoprecipitation (Co-IP), and ubiquitination assays to elucidate the molecular pathways involving circCOL3A1.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCircCOL3A1 expression was markedly elevated in both ESCC tissues and cell lines, exhibiting a significant association with advanced TNM stages and reduced overall survival. Functionally, circCOL3A1 facilitated ESCC cell proliferation, migration, and invasion in vitro, while also promoting tumor growth in vivo. Mechanistic investigations demonstrated that circCOL3A1 directly bound to the transcription factor YBX1, acting as a protein scaffold to strengthen its interaction with the deubiquitinase USP10. Consequently, this interaction enhanced YBX1 deubiquitination, increased its protein stability, and led to YBX1 accumulation, thereby accelerating ESCC progression. Rescue experiments further validated YBX1 as a pivotal downstream mediator of circCOL3A1.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study elucidates that circCOL3A1 exacerbates ESCC malignancy by stabilizing YBX1 via USP10-mediated deubiquitination. These findings suggest that circCOL3A1 could serve as both a prognostic biomarker and a therapeutic target in ESCC.\u003c/p\u003e","manuscriptTitle":"circCOL3A1 Facilitates Esophageal Squamous Cell Carcinoma Progression by Stabilizing YBX1 through Enhanced USP10-Mediated Deubiquitination","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-14 07:53:30","doi":"10.21203/rs.3.rs-9378154/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4da154ab-a3aa-4d91-9227-7c48f844693b","owner":[],"postedDate":"April 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T16:40:36+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-14 07:53:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9378154","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9378154","identity":"rs-9378154","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}