CircCOPA promotes hepatocellular carcinoma progression through the HNRNPD/SPRED2 axis-mediated activation of ERK/MAPK signaling pathway

CircCOPA promotes hepatocellular carcinoma progression through the HNRNPD/SPRED2 axis-mediated activation of ERK/MAPK signaling pathway | 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 Article CircCOPA promotes hepatocellular carcinoma progression through the HNRNPD/SPRED2 axis-mediated activation of ERK/MAPK signaling pathway Lianbao Kong, Zhiwen Feng, Qingpeng Lv, Kuan Li, Guoqing Liu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9321668/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract CircRNAs are critically involved in the progression of numerous cancers, including hepatocellular carcinoma (HCC). While circCOPA has been reported to function as a tumor suppressor in glioblastoma, its role and underlying mechanisms in HCC remain largely unexplored. By analyzing the Gene Expression Omnibus (GEO), we identified a new oncogenic circRNA, hsa_circ_0008661 (circCOPA), which was significantly upregulated in HCC tissues. In a cohort of 80 HCC patients, circCOPA upregulation was associated with poor prognosis and worse clinicopathological characteristics. Functional assays revealed that circCOPA depletion suppressed HCC proliferation and metastasis, whereas circCOPA overexpression exerted opposite effects. RNA sequencing analysis confirmed that circCOPA activated the ERK/MAPK signaling pathway through the downregulation of SPRED2. Mechanistically, RNA pulldown and mass spectrometry analysis demonstrated that circCOPA directly interacted with HNRNPD. This interaction promoted SPRED2 mRNA degradation through binding of HNRNPD to the UGUUU motif within its 3'UTR, leading to SPRED2 downregulation and subsequent activation of the ERK/MAPK signaling pathway. Notably, the oncogenic phenotypes driven by circCOPA were effectively rescued by SPRED2 overexpression. Collectively, our findings delineate a novel circCOPA/HNRNPD/SPRED2 axis that activates the ERK/MAPK signaling pathway to drive HCC progression, highlighting its promise as a novel therapeutic target. Biological sciences/Cancer/Tumour biomarkers Biological sciences/Cancer/Oncogenes circRNA hepatocellular carcinoma RNA-binding protein mRNA stability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Hepatocellular carcinoma poses a significant global health threat, ranking sixth among the most prevalent cancers and third as a cause of cancer-related deaths worldwide [ 1 ]. Due to the insidious emerging, a significant proportion of HCC patients receive their diagnosis at a late stage, rendering them unsuitable for surgery [ 2 ]. Although there have been considerable progress of HCC treatment, the prognosis remains poor [ 3 ]. Therefore, unraveling the molecular mechanisms of HCC is imperative to discover therapeutic strategies and improve patient outcomes. Circular RNAs constitute a prevalent category of non-coding RNAs formed through the back-splicing of pre-mRNA, resulting in stable, single-stranded covalently closed loops [ 4 , 5 ]. Plentiful studies have established a strong association between aberrant circRNAs expression and numerous human cancers [ 6 ], such as non-small cell lung cancer [ 7 ] and gastric cancer [ 8 ]. Mechanistically, circRNAs exert their regulatory roles via several distinct pathways, including acting as miRNA decoys, modulating protein activities, and functioning as scaffolds for peptide translation. For example, hsa_circ_0119412 was shown to upregulate the ZBED3 gene by sponging miR-1298-5p, which subsequently contributed to the progression of gastric cancer [ 9 ]. Similarly, circACTN4 promotes breast cancer progression by directly binding to FUBP1 and upregulating MYC expression [ 10 ]. Zhang et al. reported that circMET drives glioblastoma tumorigenesis by encoding MET404 [ 11 ]. Despite these studies, the roles of circRNAs need to be explored in HCC. Bioinformatic analysis of GEO datasets indicate that circCOPA is elevated in HCC tissues and associated with poor prognosis in a cohort of 80 HCC patients. Functional experiments show that circCOPA acts as an oncogenic driver, promoting HCC proliferation and metastasis. Mechanistic studies demonstrate that circCOPA directly interacts with HNRNPD to promoting SPRED2 mRNA decay by binding to the UGUUU motif in its 3’UTR region, thereby reducing SPRED2 expression and ultimately facilitating HCC progression. Thus, our findings establish the pivotal role of the circCOPA/HNRNPD/SPRED2 axis in driving HCC progression and highlight the clinical value of circCOPA as a promising treatment target. MATERIALS AND METHODS Clinical samples Eighty paired HCC samples were collected from First Affiliated Hospital of Nanjing Medical University. All patients were undergoing primary surgery and had not received any prior antitumor therapies. The study protocol was approved by the Institutional Ethics Committee (Approval number: 2024-SRFA-649). Written informed consent of each participant was obtained. RNA extraction, Nucleocytoplasmic separation and qRT-PCR Total cellular and tissue-derived RNA were isolated using the TRIzol reagent (Invitrogen, USA), while the PARIS Kit (Invitrogen, USA) was employed for the partitioning of nuclear and cytoplasmic fractions. Following extraction, cDNA synthesis was conducted with a reverse transcription system (Vazyme Biotech, China). We performed quantitative real-time PCR (qRT-PCR) using an ABI 7900 platform (Applied Biosystems, USA) and SYBR Green master mix (Vazyme Biotech, China). Relative transcript abundance was determined via the 2 − ρρCt method, normalized against GAPDH. Detailed information regarding the primer sequences is provided in Supplementary Table S2 (Supplementary Information). Cell culture Cell lines acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were utilized in this study, including human HCC lines such as MHCC-97L, MHCC-97H, MHCC-LM3, HepG2, Hep3B, and Huh7, the immortalized hepatocyte line HHL-5, and HEK-293T cells. Standard culture conditions involved DMEM (Gibco, USA) with 10% FBS (Umedium, China) and 1% penicillin-streptomycin, at 37°C in a 5% CO₂ atmosphere. Cells were harvested for passaging at a 1:3 ratio when achieving 85–90% confluence. RNase R treatment and Actinomycin D (ActD) assays Total RNA (4 µg) was treated with or without RNase R (Lucigen, USA; 3 U/µg) to assess RNA stability. The two aliquots (RNase R-treated and an untreated control) were exposed to 37°C for 30 minutes. Subsequently, the levels of circCOPA and linear COPA mRNA were quantified using qRT-PCR. For the actinomycin D (ActD) assays, transcription was blocked in MHCC-LM3 and Hep3B cells using 2 µg/mL ActD (Sigma-Aldrich, USA). RNA collected at the specific time points was analyzed via qRT-PCR to evaluate the time-dependent degradation of circCOPA, COPA mRNA, and SPRED2 mRNA. Fluorescence In Situ Hybridization ( FISH) To confirm the subcellular localization of circCOPA, Cy3-labeled probes of circCOPA were obtained from GenePharma (Shanghai, China). Subsequently, FISH assays were performed, followed by image acquisition using an Olympus fluorescence microscope (Tokyo, Japan). Plasmids, siRNA, and lentivirus transfection All siRNAs were provided by RiboBio Co., Ltd. (Guangzhou, China). Plasmid constructs for overexpression studies were cloned into pCDH vectors and supplied by GenePharma Co., Ltd. (Shanghai, China). Transient transfection was carried out by introducing plasmids or siRNAs into cells cultured in 6-well plates using Lipofectamine 3000 (Invitrogen, USA). For stable expression, cells were infected with lentiviral particles and subsequently subjected to antibiotic selection with 2 µg/mL puromycin. All sequences are provided in Table S2 (Supporting Information). Cell proliferation assay CCK-8 Assay We performed the CCK-8 assay by seeding transfected cells in 96-well plates at 2×10³ cells per well. After overnight incubation, the CCK-8 working solution was added daily for 5 days, followed by 3-hour incubation and measurement of OD450 with a microplate reader (Thermo Scientific Multiskan MK3, USA). Colony Formation Assay 1×10³ transfected cells were plated per well in 6-well plates and cultured for two weeks. The resulting colonies were then fixed with 4% paraformaldehyde (Servicebio, Wuhan, China), stained with 0.3% crystal violet (Beyotime, Shanghai, China), and imaged for subsequent quantification. EdU Assay DNA synthesis was measured using an EdU (Cy3) kit (Beyotime, Shanghai, China). After a 2-hour pulse with EdU, cells were fixed, permeabilized with 0.5% Triton X-100 (Servicebio, Wuhan, China), and subjected to the click reaction. After DAPI staining (Beyotime, Shanghai, China), images were captured using an Olympus IX73 microscope for subsequent analysis. Cell migration assay Cellular motility was assessed using Transwell inserts (Jet Bio-Filtration, Guangzhou, China). Specifically, 1x10 4 cells were suspended in serum-depleted medium and seeded into the upper compartments. These were then positioned over lower reservoirs containing complete medium supplemented with 20% fetal bovine serum (FBS) as a chemoattractant. After a one-day incubation, cells that successfully invaded the underside of the membrane underwent fixation in 4% paraformaldehyde and visualization via 0.3% crystal violet staining for subsequent quantification. Wound assay was performed by growing transfected cells to 90–100% confluence in 6-well plates. After washing, cells were maintained in medium with 2% FBS. Wound width was photographed and measured at 0 h and 48 h. Cell invasion assay The invasion assay was assessed using Matrigel-coated Transwell inserts (Corning, NewYork, USA). Each insert was coated with 200 µL of Matrigel diluted 1:20 in DMEM and allowed to polymerize at 37°C overnight. The subsequent procedures for cell seeding, incubation, and staining were consistent with the standard transwell migration protocol. Western blot (WB) Total protein fractions were isolated using RIPA lysis buffer (Beyotime, Shanghai, China). Following concentration determination, proteins were fractionated via SDS-PAGE and subsequently immobilized onto PVDF membranes. To prevent non-specific binding, membranes were treated with Quick Blocking Buffer (Beyotime, Shanghai, China) before being incubated with primary antibodies at 4°C overnight. After thorough TBST rinsing, HRP-conjugated secondary antibodies were applied for a 2-hour period at ambient temperature. Protein bands were finally visualized using an ECL chemiluminescence system. A comprehensive list of antibodies is available in Supplementary Table S3 (Supplementary Information). RNA sequencing RNA sequencing analysis was supported by Tsingke Biotechnology Co., Ltd. (Beijing, China) to compare transcriptomic profiles between circCOPA overexpression and control cells. The service included library preparation, sequencing on an Illumina HiSeq 2500 platform, and bioinformatic analysis. CircRNA pulldown and mass spectrometry (MS) RNA-interacting proteins were identified with an RNA pulldown assay with biotin-labeled circCOPA-specific and control probes (GenePharma, Shanghai, China). Streptavidin magnetic beads were incubated with 50 pmol of each probe for 2 h, then incubated overnight with lysates from 1×10⁷ Hep3B cells. Following washing, bound proteins were eluted, subjected to SDS-PAGE separation, and detected with a Fast Silver Stain Kit (Beyotime, Shanghai, China). Specifically enriched protein bands from the circCOPA probe group were excised and analyzed by Q Exactive mass spectrometry (Thermo Fisher Scientific, USA). All mass spectrometry results are provided in Table S4 . RNA immunoprecipitation (RIP) RNA immunoprecipitation (RIP) was examined using an anti-HNRNPD antibody and a commercial kit (BersinBio, Guangzhou, China). Cell lysates from MHCC-LM3 and Hep3B cells (2×10⁷ cells per sample) were incubated overnight at 4°C with magnetic beads pre-conjugated with HNRNPD antibody or IgG antibody. After washing, the co-precipitated RNAs were extracted by digestion with proteinase K, purified, and quantified by qRT-PCR. Dual-Luciferase Reporter Assay To quantify promoter activity, we employed a dual-luciferase reporter system (IBSBIO, Shanghai, China). Post-transfection, HEK293T cells were lysed, and the resulting supernatants were isolated via high-speed centrifugation (10,000–15,000 rpm) for 5 minutes. Luminescence was recorded by sequentially mixing 20 µL of cell lysate with equal volumes of Firefly and Renilla luciferase reagents, respectively. The final reporter efficiency was determined by normalizing the Firefly RLU against the Renilla-derived internal control RLU. Immunofluorescence (IF) Staining For immunostaining procedures, HCC cells underwent fixation and permeabilization, followed by a 30-minute blocking step with 10% normal goat serum. The cells were then probed overnight with primary antibodies at 4°C. On the second day, they were exposed to species-matched fluorescent secondary antibodies for 2 hours at ambient temperature. Nuclei were counterstained using DAPI, and fluorescence signals were visualized and captured via an Olympus imaging system (Tokyo, Japan). In Vivo nude mouse model Subcutaneous xenograft models were established using 4-week-old male nude mice obtained from the Animal Core Facility of Nanjing Medical University. Following an acclimatization period in an SPF environment, mice were randomized into four cohorts. Specifically, 5 × 10 6 stably transfected MHCC-LM3 or Hep3B cells were inoculated into the right flank of each mouse. Tumor growth was monitored quadrennially, with volumes determined by the formula: Volume = (length × width 2 )/2. Approximately four weeks later, mice were euthanized for tumor excision and digital documentation. For the lung metastasis model, 100 µL of cell suspension (1×10⁷ cells/mL) was administered via tail vein injection. After four weeks, metastatic progression was assessed through bioluminescence imaging where applicable. Lungs were subsequently harvested for nodule quantification and H&E histological validation. All procedures received ethical clearance from the Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-2507092). Immunohistochemistry (IHC) and hematoxylin-eosin (HE) staining For immunohistochemical (IHC) analysis, 5-µm paraffin sections were prepared from tissues initially fixed in 4% PFA. Following xylene-mediated clearing and rehydration through graded ethanol, 3% H2O2 was applied to neutralize endogenous peroxidase activity. Antigenic sites were exposed via microwave-based heat retrieval. Sections were subsequently probed overnight (4°C) with primary antibodies (1:100), followed by a 30-minute incubation with HRP-linked secondary antibodies at ambient temperature. Digital images were captured for further evaluation. Parallelly, morphological characteristics were assessed using H&E staining (Beyotime, Shanghai, China) following the manufacturer’s protocol. Statistical analysis Experimental data, derived from at least three independent biological replicates, are presented as mean ± standard deviation (SD). All statistical evaluations were conducted using GraphPad Prism 8.0 (GraphPad Software, Inc., USA). To determine significance, unpaired Student’s t-tests were utilized for binary comparisons, while one-way analysis of variance (ANOVA) was applied for cohorts with three or more groups. Kaplan–Meier curves were generated for survival analysis, and the Pearson coefficient was calculated to evaluate the inter-expression relationship between circCOPA and SPRED2. Statistical significance was defined as p < 0.05. RESULTS Identification and characterization of circCOPA in HCC By analyzing two Gene Expression Omnibus (GEO) datasets (GSE155949 and GSE94508), we identified several circRNAs that were aberrantly expressed in HCC (Fig. S1 A,1B) using the threshold of |logFC| > 1 and adjusted p-value < 0.05. CircCOPA (hsa_circ_0008661) captured our attention as the only candidate consistently upregulated in both datasets ( Fig. 1 A ) . qRT-PCR analysis confirmed significant upregulation of circCOPA in a cohort of 80 HCC patients ( Fig. 1 B, C ) . Similarly, analysis of The Gene Expression Profiling Interactive Analysis (GEPIA) database revealed that the expression of its parental gene, COPA, was also upregulated in HCC tissues (Fig. S1 C) . Among the six HCC cell lines examined, circCOPA expression was highest in MHCC-LM3 and lowest in Hep3B, relative to the normal hepatocyte cell line HHL-5 ( Fig. 1 D ) . Thus, MHCC-LM3 and Hep3B cells, representing the extremes of circCOPA expression, were selected for subsequent functional experiments. To assess clinical significance, HCC cohorts were dichotomized into high and low expression groups (n = 40 each) based on median circCOPA levels. Our findings indicated that elevated circCOPA significantly correlated with aggressive clinicopathological features, including increased tumor dimensions, advanced TNM staging, and higher Edmondson grades ( Table S1 ). Kaplan–Meier analysis revealed that patients with higher circCOPA levels experienced markedly diminished overall survival (OS) ( Fig. 1 E). Consistently, GEPIA-based validation confirmed that COPA upregulation conferred a dismal prognosis, characterized by truncated OS and disease-free survival (DFS) ( Fig. S1 D, E ). According to circBase, circCOPA is derived from exons 6–8 of the COPA gene on chromosome 1 (1q23.2) [ 12 ]. Analysis of Sanger sequencing revealed the annotation of circCOPA and its characteristic back-splicing junction (Fig. 1 F, G ) . Agarose gel electrophoresis was performed to verify its circular structure, demonstrating that divergent primers specifically amplified circCOPA from cDNA, but not from gDNA (Fig. 1 H). Additionally, RNase R digestion and actinomycin D treatment assays were conducted to examine circCOPA stability. CircCOPA exhibited significantly greater resistance to both treatments compared with linear COPA mRNA, confirming its enhanced RNA stability (Fig. 1 I-L). Furthermore, fluorescence in situ hybridization (FISH) and subcellular fractionation assays indicated that circCOPA was predominantly localized in the cytoplasm of MHCC-LM3 and Hep3B cells (Fig. 1 M-O). In summary, these data demonstrate that circCOPA is upregulated in HCC tissues and its high expression correlates with poor prognosis. circCOPA promotes proliferation, migration, and invasion of HCC cells in vitro We performed loss-of-function experiments to investigate the regulatory role of circCOPA in MHCC-LM3 cells. qRT-PCR analysis demonstrated that these designed siRNAs effectively downregulated circCOPA expression without affecting linear COPA mRNA levels (Fig. 2 A). We performed the gain-of-function assays on Hep3B cells due to its lower endogenous circCOPA expression (Fig. 1 D). qRT-PCR analysis verified the successful overexpression of circCOPA (Fig. 2 B). Functional assays revealed that circCOPA knockdown significantly suppressed the proliferation of MHCC-LM3 cells, as assessed by CCK-8 and EdU assays, whereas circCOPA overexpression promoted proliferation in Hep3B cells (Fig. 2 C-G). This pro-proliferative effect was further corroborated by colony formation assays (Fig. 2 H-J). Furthermore, transwell assays demonstrated that circCOPA depletion significantly inhibited the migration and invasion of MHCC-LM3 cells, while circCOPA overexpression exhibited the opposite effects in Hep3B cells (Fig. 2 K-M). Consistently, wound healing assays further confirmed the pro-migratory role of circCOPA (Fig. 2 N-P). In summary, these in vitro findings establish circCOPA as an oncogenic driver in HCC. circCOPA facilitates HCC tumor growth and metastasis in vivo To investigate the oncogenic role of circCOPA in vivo , we established subcutaneous xenograft mice models using MHCC-LM3 cells with circCOPA stable depletion (sh-circCOPA) and Hep3B cells with circCOPA stable overexpression (circCOPA-OE). Compared with the sh-NC control group, tumors in the sh-circCOPA group exhibited significantly smaller volumes, lower weight, and reduced growth rates. Conversely, the circCOPA-OE group showed a significant promotion of tumor growth in these parameters (Fig. 3 A-C). Moreover, immunohistochemistry (IHC) and hematoxylin and eosin (H&E) of tumor sections showed that circCOPA silencing decreased Ki-67 expression, whereas circCOPA overexpression increased Ki-67 expression (Fig. 3 D). In the lung metastasis models, circCOPA depletion substantially reduced the number of metastatic nodules, while circCOPA overexpression produced the opposite effects (Fig. 3 E, F). Furthermore, mice in the sh-circCOPA group exhibited prolonged overall survival, whereas those in the circCOPA-OE group showed a significant reduction in survival time (Fig. 3 G, H). Collectively, these in vivo findings establish a critical role of circCOPA in facilitating HCC tumor growth and metastasis. circCOPA activates the ERK/MAPK signaling pathway by downregulating SPRED2 To investigate the downstream regulatory network, we performed RNA-sequencing on Hep3B cells following circCOPA induction (Fig. 4 A). KEGG pathway enrichment analysis highlighted the MAPK signaling pathway as a primary target (Fig. 4 B). Parallel Gene Ontology (GO) annotations further implicated circCOPA in modulating the ERK1/2 cascade, a pivotal constituent of the MAPK framework ( Fig. S2 A ). This activation was corroborated by GSEA results, which showed a robust positive correlation between circCOPA levels and MAPK pathway signatures ( Fig. S2 B ). Given the established influence of MAPK signaling on cellular growth and motility [ 13 ], we speculated that circCOPA drives HCC malignancy via the activation of this specific signaling axis. Consistently, circCOPA overexpression enhanced ERK phosphorylation, whereas circCOPA knockdown decreased it. Notably, JNK and p38 phosphorylation remained unaffected ( Fig. 4 C ) . This specific activation of ERK phosphorylation, consistent with the GO analysis, indicates that circCOPA drives HCC progression via the ERK/MAPK signaling pathway. To investigate the role of the ERK/MAPK pathway in circCOPA-mediated HCC progression, we treated HCC cells with SCH772984, a highly selective ERK pathway inhibitor, to suppress ERK phosphorylation. Western blot analysis confirmed that circCOPA overexpression rescued the reduction in ERK phosphorylation induced by SCH772984 in HCC cells (Fig. S2 C, D) . Based on the most significantly changed genes and those involved in the MAPK pathway, 15 candidate genes were subsequently selected (Fig. 4 D). qRT-PCR and western blot results identified SPRED2 as a downstream target, showing an inverse correlation with circCOPA at both the transcriptional and translational levels (Fig. 4 E, F, J and Fig. S2 E, F) . This finding was further confirmed using two independent circCOPA-targeting siRNAs (Fig. S2 G, H) . Additionally, western blot and qRT-PCR analyses revealed a significant reduction of SPRED2 expression in HCC tissues ( Fig. 4 G, H ) . Furthermore, correlation analysis of 80 HCC clinical samples showed a negative correlation between circCOPA and SPRED2 mRNA expression ( Fig. 4 I ) . Lentiviral plasmids for stable SPRED2 knockdown or overexpression were constructed and successfully modulated SPRED2 expression levels (Fig. S2 I, J) . The phosphorylation levels of ERK and MEK were elevated following SPRED2 depletion, whereas the opposite effect was observed following SPRED2 overexpression ( Fig. 4 K ) . In summary, these findings suggest that circCOPA activates the ERK/MAPK signaling pathway through the downregulation of SPRED2. circCOPA directly interacts with HNRNPD in HCC cells We next sought to elucidate the mechanistic basis underlying the negative regulation of SPRED2 by circCOPA in HCC. Therefore, we investigated whether circCOPA could directly bind to proteins or function as a miRNA sponge in HCC. RNA pulldown assays were performed using a circCOPA-specific probe to examine its association with Argonaute-2 (Ago2), a core RNA-induced silencing complex component essential for miRNA sponge [ 14 ]. However, Ago2 was not enriched in the circCOPA pulldown fractions ( Fig. S3 A) , suggesting that a ceRNA mechanism is unlikely. We then performed RNA pulldown assays coupled with mass spectrometry to identify circCOPA-binding proteins. Silver staining revealed a distinct protein band at approximately 40 kDa specifically pulled down by the circCOPA probe ( Fig. 5 A ) . Mass spectrometry (MS) analysis identified Heterogeneous Nuclear Ribonucleoprotein D (HNRNPD) as the top candidate, based on its highest Sequest scores and the greatest number of unique peptides ( Fig. S3 B and Table S4 ) . This specific interaction was validated by RNA pulldown assays in both MHCC-LM3 and Hep3B cells ( Fig. 5 B ) . RNA immunoprecipitation (RIP) analysis using an anti-HNRNPD antibody showed significant enrichment of circCOPA ( Fig. 5 C, D ) . Furthermore, combined fluorescence in situ hybridization (FISH) and immunofluorescence (IF) assays demonstrated the cytoplasmic colocalization of circCOPA and HNRNPD in MHCC-LM3 and Hep3B cells ( Fig. 5 E ) . The RNA-binding capacity of HNRNPD is mediated by its two distinct RNA recognition motif (RRM) domains [ 15 ]. To identify the interaction domain, we generated a series of FLAG-tagged HNRNPD truncation mutants. Western blot analysis confirmed the successful expression and expected molecular weights of the truncated proteins ( Fig. 5 F ). RIP assays followed by qRT-PCR analysis demonstrated that circCOPA was significantly enriched by wild-type HNRNPD and the RRM1 domain mutant, but not by the RRM2 domain mutant ( Fig. 5 G ) . We next designed three biotin-labeled RNA segment probes spanning the full-length circCOPA sequence. RNA pulldown assays identified segment 1 of circCOPA as the primary region responsible for HNRNPD binding ( Fig. 5 H ) . We next investigated whether circCOPA and HNRNPD mutually regulate each other's expression. qRT-PCR and western blot analyses showed no evidence of mutual regulation: circCOPA did not change HNRNPD mRNA or protein levels ( Fig. 5 I, L and S3C ) . Consistently, HNRNPD knockdown or overexpression did not affect circCOPA levels ( Fig. 5 J, K and S3D, E ) . Collectively, these data demonstrate a direct and specific interaction between HNRNPD and circCOPA. circCOPA promotes SPRED2 mRNA decay in an HNRNPD-dependent manner Having identified SPRED2 as a downstream effector, we next explored the relationship between HNRNPD and SPRED2. Knockdown of either circCOPA or HNRNPD upregulated SPRED2 expression, while overexpression of either downregulated SPRED2 expression (Fig. 4 J, 6 A-C, and S4A-B), indicating their cooperative role in repressing SPRED2. Given the established role of HNRNPD in binding and destabilizing target mRNAs, such as ATF3 [ 16 ], we hypothesized that circCOPA promotes HCC malignancy by recruiting HNRNPD to facilitate SPRED2 mRNA degradation. Supporting this hypothesis, western blot analysis demonstrated that circCOPA depletion reversed the downregulation of SPRED2 induced by HNRNPD overexpression in MHCC-LM3 cells (Fig. 6 D), indicating the functional indispensability of circCOPA in the HNRNPD-mediated SPRED2 degradation. Moreover, HNRNPD knockdown rescued the suppression of SPRED2 caused by circCOPA overexpression in Hep3B cells (Fig. 6 E), suggesting that the regulating role of circCOPA on SPRED2 is dependent on HNRNPD. Next, RNA stability assays were performed to examine SPRED2 mRNA decay rates following modulation of circCOPA or HNRNPD expression. Knockdown of circCOPA significantly extended the half-life of SPRED2 mRNA (Fig. 6 F, S4C). Conversely, circCOPA overexpression accelerated SPRED2 mRNA decay, an effect that was abolished by HNRNPD knockdown (Fig. 6 G, S4D), confirming the circCOPA dependence of HNRNPD-mediated mRNA destabilization. RNA immunoprecipitation (RIP) assays were conducted to examine whether circCOPA promoted the interaction between HNRNPD and SPRED2 mRNA. The results revealed that circCOPA silencing attenuated the binding of HNRNPD to SPRED2 mRNA (Fig. 6 H, S4E). Conversely, circCOPA overexpression enhanced this interaction (Fig. 6 I, S4F). Collectively, these data suggest that circCOPA recruits HNRNPD to promote its binding to SPRED2 mRNA. To identify the specific HNRNPD binding sites on SPRED2 mRNA, we analyzed the sequence of its 3’UTR, including GU-/UG-rich nonclassical motifs (UGUUU and GUUUG) previously identified as potential HNRNPD binding sites [ 17 ]. RNA pulldown assays were performed using the biotinylated SPRED2 RNA probes spanning different regions, and the results demonstrated that HNRNPD exhibited a specific binding affinity for the 3'UTR of SPRED2 mRNA compared to the coding sequence (CDS) or 5'UTR regions (Fig. 6 J). Furthermore, we analyzed the 3'UTR sequence of SPRED2 mRNA and identified three candidate binding motifs: Motif 1(26-30nt: UGUUU), Motif 2(474-478nt: UGUUU) and Motif 3(761-765nt: UGUUU). Plasmids expressing each candidate sequence along with its 200-bp flanking regions on both sides were co-transfected into HEK293T cells together with HNRNPD plasmid. RIP-qPCR analysis showed that HNRNPD selectively bound to Motif 3 compared to IgG control. In contrast, no significant binding was detected for Motif 1 or Motif 2(Fig. 6 K). Subsequently, the 3'UTR containing either wild-type Motif 3 or its mutant (UGUUU→ACAAA) was inserted downstream of a firefly luciferase reporter. These constructs were co-transfected into HEK293T cells along with HNRNPD and Renilla luciferase control plasmids. Luciferase activity assays revealed that HNRNPD overexpression enhanced reporter activity for the wild-type Motif 3, whereas this effect was not observed for the mutant Motif 3(Fig. 6 L). Consistently, mutation of Motif 3 also prolonged the half-life of SPRED2 mRNA (Fig. 6 M). Collectively, these findings elucidate that HNRNPD directly binds the UGUUU motif within the SPRED2 3'UTR, thereby promoting SPRED2 mRNA decay. The circCOPA-SPRED2 axis drives HCC malignancy To establish SPRED2 as the critical downstream mediator of circCOPA, we performed rescue experiments to determine whether circCOPA drives HCC progression through suppression of SPRED2. CCK-8 assays, EdU staining and colony formation assays revealed that circCOPA depletion suppressed cell proliferation, an effect that was rescued by concurrent SPRED2 silencing ( Fig. 7 A-C and S5A ) . Conversely, circCOPA overexpression enhanced the proliferation of Hep3B cells, and this pro-proliferative effect was reversed by SPRED2 overexpression ( Fig. 7 D-F and S5B ) . Furthermore, transwell assays and wound healing assays demonstrated that SPRED2 knockdown rescued the impairment of migratory and invasive capacities in MHCC-LM3 cells resulting from circCOPA silencing ( Fig. 7 G, H and S5C, D ) . Consistently, the enhancement of invasion and migration in Hep3B cells caused by circCOPA overexpression were counteracted by SPRED2 overexpression ( Fig. 7 I, J and S5E, F ) . In summary, these rescue experiments confirm that circCOPA exerts its oncogenic roles in HCC cells by suppressing SPRED2, highlighting the circCOPA–SPRED2 axis as a promising therapeutic target. DISCUSSION Due to the prevalence of late-stage diagnoses and subsequent dismal clinical outcomes, there is a compelling need to decode the molecular drivers of HCC and uncover effective therapeutic vulnerabilities [ 18 , 19 ]. Recent studies have underscored the functional versatility of circular RNAs (circRNAs) in driving HCC malignancy [ 20 – 22 ]. Illustrative examples include EIF4A3-mediated circTOLLIP, which accelerates tumor progression by modulating the miR-516a/PBX3 axis [ 23 ]. Furthermore, exosome-derived circRNA-100338 has been implicated in enhancing the invasive and metastatic potential of HCC cells [ 24 ], while m6A-modified circCPSF6 exerts its oncogenic effects through the upregulation of YAP1 [ 25 ]. In this context, the present study identified circCOPA as a novel overexpressed transcript in HCC, whose elevation served as a marker for poor patient prognosis. Functional experiments established circCOPA as a potent oncogenic driver, promoting malignant phenotypes including proliferation and metastasis. Mechanistic studies further revealed that the circCOPA-HNRNPD interaction promoted SPRED2 mRNA decay and subsequent ERK/MAPK pathway activation, ultimately driving HCC pathogenesis. Taken together, our findings establish the circCOPA/HNRNPD/SPRED2 axis as a new therapeutic target in HCC. CircRNAs are known to exert their functions through three principal mechanisms: encoding peptides, engaging in protein interactions, and serving as miRNA sponges [ 26 , 27 ]. In the present study, RNA pulldown assays revealed no Ago2 enrichment, suggesting that circCOPA is unlikely to function via the canonical miRNA sponge mechanism. Instead, we found that circCOPA functioned by directly binding to HNRNPD. HNRNPD is an RNA-binding protein that functions in both the cytoplasm and nucleus [ 28 , 29 ]. Within the cytoplasm, it modulates mRNA stability—such as that of CCND1 mRNA—by binding to AU-rich elements in the 3'UTR [ 28 – 30 ]. Combined FISH and IF assays demonstrated the cytoplasmic colocalization of circCOPA and HNRNPD in HCC cells. Based on these findings, circCOPA was identified as a molecular scaffold that directly binds to HNRNPD, thereby promoting SPRED2 mRNA decay. HNRNPD, alternatively designated as AUF1, belongs to the RNA-binding protein superfamily and is characterized by its four distinct isoforms (p37, p40, p42, and p45) [ 15 , 31 ]. These variants emerge from the differential splicing of a single pre-mRNA precursor. Structurally, these core HNRNPD members are defined by a conserved architecture comprising dual non-identical RNA recognition motifs (RRMs), tandem repeat sequences, and an abundance of glutamine-rich regions (specifically eight clusters)[ 15 , 28 ]. However, the precise subcellular localization of HNRNPD isoforms remains contentious. The lack of isoform-specific antibodies has hindered our efforts to determine which isoform interacts with circCOPA. Given that both RRM domains are essential for RNA binding [ 32 ], we generated HNRNPD truncation mutants and identified the RRM1 domain as essential for binding circCOPA. Furthermore, circCOPA truncations and RNA pulldown assays identified segment 1 of circCOPA as the critical binding site for HNRNPD. Collectively, These findings provide new insights into the circCOPA-HNRNPD interaction. SPRED2, a member of the SPRED family, functions by suppressing the activation of the ERK/MAPK signaling cascade [ 33 ]. Previous studies have showed that downregulation of SPRED2 is associated with enhanced EMT and stemness, exerting these effects through ERK pathway activation in HCC [ 34 , 35 ]. However, the circRNA-mediated regulatory mechanisms underlying SPRED2 expression remain to be elucidated. HNRNPD functions as a key regulator of mRNA decay [ 36 ]. For example, HNRNPD was identified to be a negative regulator of ferroptosis, exerting its effects by binding to the ATF3 mRNA 3'-UTR and promoting its degradation [ 37 ]. Consistently, our study also revealed that circCOPA promoted HNRNPD-mediated decay of SPRED2 mRNA. Furthermore, RNA pulldown assays and dual-luciferase reporter assays demonstrated that HNRNPD directly bound the UGUUU motif within the SPRED2 3'UTR, thereby promoting SPRED2 mRNA decay. Collectively, our findings expand the current understanding of SPRED2 regulation by elucidating a novel circCOPA-HNRNPD scaffolding mechanism. Several limitations should be acknowledged in our study. First, the upstream mechanisms governing circCOPA biogenesis remain to be elucidated. Second, although SPRED2 was identified as a key downstream target, other potential effectors warrant further investigation. CONCLUSION Our findings demonstrate that circCOPA is upregulated in HCC and functions as both a driver of tumor progression and a biomarker of poor prognosis. Mechanistically, circCOPA directly interacts with HNRNPD. This interaction promotes SPRED2 mRNA degradation by binding to the UGUUU motif in its 3’UTR region, subsequently activating the ERK/MAPK signaling pathway. These results establish the circCOPA/HNRNPD/SPRED2 axis as a potential therapeutic target for HCC. Declarations DATA AVAILABILITY STATEMENT All data used to support the findings of this study are available from the corresponding author upon request. ACKNOWLEDGEMENTS We would like to appreciate the Core Facility of the First Affiliated Hospital of Nanjing Medical University for the detection of HCC samples. AUTHOR CONTRIBUTIONS Zhiwen Feng and Lianbao Kong designed this study. Zhiwen Feng, Qingpeng Lv and Kuan Li performed the major experiments and drafted the manuscript. Lianbao Kong, Chao Yang and Guoqing Liu supervised this study. Wenzhou Ding, Wenhu Zhao, Yanzhi Feng, Deming Zhu, Xiangyu Ling and Xiangyu Qu collected the clinical samples. All authors read and approved the final manuscript. FUNDING This work was supported by grants from the National Natural Science Foundation of China (81871260); Young Scholars Fostering Fund of the First Affiliated Hospital of Nanjing Medical University (PY2022007); Key Program of the Youth Fund of Wannan Medical College (WK2022ZF21); National Key Clinical Specialty Construction Fund for General Surgery (KGZ002). COMPETING INTERESTS The authors declare no competing interests. ETHICS APPROVAL AND CONSENT TO PARTICIPATE This study was conducted in compliance with the ethical principles of the Declaration of Helsinki. The protocol was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University, and written informed consent was obtained from all participants. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin.2021; 71:209–49. 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Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors. Oncogene.2006; 25:6056–66. Sidali A, Teotia V, Solaiman NS, Bashir N, Kanagaraj R, Murphy JJ, et al. AU-Rich Element RNA Binding Proteins: At the Crossroads of Post-Transcriptional Regulation and Genome Integrity. Int J Mol Sci.2021; 23. Wang Y, Chen D, Xie H, Jia M, Sun X, Peng F, et al. AUF1 protects against ferroptosis to alleviate sepsis-induced acute lung injury by regulating NRF2 and ATF3. Cell Mol Life Sci.2022; 79:228. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupplementaryInformation.pdf Supplementary Information SupplementalTableS4.xlsx Mass spectrometry (MS) analysis results following circCOPA probe pulldown originaldatafilesforqPCR.xlsx original data files for qPCR Uncroppedoriginalwesternblots.doc Uncropped original western blots Cite Share Download PDF Status: Under Review Version 1 posted Reviewer # 1 agreed at journal 07 May, 2026 Reviewers invited by journal 14 Apr, 2026 Submission checks completed at journal 10 Apr, 2026 First submitted to journal 08 Apr, 2026 Unknown event 08 Apr, 2026 Editor assigned by journal 04 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-9321668","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":622837922,"identity":"f5406da5-1db3-4c55-b662-fb1c3e73081a","order_by":0,"name":"Lianbao Kong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACPgk2EGXDwNgAotmI0MIG0ZJGupbDMC4xWqTbEh8X/DqfxzztjAHDh7LDDPyzGwhokTl22Hhm3+1ixtk5Bowzzh1mkLhzgJDD0tukeXtuJzYCtTDzth1mMJBIIErLOYiWv8RpSTsmzfPjAEQLI1FaZI4lG/M2JAO1pBUc7DmXziNxg4AWfuk2w8c8f+wSN85O3vjgR5m1HP8MAlrAgLGNgcGwgYHhAJDNQ4R6EPjDwCBPpNJRMApGwSgYgQAAEJdAip2OL+IAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2508-1321","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Lianbao","middleName":"","lastName":"Kong","suffix":""},{"id":622837923,"identity":"9a71b8e2-e100-4161-a1f8-b59f6ecfbdbc","order_by":1,"name":"Zhiwen Feng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhiwen","middleName":"","lastName":"Feng","suffix":""},{"id":622837924,"identity":"c2081280-0319-4c97-93b4-59aeaf1543c8","order_by":2,"name":"Qingpeng Lv","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qingpeng","middleName":"","lastName":"Lv","suffix":""},{"id":622837925,"identity":"18373cd0-67de-4355-a14f-877eca7e203f","order_by":3,"name":"Kuan Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kuan","middleName":"","lastName":"Li","suffix":""},{"id":622837926,"identity":"ed7f7b19-cb03-46aa-944e-1c0d3fd4dc82","order_by":4,"name":"Guoqing Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Guoqing","middleName":"","lastName":"Liu","suffix":""},{"id":622837927,"identity":"a60e4d76-e0b8-4962-b960-02acd9527b02","order_by":5,"name":"Chao Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Yang","suffix":""},{"id":622837928,"identity":"9188ab61-ef02-421c-9416-80b2dfb1200f","order_by":6,"name":"Wenzhou Ding","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wenzhou","middleName":"","lastName":"Ding","suffix":""},{"id":622837929,"identity":"36689e0c-c3ee-47bb-8007-ed2eb7dbe521","order_by":7,"name":"Deming Zhu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Deming","middleName":"","lastName":"Zhu","suffix":""},{"id":622837930,"identity":"df87eca9-4bba-49ef-b7e9-01d5e38153f5","order_by":8,"name":"Wenhu Zhao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wenhu","middleName":"","lastName":"Zhao","suffix":""},{"id":622837931,"identity":"39e0206f-d043-4527-a84a-686bc7eccf0a","order_by":9,"name":"Yanzhi Feng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yanzhi","middleName":"","lastName":"Feng","suffix":""},{"id":622837932,"identity":"5373fb91-8c8e-41b6-8880-e2a5f14d57c1","order_by":10,"name":"Xiangyu Ling","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiangyu","middleName":"","lastName":"Ling","suffix":""},{"id":622837933,"identity":"dec3d7ae-f93e-4720-af12-d5a2bc638f7a","order_by":11,"name":"Xiangyu Qu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiangyu","middleName":"","lastName":"Qu","suffix":""}],"badges":[],"createdAt":"2026-04-04 15:40:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9321668/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9321668/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107453885,"identity":"15b37b8f-79f9-4e7b-8e4d-0ec492e02313","added_by":"auto","created_at":"2026-04-21 15:36:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":480447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification and characterization of circCOPA in HCC. (A) \u003c/strong\u003eVenn diagram showing overlapping upregulated circRNAs from the GSE155949 and GSE94508 datasets. Hsa_circ_0008661 (circCOPA) was the only circRNA identified in both datasets. (\u003cstrong\u003eB,C)\u003c/strong\u003e qRT-PCR analysis of circCOPA in 80 paired HCC samples. (\u003cstrong\u003eD)\u003c/strong\u003eExpression levels of circCOPA in HCC cells and the immortalized hepatocyte HHL-5. (\u003cstrong\u003eE)\u003c/strong\u003e Overall survival (OS) analysis of 80 HCC patients stratified by circCOPA expression. (\u003cstrong\u003eF,G) \u003c/strong\u003eIllustration of the genomic locus of circCOPA, and the back-splice junction confirmed by Sanger sequencing. (\u003cstrong\u003eH)\u003c/strong\u003e Detection of circCOPA and linear COPA mRNA by qRT-PCR and agarose gel electrophoresis.(\u003cstrong\u003eI,J)\u003c/strong\u003e Expression levels of circCOPA and linear COPA mRNA in MHCC-LM3 and Hep3B cells after RNase R treatment. (\u003cstrong\u003eK,L) \u003c/strong\u003eExpression levels of circCOPA and linear COPA mRNA in MHCC-LM3 and Hep3B cells following actinomycin D treatment. (\u003cstrong\u003eM)\u003c/strong\u003e Fluorescence in situ hybridization (FISH) assays in HCC cells. Scale bar: 20μm. (\u003cstrong\u003eN,O) \u003c/strong\u003eNuclear-cytoplasmic fractionation assays demonstrating the predominant cytoplasmic localization of circCOPA in HCC cells. Data are expressed as mean ± SD of three independent experiments. **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-9321668/v1/9823df45641d056b481a032e.png"},{"id":107489432,"identity":"09f3873d-b154-4c5e-88d0-099596358144","added_by":"auto","created_at":"2026-04-22 02:47:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1094022,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecircCOPA promotes proliferation, migration, and invasion of HCC cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (\u003cstrong\u003eA,B) \u003c/strong\u003eMHCC-LM3 cells with circCOPA knockdown and Hep3B cells with circCOPA overexpression were established. qRT-PCR confirmed circCOPA knockdown or overexpression, with no significant effect on linear COPA mRNA levels. (\u003cstrong\u003eC,D) \u003c/strong\u003eCCK-8 assays performed after circCOPA knockdown or overexpression. (\u003cstrong\u003eE,F,G) \u003c/strong\u003eEdU staining assays following circCOPA knockdown or overexpression. (\u003cstrong\u003eH,I,J) \u003c/strong\u003eColony formation assays after circCOPA knockdown or overexpression. (\u003cstrong\u003eK,L,M) \u003c/strong\u003eTranswell assays for migration and invasion with circCOPA knockdown or overexpression. (\u003cstrong\u003eN,O,P) \u003c/strong\u003eWound healing assays following circCOPA knockdown or overexpression. Data are expressed as mean ± SD of three independent experiments. **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-9321668/v1/8fecc2d3c8644ff426a8ff78.png"},{"id":107490237,"identity":"3b56d16b-70a5-48b2-ab19-c9f74a4550d2","added_by":"auto","created_at":"2026-04-22 02:51:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":658850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecircCOPA facilitates HCC tumor growth and metastasis \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A,B) \u003c/strong\u003eTumor volumes and weights were measured in subcutaneous xenograft models established with MHCC-LM3 cells (sh-NC vs. sh-circCOPA) and Hep3B cells (Vector vs. circCOPA-OE) (n = 5 mice per group). (\u003cstrong\u003eC) \u003c/strong\u003eTumor growth curves in each group. (\u003cstrong\u003eD) \u003c/strong\u003eH\u0026amp;E and Ki-67 IHC staining in each group. Scale bar:50μm. (\u003cstrong\u003eE)\u003c/strong\u003e Images of lung metastatic nodules.\u003cstrong\u003e \u003c/strong\u003eScale bar:2mm. H\u0026amp;E staining of lung sections. Scale bar:50μm. (\u003cstrong\u003eF) \u003c/strong\u003eQuantification of lung metastatic nodules in each group. (\u003cstrong\u003eG,H) \u003c/strong\u003eSurvival analysis \u0026nbsp;of mice in each group(n = 10 mice per group). Data are expressed as mean ± SD of three independent experiments. **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-9321668/v1/9e5da8f05feade09cf7b5083.png"},{"id":107453891,"identity":"fe434b13-bf1b-4a23-9d25-28a1f7dbb03c","added_by":"auto","created_at":"2026-04-21 15:36:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":618042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecircCOPA activates the ERK/MAPK pathway by downregulating SPRED2. (A) \u003c/strong\u003eHeatmap showing RNA sequencing transcriptome profiles of Hep3B cells transfected with circCOPA overexpression (circCOPA-OE) or vector (Vector). (\u003cstrong\u003eB)\u003c/strong\u003e KEGG pathway enrichment analysis of differentially expressed genes identified from RNA-seq data. (\u003cstrong\u003eC) \u003c/strong\u003eWestern blot analysis of phosphorylated and total ERK, JNK, and p38 levels in MHCC‑LM3 and Hep3B cells following stable circCOPA knockdown or overexpression. (\u003cstrong\u003eD) \u003c/strong\u003eHeatmap displaying the expression of 15 candidate genes in Hep3B cells transfected with circCOPA-OE or Vector. Red and blue bars indicate upregulated and downregulated genes, respectively. (\u003cstrong\u003eE,F) \u003c/strong\u003eqRT‑PCR results of the 15 candidate genes in MHCC-LM3 cells following circCOPA knockdown or overexpression. (\u003cstrong\u003eG) \u003c/strong\u003eWestern blot analysis of SPRED2 expression in 10 paired tumor (T) and adjacent non-tumor (N) tissues. (\u003cstrong\u003eH)\u003c/strong\u003eSPRED2 mRNA expression levels in 80 paired HCC samples. (\u003cstrong\u003eI)\u003c/strong\u003e Pearson correlation analysis between circCOPA and SPRED2 mRNA expression levels in 80 HCC tissue samples. (\u003cstrong\u003eJ) \u003c/strong\u003eWestern blot analysis of SPRED2 protein expression following stable circCOPA knockdown or overexpression. (\u003cstrong\u003eK) \u003c/strong\u003eWestern blot analysis of MEK, phosphorylated MEK (p-MEK), ERK1/2, and phosphorylated ERK1/2 (p-ERK1/2) levels in HCC cells following stable SPRED2 knockdown or overexpression. Data are expressed as mean ± SD of three independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05,**\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-9321668/v1/65f4bf480e6e406ad86d0131.png"},{"id":107490378,"identity":"280ae33d-7b6a-4ad4-bdb6-fbf1e766547a","added_by":"auto","created_at":"2026-04-22 02:52:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":506934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecircCOPA directly interacts with HNRNPD in HCC cells. (A) \u003c/strong\u003eRNA pulldown assays were performed using biotinylated circCOPA or negative control (NC) probes. Specific protein bands pulled down by circCOPA were visualized by silver staining and subjected to mass spectrometry analysis. (\u003cstrong\u003eB) \u003c/strong\u003eRNA pulldown followed by western blot confirmed the interaction between circCOPA and HNRNPD in MHCC‑LM3 and Hep3B cells. (\u003cstrong\u003eC,D)\u003c/strong\u003e RNA immunoprecipitation (RIP) assays using an anti‑HNRNPD antibody or control IgG, followed by qRT‑PCR analysis of circCOPA enrichment in HCC cells. (\u003cstrong\u003eE) \u003c/strong\u003eCombined\u003cstrong\u003e \u003c/strong\u003eFISH and IF assays showing the co-localization of circCOPA (red) and HNRNPD (green) in the cytoplasm of MHCC-LM3 and Hep3B cells. Scale bar: 20μm. (\u003cstrong\u003eF) \u003c/strong\u003eSchematic representation and western blot analysis showing the expression of FLAG‑tagged HNRNPD wild‑type (WT) and truncation mutants (RRM1, RRM2) in HEK‑293T cells. (\u003cstrong\u003eG) \u003c/strong\u003eRIP assays performed in HEK‑293T cells expressing each HNRNPD truncation mutant, followed by qRT‑PCR analysis of circCOPA enrichment. (\u003cstrong\u003eH) \u003c/strong\u003eSchematic illustration of circCOPA divided into three segments. RNA pulldown assays were performed in MHCC-LM3 and Hep3B cells using biotinylated probes targeting the full-length (FL) circCOPA or each individual segment. (\u003cstrong\u003eI) \u003c/strong\u003eqRT‑PCR\u003cstrong\u003e \u003c/strong\u003eanalysis\u003cstrong\u003e \u003c/strong\u003eof HNRNPD mRNA levels following circCOPA knockdown or overexpression in MHCC‑LM3 cells. (\u003cstrong\u003eJ,K) \u003c/strong\u003eqRT‑PCR analysis of circCOPA expression following HNRNPD knockdown or overexpression in MHCC‑LM3 cells. (\u003cstrong\u003eL) \u003c/strong\u003eWestern blot analysis of HNRNPD protein levels following stable circCOPA knockdown or overexpression. Data are expressed as mean ± SD of three independent experiments. ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-9321668/v1/19759a9f543b0a80694c33fa.png"},{"id":107487665,"identity":"8b9387f6-6c8f-4990-8487-14bec5b38b68","added_by":"auto","created_at":"2026-04-22 02:42:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":524187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecircCOPA promotes SPRED2 mRNA decay in an HNRNPD-dependent manner. (A,B) \u003c/strong\u003eqRT‑PCR analysis of SPRED2 mRNA expression after HNRNPD knockdown in MHCC‑LM3 and Hep3B cells. (\u003cstrong\u003eC)\u003c/strong\u003e Western blot analysis of HNRNPD and SPRED2 protein levels following HNRNPD knockdown or overexpression. (\u003cstrong\u003eD,E) \u003c/strong\u003eWestern blot analysis of SPRED2 and HNRNPD protein expression in MHCC‑LM3 and Hep3B cells with combined modulation of circCOPA and HNRNPD. (\u003cstrong\u003eF)\u003c/strong\u003e SPRED2 mRNA stability was assessed by qRT-PCR at specific time points after actinomycin D treatment (10 μg/mL) in MHCC-LM3 cells with circCOPA knockdown. (\u003cstrong\u003eG) \u003c/strong\u003eSPRED2 mRNA stability was measured by qRT-PCR at specific time points following actinomycin D treatment (10μg/mL) in MHCC-LM3 cells with circCOPA overexpression alone or in combination with HNRNPD knockdown. (\u003cstrong\u003eH,I)\u003c/strong\u003e HNRNPD RIP assays were performed in MHCC‑LM3 cells with circCOPA knockdown or overexpression, followed by qRT‑PCR analysis of SPRED2 mRNA enrichment to determine the effect of circCOPA on the binding affinity of HNRNPD for SPRED2 mRNA.\u003cstrong\u003e (J) \u003c/strong\u003eThe schematic representation of full-length SPRED2 mRNA was presented. RNA pull-down assays were performed using biotinylated SPRED2 RNA segments, and the bound proteins were analyzed by western blot. \u003cstrong\u003e(K) \u003c/strong\u003eMotif specific RIP-qPCR analysis of HNRNPD binding to SPRED2 3'UTR fragments. \u003cstrong\u003e(L) \u003c/strong\u003eLuciferase reporter assays assessing the effect of HNRNPD overexpression on wild-type or mutant Motif 3. \u003cstrong\u003e(M) \u003c/strong\u003emRNA stability assays comparing wild-type and mutant Motif 3 constructs. Data are expressed as mean ± SD of three independent experiments. **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-9321668/v1/fd38b8c189c09db4fdfdec0b.png"},{"id":107453892,"identity":"163d5f3d-fa44-47f6-9099-e60f53b7eca3","added_by":"auto","created_at":"2026-04-21 15:37:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1111401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe circCOPA-SPRED2 axis drives HCC malignancy. (A-C) \u003c/strong\u003eRescue experiments based on colony formation and EdU assays were performed in circCOPA and SPRED2-modulated MHCC-LM3 cells. Scale bar for EdU assays : 100μm. (\u003cstrong\u003eD-F) \u003c/strong\u003eRescue experiments based on colony formation and EdU assays were performed in circCOPA and SPRED2-modulated Hep3B cells. Scale bar for EdU assays : 100μm. (\u003cstrong\u003eG,H) \u003c/strong\u003eTranswell-based rescue assays were performed in circCOPA and SPRED2-modulated MHCC-LM3 cells. (\u003cstrong\u003eI,J)\u003c/strong\u003e Transwell-based rescue assays were performed in circCOPA and SPRED2-modulated Hep3B cells. Data are expressed as mean ± SD of three independent experiments. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05,**\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-9321668/v1/28e515bcb8e071969df2205f.png"},{"id":107705526,"identity":"74b7b084-3be1-4ce0-9af9-26011b47d08b","added_by":"auto","created_at":"2026-04-24 09:13:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4801005,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9321668/v1/651513e9-7498-4747-91db-db867a54c419.pdf"},{"id":107453886,"identity":"fbc503db-293b-4105-bfd0-220a5fecc250","added_by":"auto","created_at":"2026-04-21 15:36:59","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1526422,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9321668/v1/577297727b75055ae0923db2.pdf"},{"id":107453888,"identity":"fc2eff8a-5ac8-4495-bbc1-0dddb6c0486e","added_by":"auto","created_at":"2026-04-21 15:36:59","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18931,"visible":true,"origin":"","legend":"Mass spectrometry (MS) analysis results following circCOPA probe pulldown","description":"","filename":"SupplementalTableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9321668/v1/eec4eee62040b5b2eb7292d3.xlsx"},{"id":107490409,"identity":"20c6e5c2-b859-4efb-a751-b823b02d722b","added_by":"auto","created_at":"2026-04-22 02:52:29","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":95953,"visible":true,"origin":"","legend":"original data files for qPCR","description":"","filename":"originaldatafilesforqPCR.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9321668/v1/6676fb50dc7e627d7f9d0025.xlsx"},{"id":107453894,"identity":"1f49acbf-4e5a-4608-b6fb-f6af0c081934","added_by":"auto","created_at":"2026-04-21 15:37:00","extension":"doc","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18264064,"visible":true,"origin":"","legend":"Uncropped original western blots","description":"","filename":"Uncroppedoriginalwesternblots.doc","url":"https://assets-eu.researchsquare.com/files/rs-9321668/v1/f20d7037eeb0b1f178ea9a8a.doc"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"CircCOPA promotes hepatocellular carcinoma progression through the HNRNPD/SPRED2 axis-mediated activation of ERK/MAPK signaling pathway","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHepatocellular carcinoma poses a significant global health threat, ranking sixth among the most prevalent cancers and third as a cause of cancer-related deaths worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Due to the insidious emerging, a significant proportion of HCC patients receive their diagnosis at a late stage, rendering them unsuitable for surgery [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although there have been considerable progress of HCC treatment, the prognosis remains poor [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, unraveling the molecular mechanisms of HCC is imperative to discover therapeutic strategies and improve patient outcomes.\u003c/p\u003e \u003cp\u003eCircular RNAs constitute a prevalent category of non-coding RNAs formed through the back-splicing of pre-mRNA, resulting in stable, single-stranded covalently closed loops [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Plentiful studies have established a strong association between aberrant circRNAs expression and numerous human cancers [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], such as non-small cell lung cancer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and gastric cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Mechanistically, circRNAs exert their regulatory roles via several distinct pathways, including acting as miRNA decoys, modulating protein activities, and functioning as scaffolds for peptide translation. For example, hsa_circ_0119412 was shown to upregulate the ZBED3 gene by sponging miR-1298-5p, which subsequently contributed to the progression of gastric cancer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Similarly, circACTN4 promotes breast cancer progression by directly binding to FUBP1 and upregulating MYC expression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Zhang et al. reported that circMET drives glioblastoma tumorigenesis by encoding MET404 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Despite these studies, the roles of circRNAs need to be explored in HCC.\u003c/p\u003e \u003cp\u003eBioinformatic analysis of GEO datasets indicate that circCOPA is elevated in HCC tissues and associated with poor prognosis in a cohort of 80 HCC patients. Functional experiments show that circCOPA acts as an oncogenic driver, promoting HCC proliferation and metastasis. Mechanistic studies demonstrate that circCOPA directly interacts with HNRNPD to promoting SPRED2 mRNA decay by binding to the UGUUU motif in its 3\u0026rsquo;UTR region, thereby reducing SPRED2 expression and ultimately facilitating HCC progression. Thus, our findings establish the pivotal role of the circCOPA/HNRNPD/SPRED2 axis in driving HCC progression and highlight the clinical value of circCOPA as a promising treatment target.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical samples\u003c/h2\u003e \u003cp\u003eEighty paired HCC samples were collected from First Affiliated Hospital of Nanjing Medical University. All patients were undergoing primary surgery and had not received any prior antitumor therapies. The study protocol was approved by the Institutional Ethics Committee (Approval number: 2024-SRFA-649). Written informed consent of each participant was obtained.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA extraction, Nucleocytoplasmic separation and qRT-PCR\u003c/h3\u003e\n\u003cp\u003eTotal cellular and tissue-derived RNA were isolated using the TRIzol reagent (Invitrogen, USA), while the PARIS Kit (Invitrogen, USA) was employed for the partitioning of nuclear and cytoplasmic fractions. Following extraction, cDNA synthesis was conducted with a reverse transcription system (Vazyme Biotech, China). We performed quantitative real-time PCR (qRT-PCR) using an ABI 7900 platform (Applied Biosystems, USA) and SYBR Green master mix (Vazyme Biotech, China). Relative transcript abundance was determined via the 2\u003csup\u003e\u0026minus;\u0026thinsp;ρρCt\u003c/sup\u003e method, normalized against GAPDH. Detailed information regarding the primer sequences is provided in Supplementary \u003cb\u003eTable S2\u003c/b\u003e(Supplementary Information).\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eCell lines acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were utilized in this study, including human HCC lines such as MHCC-97L, MHCC-97H, MHCC-LM3, HepG2, Hep3B, and Huh7, the immortalized hepatocyte line HHL-5, and HEK-293T cells. Standard culture conditions involved DMEM (Gibco, USA) with 10% FBS (Umedium, China) and 1% penicillin-streptomycin, at 37\u0026deg;C in a 5% CO₂ atmosphere. Cells were harvested for passaging at a 1:3 ratio when achieving 85\u0026ndash;90% confluence.\u003c/p\u003e\n\u003ch3\u003eRNase R treatment and Actinomycin D (ActD) assays\u003c/h3\u003e\n\u003cp\u003eTotal RNA (4 \u0026micro;g) was treated with or without RNase R (Lucigen, USA; 3 U/\u0026micro;g) to assess RNA stability. The two aliquots (RNase R-treated and an untreated control) were exposed to 37\u0026deg;C for 30 minutes. Subsequently, the levels of circCOPA and linear COPA mRNA were quantified using qRT-PCR. For the actinomycin D (ActD) assays, transcription was blocked in MHCC-LM3 and Hep3B cells using 2 \u0026micro;g/mL ActD (Sigma-Aldrich, USA). RNA collected at the specific time points was analyzed via qRT-PCR to evaluate the time-dependent degradation of circCOPA, COPA mRNA, and SPRED2 mRNA.\u003c/p\u003e\n\u003ch3\u003eFluorescence In Situ Hybridization ( FISH)\u003c/h3\u003e\n\u003cp\u003eTo confirm the subcellular localization of circCOPA, Cy3-labeled probes of circCOPA were obtained from GenePharma (Shanghai, China). Subsequently, FISH assays were performed, followed by image acquisition using an Olympus fluorescence microscope (Tokyo, Japan).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids, siRNA, and lentivirus transfection\u003c/h2\u003e \u003cp\u003eAll siRNAs were provided by RiboBio Co., Ltd. (Guangzhou, China). Plasmid constructs for overexpression studies were cloned into pCDH vectors and supplied by GenePharma Co., Ltd. (Shanghai, China). Transient transfection was carried out by introducing plasmids or siRNAs into cells cultured in 6-well plates using Lipofectamine 3000 (Invitrogen, USA). For stable expression, cells were infected with lentiviral particles and subsequently subjected to antibiotic selection with 2 \u0026micro;g/mL puromycin. All sequences are provided in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e(Supporting Information).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell proliferation assay\u003c/h3\u003e\n\u003cp\u003e \u003cstrong\u003eCCK-8 Assay\u003c/strong\u003e \u003cp\u003eWe performed the CCK-8 assay by seeding transfected cells in 96-well plates at 2\u0026times;10\u0026sup3; cells per well. After overnight incubation, the CCK-8 working solution was added daily for 5 days, followed by 3-hour incubation and measurement of OD450 with a microplate reader (Thermo Scientific Multiskan MK3, USA).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eColony Formation Assay\u003c/strong\u003e \u003cp\u003e1\u0026times;10\u0026sup3; transfected cells were plated per well in 6-well plates and cultured for two weeks. The resulting colonies were then fixed with 4% paraformaldehyde (Servicebio, Wuhan, China), stained with 0.3% crystal violet (Beyotime, Shanghai, China), and imaged for subsequent quantification.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEdU Assay\u003c/strong\u003e \u003cp\u003eDNA synthesis was measured using an EdU (Cy3) kit (Beyotime, Shanghai, China). After a 2-hour pulse with EdU, cells were fixed, permeabilized with 0.5% Triton X-100 (Servicebio, Wuhan, China), and subjected to the click reaction. After DAPI staining (Beyotime, Shanghai, China), images were captured using an Olympus IX73 microscope for subsequent analysis.\u003c/p\u003e \u003c/p\u003e\n\u003ch3\u003eCell migration assay\u003c/h3\u003e\n\u003cp\u003eCellular motility was assessed using Transwell inserts (Jet Bio-Filtration, Guangzhou, China). Specifically, 1x10\u003csup\u003e4\u003c/sup\u003e cells were suspended in serum-depleted medium and seeded into the upper compartments. These were then positioned over lower reservoirs containing complete medium supplemented with 20% fetal bovine serum (FBS) as a chemoattractant. After a one-day incubation, cells that successfully invaded the underside of the membrane underwent fixation in 4% paraformaldehyde and visualization via 0.3% crystal violet staining for subsequent quantification. Wound assay was performed by growing transfected cells to 90\u0026ndash;100% confluence in 6-well plates. After washing, cells were maintained in medium with 2% FBS. Wound width was photographed and measured at 0 h and 48 h.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell invasion assay\u003c/h2\u003e \u003cp\u003eThe invasion assay was assessed using Matrigel-coated Transwell inserts (Corning, NewYork, USA). Each insert was coated with 200 \u0026micro;L of Matrigel diluted 1:20 in DMEM and allowed to polymerize at 37\u0026deg;C overnight. The subsequent procedures for cell seeding, incubation, and staining were consistent with the standard transwell migration protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot (WB)\u003c/h2\u003e \u003cp\u003eTotal protein fractions were isolated using RIPA lysis buffer (Beyotime, Shanghai, China). Following concentration determination, proteins were fractionated via SDS-PAGE and subsequently immobilized onto PVDF membranes. To prevent non-specific binding, membranes were treated with Quick Blocking Buffer (Beyotime, Shanghai, China) before being incubated with primary antibodies at 4\u0026deg;C overnight. After thorough TBST rinsing, HRP-conjugated secondary antibodies were applied for a 2-hour period at ambient temperature. Protein bands were finally visualized using an ECL chemiluminescence system. A comprehensive list of antibodies is available in Supplementary \u003cb\u003eTable S3\u003c/b\u003e(Supplementary Information).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing\u003c/h2\u003e \u003cp\u003eRNA sequencing analysis was supported by Tsingke Biotechnology Co., Ltd. (Beijing, China) to compare transcriptomic profiles between circCOPA overexpression and control cells. The service included library preparation, sequencing on an Illumina HiSeq 2500 platform, and bioinformatic analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCircRNA pulldown and mass spectrometry (MS)\u003c/h2\u003e \u003cp\u003eRNA-interacting proteins were identified with an RNA pulldown assay with biotin-labeled circCOPA-specific and control probes (GenePharma, Shanghai, China). Streptavidin magnetic beads were incubated with 50 pmol of each probe for 2 h, then incubated overnight with lysates from 1\u0026times;10⁷ Hep3B cells. Following washing, bound proteins were eluted, subjected to SDS-PAGE separation, and detected with a Fast Silver Stain Kit (Beyotime, Shanghai, China). Specifically enriched protein bands from the circCOPA probe group were excised and analyzed by Q Exactive mass spectrometry (Thermo Fisher Scientific, USA). All mass spectrometry results are provided in Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA immunoprecipitation (RIP)\u003c/h2\u003e \u003cp\u003eRNA immunoprecipitation (RIP) was examined using an anti-HNRNPD antibody and a commercial kit (BersinBio, Guangzhou, China). Cell lysates from MHCC-LM3 and Hep3B cells (2\u0026times;10⁷ cells per sample) were incubated overnight at 4\u0026deg;C with magnetic beads pre-conjugated with HNRNPD antibody or IgG antibody. After washing, the co-precipitated RNAs were extracted by digestion with proteinase K, purified, and quantified by qRT-PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDual-Luciferase Reporter Assay\u003c/h2\u003e \u003cp\u003eTo quantify promoter activity, we employed a dual-luciferase reporter system (IBSBIO, Shanghai, China). Post-transfection, HEK293T cells were lysed, and the resulting supernatants were isolated via high-speed centrifugation (10,000\u0026ndash;15,000 rpm) for 5 minutes. Luminescence was recorded by sequentially mixing 20 \u0026micro;L of cell lysate with equal volumes of Firefly and Renilla luciferase reagents, respectively. The final reporter efficiency was determined by normalizing the Firefly RLU against the Renilla-derived internal control RLU.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence (IF) Staining\u003c/h2\u003e \u003cp\u003eFor immunostaining procedures, HCC cells underwent fixation and permeabilization, followed by a 30-minute blocking step with 10% normal goat serum. The cells were then probed overnight with primary antibodies at 4\u0026deg;C. On the second day, they were exposed to species-matched fluorescent secondary antibodies for 2 hours at ambient temperature. Nuclei were counterstained using DAPI, and fluorescence signals were visualized and captured via an Olympus imaging system (Tokyo, Japan).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003enude mouse model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSubcutaneous xenograft models were established using 4-week-old male nude mice obtained from the Animal Core Facility of Nanjing Medical University. Following an acclimatization period in an SPF environment, mice were randomized into four cohorts. Specifically, 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e stably transfected MHCC-LM3 or Hep3B cells were inoculated into the right flank of each mouse. Tumor growth was monitored quadrennially, with volumes determined by the formula: Volume = (length \u0026times; width\u003csup\u003e2\u003c/sup\u003e)/2. Approximately four weeks later, mice were euthanized for tumor excision and digital documentation. For the lung metastasis model, 100 \u0026micro;L of cell suspension (1\u0026times;10⁷ cells/mL) was administered via tail vein injection. After four weeks, metastatic progression was assessed through bioluminescence imaging where applicable. Lungs were subsequently harvested for nodule quantification and H\u0026amp;E histological validation. All procedures received ethical clearance from the Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-2507092).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC) and hematoxylin-eosin (HE) staining\u003c/h2\u003e \u003cp\u003eFor immunohistochemical (IHC) analysis, 5-\u0026micro;m paraffin sections were prepared from tissues initially fixed in 4% PFA. Following xylene-mediated clearing and rehydration through graded ethanol, 3% H2O2 was applied to neutralize endogenous peroxidase activity. Antigenic sites were exposed via microwave-based heat retrieval. Sections were subsequently probed overnight (4\u0026deg;C) with primary antibodies (1:100), followed by a 30-minute incubation with HRP-linked secondary antibodies at ambient temperature. Digital images were captured for further evaluation. Parallelly, morphological characteristics were assessed using H\u0026amp;E staining (Beyotime, Shanghai, China) following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eExperimental data, derived from at least three independent biological replicates, are presented as mean \u0026plusmn; standard deviation (SD). All statistical evaluations were conducted using GraphPad Prism 8.0 (GraphPad Software, Inc., USA). To determine significance, unpaired Student\u0026rsquo;s t-tests were utilized for binary comparisons, while one-way analysis of variance (ANOVA) was applied for cohorts with three or more groups. Kaplan\u0026ndash;Meier curves were generated for survival analysis, and the Pearson coefficient was calculated to evaluate the inter-expression relationship between circCOPA and SPRED2. Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and characterization of circCOPA in HCC\u003c/h2\u003e \u003cp\u003eBy analyzing two Gene Expression Omnibus (GEO) datasets (GSE155949 and GSE94508), we identified several circRNAs that were aberrantly expressed in HCC \u003cb\u003e(Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA,1B)\u003c/b\u003e using the threshold of |logFC| \u0026gt; 1 and adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. CircCOPA (hsa_circ_0008661) captured our attention as the only candidate consistently upregulated in both datasets \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. qRT-PCR analysis confirmed significant upregulation of circCOPA in a cohort of 80 HCC patients \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C\u003cb\u003e)\u003c/b\u003e. Similarly, analysis of The Gene Expression Profiling Interactive Analysis (GEPIA) database revealed that the expression of its parental gene, COPA, was also upregulated in HCC tissues \u003cb\u003e(Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC)\u003c/b\u003e. Among the six HCC cell lines examined, circCOPA expression was highest in MHCC-LM3 and lowest in Hep3B, relative to the normal hepatocyte cell line HHL-5 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Thus, MHCC-LM3 and Hep3B cells, representing the extremes of circCOPA expression, were selected for subsequent functional experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess clinical significance, HCC cohorts were dichotomized into high and low expression groups (n\u0026thinsp;=\u0026thinsp;40 each) based on median circCOPA levels. Our findings indicated that elevated circCOPA significantly correlated with aggressive clinicopathological features, including increased tumor dimensions, advanced TNM staging, and higher Edmondson grades (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Kaplan\u0026ndash;Meier analysis revealed that patients with higher circCOPA levels experienced markedly diminished overall survival (OS) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Consistently, GEPIA-based validation confirmed that COPA upregulation conferred a dismal prognosis, characterized by truncated OS and disease-free survival (DFS) (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD, E\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAccording to circBase, circCOPA is derived from exons 6\u0026ndash;8 of the COPA gene on chromosome 1 (1q23.2) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Analysis of Sanger sequencing revealed the annotation of circCOPA and its characteristic back-splicing junction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G\u003cb\u003e)\u003c/b\u003e. Agarose gel electrophoresis was performed to verify its circular structure, demonstrating that divergent primers specifically amplified circCOPA from cDNA, but not from gDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Additionally, RNase R digestion and actinomycin D treatment assays were conducted to examine circCOPA stability. CircCOPA exhibited significantly greater resistance to both treatments compared with linear COPA mRNA, confirming its enhanced RNA stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-L). Furthermore, fluorescence in situ hybridization (FISH) and subcellular fractionation assays indicated that circCOPA was predominantly localized in the cytoplasm of MHCC-LM3 and Hep3B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM-O). In summary, these data demonstrate that circCOPA is upregulated in HCC tissues and its high expression correlates with poor prognosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003ecircCOPA promotes proliferation, migration, and invasion of HCC cells\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe performed loss-of-function experiments to investigate the regulatory role of circCOPA in MHCC-LM3 cells. qRT-PCR analysis demonstrated that these designed siRNAs effectively downregulated circCOPA expression without affecting linear COPA mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We performed the gain-of-function assays on Hep3B cells due to its lower endogenous circCOPA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). qRT-PCR analysis verified the successful overexpression of circCOPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFunctional assays revealed that circCOPA knockdown significantly suppressed the proliferation of MHCC-LM3 cells, as assessed by CCK-8 and EdU assays, whereas circCOPA overexpression promoted proliferation in Hep3B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-G). This pro-proliferative effect was further corroborated by colony formation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-J). Furthermore, transwell assays demonstrated that circCOPA depletion significantly inhibited the migration and invasion of MHCC-LM3 cells, while circCOPA overexpression exhibited the opposite effects in Hep3B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-M). Consistently, wound healing assays further confirmed the pro-migratory role of circCOPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN-P). In summary, these \u003cem\u003ein vitro\u003c/em\u003e findings establish circCOPA as an oncogenic driver in HCC.\u003c/p\u003e \u003cp\u003e \u003cb\u003ecircCOPA facilitates HCC tumor growth and metastasis\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the oncogenic role of circCOPA \u003cem\u003ein vivo\u003c/em\u003e, we established subcutaneous xenograft mice models using MHCC-LM3 cells with circCOPA stable depletion (sh-circCOPA) and Hep3B cells with circCOPA stable overexpression (circCOPA-OE). Compared with the sh-NC control group, tumors in the sh-circCOPA group exhibited significantly smaller volumes, lower weight, and reduced growth rates. Conversely, the circCOPA-OE group showed a significant promotion of tumor growth in these parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Moreover, immunohistochemistry (IHC) and hematoxylin and eosin (H\u0026amp;E) of tumor sections showed that circCOPA silencing decreased Ki-67 expression, whereas circCOPA overexpression increased Ki-67 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the lung metastasis models, circCOPA depletion substantially reduced the number of metastatic nodules, while circCOPA overexpression produced the opposite effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). Furthermore, mice in the sh-circCOPA group exhibited prolonged overall survival, whereas those in the circCOPA-OE group showed a significant reduction in survival time (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H). Collectively, these \u003cem\u003ein vivo\u003c/em\u003e findings establish a critical role of circCOPA in facilitating HCC tumor growth and metastasis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ecircCOPA activates the ERK/MAPK signaling pathway by downregulating SPRED2\u003c/h2\u003e \u003cp\u003eTo investigate the downstream regulatory network, we performed RNA-sequencing on Hep3B cells following circCOPA induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). KEGG pathway enrichment analysis highlighted the MAPK signaling pathway as a primary target (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Parallel Gene Ontology (GO) annotations further implicated circCOPA in modulating the ERK1/2 cascade, a pivotal constituent of the MAPK framework (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA\u003c/b\u003e). This activation was corroborated by GSEA results, which showed a robust positive correlation between circCOPA levels and MAPK pathway signatures (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB\u003c/b\u003e). Given the established influence of MAPK signaling on cellular growth and motility [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], we speculated that circCOPA drives HCC malignancy via the activation of this specific signaling axis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsistently, circCOPA overexpression enhanced ERK phosphorylation, whereas circCOPA knockdown decreased it. Notably, JNK and p38 phosphorylation remained unaffected \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. This specific activation of ERK phosphorylation, consistent with the GO analysis, indicates that circCOPA drives HCC progression via the ERK/MAPK signaling pathway. To investigate the role of the ERK/MAPK pathway in circCOPA-mediated HCC progression, we treated HCC cells with SCH772984, a highly selective ERK pathway inhibitor, to suppress ERK phosphorylation. Western blot analysis confirmed that circCOPA overexpression rescued the reduction in ERK phosphorylation induced by SCH772984 in HCC cells \u003cb\u003e(Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC, D)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eBased on the most significantly changed genes and those involved in the MAPK pathway, 15 candidate genes were subsequently selected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). qRT-PCR and western blot results identified SPRED2 as a downstream target, showing an inverse correlation with circCOPA at both the transcriptional and translational levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F, J \u003cb\u003eand Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eE, F)\u003c/b\u003e. This finding was further confirmed using two independent circCOPA-targeting siRNAs \u003cb\u003e(Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eG, H)\u003c/b\u003e. Additionally, western blot and qRT-PCR analyses revealed a significant reduction of SPRED2 expression in HCC tissues \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, H\u003cb\u003e)\u003c/b\u003e. Furthermore, correlation analysis of 80 HCC clinical samples showed a negative correlation between circCOPA and SPRED2 mRNA expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e. Lentiviral plasmids for stable SPRED2 knockdown or overexpression were constructed and successfully modulated SPRED2 expression levels \u003cb\u003e(Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eI, J)\u003c/b\u003e. The phosphorylation levels of ERK and MEK were elevated following SPRED2 depletion, whereas the opposite effect was observed following SPRED2 overexpression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK\u003cb\u003e)\u003c/b\u003e. In summary, these findings suggest that circCOPA activates the ERK/MAPK signaling pathway through the downregulation of SPRED2.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003ecircCOPA directly interacts with HNRNPD in HCC cells\u003c/h2\u003e \u003cp\u003eWe next sought to elucidate the mechanistic basis underlying the negative regulation of SPRED2 by circCOPA in HCC. Therefore, we investigated whether circCOPA could directly bind to proteins or function as a miRNA sponge in HCC. RNA pulldown assays were performed using a circCOPA-specific probe to examine its association with Argonaute-2 (Ago2), a core RNA-induced silencing complex component essential for miRNA sponge [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, Ago2 was not enriched in the circCOPA pulldown fractions (\u003cb\u003eFig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA)\u003c/b\u003e, suggesting that a ceRNA mechanism is unlikely. We then performed RNA pulldown assays coupled with mass spectrometry to identify circCOPA-binding proteins. Silver staining revealed a distinct protein band at approximately 40 kDa specifically pulled down by the circCOPA probe \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Mass spectrometry (MS) analysis identified Heterogeneous Nuclear Ribonucleoprotein D (HNRNPD) as the top candidate, based on its highest Sequest scores and the greatest number of unique peptides (\u003cb\u003eFig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB and Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e)\u003c/b\u003e. This specific interaction was validated by RNA pulldown assays in both MHCC-LM3 and Hep3B cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. RNA immunoprecipitation (RIP) analysis using an anti-HNRNPD antibody showed significant enrichment of circCOPA \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D\u003cb\u003e)\u003c/b\u003e. Furthermore, combined fluorescence in situ hybridization (FISH) and immunofluorescence (IF) assays demonstrated the cytoplasmic colocalization of circCOPA and HNRNPD in MHCC-LM3 and Hep3B cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe RNA-binding capacity of HNRNPD is mediated by its two distinct RNA recognition motif (RRM) domains [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To identify the interaction domain, we generated a series of FLAG-tagged HNRNPD truncation mutants. Western blot analysis confirmed the successful expression and expected molecular weights of the truncated proteins \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u003cb\u003e).\u003c/b\u003e RIP assays followed by qRT-PCR analysis demonstrated that circCOPA was significantly enriched by wild-type HNRNPD and the RRM1 domain mutant, but not by the RRM2 domain mutant \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. We next designed three biotin-labeled RNA segment probes spanning the full-length circCOPA sequence. RNA pulldown assays identified segment 1 of circCOPA as the primary region responsible for HNRNPD binding \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWe next investigated whether circCOPA and HNRNPD mutually regulate each other's expression. qRT-PCR and western blot analyses showed no evidence of mutual regulation: circCOPA did not change HNRNPD mRNA or protein levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, L and S3C\u003cb\u003e)\u003c/b\u003e. Consistently, HNRNPD knockdown or overexpression did not affect circCOPA levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ, K and S3D, E\u003cb\u003e)\u003c/b\u003e. Collectively, these data demonstrate a direct and specific interaction between HNRNPD and circCOPA.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003ecircCOPA promotes SPRED2 mRNA decay in an HNRNPD-dependent manner\u003c/h2\u003e \u003cp\u003eHaving identified SPRED2 as a downstream effector, we next explored the relationship between HNRNPD and SPRED2. Knockdown of either circCOPA or HNRNPD upregulated SPRED2 expression, while overexpression of either downregulated SPRED2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C, and S4A-B), indicating their cooperative role in repressing SPRED2. Given the established role of HNRNPD in binding and destabilizing target mRNAs, such as ATF3 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], we hypothesized that circCOPA promotes HCC malignancy by recruiting HNRNPD to facilitate SPRED2 mRNA degradation. Supporting this hypothesis, western blot analysis demonstrated that circCOPA depletion reversed the downregulation of SPRED2 induced by HNRNPD overexpression in MHCC-LM3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), indicating the functional indispensability of circCOPA in the HNRNPD-mediated SPRED2 degradation. Moreover, HNRNPD knockdown rescued the suppression of SPRED2 caused by circCOPA overexpression in Hep3B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), suggesting that the regulating role of circCOPA on SPRED2 is dependent on HNRNPD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, RNA stability assays were performed to examine SPRED2 mRNA decay rates following modulation of circCOPA or HNRNPD expression. Knockdown of circCOPA significantly extended the half-life of SPRED2 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, S4C). Conversely, circCOPA overexpression accelerated SPRED2 mRNA decay, an effect that was abolished by HNRNPD knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, S4D), confirming the circCOPA dependence of HNRNPD-mediated mRNA destabilization. RNA immunoprecipitation (RIP) assays were conducted to examine whether circCOPA promoted the interaction between HNRNPD and SPRED2 mRNA. The results revealed that circCOPA silencing attenuated the binding of HNRNPD to SPRED2 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, S4E). Conversely, circCOPA overexpression enhanced this interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI, S4F). Collectively, these data suggest that circCOPA recruits HNRNPD to promote its binding to SPRED2 mRNA.\u003c/p\u003e \u003cp\u003eTo identify the specific HNRNPD binding sites on SPRED2 mRNA, we analyzed the sequence of its 3\u0026rsquo;UTR, including GU-/UG-rich nonclassical motifs (UGUUU and GUUUG) previously identified as potential HNRNPD binding sites [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. RNA pulldown assays were performed using the biotinylated SPRED2 RNA probes spanning different regions, and the results demonstrated that HNRNPD exhibited a specific binding affinity for the 3'UTR of SPRED2 mRNA compared to the coding sequence (CDS) or 5'UTR regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Furthermore, we analyzed the 3'UTR sequence of SPRED2 mRNA and identified three candidate binding motifs: Motif 1(26-30nt: UGUUU), Motif 2(474-478nt: UGUUU) and Motif 3(761-765nt: UGUUU). Plasmids expressing each candidate sequence along with its 200-bp flanking regions on both sides were co-transfected into HEK293T cells together with HNRNPD plasmid. RIP-qPCR analysis showed that HNRNPD selectively bound to Motif 3 compared to IgG control. In contrast, no significant binding was detected for Motif 1 or Motif 2(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eSubsequently, the 3'UTR containing either wild-type Motif 3 or its mutant (UGUUU\u0026rarr;ACAAA) was inserted downstream of a firefly luciferase reporter. These constructs were co-transfected into HEK293T cells along with HNRNPD and Renilla luciferase control plasmids. Luciferase activity assays revealed that HNRNPD overexpression enhanced reporter activity for the wild-type Motif 3, whereas this effect was not observed for the mutant Motif 3(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). Consistently, mutation of Motif 3 also prolonged the half-life of SPRED2 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). Collectively, these findings elucidate that HNRNPD directly binds the UGUUU motif within the SPRED2 3'UTR, thereby promoting SPRED2 mRNA decay.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eThe circCOPA-SPRED2 axis drives HCC malignancy\u003c/h2\u003e \u003cp\u003eTo establish SPRED2 as the critical downstream mediator of circCOPA, we performed rescue experiments to determine whether circCOPA drives HCC progression through suppression of SPRED2. CCK-8 assays, EdU staining and colony formation assays revealed that circCOPA depletion suppressed cell proliferation, an effect that was rescued by concurrent SPRED2 silencing \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C and S5A\u003cb\u003e)\u003c/b\u003e. Conversely, circCOPA overexpression enhanced the proliferation of Hep3B cells, and this pro-proliferative effect was reversed by SPRED2 overexpression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-F and S5B\u003cb\u003e)\u003c/b\u003e. Furthermore, transwell assays and wound healing assays demonstrated that SPRED2 knockdown rescued the impairment of migratory and invasive capacities in MHCC-LM3 cells resulting from circCOPA silencing \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG, H and S5C, D\u003cb\u003e)\u003c/b\u003e. Consistently, the enhancement of invasion and migration in Hep3B cells caused by circCOPA overexpression were counteracted by SPRED2 overexpression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI, J and S5E, F\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, these rescue experiments confirm that circCOPA exerts its oncogenic roles in HCC cells by suppressing SPRED2, highlighting the circCOPA\u0026ndash;SPRED2 axis as a promising therapeutic target.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eDue to the prevalence of late-stage diagnoses and subsequent dismal clinical outcomes, there is a compelling need to decode the molecular drivers of HCC and uncover effective therapeutic vulnerabilities [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Recent studies have underscored the functional versatility of circular RNAs (circRNAs) in driving HCC malignancy [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Illustrative examples include EIF4A3-mediated circTOLLIP, which accelerates tumor progression by modulating the miR-516a/PBX3 axis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, exosome-derived circRNA-100338 has been implicated in enhancing the invasive and metastatic potential of HCC cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], while m6A-modified circCPSF6 exerts its oncogenic effects through the upregulation of YAP1 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this context, the present study identified circCOPA as a novel overexpressed transcript in HCC, whose elevation served as a marker for poor patient prognosis. Functional experiments established circCOPA as a potent oncogenic driver, promoting malignant phenotypes including proliferation and metastasis. Mechanistic studies further revealed that the circCOPA-HNRNPD interaction promoted SPRED2 mRNA decay and subsequent ERK/MAPK pathway activation, ultimately driving HCC pathogenesis. Taken together, our findings establish the circCOPA/HNRNPD/SPRED2 axis as a new therapeutic target in HCC.\u003c/p\u003e \u003cp\u003eCircRNAs are known to exert their functions through three principal mechanisms: encoding peptides, engaging in protein interactions, and serving as miRNA sponges [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the present study, RNA pulldown assays revealed no Ago2 enrichment, suggesting that circCOPA is unlikely to function via the canonical miRNA sponge mechanism. Instead, we found that circCOPA functioned by directly binding to HNRNPD. HNRNPD is an RNA-binding protein that functions in both the cytoplasm and nucleus [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Within the cytoplasm, it modulates mRNA stability\u0026mdash;such as that of CCND1 mRNA\u0026mdash;by binding to AU-rich elements in the 3'UTR [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Combined FISH and IF assays demonstrated the cytoplasmic colocalization of circCOPA and HNRNPD in HCC cells. Based on these findings, circCOPA was identified as a molecular scaffold that directly binds to HNRNPD, thereby promoting SPRED2 mRNA decay.\u003c/p\u003e \u003cp\u003eHNRNPD, alternatively designated as AUF1, belongs to the RNA-binding protein superfamily and is characterized by its four distinct isoforms (p37, p40, p42, and p45) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These variants emerge from the differential splicing of a single pre-mRNA precursor. Structurally, these core HNRNPD members are defined by a conserved architecture comprising dual non-identical RNA recognition motifs (RRMs), tandem repeat sequences, and an abundance of glutamine-rich regions (specifically eight clusters)[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the precise subcellular localization of HNRNPD isoforms remains contentious. The lack of isoform-specific antibodies has hindered our efforts to determine which isoform interacts with circCOPA. Given that both RRM domains are essential for RNA binding [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], we generated HNRNPD truncation mutants and identified the RRM1 domain as essential for binding circCOPA. Furthermore, circCOPA truncations and RNA pulldown assays identified segment 1 of circCOPA as the critical binding site for HNRNPD. Collectively, These findings provide new insights into the circCOPA-HNRNPD interaction.\u003c/p\u003e \u003cp\u003eSPRED2, a member of the SPRED family, functions by suppressing the activation of the ERK/MAPK signaling cascade [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Previous studies have showed that downregulation of SPRED2 is associated with enhanced EMT and stemness, exerting these effects through ERK pathway activation in HCC [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, the circRNA-mediated regulatory mechanisms underlying SPRED2 expression remain to be elucidated. HNRNPD functions as a key regulator of mRNA decay [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. For example, HNRNPD was identified to be a negative regulator of ferroptosis, exerting its effects by binding to the ATF3 mRNA 3'-UTR and promoting its degradation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Consistently, our study also revealed that circCOPA promoted HNRNPD-mediated decay of SPRED2 mRNA. Furthermore, RNA pulldown assays and dual-luciferase reporter assays demonstrated that HNRNPD directly bound the UGUUU motif within the SPRED2 3'UTR, thereby promoting SPRED2 mRNA decay. Collectively, our findings expand the current understanding of SPRED2 regulation by elucidating a novel circCOPA-HNRNPD scaffolding mechanism.\u003c/p\u003e \u003cp\u003eSeveral limitations should be acknowledged in our study. First, the upstream mechanisms governing circCOPA biogenesis remain to be elucidated. Second, although SPRED2 was identified as a key downstream target, other potential effectors warrant further investigation.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eOur findings demonstrate that circCOPA is upregulated in HCC and functions as both a driver of tumor progression and a biomarker of poor prognosis. Mechanistically, circCOPA directly interacts with HNRNPD. This interaction promotes SPRED2 mRNA degradation by binding to the UGUUU motif in its 3\u0026rsquo;UTR region, subsequently activating the ERK/MAPK signaling pathway. These results establish the circCOPA/HNRNPD/SPRED2 axis as a potential therapeutic target for HCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA \u0026nbsp;AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to appreciate the Core Facility of the First Affiliated Hospital of Nanjing Medical University for the detection of HCC samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhiwen Feng and Lianbao Kong designed this study. Zhiwen Feng, Qingpeng Lv and Kuan Li performed the major experiments and drafted the manuscript. Lianbao Kong, Chao Yang and Guoqing Liu supervised this study. Wenzhou Ding, Wenhu Zhao, Yanzhi Feng, Deming Zhu, Xiangyu Ling and Xiangyu Qu collected the clinical samples. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (81871260); Young Scholars Fostering Fund of the First Affiliated Hospital of Nanjing Medical University (PY2022007); Key Program of the Youth Fund of Wannan Medical College (WK2022ZF21); National Key Clinical Specialty Construction Fund for General Surgery (KGZ002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in compliance with the ethical principles of the Declaration of Helsinki. The protocol was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University, and written informed consent was obtained from all participants.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin.2021; 71:209\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForner A, Reig M, Bruix J. Hepatocellular carcinoma. Lancet.2018; 391:1301\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFinn RS, Zhu AX, Farah W, Almasri J, Zaiem F, Prokop LJ, et al. Therapies for advanced stage hepatocellular carcinoma with macrovascular invasion or metastatic disease: A systematic review and meta-analysis. 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Cell Mol Life Sci.2022; 79:228.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cgt","sideBox":"Learn more about [Cancer Gene Therapy](http://www.nature.com/cgt/)","snPcode":"41417","submissionUrl":"https://mts-cgt.nature.com/cgi-bin/main.plex","title":"Cancer Gene Therapy","twitterHandle":"@cgtnature","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"circRNA, hepatocellular carcinoma, RNA-binding protein, mRNA stability","lastPublishedDoi":"10.21203/rs.3.rs-9321668/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9321668/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCircRNAs are critically involved in the progression of numerous cancers, including hepatocellular carcinoma (HCC). While circCOPA has been reported to function as a tumor suppressor in glioblastoma, its role and underlying mechanisms in HCC remain largely unexplored. By analyzing the Gene Expression Omnibus (GEO), we identified a new oncogenic circRNA, hsa_circ_0008661 (circCOPA), which was significantly upregulated in HCC tissues. In a cohort of 80 HCC patients, circCOPA upregulation was associated with poor prognosis and worse clinicopathological characteristics. Functional assays revealed that circCOPA depletion suppressed HCC proliferation and metastasis, whereas circCOPA overexpression exerted opposite effects. RNA sequencing analysis confirmed that circCOPA activated the ERK/MAPK signaling pathway through the downregulation of SPRED2. Mechanistically, RNA pulldown and mass spectrometry analysis demonstrated that circCOPA directly interacted with HNRNPD. This interaction promoted SPRED2 mRNA degradation through binding of HNRNPD to the UGUUU motif within its 3'UTR, leading to SPRED2 downregulation and subsequent activation of the ERK/MAPK signaling pathway. Notably, the oncogenic phenotypes driven by circCOPA were effectively rescued by SPRED2 overexpression. Collectively, our findings delineate a novel circCOPA/HNRNPD/SPRED2 axis that activates the ERK/MAPK signaling pathway to drive HCC progression, highlighting its promise as a novel therapeutic target.\u003c/p\u003e","manuscriptTitle":"CircCOPA promotes hepatocellular carcinoma progression through the HNRNPD/SPRED2 axis-mediated activation of ERK/MAPK signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 15:36:54","doi":"10.21203/rs.3.rs-9321668/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-07T10:05:18+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-04-14T08:56:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-10T09:50:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Gene Therapy","date":"2026-04-08T14:59:38+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-04-08T12:01:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-04T15:39:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cgt","sideBox":"Learn more about [Cancer Gene Therapy](http://www.nature.com/cgt/)","snPcode":"41417","submissionUrl":"https://mts-cgt.nature.com/cgi-bin/main.plex","title":"Cancer Gene Therapy","twitterHandle":"@cgtnature","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"71971a22-2fcb-4120-a9ab-afddbffabcc1","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-07T10:05:18+00:00","index":1,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66278564,"name":"Biological sciences/Cancer/Tumour biomarkers"},{"id":66278565,"name":"Biological sciences/Cancer/Oncogenes"}],"tags":[],"updatedAt":"2026-04-21T15:36:54+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 15:36:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9321668","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9321668","identity":"rs-9321668","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}