CircARHGAP12 suppresses cisplatin-induced alkaliptosis in nasopharyngeal carcinoma through G3BP1-mediated upregulation of CA9 in an RNA G-quadruplex-dependent manner

CircARHGAP12 suppresses cisplatin-induced alkaliptosis in nasopharyngeal carcinoma through G3BP1-mediated upregulation of CA9 in an RNA G-quadruplex-dependent manner | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CircARHGAP12 suppresses cisplatin-induced alkaliptosis in nasopharyngeal carcinoma through G3BP1-mediated upregulation of CA9 in an RNA G-quadruplex-dependent manner Tiansheng Li, Yixuan Liu, Hongke Qu, Dan Wang, Junshang Ge, Lei Shi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7984659/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor with a high prevalence in Southern China, and cisplatin remains a commonly used first-line chemotherapeutic agent. However, the effectiveness of cisplatin-based therapy is often compromised by the development of drug resistance, posing a major clinical challenge. Our previous studies demonstrated that circular RNA circARHGAP12 is highly expressed in NPC and promotes tumor cell migration and invasion, although its other potential functions remain unclear. Methods To evaluate the effect of circARHGAP12 on cisplatin resistance, MTT assays, colony formation assays, and nude mouse xenograft models were utilized in vitro and in vivo. Targeted genes of circARHGAP 12 were screened by RNA sequencing, combined with RT-qPCR, western blotting, and rescue experiments to validate their functions. RNA pulldown, RNA immunoprecipitation (RIP), dual-luciferase reporter assays, and actinomycin D experiments were used to elucidate the circARHGAP12/G3BP1/CA9 regulatory axis. Analysis of stress granule dynamics using immunofluorescence, fluorescence in situ hybridization, and fluorescence recovery after photobleaching (FRAP). Results In this study, we found that circARHGAP12 confers cisplatin resistance in NPC by suppressing cisplatin-induced alkaliptosis, a process mediated by the upregulation of carbonic anhydrase 9 (CA9) and reversed upon CA9 knockdown. Mechanistically, circARHGAP12 stabilizes CA9 mRNA in an RNA G-quadruplex (rG4)-dependent manner through interaction with the RNA-binding protein G3BP1. In addition, circARHGAP12 enhances the assembly of cisplatin-induced stress granules, an effect abolished by either G3BP1 silencing or treatment with pyridostatin (PDS), which disrupts the G3BP1-rG4 interaction. Conclusions Collectively, our findings reveal a novel role of circARHGAP12 in mediating cisplatin resistance in NPC through G3BP1-dependent stabilization of CA9 mRNA and modulation of stress granule dynamics. Targeting the circARHGAP12–G3BP1–CA9 axis may therefore represent a promising therapeutic strategy to overcome chemotherapy resistance in NPC. Nasopharyngeal carcinoma CircARHGAP12 Alkaliptosis RNA G-quadruplex Stress granule Cisplatin resistance Phase separation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Circular RNAs (circRNAs) have attracted increasing attention because of their unique covalently closed-loop structure and crucial roles in regulating cellular homeostasis [ 1 , 2 ] . Although numerous studies have demonstrated that aberrant expression of specific circRNAs is closely associated with tumor cell proliferation, invasion, and chemotherapy resistance [ 3 – 7 ] , their precise mechanisms of action—particularly the underlying molecular regulatory networks—remain to be fully elucidated. Our previous study revealed that circARHGAP12 is highly expressed in nasopharyngeal carcinoma (NPC) tissues, where it specifically binds to the 3′ untranslated region (3′UTR) of EZR mRNA, enhances its stability, strengthens the interaction among EZR, TPM3, and RhoA proteins, and consequently promotes NPC cell invasion and metastasis [ 3 ] . However, whether circARHGAP12 exerts additional functions in NPC remains unclear. NPC is a malignant tumor originating from the epithelial cells of the nasopharyngeal mucosa and is highly prevalent in Southeast Asia and Southern China [ 8 ] . Cis-diamminedichloroplatinum (II) (DDP, commonly known as cisplatin) remains the first-line chemotherapeutic agent for NPC treatment. Although cisplatin-based chemotherapy combined with radiotherapy has markedly improved the prognosis of patients with early-stage NPC, disease recurrence still occurs in 20%–30% of those with advanced disease [ 9 ] . Notably, the emergence of cisplatin resistance severely compromises the efficacy of chemotherapy, resulting in higher tumor recurrence rates and poor clinical outcomes [ 10 – 15 ] . Therefore, elucidating the molecular mechanisms underlying cisplatin resistance is essential for optimizing therapeutic strategies and developing novel anticancer agents. Interestingly, we found that circARHGAP12 promotes cisplatin resistance in NPC—a function that has not been previously reported [ 16 , 17 ] . However, the precise mechanisms by which circARHGAP12 contributes to cisplatin resistance in NPC remain unexplored. Alkaliptosis is a distinct form of cell death triggered by intracellular alkalinization [ 18 ] . Unlike other types of programmed cell death, it cannot be inhibited by conventional cell death blockers, as exemplified by JTC801-induced cell death [ 19 ] . JTC801 suppresses the expression of carbonic anhydrase 9 (CA9), an enzyme that dynamically catalyzes the reversible hydration of carbon dioxide to bicarbonate and protons, thereby maintaining intracellular and extracellular pH homeostasis and acting as a negative regulator of alkaliptosis [ 19 ] . Although previous studies have shown that CA9 modulates the tumor microenvironment, counteracts cell death, and promotes tumor cell adaptation and survival [ 20 – 22 ] , the regulatory mechanisms governing CA9 expression and its specific relationship with chemotherapy resistance in NPC are unknown. In this study, we demonstrated for the first time, both in vitro and in vivo , that circARHGAP12 induces cisplatin resistance in NPC cells. Further mechanistic analyses revealed that this effect is mediated by the suppression of alkaliptosis. We found that circARHGAP12 binds to G3BP stress granule assembly factor 1 (G3BP1) to promote stress granule formation, thereby stabilizing CA9 expression in an RNA G-quadruplex (rG4)-dependent manner and establishing a signaling axis that suppresses alkaliptosis. These findings uncover a novel role of circARHGAP12 in mediating cisplatin resistance in NPC and highlight a potential therapeutic target for overcoming drug resistance in cancer treatment. Methods Cell culture and transfection At the institute of Cancer Research, Central South University, the NPC cell lines (CNE2, HNE2) were maintained. And all cell lines were confirmed to be free of mycoplasma contamination by using mycoplasma detection kit (GENESEED). NPC cells were cultured in a humidified incubator at 37°C with 5% CO₂ in RPMI-1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (OriCell, Guangzhou, China) and 1% penicillin–streptomycin (Life Technologies). Plasmid transfections were performed using Neofect (Neofect Biotech, Beijing, China), while siRNA transfections were carried out using HiPerFect (Qiagen, Shanghai, China). The sequences of the siRNAs are listed in Supplementary Table 1. Animal experiments Female BALB/c nude mice (4 weeks old, weighing 15 ± 2 g) used in this study were purchased from the Experimental Animal Center of Central South University. All mice were maintained under specific pathogen-free (SPF) conditions at the Central South University animal facility. The experimental protocol was approved by the Animal Ethics Committee of Central South University. A total of 28 mice were randomly divided into four groups (n = 7 per group). Two groups were subcutaneously injected with CNE2 cells overexpressing circARHGAP12, while the other two groups received CNE2 cells transfected with the empty vector (2 × 10⁶ cells per mouse). When tumor volumes reached 100–150 mm³ (approximately day 7), one circARHGAP12 group and one control group were treated with cisplatin (4 mg/kg, intraperitoneally) every 3 days, and the remaining two groups received saline. Tumor volume and body weight were recorded every 3 days. After 21 days of cisplatin treatment, the mice were euthanized, and tumors were excised, weighed, and subjected to histopathological analysis. Plasmids The circARHGAP12 (has_circ_0000231) overexpression plasmid was constructed by inserting the full-length sequence of circARHGAP12 into the pcDNA3.1(+) CircRNA Mini Vector (a gift from Professor Yong Li, Baylor College of Medicine). The insert was flanked by complementary sequences to promote back-splicing and enhance circular RNA expression efficiency. The plasmids pEGFP-G3BP1, pMIR-Report Luciferase-CA9-3′UTR (WT), and pMIR-Report Luciferase-CA9-3′UTR (MUT) were purchased from Tsingke (Beijing, China). The sequences of the PCR primers are listed in Supplementary Table 1. RNA extraction and RT-qPCR Total RNA was extracted from NPC cells using TRIzol (Life, USA). Reverse trancription was carried out with the HiScript II Q RT SuperMix for qPCR kit (Vazyme, Nanjing, China). Quantitative real-time PCR (RT-qPCR) was then performed on a CFX96™ Real-Time PCR Detection System (Bio-Rad, USA) using 2× SYBR Green qPCR Master Mix (Bimake, USA). Ct values of target genes were normalized to those of the reference gene, and relative expression levels were calculated using the 2⁻ΔΔCt method. The sequences of all primers used are listed in Supplementary Table 1. Western blotting Cells were lysed 48 hours after transfection using RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (KeyGen, Jiangsu, China). Following centrifugation, protein concentrations were determined using a BCA assay kit. Equal amounts of protein were separated by SDS-PAGE and transferred onto 0.2 µm PVDF membranes. After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with primary antibodies against CA9, G3BP1, NONO, PTBP1, or GAPDH (Proteintech, Wuhan, China). The membranes were then incubated with the appropriate HRP-conjugated secondary antibody (Proteintech) for approximately 2 hours at room temperature. Protein bands were visualized using an ECL detection device (Millipore, USA). All antibodies used are listed in Supplementary Table 2. RNA immunoprecipitation (RIP) Cells were washed with PBS and lysed in RIP buffer (Merck, Germany) supplemented with protease inhibitors. The lysates were incubated with magnetic beads (Merck, Germany) conjugated to either anti-G3BP1 or control IgG antibodies. After four washes with high-salt buffer (700 mM NaCl), RNA was extracted from the immunoprecipitated protein–RNA complexes using TRIzol reagent and subsequently analyzed by RT-qPCR. RNA pulldown Cells were lysed 48 hours after transfection using RIP-buffer (150mM KCl, 25mM Tris-HCl, 0.5% NP40, 0.5mM DDT). Biotin-labeled circARHGAP12 probes (Sangon Biotech, Shanghai, China) and streptavidin magnetic beads (Thermo Fisher, USA) were incubated with cell lysates at 4°C overnight. After six washes, the pulled-down proteins were analyzed by LC-MS/MS (PTM Bio, Hangzhou, China) or Western blotting. The probe sequences are listed in Supplementary Table 1, and the identified interacting proteins are shown in Supplementary Table 3. Liquid chromatography‑mass spectrometry (LC-MS/MS) To identify circARHGAP12-interacting proteins, cell lysates were incubated with biotin-labeled probes and biotin-affinity magnetic beads overnight at 4°C to pull down circARHGAP12-associated proteins. The immunoprecipitated proteins were separated by SDS-PAGE and analyzed by LC-MS/MS using an Ultimate 3000 RSLC nanoliter liquid chromatography system (Dionex, CA, USA) coupled to an LTQ Orbitrap Velos Pro mass spectrometer (Thermo Scientific, USA). The resulting data were searched against the SwissProt human database using Proteome Discoverer 2.4. Trypsin digestion was specified, allowing up to two missed cleavages. The mass error tolerance was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Cysteine alkylation was set as a fixed modification, while methionine oxidation and N-terminal acetylation were set as variable modifications. Peptides were required to have a score greater than 20, and only identifications with a high confidence level were considered. Actinomycin D treatment NPC cells were seeded in 12-well plates and cultured to an appropriate density to ensure growth in the logarithmic phase. To inhibit new RNA synthesis, actinomycin D (Abmole, Shanghai, China) was added to fresh culture medium at a final concentration of 5 µg/mL. Cells were collected at 0, 2, 4, and 6 hours after treatment. Total RNA was extracted using TRIzol, and equal amounts of RNA were subjected to reverse transcription for RT-qPCR analysis. The remaining RNA at each time point was normalized to the initial amount at 0 hours to assess RNA stability, and data were analyzed using nonlinear regression with a one-phase exponential decay model in GraphPad Prism. Colony formation assay Cells were transfected with either a circARHGAP12 overexpression plasmid or circARHGAP12 siRNA, with empty vector or scrambled siRNA serving as controls, respectively. Subsequently, 2,000 cells were seeded into 12-well plates and cultured at 37°C for 24 hours. Cells were then treated with cisplatin at final concentrations of 0, 0.5, 1, 2, or 4 µM, followed by incubation at 37°C for 8–12 days. After staining with 0.1% crystal violet, the number of colonies in each group was counted using ImageJ. Survival curves were generated by fitting the data with a nonlinear regression model. MTT Cells were transfected with specific plasmids or siRNAs. At 36 hours post-transfection, 4,000 cells per well were seeded into 96-well plates. After cell adhesion, cells were treated for 48 hours with the following inducers: the apoptosis inducer ABT-263 (AbMole, Shanghai, China, 1 µM), the necroptosis inducer 5-FU (AbMole, 1 µM), the autophagy inducer Rapamycin (AbMole, 1 µM), the ferroptosis inducer RSL3 (Selleck, Shanghai, China, 2 µM), and the alkaliptosis inducer JTC-801 (TargetMol, Shanghai, China, 1 µM). For cisplatin sensitivity assays, cells were treated with cisplatin at concentrations of 0, 2, 4, 8, 16, and 32 µM for 48 hours. Following treatment, MTT reagent was added for 4 hours, after which the medium was removed and DMSO was added to solubilize the formazan crystals. Absorbance was measured at 490 nm using a microplate reader, and IC₅₀ curves were generated to assess cell sensitivity to cisplatin. Fluorescence in situ hybridization (FISH) Cells were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, and blocked with 5% BSA. Cells were then incubated overnight at 4°C with primary antibodies (anti-G3BP1, anti-BG4) or fluorescent probes (Cy3-circARHGAP12, Cy5-CA9; 5 ng/µL each), followed by incubation with species-matched secondary antibodies (Alexa Fluor 488 Anti-Mouse, Alexa Fluor 594 Anti-Mouse, FITC Anti-Goat) for 1 hour at 37°C. Nuclei were stained with DAPI for 10 minutes. Images were captured using a confocal laser scanning microscope. Probe sequences are provided in Supplementary Table 1. Live cell immunofluorescence Cells were transfected with the EGFP-G3BP1 plasmid. After 24 hours, cells were seeded into 35 mm glass-bottom dishes. Once the cells adhered and spread fully, they were treated with 250 µM cisplatin (DDP) for 1 hour. The dishes were then placed on the stage of a confocal laser scanning microscope, and 10% 1,6-hexanediol was added to monitor the dynamic changes of fluorescent puncta in real time. Fluorescence recovery after photobleaching (FRAP) Cells expressing EGFP-G3BP1 were seeded in 35 mm glass-bottom dishes and treated with 250 µM cisplatin for 1 hour after full adhesion. Fluorescent puncta were then photobleached using a 488 nm laser at 50% power on a Leica confocal microscope. Images were acquired every 3 seconds following bleaching. Recovery data were subsequently quantified and analyzed using GraphPad Prism 10. In situ hybridization (ISH) and Immunohistochemistry (IHC) The expression of circARHGAP12 in tumor tissues from animals was detected using an in situ hybridization assay kit (Boster, Wuhan, China) following the manufacturer’s instructions. The digoxin-labeled probe was diluted to 10 µM and incubated with tumor sections overnight at 37°C. Nonspecific binding was removed by washing with saline-sodium citrate (SSC) buffer. Staining was developed using DAB, followed by hematoxylin counterstaining. Sections were then dehydrated through a graded alcohol series, mounted, and examined under an inverted microscope. Probe sequences are provided in Supplementary Table 1. Paraffin-embedded tissue sections were dewaxed and rehydrated through a graded ethanol series. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide, and antigen retrieval was performed in boiling water. After rinsing three times with PBS, sections were blocked with goat serum for 1 hour and then incubated overnight at 4°C with primary antibodies against Ki67 or CA9. Following PBS washes, sections were incubated with the appropriate secondary antibody at 37°C for 10 minutes, developed with DAB, and counterstained with hematoxylin. The sections were then dehydrated through a graded alcohol series, mounted, and examined under an optical microscope. A semi-quantitative scoring system was used to evaluate staining based on intensity and the percentage of positive cells. Staining intensity was scored as 0 (no staining), 1 (light yellow), 2 (light brown), or 3 (dark brown), and the percentage of positive cells was scored as 0 ( 75%). The final composite score was calculated by multiplying the intensity score by the percentage score. Samples with a composite score > 5 were classified as high expression, while those with a score ≤ 5 were considered low expression. RNA sequencing (RNA-seq) RNA-Seq analysis was performed on CNE2 cells transfected with siNC or sicircARHGAP12 (n = 2) to identify mRNAs regulated by circARHGAP12. Total RNA was extracted from cell samples using TRIzol reagent, and residual DNA was removed with the Turbo DNase kit (Ambion). RNA was subsequently purified using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). RNA purity and integrity were assessed by NanoDrop spectrophotometry (Thermo Fisher Scientific, CA, USA) and agarose gel electrophoresis. Library construction for RNA-Seq was performed by Oebiotech Inc. (Shanghai, China) using the NEBNext® Ultra™ Directional RNA Library Preparation Kit. RNA samples were fragmented at high temperature in the presence of divalent cations. Ribosomal RNA was removed by comparison against multiple databases using SortMeRNA. Differentially expressed genes were identified using DESeq2. Statistical analysis All data analyses and graph generation were performed using GraphPad Prism 10.0. Data are presented as mean ± standard deviation (SD). Differences between two groups were evaluated using Student’s t -test, while comparisons among multiple groups were conducted using one-way ANOVA followed by appropriate post-hoc tests. A p-value of < 0.05 was considered statistically significant. Results CircARHGAP12 enhances cisplatin resistance in NPC cells Cisplatin-based chemotherapy remains the primary clinical treatment for NPC, with multiple clinical trials demonstrating favorable therapeutic outcomes [ 8 , 11 , 13 , 14 ] . However, a subset of patients still develops chemotherapy resistance under current treatment regimens [ 10 , 12 ] . Our previous work showed that circARHGAP12 promoted migration and invasion of NPC [ 3 ] , nevertheless, its broader biological functions remain incompletely characterized. To investigate whether circARHGAP12 contributes to cisplatin resistance in NPC, we knocked down or overexpressed circARHGAP12 in CNE2 and HNE2 cells and treated them with cisplatin at concentrations of 0, 2, 4, 8, 16, and 32 µM. Cell survival was assessed using MTT assays, and IC₅₀ values were calculated by fitting survival curves with a dose–response nonlinear regression model. The results showed that, compared to controls, IC₅₀ values significantly decreased following circARHGAP12 knockdown and increased markedly upon circARHGAP12 overexpression (Fig. 1 A). We next performed a colony formation assay to evaluate the effect of circARHGAP12 on the clonogenic potential of NPC cells under cisplatin treatment. CNE2 and HNE2 cells were treated with low concentrations of cisplatin (0, 0.5, 1, 2, 4 µM), and survival curves were fitted using a dose–response nonlinear regression model. In the absence of cisplatin (0 µM), circARHGAP12 had minimal impact on colony formation. However, under cisplatin treatment, circARHGAP12 knockdown significantly reduced clonogenic capacity, whereas its overexpression produced the opposite effect (Fig. 1 B-C). These results indicate that circARHGAP12 promotes cisplatin resistance in NPC in vitro . CircARHGAP12 promotes cisplatin resistance in NPC in vivo To further validate the role of circARHGAP12 in promoting cisplatin resistance in NPC, we established a subcutaneous xenograft model in nude mice using CNE2 cells. Mice were randomly divided into four groups (n = 7) and subcutaneously injected with CNE2 cells overexpressing circARHGAP12 or empty vector (2 × 10⁶ cells per mouse). Once palpable tumors formed, both circARHGAP12-overexpressing and control groups were further subdivided to receive either cisplatin treatment (4 mg/kg) or saline. Mice were administered intraperitoneal injections every three days, and tumor growth was monitored throughout the treatment period. The results showed that circARHGAP12 overexpression alone did not affect tumor growth. However, under cisplatin treatment, circARHGAP12-overexpressing tumors exhibited significantly larger volumes and weights compared to controls, markedly attenuating cisplatin-induced tumor growth inhibition (Fig. 2 A-C). In situ hybridization confirmed higher circARHGAP12 expression in the overexpression group, while immunohistochemistry revealed that circARHGAP12 overexpression did not affect the proliferation marker Ki67 in the absence of cisplatin but substantially suppressed Ki67 expression under cisplatin therapy (Fig. 2 D). These findings demonstrate that circARHGAP12 promotes cisplatin resistance in NPC in vivo . CircARHGAP12 inhibits cisplatin-induced alkaliptosis by upregulating CA9 Cisplatin-induced cell death has been reported to occur via apoptosis, necroptosis, ferroptosis, autophagy, and alkaliptosis [ 23 – 27 ] . To explore the specific mechanism by which circARHGAP12 regulates cisplatin resistance, NPC cells with circARHGAP12 knockdown or overexpression were treated with specific inducers: ABT-263 (apoptosis), 5-FU (necroptosis), Rapamycin (autophagy), RSL3 (ferroptosis), and JTC-801 (alkaliptosis) [ 19 ] , followed by assessment of cell survival using MTT assays. The results showed that circARHGAP12 modulation did not affect apoptosis, necroptosis, autophagy, or ferroptosis. However, upon treatment with the alkaliptosis inducer JTC-801, circARHGAP12 knockdown significantly increased cell death, whereas overexpression produced the opposite effect ( Fig. S1 A ). To determine whether circARHGAP12 promotes cisplatin resistance by suppressing alkaliptosis, we examined its effect on cisplatin-induced cytotoxicity in the presence of an alkaliptosis inducer or inhibitor. MTT assays revealed that circARHGAP12 knockdown enhanced cisplatin-induced cell death, which was rescued by the alkaliptosis inhibitor NAC [ 19 ] . Conversely, circARHGAP12 overexpression attenuated cisplatin-induced cytotoxicity, and this effect was reversed by treatment with the alkaliptosis inducer JTC-801 (Fig. 3 A). These findings indicate that circARHGAP12 enhances cisplatin resistance in NPC cells by inhibiting alkaliptosis. To investigate how circARHGAP12 inhibits alkaliptosis in NPC cells, we knocked down circARHGAP12 in CNE2 cells (using si-scramble as control) and performed RNA-seq (n = 2). KEGG pathway enrichment analysis of differentially expressed genes (|log₂FC| >1, p < 0.05) revealed that genes in the nitrogen metabolism pathway (mainly consist of carbonic anhydrase family) were most significantly affected by circARHGAP12 ( Fig. S1 B-C ). Further analysis of this pathway identified CA9 as the gene most significantly regulated by circARHGAP12 knockdown ( Fig. S1 D ). Analysis of CA9 expression in head and neck squamous cell carcinoma (HNSC) using the TCGA database via GEPIA2 showed that CA9 was significantly upregulated in HNSC tissues compared to normal controls ( Fig. S1 E ), suggesting a potential pro-tumorigenic role. RT-qPCR and western blotting confirmed that circARHGAP12 knockdown reduced CA9 mRNA and protein levels, whereas overexpression increased CA9 expression, consistent with RNA-seq results (Fig. 3 B-C). Immunohistochemistry of subcutaneous tumors in nude mice further demonstrated that circARHGAP12 overexpression promoted CA9 expression, while cisplatin treatment reduced it. Semi-quantitative scoring indicated a positive correlation between circARHGAP12 and CA9 expression (Fig. 3 D). As a member of the carbonic anhydrase family, CA9 regulates cellular acid–base balance by catalyzing the reversible reaction CO₂ + H₂O ⇌ H⁺ + HCO₃⁻, thereby suppressing alkaliptosis induced by intracellular and extracellular pH dysregulation [ 19 , 28 , 29 ] . BCECF, a neutral lipophilic bis-carboxyfluorescein derivative, freely diffuses across the plasma membrane and is hydrolyzed by intracellular esterases to retain fluorescence within the cytoplasm. Its fluorescence intensity is inversely correlated with pH [ 30 ] . Using both a pH meter and BCECF-AM probe, we measured intracellular and extracellular pH. CircARHGAP12 knockdown increased intracellular pH and decreased extracellular pH, whereas overexpression had the opposite effect, decreasing intracellular pH and increasing extracellular pH (Fig. 3 . E-F ). To determine whether circARHGAP12 suppresses cisplatin-induced alkaliptosis in NPC cells via CA9, we performed colony formation assays to assess cell proliferation. Compared to the control group (siNC + Vector), CA9 knockdown significantly reduced the clonogenic ability of NPC cells, whereas circARHGAP12 overexpression enhanced it. Notably, simultaneous circARHGAP12 overexpression and CA9 knockdown restored clonogenic capacity to levels comparable with the control group (siNC + Vector) ( Fig. S2A ). MTT assay yielded consistent results. ( Fig. S2B ). These findings indicate that circARHGAP12 inhibits cisplatin-induced alkaliptosis in NPC cells by upregulating CA9 expression. CircARHGAP12 upregulates CA9 through binding to G3BP1 The most well-established function of circRNAs is acting as competing endogenous RNAs (ceRNAs) that sequester miRNAs to regulate target gene expression [ 1 , 31 , 32 ] . To explore the mechanism by which circARHGAP12 regulates CA9 mRNA, we used the online databases circinteractome and miRcode to predict miRNAs that could bind both circARHGAP12 and the CA9 3′UTR. No shared miRNAs were identified ( Fig. S3A ), suggesting that circARHGAP12 likely regulates CA9 through alternative mechanisms, such as interactions with RNA-binding proteins (RBPs) [ 2 ] . We next performed RNA pulldown coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify potential circARHGAP12-binding proteins ( Fig. S3B-C ). Among the top candidates, G3BP1, NONO, and PTBP1 were confirmed to bind biotin-labeled circARHGAP12 by RNA pulldown validation (Fig. 4 A). Western blotting indicated that circARHGAP12 did not affect the expression levels of these RBPs ( Fig. S3D ), and RT-qPCR showed that knockdown of G3BP1, NONO, or PTBP1 did not alter circARHGAP12 expression ( Fig. S3E ), suggesting that these proteins do not influence circARHGAP12 biogenesis. Collectively, these findings indicate that circARHGAP12 and its binding proteins do not directly regulate each other. To investigate whether G3BP1, NONO, or PTBP1 might serve as molecular scaffolds through which circARHGAP12 regulates CA9 mRNA, we first assessed CA9 expression following modulation of these RBPs. RT-qPCR and western blotting showed that G3BP1 knockdown reduced both CA9 mRNA and protein levels, whereas G3BP1 overexpression increased CA9 expression (Fig. 4 B-C). In contrast, knockdown of NONO or PTBP1 had no effect on CA9 expression ( Fig. S3F-G ), indicating G3BP1 as the primary downstream candidate. Nuclear-cytoplasmic fractionation assays revealed that circARHGAP12 overexpression or knockdown did not alter G3BP1 subcellular localization ( Fig. S3H ), supporting its potential role as a scaffold for CA9 regulation. RIP assays further demonstrated that G3BP1 antibody enriched significantly more circARHGAP12 than the IgG control (Fig. 4 D), consistent with the RNA pulldown results. RT-qPCR and western blotting showed that, compared with the control group (siNC + Vector), G3BP1 knockdown reduced CA9 mRNA and protein levels, whereas circARHGAP12 overexpression increased them. Notably, simultaneous circARHGAP12 overexpression and G3BP1 knockdown restored CA9 expression to levels comparable with the control group (siNC + Vector) (Fig. 4 E, Fig. S3I ). To assess whether circARHGAP12 suppresses cisplatin-induced alkaliptosis in NPC cells via G3BP1, cell viability was measured by MTT assay. G3BP1 knockdown decreased the IC₅₀ of NPC cells, while circARHGAP12 overexpression increased it. Concurrent circARHGAP12 overexpression and G3BP1 knockdown restored the IC₅₀ to levels similarly to the control group (siNC + Vector) (Fig. 4 F). Colony formation assays yielded consistent results (Fig. 4 G). These findings indicate that circARHGAP12 inhibits cisplatin-induced alkaliptosis in NPC cells by binding to G3BP1 and upregulating CA9 expression. G3BP1 binds to and stabilizes CA9 mRNA in an rG4-dependent manner G-quadruplexes are three-dimensional structures formed by guanine-rich sequences, present in both DNA and RNA, collectively referred to as G4 structures. Those specifically in RNA are termed rG4s [ 33 ] . G3BP1 exhibits high selectivity and affinity for rG4s, enabling it to recognize and bind rG4 motifs in the 3′UTR of target mRNAs, thereby preventing their degradation [ 34 ] . Based on these properties, we used the online tool QGRS to predict potential rG4 structures in the CA9 3′UTR ( Fig. S4A-B ). Pyridostatin (PDS) is a small-molecule ligand that binds and stabilizes G4 structures [ 34 ] . PDS can disrupt the interaction between G3BP1 and rG4, displacing G3BP1 from target mRNAs and promoting mRNA degradation due to loss of G3BP1-mediated protection [ 11 , 34 , 35 ] . To determine whether G3BP1 regulates CA9 mRNA via rG4s, we performed actinomycin D-based RNA stability assays. G3BP1 overexpression enhanced CA9 mRNA stability, whereas PDS treatment reduced it. Importantly, CA9 mRNA stability was restored to control levels when G3BP1 was overexpressed following PDS treatment (Fig. 5 A). To confirm rG4 dependence, we designed a mutant CA9 3′UTR in which guanine-to-adenine substitutions (GG → AA) disrupted the rG4-forming region ( Fig. S4C ) and cloned it into a dual-luciferase reporter system. Dual-luciferase assays showed that G3BP1’s regulatory effect on CA9 mRNA was abolished with the mutant 3′UTR, indicating that G3BP1 stabilizes CA9 mRNA in an rG4-dependent manner (Fig. 5 B). Furthermore, RT-qPCR-based actinomycin D assays under cisplatin treatment revealed that circARHGAP12 enhanced CA9 mRNA stability, whereas circARHGAP12 knockdown decreased it ( Fig. S4D ). To determine whether circARHGAP12 influences the binding of G3BP1 to CA9 mRNA, we assessed G3BP1–CA9 mRNA interactions following circARHGAP12 knockdown or overexpression in NPC cells. RIP assays revealed that circARHGAP12 enhanced the association between G3BP1 and CA9 mRNA (Fig. 5 C). Additionally, FISH analysis showed clear colocalization of G3BP1, circARHGAP12, and CA9 mRNA (Fig. 5 D). These results indicate that G3BP1 binds to the CA9 3′UTR in an rG4-dependent manner to stabilize CA9 expression, and that circARHGAP12 facilitates this interaction. CircARHGAP12 promotes cisplatin-induced Stress Granule (SG) assembly and stabilizes rG4 structures G3BP1 is the core assembly component of SGs [ 36 ] . SGs are dynamic, membraneless organelles formed through liquid-liquid phase separation (LLPS) in response to various cellular stresses. The components within SGs are transiently segregated from the cytoplasm and can be re-released once the stress subsides [ 37 – 40 ] . Previous studies have shown that SGs play a crucial role in helping tumor cells adapt to diverse stress conditions [ 41 – 43 ] . To investigate whether circARHGAP12 influences SG assembly by binding to G3BP1, FISH analysis revealed that in the absence of cisplatin, knockdown or overexpression of circARHGAP12 did not alter the diffuse cytoplasmic distribution of G3BP1 (Fig. 6 A, Fig. S5A ). However, upon cisplatin treatment, SGs—characterized by G3BP1-positive LLPS condensates—were assembled within cells, where circARHGAP12 exhibited strong colocalization with these granules and promoted their formation. Using sodium arsenite-induced SGs as a positive control [ 44 , 45 ] , we further confirmed that circARHGAP12 colocalized with G3BP1 within SGs and enhanced their assembly ( Fig. S5B ). As reported, RNA G-quadruplex (rG4) structures are often enriched within SGs, and RBP–rG4 interactions are essential for SG formation [ 46 – 49 ] . BG4, a specific antibody recognizing rG4 structures, can be used to visualize rG4-containing mRNAs in cells. To explore the relationship between rG4 and cisplatin-induced SG assembly, FISH assays demonstrated that CA9 mRNA and its associated rG4 structures showed marked colocalization with G3BP1 in cisplatin-induced SGs (Fig. 6 B). These findings suggest that SGs may play a critical role in sequestering and stabilizing CA9 mRNA under cisplatin stress. As SGs are membraneless organelles formed in the cytoplasm through LLPS under stress conditions, they can transiently sequester mRNAs and proteins [ 50 , 51 ] . To visualize SG dynamics, we constructed an EGFP-G3BP1 vector for live-cell imaging. Under cisplatin treatment, EGFP-G3BP1 formed distinct condensates in NPC cells. Treatment with 1,6-hexanediol, a chemical disruptor of phase separation, led to the gradual dissolution of these EGFP-G3BP1 foci, confirming that cisplatin induces G3BP1-dependent phase separation ( Fig. S5C ). FRAP assays further revealed that the fluorescence recovery rate of EGFP-G3BP1 foci was significantly faster in cisplatin-treated circARHGAP12-overexpressing cells compared with controls, indicating enhanced SG dynamics (Fig. 6 C, Fig. S5D ). Moreover, FISH analysis showed that circARHGAP12 promoted the assembly of G3BP1-marked SGs upon cisplatin induction, whereas this effect was reversed by PDS—a small molecule that competitively binds rG4 structures and disrupts their interaction with G3BP1 (Fig. 6 D, Fig. S5E ). Under cisplatin treatment, G3BP1, rG4 structures, and CA9 mRNA exhibited strong colocalization within SGs; this colocalization was markedly reduced following G3BP1 knockdown, which also impaired SG formation ( Fig. S5F ). Together, these results indicate that under cisplatin-induced stress, circARHGAP12 promotes the assembly and phase separation of G3BP1-marked SGs, thereby protecting and stabilizing CA9 mRNA within SGs in an rG4-dependent manner through G3BP1. Discussion The treatment of NPC, particularly in patients with advanced disease, relies heavily on cisplatin-based chemotherapy regimens [ 11 ] . However, the development of cisplatin resistance frequently results in treatment failure and tumor recurrence, posing a major clinical challenge [ 9 , 15 ] . Therefore, elucidating the molecular mechanisms underlying cisplatin resistance is of great importance for overcoming this obstacle. Traditionally, the antitumor effects of cisplatin have been attributed to its ability to induce DNA damage and trigger apoptosis [ 52 ] . Recent studies, however, have demonstrated that cisplatin can also exert cytotoxicity through alternative cell death pathways such as ferroptosis and autophagy [ 24 , 53 ] . In this study, we provide the first evidence that cisplatin can induce alkaliptosis in NPC cells. Furthermore, we identify circARHGAP12 as a novel and essential driver of cisplatin resistance in NPC, acting through the upregulation of CA9 to suppress alkaliptosis. Alkaliptosis is a recently identified form of regulated cell death characterized by an increase in intracellular pH [ 19 ] . Unlike other programmed cell death pathways, alkaliptosis induced by elevated intracellular pH cannot be prevented by existing cell death inhibitors. The compound JTC801 triggers alkaliptosis by suppressing CA9 expression, thereby disrupting intracellular and extracellular pH homeostasis. CA9 functions as a pH regulator by catalyzing the reversible hydration of carbon dioxide to form bicarbonate and protons, helping to maintain acid-base balance and prevent alkaliptosis. In this study, we found that cisplatin treatment induces alkaliptosis in NPC cells. Moreover, we demonstrated that circARHGAP12 upregulates CA9 expression to suppress cisplatin-induced alkaliptosis. As a newly defined form of cell death, the relationship between alkaliptosis and chemotherapy remains poorly understood [ 28 ] . Our findings suggest that pharmacological induction of alkaliptosis—for instance, through JTC801—may enhance the antitumor efficacy of cisplatin, providing a potential therapeutic strategy for NPC. However, whether alkaliptosis represents a direct target of cisplatin or interacts with other cell death pathways warrants further investigation. Mechanistically, we identified a novel regulatory axis in which circARHGAP12 cooperates with G3BP1 under cisplatin stress to promote the assembly of SGs. These granules create a protective microenvironment that enriches rG4 structures within the CA9 mRNA 3′UTR. Acting as a molecular scaffold, G3BP1 stabilizes CA9 mRNA within SGs, leading to elevated CA9 expression, suppression of alkaliptosis, and ultimately enhanced cisplatin resistance. Stress granules (SGs) serve as a crucial adaptive mechanism that enables cells to withstand environmental stress, and their emerging link to chemotherapy resistance has garnered increasing attention. SGs are dynamic, membrane-less condensates that transiently assemble through LLPS in response to stress and are primarily composed of untranslated mRNAs and RBPs [ 37 – 40 , 54 ] . By transiently inhibiting translation and preventing mRNA degradation, SGs facilitate cellular adaptation to adverse conditions [ 55 ] . Dysregulation of G3BP1, a core SG assembly factor, has been implicated in tumor initiation, progression, and therapeutic resistance [ 41 – 43 ] . In this study, we found that circARHGAP12 binds to G3BP1 and cooperates with cisplatin to promote SG assembly, revealing a novel role for circARHGAP12 in cisplatin-induced SG formation. Moreover, we observed enrichment of G3BP1-bound CA9 3′UTR rG4 structures within SGs. This finding suggests that, beyond their classical role in translational repression, SGs may contribute to chemotherapy resistance in NPC by sequestering RNA–protein complexes. Such sequestration could protect key cisplatin resistance–related mRNAs, such as CA9, from degradation during stress, thereby maintaining CA9 expression and suppressing alkaliptosis. Notably, cisplatin-induced SGs exhibited slower disassembly kinetics compared with classical SGs (e.g., those formed under oxidative stress) [ 56 ] . Consequently, the fate of these SGs—and how their delayed disassembly influences the reinitiation of translation once cisplatin stress subsides—warrants further investigation. RNA G-quadruplexes (rG4s) are unique secondary structures formed by the stacking of guanine tetrads within guanine-rich RNA sequences. These structures can form in various regions of mRNAs, including the 5′UTR, 3′UTR, and coding sequences, where they play pivotal roles in regulating gene expression [ 33 ] . The formation and function of rG4s are typically modulated by rG4-binding proteins (G4BPs), such as TMPRSS2 and G3BP1 [ 57 ] . Notably, G3BP1 exhibits high affinity and specificity for rG4 structures located in mRNA 3′UTRs. This interaction is mediated by its C-terminal RGG domain and further stabilized by the RNA recognition motif (RRM) domain [ 11 ] . In this study, we identified the rG4 structure within the CA9 mRNA 3′UTR as a critical site for G3BP1 binding and for maintaining CA9 mRNA stability—a mechanism reminiscent of the rG4-dependent regulation of PITX1 mRNA by G3BP1 [ 11 ] . Importantly, our findings reveal for the first time that circRNAs can enhance the recognition and binding of rG4 structures by RBPs, thereby stabilizing downstream target mRNAs. This discovery expands the biological significance of rG4s: beyond serving as independent regulatory elements, rG4s may act as scaffolding platforms for circRNA–protein–mRNA ternary complex formation. Such a mechanism suggests that rG4-dependent G3BP1-mediated transcriptomic regulation may be far more extensive than previously recognized. Furthermore, small-molecule compounds targeting rG4 structures—such as 2a and 2b, which suppress KRAS translation and inhibit tumor progression in pancreatic cancer [ 58 ] —highlight the therapeutic potential of this regulatory mechanism. Analogously, small molecules that specifically target the CA9 3′UTR rG4 structure (e.g., PDS) may offer promising strategies to overcome cisplatin resistance in NPC. Conclusions In summary, this study demonstrates that circARHGAP12 promotes cisplatin resistance in NPC. Mechanistically, circARHGAP12 directly binds to G3BP1 and, in an rG4-dependent manner, interacts with the 3′UTR of CA9 mRNA to enhance its stability. Further investigations revealed that circARHGAP12 facilitates cisplatin-induced stress granule (SG) assembly through G3BP1, resulting in the colocalization of circARHGAP12, G3BP1, and CA9 mRNA within SGs. This spatial organization protects CA9 mRNA from degradation under stress conditions (Fig. 7 ). Collectively, these findings uncover a novel regulatory axis—circARHGAP12–G3BP1–CA9—that contributes to cisplatin resistance in NPC, and suggest that targeting this pathway to modulate SG dynamics may represent a promising therapeutic strategy for overcoming chemoresistance. Abbreviations Circular RNA (circRNA) Carbonic Anhydrase 9 (CA9) Cis-diamminedichloroplatinum (II) (DDP) Fluorescence recovery after photobleaching (FRAP) G3BP Stress Granule Assembly Factor 1 (G3BP1) Liquid-Liquid Phase Separation (LLPS) Nasopharyngeal carcinoma (NPC) RNA immunoprecipitation (RIP) RNA binding proteins (RBPs) RNA G-quadruplex (rG4) Pyridostatin (PDS) Stress Granules (SGs) Fluorescence in situ hybridization (FISH) In situ hybridization (ISH) Immunohistochemistry (IHC) Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of Central South University (CSU-2024-0335). Consent for publication All authors have critically reviewed the manuscript and approved its submission. Competing interests The authors declare no competing interests. Funding This work has been supported by the National Natural Science Foundation of China (82203163), the Natural Science Foundation of Hunan Province (2025JJ50544), Young Elite Scientists Sponsorship Program by CAST (2022QNRC001), and grant from The Scientific Research Program of FuRong Laboratory (No. 2024PT5102). Author Contribution T.L. and Y.L. contributed to experiment execution, data analysis, and manuscript drafting. H.Q., D.W., J.G. performed some of the experiments. L.S., Q.Y. and W.X. participated in project design. W.X., C.F. and Z.Z. revised the manuscript. C.F. and Z.Z. were responsible for overall study design and manuscript revision. Acknowledgements Not applicable. Data Availability The datasets generated during this study are available from the corresponding author upon reasonable request. References CHEN LL. The expanding regulatory mechanisms and cellular functions of circular RNAs [J]. Nat Rev Mol Cell Biol. 2020;21(8):475–90. ZHOU W Y, CAI Z R, LIU J, et al. Circular RNA: metabolism, functions and interactions with proteins [J]. Mol Cancer. 2020;19(1):172. FAN C, QU H, XIONG F, et al. CircARHGAP12 promotes nasopharyngeal carcinoma migration and invasion via ezrin-mediated cytoskeletal remodeling [J]. Cancer Lett. 2021;496:41–56. LI Q, ZHAO Y H, XU C, et al. Chemotherapy-Induced Senescence Reprogramming Promotes Nasopharyngeal Carcinoma Metastasis by circRNA-Mediated PKR Activation [J]. Adv Sci (Weinh). 2023;10(8):e2205668. HONG X, LI Q, LI J, et al. CircIPO7 Promotes Nasopharyngeal Carcinoma Metastasis and Cisplatin Chemoresistance by Facilitating YBX1 Nuclear Localization [J]. Clin Cancer Res. 2022;28(20):4521–35. MO Y, WANG Y, ZHANG S, et al. Circular RNA circRNF13 inhibits proliferation and metastasis of nasopharyngeal carcinoma via SUMO2 [J]. Mol Cancer. 2021;20(1):112. GE J, WANG J, XIONG F, et al. Epstein-Barr Virus-Encoded Circular RNA CircBART2.2 Promotes Immune Escape of Nasopharyngeal Carcinoma by Regulating PD-L1 [J]. Cancer Res. 2021;81(19):5074–88. CHEN Y P, CHAN A T C, LE Q T, et al. Nasopharyngeal carcinoma [J]. Lancet. 2019;394(10192):64–80. LIANG Y-L, LIU X, SHEN L-F, et al. Adjuvant PD-1 Blockade With Camrelizumab for Nasopharyngeal Carcinoma: The DIPPER Randomized Clinical Trial [J]. JAMA. 2025;333(18):1589–98. DAI J, ZHANG B, SU Y, et al. Induction Chemotherapy Followed by Radiotherapy vs Chemoradiotherapy in Nasopharyngeal Carcinoma: A Randomized Clinical Trial [J]. JAMA Oncol. 2024;10(4):456–63. TANG L L, CHEN Y P, CHEN C B, et al. The Chinese Society of Clinical Oncology (CSCO) clinical guidelines for the diagnosis and treatment of nasopharyngeal carcinoma [J]. Cancer Commun (Lond). 2021;41(11):1195–227. GUO TANGLL, ZHANG R. Effect of Radiotherapy Alone vs Radiotherapy With Concurrent Chemoradiotherapy on Survival Without Disease Relapse in Patients With Low-risk Nasopharyngeal Carcinoma: A Randomized Clinical Trial [J]. JAMA. 2022;328(8):728–36. ZHANG Y, CHEN L, HU G Q, et al. Gemcitabine and Cisplatin Induction Chemotherapy in Nasopharyngeal Carcinoma [J]. N Engl J Med. 2019;381(12):1124–35. ZHANG Y, SUN Y. Induction gemcitabine and cisplatin in locoregionally advanced nasopharyngeal carcinoma [J]. Cancer Commun (Lond). 2019;39(1):39. TANG L-L, GUO R, ZHANG N, et al. Effect of Radiotherapy Alone vs Radiotherapy With Concurrent Chemoradiotherapy on Survival Without Disease Relapse in Patients With Low-risk Nasopharyngeal Carcinoma: A Randomized Clinical Trial [J]. JAMA. 2022;328(8):728–36. JI F, LU Y, CHEN S, et al. IGF2BP2-modified circular RNA circARHGAP12 promotes cervical cancer progression by interacting m6A/FOXM1 manner [J]. Cell Death Discovery. 2021;7(1):215. ZHANG D, GUO Q, YOU K, et al. m6A-modified circARHGAP12 promotes the aerobic glycolysis of doxorubicin-resistance osteosarcoma by targeting c-Myc [J]. J Orthop Surg Res. 2024;19(1):33. MAO C, WANG M. ZHUANG L, et al. Metabolic cell death in cancer: ferroptosis, cuproptosis, disulfidptosis, and beyond [J]. Volume 15. Protein & Cell; 2024. pp. 642–60. 9. SONG X, ZHU S, XIE Y, et al. JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice [J]. Gastroenterology. 2018;154(5):1480–93. MCDONALD P C, CHAFE S C, BROWN W S, et al. Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia [J]. Gastroenterology. 2019;157(3):823–37. LI Z, JIANG L, CHEW SH et al. Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia [J]. Redox Biol, 2019, 26(101297. KOUKOURAKIS M I, GIATROMANOLAKI A, SIVRIDIS E, et al. Hypoxia-regulated carbonic anhydrase-9 (CA9) relates to poor vascularization and resistance of squamous cell head and neck cancer to chemoradiotherapy [J]. Clin Cancer Res. 2001;7(11):3399–403. MACKAY C, CARROLL E, IBRAHIM A F M, et al. E3 ubiquitin ligase HOIP attenuates apoptotic cell death induced by cisplatin [J]. Cancer Res. 2014;74(8):2246–57. YANG Y, LIU L, TIAN Y et al. Autophagy-driven regulation of cisplatin response in human cancers: Exploring molecular and cell death dynamics [J]. Cancer Lett, 2024, 587(216659. GUO J, XU B, HAN Q, et al. Ferroptosis: A Novel Anti-tumor Action for Cisplatin [J]. Cancer Res Treat. 2017;50(2):445–60. XU Y, MA H, SHAO J, et al. A Role for Tubular Necroptosis in Cisplatin-Induced AKI [J]. J Am Soc Nephrol. 2015;26(11):2647–58. LIU C, LI D, WANG J, et al. Arenobufagin increases the sensitivity of gastric cancer to cisplatin via alkaliptosis [J]. Heliyon. 2023;9(11):e21110. TANG D, KANG R, BERGHE T V, et al. The molecular machinery of regulated cell death [J]. Cell Res. 2019;29(5):347–64. MAO C, WANG M. Metabolic cell death in cancer: ferroptosis, cuproptosis, disulfidptosis, and beyond [J]. Protein Cell. 2024;15(9):642–60. ZHENG G, PENG C, JIA X et al. ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH [J]. Molecular cancer, 2015, 14(84. CONN V M, CHINNAIYAN A M, CONN S J. Circular RNA in cancer [J]. Nat Rev Cancer. 2024;24(9):597–613. LI J, SUN D, PU W, et al. Circular RNAs in Cancer: Biogenesis, Function, and Clinical Significance [J]. Trends Cancer. 2020;6(4):319–36. LYU K, CHOW E Y, MOU X, et al. RNA G-quadruplexes (rG4s): genomics and biological functions [J]. Nucleic Acids Res. 2021;49(10):5426–50. HE X, YUAN J, WANG Y. G3BP1 binds to guanine quadruplexes in mRNAs to modulate their stabilities [J]. Nucleic Acids Res. 2021;49(19):11323–36. TURNER M, DANINO Y M, BARSHAI M, et al. rG4detector, a novel RNA G-quadruplex predictor, uncovers their impact on stress granule formation [J]. Nucleic Acids Res. 2022;50(20):11426–41. YANG P, MATHIEU C, KOLAITIS R M et al. G3BP1 Is a Tunable Switch that Triggers Phase Separation to Assemble Stress Granules [J]. Cell, 2020, 181(2): 325 – 45.e28. YOUN J Y, DYAKOV B J A, ZHANG J, et al. Properties of Stress Granule and P-Body Proteomes [J]. Mol Cell. 2019;76(2):286–94. PROTTER D S W PARKERR. Principles and Properties of Stress Granules [J]. Trends Cell Biol. 2016;26(9):668–79. KEDERSHA N, IVANOV P, ANDERSON P. Stress granules and cell signaling: more than just a passing phase? [J]. Trends Biochem Sci. 2013;38(10):494–506. WHEELER J R JAINS, WALTERS R W, et al. ATPase-Modulated Stress Granules Contain a Diverse Proteome and Substructure [J]. Cell. 2016;164(3):487–98. REDDING A, GRABOCKA E. Stress granules and hormetic adaptation of cancer [J]. Trends Cancer. 2023;9(12):995–1005. LI T, ZENG Z, FAN C, et al. Role of stress granules in tumorigenesis and cancer therapy [J]. Biochim Biophys Acta Rev Cancer. 2023;1878(6):189006. XING F, QIN Y, XU J, et al. Stress granules dynamics and promising functions in pancreatic cancer [J]. Biochim Biophys Acta Rev Cancer. 2023;1878(3):188885. ZHAO T, XIONG W, CAI J et al. YTHDF2 phase separation promotes arsenic-induced keratinocyte transformation in a poly-m6A-dependent manner by inhibiting translational initiation of the key tumor suppressor PTEN [J]. J Hazard Mater, 2024, 480(136243. LI Q, LIU J, GUO L, et al. Decoding the interplay between m6A modification and stress granule stability by live-cell imaging [J]. Sci Adv. 2024;10(46):eadp5689. ASAMITSU S, YABUKI Y, MATSUO K et al. RNA G-quadruplex organizes stress granule assembly through DNAPTP6 in neurons [J]. Sci Adv, 2023, 9(8): eade2035. KHAREL P. RNA G-quadruplexes and stress: emerging mechanisms and functions [J]. Trends Cell Biol. 2024;34(9):771–84. TURNER M, DANINO Y M, BARSHAI M, et al. rG4detector, a novel RNA G-quadruplex predictor, uncovers their impact on stress granule formation [J]. Nucleic Acids Res. 2022;50(20):11426–41. DANINO YM, ROSENBAUM-COHEN T MOLITORL, et al. BLM helicase protein negatively regulates stress granule formation through unwinding RNA G-quadruplex structures [J]. Nucleic Acids Res. 2023;51(17):9369–84. MAO L-H YUANL, HUANG Y-Y, et al. Stress granules: emerging players in neurodegenerative diseases [J]. Transl Neurodegener. 2025;14(1):22. FANG M, LUO L, CHEN Y et al. Perillaldehyde Improves Parkinson-Like Deficits by Targeting G3BP Mediated Stress Granule Assembly in Preclinical Models [J]. Advanced Science (Weinheim, Baden-Wurttemberg, Germany), 2025, 12(14): e2412152. LIU C, ZHOU L, CHEN J et al. Galectin-7 promotes cisplatin efficacy by facilitating apoptosis and G3BP1 degradation in cervical cancer [J]. Biochem Pharmacol, 2023, 217(115834. TUFO G, JONES A W, WANG Z, et al. The protein disulfide isomerases PDIA4 and PDIA6 mediate resistance to cisplatin-induced cell death in lung adenocarcinoma [J]. Cell Death Differ. 2014;21(5):685–95. LEE C Y SEYDOUXG. Dynamics of mRNA entry into stress granules [J]. Nat Cell Biol. 2019;21(2):116–7. ZHAO Z, QING Y. QKI shuttles internal m(7)G-modified transcripts into stress granules and modulates mRNA metabolism [J]. Cell. 2023;186(15):3208–e2627. PIETRAS P, AULAS A, FAY M M et al. Translation inhibition and suppression of stress granules formation by cisplatin [J]. Biomed Pharmacother, 2022, 145(112382. LIU G, DU W. RNA G-quadruplex in TMPRSS2 reduces SARS-CoV-2 infection [J]. Nat Commun. 2022;13(1):1444. MIGLIETTA G, COGOI S, MARINELLO J, et al. RNA G-Quadruplexes in Kirsten Ras (KRAS) Oncogene as Targets for Small Molecules Inhibiting Translation [J]. J Med Chem. 2017;60(23):9448–61. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.xlsx SupplementaryTable2.xlsx SupplementaryTable3.xlsx Supplementaryfigure.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7984659","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":554960869,"identity":"89fc4fca-2190-47c6-9d78-8e9d2aa1951d","order_by":0,"name":"Tiansheng Li","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Tiansheng","middleName":"","lastName":"Li","suffix":""},{"id":554960871,"identity":"f0dadfa6-c1c6-4edf-81ae-1def08031643","order_by":1,"name":"Yixuan Liu","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Yixuan","middleName":"","lastName":"Liu","suffix":""},{"id":554960873,"identity":"f3dde288-d4e9-4f20-a5ed-96510861fa02","order_by":2,"name":"Hongke Qu","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Hongke","middleName":"","lastName":"Qu","suffix":""},{"id":554960874,"identity":"eaf8f113-5ec7-47d8-bc62-a92d43ae6e29","order_by":3,"name":"Dan Wang","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Wang","suffix":""},{"id":554960875,"identity":"f7814d24-6aee-4952-ba91-a3dc16b855c9","order_by":4,"name":"Junshang Ge","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Junshang","middleName":"","lastName":"Ge","suffix":""},{"id":554960878,"identity":"87ddc480-b1c3-492c-8f3d-42158b3a8528","order_by":5,"name":"Lei Shi","email":"","orcid":"","institution":"Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Shi","suffix":""},{"id":554960879,"identity":"b0ddded0-0afb-477a-ae48-ab11de5effc1","order_by":6,"name":"Qijia Yan","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Qijia","middleName":"","lastName":"Yan","suffix":""},{"id":554960880,"identity":"210d4d23-1b84-4bd5-a5ce-1cc606e5cc0e","order_by":7,"name":"Wei Xiong","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Xiong","suffix":""},{"id":554960882,"identity":"389939d2-06bb-4c73-bfe9-6bd539709cee","order_by":8,"name":"Chunmei Fan","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Chunmei","middleName":"","lastName":"Fan","suffix":""},{"id":554960884,"identity":"64589c2a-dfa6-4173-8541-f4b2dd0dd46f","order_by":9,"name":"Zhaoyang Zeng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYLACxgYo4wOEMiBeC+MMkrUw8xCjRb699/DLnzvs5AyOnz382qamLrGBvXmbBEPNHdwW9JxLs5A8k2xscCYvzTrn2OHEBp5jZRIMx57h1MIskWNmYNjGnLjhQI6ZcW7DgcQGoIgEY8NhnFrY5N+YGSS21SduOP/GzNiyAegwoAheLTwSPMYPDrYdTtxwI8f4MWMDM9AWHvxaJHhyzBgb244bS954Y8bYc+ywcRtPWrFFwjHcWuTbzxh//NlWLcd3Psf4w4+aOtl+9sMbb3yowa0F5B0JEKlwAMpgAxEJ+DQAAw2cTOQboIxRMApGwSgYBegAAAqPVq4m2AlGAAAAAElFTkSuQmCC","orcid":"","institution":"Central South University","correspondingAuthor":true,"prefix":"","firstName":"Zhaoyang","middleName":"","lastName":"Zeng","suffix":""}],"badges":[],"createdAt":"2025-10-30 03:38:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7984659/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7984659/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97672926,"identity":"043bca51-3b39-4abc-8d3f-6e9eee523ac5","added_by":"auto","created_at":"2025-12-08 09:39:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":217934,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/d30eed002098f43b1597d20d.docx"},{"id":97673127,"identity":"0a14cce0-50b8-4406-9779-3423d2bb15ab","added_by":"auto","created_at":"2025-12-08 09:39:29","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9945,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/1551790a5f55a13e2df9192a.xlsx"},{"id":97549359,"identity":"eb1ca0be-f721-4b0d-ad4c-7a3330e9a787","added_by":"auto","created_at":"2025-12-05 16:46:33","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9773,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/b02ea758839d27f57dd3f96f.xlsx"},{"id":97549367,"identity":"b84298b3-60bd-432e-9c82-37201b73e4cc","added_by":"auto","created_at":"2025-12-05 16:46:34","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":158951,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/c7b159504fe80ee42596312a.pdf"},{"id":97672714,"identity":"eca6f44a-d0c9-4423-893e-5e7e624bc2ca","added_by":"auto","created_at":"2025-12-08 09:38:38","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9593,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/6e4be0a81db68487b7b01d98.xlsx"},{"id":97672232,"identity":"82c8bec2-1c60-45e2-aa10-7ff1e0d5d75f","added_by":"auto","created_at":"2025-12-08 09:34:55","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":525740,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/485a09c3f46b990db082ca07.pdf"},{"id":97549374,"identity":"b105a935-0701-4761-8791-d2a7f1001363","added_by":"auto","created_at":"2025-12-05 16:46:34","extension":"pdf","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":656623,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/1013aba11f455ca1405ef797.pdf"},{"id":97549365,"identity":"314ecc9b-0f86-4644-9097-5b831738513c","added_by":"auto","created_at":"2025-12-05 16:46:33","extension":"pdf","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":305187,"visible":true,"origin":"","legend":"","description":"","filename":"Figure7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/b7ccec1bb89f20bd58dcbb07.pdf"},{"id":97672595,"identity":"e15b2ce1-27fc-4038-8afd-e38259392d19","added_by":"auto","created_at":"2025-12-08 09:38:27","extension":"json","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10948,"visible":true,"origin":"","legend":"","description":"","filename":"2123dd18d14a4cab84fa4d0c8f9363a8.json","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/10c04944342d7f80f3e8f231.json"},{"id":97549381,"identity":"9973f275-bc0a-47eb-b835-1ef9eb8943ce","added_by":"auto","created_at":"2025-12-05 16:46:34","extension":"pdf","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":487293,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/22074652139b967e599b29bd.pdf"},{"id":97549363,"identity":"5dde0f0e-5ad3-4f16-a4a3-417f3ec6c4f6","added_by":"auto","created_at":"2025-12-05 16:46:33","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138948,"visible":true,"origin":"","legend":"","description":"","filename":"2123dd18d14a4cab84fa4d0c8f9363a81enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/102bd1f07970287d95444e2b.xml"},{"id":97672603,"identity":"a2378d83-6645-47ce-b071-83548ea78d8a","added_by":"auto","created_at":"2025-12-08 09:38:29","extension":"pdf","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":335856,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/f72cacb2e8fc5a3c30376d3c.pdf"},{"id":97672769,"identity":"d4e5b8bc-bc1b-4dd3-8921-e101699830ef","added_by":"auto","created_at":"2025-12-08 09:38:46","extension":"pdf","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":363477,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/259915670fcf7959497f05a6.pdf"},{"id":97549369,"identity":"eecfa063-c52b-4371-ae0a-ebacdd28d1db","added_by":"auto","created_at":"2025-12-05 16:46:34","extension":"pdf","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":441177,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/35214e7086ae2c9c69f1232a.pdf"},{"id":97549368,"identity":"d5d05b3b-6d95-4341-baaa-eeb203310cd7","added_by":"auto","created_at":"2025-12-05 16:46:34","extension":"pdf","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":158951,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/afee848014ab9be161343677.pdf"},{"id":97549361,"identity":"b6baba3e-bf8d-4fc7-a8aa-2b335056d2ac","added_by":"auto","created_at":"2025-12-05 16:46:33","extension":"pdf","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":525740,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/8f64cf1d26a282914e71ba23.pdf"},{"id":97673090,"identity":"86e978b4-39e0-4e21-a25f-50fd0d8c5792","added_by":"auto","created_at":"2025-12-08 09:39:25","extension":"pdf","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":494286,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/f9e03f1db3ffd62f044f3bbd.pdf"},{"id":97549372,"identity":"61ad2b24-72c7-4235-a0f0-a0376210fdd0","added_by":"auto","created_at":"2025-12-05 16:46:34","extension":"pdf","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":491590,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/adbf425c960bf72677ba31f3.pdf"},{"id":97549379,"identity":"537ec9aa-385f-423c-bc1f-25a337dedc0d","added_by":"auto","created_at":"2025-12-05 16:46:34","extension":"pdf","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":493338,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/93a06f7b2c83134a325fdb1e.pdf"},{"id":97549370,"identity":"61149e4c-8b35-413d-8bd8-6fcb817cdd18","added_by":"auto","created_at":"2025-12-05 16:46:34","extension":"pdf","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":460726,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/18f86daba94d6f3337339a0f.pdf"},{"id":97549378,"identity":"3ed5320b-65ef-46c5-a626-68a3a5c79123","added_by":"auto","created_at":"2025-12-05 16:46:34","extension":"pdf","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":390183,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/78e26c68d3ab09ea51022adb.pdf"},{"id":97549377,"identity":"9eafde54-eaaf-45b1-9b5c-2296c2084e61","added_by":"auto","created_at":"2025-12-05 16:46:34","extension":"pdf","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":656623,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/3b5a41728aa2bcf5d2bebdf0.pdf"},{"id":97672288,"identity":"95ec488e-51bb-4301-8785-eb7a96edc2df","added_by":"auto","created_at":"2025-12-08 09:35:03","extension":"pdf","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":305187,"visible":true,"origin":"","legend":"","description":"","filename":"Figure7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/6063a5977004d23eb1830d4a.pdf"},{"id":97549371,"identity":"e75490c5-9d95-4889-b94d-464d268c135a","added_by":"auto","created_at":"2025-12-05 16:46:34","extension":"xml","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139738,"visible":true,"origin":"","legend":"","description":"","filename":"2123dd18d14a4cab84fa4d0c8f9363a81structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/eb3cc4ec8d94b6c5446a483a.xml"},{"id":97549376,"identity":"f5a59c86-1345-4541-b1f8-08c4d3bae3ae","added_by":"auto","created_at":"2025-12-05 16:46:34","extension":"html","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154486,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/de4a6f7fc43b80e8532864e8.html"},{"id":97549346,"identity":"aa832739-00d5-4e45-b457-81e84d969f3d","added_by":"auto","created_at":"2025-12-05 16:46:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":833406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCircARHGAP12 enhances cisplatin resistance in NPC cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e After knockdown or overexpression of circARHGAP12, NPC cells were treated with varying concentrations of cisplatin (0, 2, 4, 8, 16, and 32 μM) for 48 hours. Cell viability was assessed using the MTT assay, and the IC₅₀ values were calculated via nonlinear regression analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B–C)\u003c/strong\u003e Following knockdown or overexpression of circARHGAP12, NPC cells were exposed to different concentrations of cisplatin (0, 0.5, 1, 2, and 4 μM). Colony formation was evaluated by crystal violet staining after 7–10 days (B), and survival curves were fitted using a nonlinear regression model (C).\u003c/p\u003e\n\u003cp\u003eCisplatin, cis-diamminedichloroplatinum (II). Data are presented as mean ± standard deviation from at least 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, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; ns, not significant.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/ec18630acf91236b49336130.jpg"},{"id":97549349,"identity":"ef43eca2-3965-4537-bfa7-596e4dedc01b","added_by":"auto","created_at":"2025-12-05 16:46:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":818368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCircARHGAP12 promotes cisplatin resistance in NPC \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A–C)\u003c/strong\u003e Twenty-eight female BALB/c nude mice were randomly assigned to four groups (n = 7 per group). Two groups were subcutaneously injected with CNE2 cells overexpressing circARHGAP12, while the remaining two groups received CNE2 cells transfected with the empty vector (2 × 10⁶ cells per mouse). When tumor volumes reached 100–150 mm³, both the vector-control and circARHGAP12-overexpressing groups were further divided into cisplatin-treated (4 mg/kg) and saline-treated control subgroups. Mice were administered intraperitoneal injections of cisplatin or saline every 3 days, and tumor dimensions were monitored throughout the treatment period. After 21 days of cisplatin treatment, all mice were euthanized, and xenograft tumors were collected (A). Tumor weight (B) and tumor volume (C) were subsequently measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Tumor sections were subjected to H\u0026amp;E staining, \u003cem\u003ein situ\u003c/em\u003e hybridization to detect circARHGAP12 expression, and immunohistochemical analysis of Ki67. Scale bar = 200 μm；Scale bar = 100 μm；\u003c/p\u003e\n\u003cp\u003eCisplatin, cis-diamminedichloroplatinum (II). Data are presented as mean ± standard deviation from at least three independent experiments. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/6edf8d9dae17b20830d0870b.jpg"},{"id":97549347,"identity":"704c5ca4-8cf2-4c23-b088-29b366652053","added_by":"auto","created_at":"2025-12-05 16:46:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":754011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCircARHGAP12 inhibits cisplatin-induced alkaliptosis by upregulating CA9.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Following knockdown or overexpression of circARHGAP12, NPC cells were treated with cisplatin in combination with the alkaliptosis inducer JTC-801 or the alkaliptosis inhibitor NAC for 48 hours, and cell viability was assessed using the MTT assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e CA9 mRNA expression levels after circARHGAP12 knockdown or overexpression were quantified by RT-qPCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e CA9 protein expression under the same conditions was analyzed by western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e The expression of circARHGAP12 and CA9 in subcutaneous tumor tissues was examined using \u003cem\u003ein situ\u003c/em\u003ehybridization or immunohistochemistry, respectively. Scale bar = 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Extracellular pH was measured by a pH meter following circARHGAP12 knockdown or overexpression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Intracellular pH was determined using the BCECF-AM probe after circARHGAP12 knockdown or overexpression. The fluorescence intensity of BCECF-AM (green) was inversely correlated with intracellular pH. Cell nuclei were counterstained with Hoechst (blue). Scale bar = 10 μm.\u003c/p\u003e\n\u003cp\u003eCisplatin, cis-diamminedichloroplatinum (II). Data are presented as mean ± standard deviation from at least 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, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/6d13931d1899f2b56c073d37.jpg"},{"id":97673171,"identity":"8a5bff5d-6917-476d-a9b1-ba056e855e49","added_by":"auto","created_at":"2025-12-08 09:39:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":674128,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCircARHGAP12 upregulates CA9 through binding to G3BP1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e RNA pulldown assay was performed to identify proteins bound to biotin-labeled circARHGAP12, with unlabeled RNA (Unbio) used as a negative control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e RT-qPCR experiment was conducted to examine CA9 mRNA expression following knockdown or overexpression of G3BP1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e CA9 protein expression after G3BP1 knockdown or overexpression was analyzed by western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e RIP using an anti-G3BP1 antibody followed by RT-qPCR was carried out to assess the interaction between G3BP1 and circARHGAP12.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e RT-qPCR analysis of CA9 mRNA expression in NPC cells following circARHGAP12 overexpression and/or G3BP1 knockdown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e After circARHGAP12 overexpression and/or G3BP1 knockdown, NPC cells were treated with increasing concentrations of cisplatin (0, 2, 4, 8, 16, 32 μM) for 48 hours. Cell viability was determined using the MTT assay, and IC50 values were calculated via nonlinear regression analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Following circARHGAP12 overexpression and/or G3BP1 knockdown, NPC cells were treated with different concentrations of cisplatin (0, 0.5, 1, 2, 4 μM). Colony formation was assessed by crystal violet staining after 7–10 days (left), and survival curves were fitted using a nonlinear regression model (right).\u003c/p\u003e\n\u003cp\u003eCisplatin, cis-diamminedichloroplatinum (II). Data are presented as mean ± standard deviation from at least 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, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; ns, not significant.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/cf6cebbc50f822c316ce65e2.jpg"},{"id":97549355,"identity":"256ce042-10c3-42d6-be76-cfd86365cf84","added_by":"auto","created_at":"2025-12-05 16:46:33","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":599897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eG3BP1 binds to and stabilizes CA9 mRNA in an rG4-dependent manner.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e CA9 mRNA stability was analyzed by RT-qPCR in NPC cells treated with actinomycin D following G3BP1 overexpression and/or treatment with the rG4-binding competitor PDS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Dual-luciferase reporter assay comparing wild-type and mutant rG4 structures of CA9 under G3BP1 overexpression with or without PDS co-treatment. Firefly luciferase activity was normalized to Renilla luciferase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e After circARHGAP12 knockdown or overexpression, an RIP assay using an anti-G3BP1 antibody was performed to assess the enrichment of CA9 mRNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Colocalization of G3BP1 (green), circARHGAP12 (red), and CA9 mRNA (CA9-Cy3 probe, violet) was visualized by FISH. Cell nuclei were counterstained with DAPI (blue). Scale bar = 10 μm.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± standard deviation from at least 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, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; ns, not significant.\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/610ded3a039591e3b3e78a2f.jpg"},{"id":97670740,"identity":"9892ba21-b8bd-4b21-a5b8-c4c60c1da3a8","added_by":"auto","created_at":"2025-12-08 09:31:14","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":575454,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCircARHGAP12 promotes cisplatin-induced SG assembly.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Colocalization of circARHGAP12 (red) with G3BP1 (green), a marker of stress granules (SGs), was visualized by FISH following cisplatin treatment (250 μM). Cell nuclei were counterstained with DAPI (blue). Scale bar = 10 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Immunofluorescence analysis showing colocalization of BG4 (red), CA9 mRNA (CA9-Cy3 probe, violet), and SGs under cisplatin treatment (250 μM). Cell nuclei were counterstained with DAPI (blue). Scale bar = 10 μm. Colocalization was quantified using ImageJ. BG4, an rG4-specific antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Fluorescence recovery after photobleaching (FRAP) was performed to assess the dynamics of EGFP-G3BP1 (green) puncta under cisplatin treatment (250 μM). The effect of circARHGAP12 on G3BP1 condensate recovery was monitored in real time and quantified using ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Under cisplatin treatment, the effect of PDS on the colocalization of circARHGAP12 (red) and G3BP1 (green) was examined by FISH. Cell nuclei were counterstained with DAPI (blue). Scale bar = 10 μm. Cisplatin, cis-diamminedichloroplatinum (II).\u003c/p\u003e","description":"","filename":"16.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/d5a02568cda26c51ac9a9e0f.jpg"},{"id":97549358,"identity":"c7931891-7edc-4153-9407-cb1ec43575e1","added_by":"auto","created_at":"2025-12-05 16:46:33","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":262728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic model of circARHGAP12 promoting cisplatin resistance in NPC by suppressing alkaliptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCircARHGAP12 promotes stress granule (SG) assembly and inhibits cisplatin-induced alkaliptosis in NPC. Within SGs, circARHGAP12 exerts this function by binding to G3BP1 and, in an RNA G-quadruplex-dependent manner, upregulating CA9 expression. SGs temporarily sequester and protect mRNAs from degradation.\u003c/p\u003e","description":"","filename":"17.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/fce10a3686d269825d124c07.jpg"},{"id":100356095,"identity":"10c6856a-0a2a-4033-9807-3f505cb31fc5","added_by":"auto","created_at":"2026-01-16 06:51:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5826364,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/dd16e07f-3a9f-4251-bedc-8b53feaf5972.pdf"},{"id":97672400,"identity":"3c66d589-8258-4e30-b6aa-5c441717b77a","added_by":"auto","created_at":"2025-12-08 09:37:13","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":9945,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/0e0147f4b206ad2dc06d3d5b.xlsx"},{"id":97672712,"identity":"0e37044f-5baa-4c7c-ab59-f558a806930a","added_by":"auto","created_at":"2025-12-08 09:38:38","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9773,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/a760fa21d9b7705c076cdc35.xlsx"},{"id":97670763,"identity":"63f7246f-9b17-4137-8430-cf0299e0f002","added_by":"auto","created_at":"2025-12-08 09:31:18","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9593,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/ba6e951e07544ee3c99ba50f.xlsx"},{"id":97549356,"identity":"9289b173-16c1-4cf6-8fe5-2a3f7faf3a32","added_by":"auto","created_at":"2025-12-05 16:46:33","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":487293,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984659/v1/01a48aa36ece505a65f0bd54.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"CircARHGAP12 suppresses cisplatin-induced alkaliptosis in nasopharyngeal carcinoma through G3BP1-mediated upregulation of CA9 in an RNA G-quadruplex-dependent manner","fulltext":[{"header":"Background","content":"\u003cp\u003eCircular RNAs (circRNAs) have attracted increasing attention because of their unique covalently closed-loop structure and crucial roles in regulating cellular homeostasis\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Although numerous studies have demonstrated that aberrant expression of specific circRNAs is closely associated with tumor cell proliferation, invasion, and chemotherapy resistance\u003csup\u003e[\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, their precise mechanisms of action\u0026mdash;particularly the underlying molecular regulatory networks\u0026mdash;remain to be fully elucidated. Our previous study revealed that circARHGAP12 is highly expressed in nasopharyngeal carcinoma (NPC) tissues, where it specifically binds to the 3\u0026prime; untranslated region (3\u0026prime;UTR) of EZR mRNA, enhances its stability, strengthens the interaction among EZR, TPM3, and RhoA proteins, and consequently promotes NPC cell invasion and metastasis\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. However, whether circARHGAP12 exerts additional functions in NPC remains unclear.\u003c/p\u003e\u003cp\u003eNPC is a malignant tumor originating from the epithelial cells of the nasopharyngeal mucosa and is highly prevalent in Southeast Asia and Southern China\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Cis-diamminedichloroplatinum (II) (DDP, commonly known as cisplatin) remains the first-line chemotherapeutic agent for NPC treatment. Although cisplatin-based chemotherapy combined with radiotherapy has markedly improved the prognosis of patients with early-stage NPC, disease recurrence still occurs in 20%\u0026ndash;30% of those with advanced disease\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Notably, the emergence of cisplatin resistance severely compromises the efficacy of chemotherapy, resulting in higher tumor recurrence rates and poor clinical outcomes\u003csup\u003e[\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Therefore, elucidating the molecular mechanisms underlying cisplatin resistance is essential for optimizing therapeutic strategies and developing novel anticancer agents. Interestingly, we found that circARHGAP12 promotes cisplatin resistance in NPC\u0026mdash;a function that has not been previously reported\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. However, the precise mechanisms by which circARHGAP12 contributes to cisplatin resistance in NPC remain unexplored.\u003c/p\u003e\u003cp\u003eAlkaliptosis is a distinct form of cell death triggered by intracellular alkalinization\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Unlike other types of programmed cell death, it cannot be inhibited by conventional cell death blockers, as exemplified by JTC801-induced cell death\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. JTC801 suppresses the expression of carbonic anhydrase 9 (CA9), an enzyme that dynamically catalyzes the reversible hydration of carbon dioxide to bicarbonate and protons, thereby maintaining intracellular and extracellular pH homeostasis and acting as a negative regulator of alkaliptosis\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Although previous studies have shown that CA9 modulates the tumor microenvironment, counteracts cell death, and promotes tumor cell adaptation and survival\u003csup\u003e[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, the regulatory mechanisms governing CA9 expression and its specific relationship with chemotherapy resistance in NPC are unknown.\u003c/p\u003e\u003cp\u003eIn this study, we demonstrated for the first time, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, that circARHGAP12 induces cisplatin resistance in NPC cells. Further mechanistic analyses revealed that this effect is mediated by the suppression of alkaliptosis. We found that circARHGAP12 binds to G3BP stress granule assembly factor 1 (G3BP1) to promote stress granule formation, thereby stabilizing CA9 expression in an RNA G-quadruplex (rG4)-dependent manner and establishing a signaling axis that suppresses alkaliptosis. These findings uncover a novel role of circARHGAP12 in mediating cisplatin resistance in NPC and highlight a potential therapeutic target for overcoming drug resistance in cancer treatment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eCell culture and transfection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt the institute of Cancer Research, Central South University, the NPC cell lines (CNE2, HNE2) were maintained. And all cell lines were confirmed to be free of mycoplasma contamination by using mycoplasma detection kit (GENESEED). NPC cells were cultured in a humidified incubator at 37\u0026deg;C with 5% CO₂ in RPMI-1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (OriCell, Guangzhou, China) and 1% penicillin\u0026ndash;streptomycin (Life Technologies). Plasmid transfections were performed using Neofect (Neofect Biotech, Beijing, China), while siRNA transfections were carried out using HiPerFect (Qiagen, Shanghai, China). The sequences of the siRNAs are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eAnimal experiments\u003c/h3\u003e\n\u003cp\u003eFemale BALB/c nude mice (4 weeks old, weighing 15\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g) used in this study were purchased from the Experimental Animal Center of Central South University. All mice were maintained under specific pathogen-free (SPF) conditions at the Central South University animal facility. The experimental protocol was approved by the Animal Ethics Committee of Central South University. A total of 28 mice were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;7 per group). Two groups were subcutaneously injected with CNE2 cells overexpressing circARHGAP12, while the other two groups received CNE2 cells transfected with the empty vector (2 \u0026times; 10⁶ cells per mouse). When tumor volumes reached 100\u0026ndash;150 mm\u0026sup3; (approximately day 7), one circARHGAP12 group and one control group were treated with cisplatin (4 mg/kg, intraperitoneally) every 3 days, and the remaining two groups received saline. Tumor volume and body weight were recorded every 3 days. After 21 days of cisplatin treatment, the mice were euthanized, and tumors were excised, weighed, and subjected to histopathological analysis.\u003c/p\u003e\n\u003ch3\u003ePlasmids\u003c/h3\u003e\n\u003cp\u003eThe circARHGAP12 (has_circ_0000231) overexpression plasmid was constructed by inserting the full-length sequence of circARHGAP12 into the pcDNA3.1(+) CircRNA Mini Vector (a gift from Professor Yong Li, Baylor College of Medicine). The insert was flanked by complementary sequences to promote back-splicing and enhance circular RNA expression efficiency. The plasmids pEGFP-G3BP1, pMIR-Report Luciferase-CA9-3\u0026prime;UTR (WT), and pMIR-Report Luciferase-CA9-3\u0026prime;UTR (MUT) were purchased from Tsingke (Beijing, China). The sequences of the PCR primers are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eRNA extraction and RT-qPCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from NPC cells using TRIzol (Life, USA). Reverse trancription was carried out with the HiScript II Q RT SuperMix for qPCR kit (Vazyme, Nanjing, China). Quantitative real-time PCR (RT-qPCR) was then performed on a CFX96\u0026trade; Real-Time PCR Detection System (Bio-Rad, USA) using 2\u0026times; SYBR Green qPCR Master Mix (Bimake, USA). Ct values of target genes were normalized to those of the reference gene, and relative expression levels were calculated using the 2⁻ΔΔCt method. The sequences of all primers used are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blotting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were lysed 48 hours after transfection using RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (KeyGen, Jiangsu, China). Following centrifugation, protein concentrations were determined using a BCA assay kit. Equal amounts of protein were separated by SDS-PAGE and transferred onto 0.2 \u0026micro;m PVDF membranes. After blocking with 5% skim milk, the membranes were incubated overnight at 4\u0026deg;C with primary antibodies against CA9, G3BP1, NONO, PTBP1, or GAPDH (Proteintech, Wuhan, China). The membranes were then incubated with the appropriate HRP-conjugated secondary antibody (Proteintech) for approximately 2 hours at room temperature. Protein bands were visualized using an ECL detection device (Millipore, USA). All antibodies used are listed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA immunoprecipitation (RIP)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were washed with PBS and lysed in RIP buffer (Merck, Germany) supplemented with protease inhibitors. The lysates were incubated with magnetic beads (Merck, Germany) conjugated to either anti-G3BP1 or control IgG antibodies. After four washes with high-salt buffer (700 mM NaCl), RNA was extracted from the immunoprecipitated protein\u0026ndash;RNA complexes using TRIzol reagent and subsequently analyzed by RT-qPCR.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA pulldown\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were lysed 48 hours after transfection using RIP-buffer (150mM KCl, 25mM Tris-HCl, 0.5% NP40, 0.5mM DDT). Biotin-labeled circARHGAP12 probes (Sangon Biotech, Shanghai, China) and streptavidin magnetic beads (Thermo Fisher, USA) were incubated with cell lysates at 4\u0026deg;C overnight. After six washes, the pulled-down proteins were analyzed by LC-MS/MS (PTM Bio, Hangzhou, China) or Western blotting. The probe sequences are listed in Supplementary Table\u0026nbsp;1, and the identified interacting proteins are shown in Supplementary Table\u0026nbsp;3.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLiquid chromatography‑mass spectrometry (LC-MS/MS)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify circARHGAP12-interacting proteins, cell lysates were incubated with biotin-labeled probes and biotin-affinity magnetic beads overnight at 4\u0026deg;C to pull down circARHGAP12-associated proteins. The immunoprecipitated proteins were separated by SDS-PAGE and analyzed by LC-MS/MS using an Ultimate 3000 RSLC nanoliter liquid chromatography system (Dionex, CA, USA) coupled to an LTQ Orbitrap Velos Pro mass spectrometer (Thermo Scientific, USA). The resulting data were searched against the SwissProt human database using Proteome Discoverer 2.4. Trypsin digestion was specified, allowing up to two missed cleavages. The mass error tolerance was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Cysteine alkylation was set as a fixed modification, while methionine oxidation and N-terminal acetylation were set as variable modifications. Peptides were required to have a score greater than 20, and only identifications with a high confidence level were considered.\u003c/p\u003e\u003cp\u003e\u003cb\u003eActinomycin D treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNPC cells were seeded in 12-well plates and cultured to an appropriate density to ensure growth in the logarithmic phase. To inhibit new RNA synthesis, actinomycin D (Abmole, Shanghai, China) was added to fresh culture medium at a final concentration of 5 \u0026micro;g/mL. Cells were collected at 0, 2, 4, and 6 hours after treatment. Total RNA was extracted using TRIzol, and equal amounts of RNA were subjected to reverse transcription for RT-qPCR analysis. The remaining RNA at each time point was normalized to the initial amount at 0 hours to assess RNA stability, and data were analyzed using nonlinear regression with a one-phase exponential decay model in GraphPad Prism.\u003c/p\u003e\u003cp\u003e\u003cb\u003eColony formation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were transfected with either a circARHGAP12 overexpression plasmid or circARHGAP12 siRNA, with empty vector or scrambled siRNA serving as controls, respectively. Subsequently, 2,000 cells were seeded into 12-well plates and cultured at 37\u0026deg;C for 24 hours. Cells were then treated with cisplatin at final concentrations of 0, 0.5, 1, 2, or 4 \u0026micro;M, followed by incubation at 37\u0026deg;C for 8\u0026ndash;12 days. After staining with 0.1% crystal violet, the number of colonies in each group was counted using ImageJ. Survival curves were generated by fitting the data with a nonlinear regression model.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMTT\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were transfected with specific plasmids or siRNAs. At 36 hours post-transfection, 4,000 cells per well were seeded into 96-well plates. After cell adhesion, cells were treated for 48 hours with the following inducers: the apoptosis inducer ABT-263 (AbMole, Shanghai, China, 1 \u0026micro;M), the necroptosis inducer 5-FU (AbMole, 1 \u0026micro;M), the autophagy inducer Rapamycin (AbMole, 1 \u0026micro;M), the ferroptosis inducer RSL3 (Selleck, Shanghai, China, 2 \u0026micro;M), and the alkaliptosis inducer JTC-801 (TargetMol, Shanghai, China, 1 \u0026micro;M). For cisplatin sensitivity assays, cells were treated with cisplatin at concentrations of 0, 2, 4, 8, 16, and 32 \u0026micro;M for 48 hours. Following treatment, MTT reagent was added for 4 hours, after which the medium was removed and DMSO was added to solubilize the formazan crystals. Absorbance was measured at 490 nm using a microplate reader, and IC₅₀ curves were generated to assess cell sensitivity to cisplatin.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFluorescence\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003ehybridization (FISH)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, and blocked with 5% BSA. Cells were then incubated overnight at 4\u0026deg;C with primary antibodies (anti-G3BP1, anti-BG4) or fluorescent probes (Cy3-circARHGAP12, Cy5-CA9; 5 ng/\u0026micro;L each), followed by incubation with species-matched secondary antibodies (Alexa Fluor 488 Anti-Mouse, Alexa Fluor 594 Anti-Mouse, FITC Anti-Goat) for 1 hour at 37\u0026deg;C. Nuclei were stained with DAPI for 10 minutes. Images were captured using a confocal laser scanning microscope. Probe sequences are provided in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eLive cell immunofluorescence\u003c/h3\u003e\n\u003cp\u003eCells were transfected with the EGFP-G3BP1 plasmid. After 24 hours, cells were seeded into 35 mm glass-bottom dishes. Once the cells adhered and spread fully, they were treated with 250 \u0026micro;M cisplatin (DDP) for 1 hour. The dishes were then placed on the stage of a confocal laser scanning microscope, and 10% 1,6-hexanediol was added to monitor the dynamic changes of fluorescent puncta in real time.\u003c/p\u003e\n\u003ch3\u003eFluorescence recovery after photobleaching (FRAP)\u003c/h3\u003e\n\u003cp\u003eCells expressing EGFP-G3BP1 were seeded in 35 mm glass-bottom dishes and treated with 250 \u0026micro;M cisplatin for 1 hour after full adhesion. Fluorescent puncta were then photobleached using a 488 nm laser at 50% power on a Leica confocal microscope. Images were acquired every 3 seconds following bleaching. Recovery data were subsequently quantified and analyzed using GraphPad Prism 10.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn situ\u003c/b\u003e \u003cb\u003ehybridization (ISH) and Immunohistochemistry (IHC)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression of circARHGAP12 in tumor tissues from animals was detected using an \u003cem\u003ein situ\u003c/em\u003e hybridization assay kit (Boster, Wuhan, China) following the manufacturer\u0026rsquo;s instructions. The digoxin-labeled probe was diluted to 10 \u0026micro;M and incubated with tumor sections overnight at 37\u0026deg;C. Nonspecific binding was removed by washing with saline-sodium citrate (SSC) buffer. Staining was developed using DAB, followed by hematoxylin counterstaining. Sections were then dehydrated through a graded alcohol series, mounted, and examined under an inverted microscope. Probe sequences are provided in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003eParaffin-embedded tissue sections were dewaxed and rehydrated through a graded ethanol series. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide, and antigen retrieval was performed in boiling water. After rinsing three times with PBS, sections were blocked with goat serum for 1 hour and then incubated overnight at 4\u0026deg;C with primary antibodies against Ki67 or CA9. Following PBS washes, sections were incubated with the appropriate secondary antibody at 37\u0026deg;C for 10 minutes, developed with DAB, and counterstained with hematoxylin. The sections were then dehydrated through a graded alcohol series, mounted, and examined under an optical microscope. A semi-quantitative scoring system was used to evaluate staining based on intensity and the percentage of positive cells. Staining intensity was scored as 0 (no staining), 1 (light yellow), 2 (light brown), or 3 (dark brown), and the percentage of positive cells was scored as 0 (\u0026lt;\u0026thinsp;25%), 1 (25\u0026ndash;50%), 2 (50\u0026ndash;75%), or 3 (\u0026gt;\u0026thinsp;75%). The final composite score was calculated by multiplying the intensity score by the percentage score. Samples with a composite score\u0026thinsp;\u0026gt;\u0026thinsp;5 were classified as high expression, while those with a score\u0026thinsp;\u0026le;\u0026thinsp;5 were considered low expression.\u003c/p\u003e\n\u003ch3\u003eRNA sequencing (RNA-seq)\u003c/h3\u003e\n\u003cp\u003eRNA-Seq analysis was performed on CNE2 cells transfected with siNC or sicircARHGAP12 (n\u0026thinsp;=\u0026thinsp;2) to identify mRNAs regulated by circARHGAP12. Total RNA was extracted from cell samples using TRIzol reagent, and residual DNA was removed with the Turbo DNase kit (Ambion). RNA was subsequently purified using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). RNA purity and integrity were assessed by NanoDrop spectrophotometry (Thermo Fisher Scientific, CA, USA) and agarose gel electrophoresis. Library construction for RNA-Seq was performed by Oebiotech Inc. (Shanghai, China) using the NEBNext\u0026reg; Ultra\u0026trade; Directional RNA Library Preparation Kit. RNA samples were fragmented at high temperature in the presence of divalent cations. Ribosomal RNA was removed by comparison against multiple databases using SortMeRNA. Differentially expressed genes were identified using DESeq2.\u003c/p\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data analyses and graph generation were performed using GraphPad Prism 10.0. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences between two groups were evaluated using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, while comparisons among multiple groups were conducted using one-way ANOVA followed by appropriate post-hoc tests. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eCircARHGAP12 enhances cisplatin resistance in NPC cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCisplatin-based chemotherapy remains the primary clinical treatment for NPC, with multiple clinical trials demonstrating favorable therapeutic outcomes\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. However, a subset of patients still develops chemotherapy resistance under current treatment regimens\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Our previous work showed that circARHGAP12 promoted migration and invasion of NPC\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, nevertheless, its broader biological functions remain incompletely characterized. To investigate whether circARHGAP12 contributes to cisplatin resistance in NPC, we knocked down or overexpressed circARHGAP12 in CNE2 and HNE2 cells and treated them with cisplatin at concentrations of 0, 2, 4, 8, 16, and 32 \u0026micro;M. Cell survival was assessed using MTT assays, and IC₅₀ values were calculated by fitting survival curves with a dose\u0026ndash;response nonlinear regression model. The results showed that, compared to controls, IC₅₀ values significantly decreased following circARHGAP12 knockdown and increased markedly upon circARHGAP12 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next performed a colony formation assay to evaluate the effect of circARHGAP12 on the clonogenic potential of NPC cells under cisplatin treatment. CNE2 and HNE2 cells were treated with low concentrations of cisplatin (0, 0.5, 1, 2, 4 \u0026micro;M), and survival curves were fitted using a dose\u0026ndash;response nonlinear regression model. In the absence of cisplatin (0 \u0026micro;M), circARHGAP12 had minimal impact on colony formation. However, under cisplatin treatment, circARHGAP12 knockdown significantly reduced clonogenic capacity, whereas its overexpression produced the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). These results indicate that circARHGAP12 promotes cisplatin resistance in NPC \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCircARHGAP12 promotes cisplatin resistance in NPC\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further validate the role of circARHGAP12 in promoting cisplatin resistance in NPC, we established a subcutaneous xenograft model in nude mice using CNE2 cells. Mice were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;7) and subcutaneously injected with CNE2 cells overexpressing circARHGAP12 or empty vector (2 \u0026times; 10⁶ cells per mouse). Once palpable tumors formed, both circARHGAP12-overexpressing and control groups were further subdivided to receive either cisplatin treatment (4 mg/kg) or saline. Mice were administered intraperitoneal injections every three days, and tumor growth was monitored throughout the treatment period. The results showed that circARHGAP12 overexpression alone did not affect tumor growth. However, under cisplatin treatment, circARHGAP12-overexpressing tumors exhibited significantly larger volumes and weights compared to controls, markedly attenuating cisplatin-induced tumor growth inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). \u003cem\u003eIn situ\u003c/em\u003e hybridization confirmed higher circARHGAP12 expression in the overexpression group, while immunohistochemistry revealed that circARHGAP12 overexpression did not affect the proliferation marker Ki67 in the absence of cisplatin but substantially suppressed Ki67 expression under cisplatin therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These findings demonstrate that circARHGAP12 promotes cisplatin resistance in NPC \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eCircARHGAP12 inhibits cisplatin-induced alkaliptosis by upregulating CA9\u003c/h3\u003e\n\u003cp\u003eCisplatin-induced cell death has been reported to occur via apoptosis, necroptosis, ferroptosis, autophagy, and alkaliptosis\u003csup\u003e[\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. To explore the specific mechanism by which circARHGAP12 regulates cisplatin resistance, NPC cells with circARHGAP12 knockdown or overexpression were treated with specific inducers: ABT-263 (apoptosis), 5-FU (necroptosis), Rapamycin (autophagy), RSL3 (ferroptosis), and JTC-801 (alkaliptosis)\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, followed by assessment of cell survival using MTT assays. The results showed that circARHGAP12 modulation did not affect apoptosis, necroptosis, autophagy, or ferroptosis. However, upon treatment with the alkaliptosis inducer JTC-801, circARHGAP12 knockdown significantly increased cell death, whereas overexpression produced the opposite effect (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). To determine whether circARHGAP12 promotes cisplatin resistance by suppressing alkaliptosis, we examined its effect on cisplatin-induced cytotoxicity in the presence of an alkaliptosis inducer or inhibitor. MTT assays revealed that circARHGAP12 knockdown enhanced cisplatin-induced cell death, which was rescued by the alkaliptosis inhibitor NAC\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Conversely, circARHGAP12 overexpression attenuated cisplatin-induced cytotoxicity, and this effect was reversed by treatment with the alkaliptosis inducer JTC-801 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These findings indicate that circARHGAP12 enhances cisplatin resistance in NPC cells by inhibiting alkaliptosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate how circARHGAP12 inhibits alkaliptosis in NPC cells, we knocked down circARHGAP12 in CNE2 cells (using si-scramble as control) and performed RNA-seq (n\u0026thinsp;=\u0026thinsp;2). KEGG pathway enrichment analysis of differentially expressed genes (|log₂FC| \u0026gt;1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) revealed that genes in the nitrogen metabolism pathway (mainly consist of carbonic anhydrase family) were most significantly affected by circARHGAP12 (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB-C\u003c/b\u003e). Further analysis of this pathway identified CA9 as the gene most significantly regulated by circARHGAP12 knockdown (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD\u003c/b\u003e). Analysis of CA9 expression in head and neck squamous cell carcinoma (HNSC) using the TCGA database via GEPIA2 showed that CA9 was significantly upregulated in HNSC tissues compared to normal controls (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE\u003c/b\u003e), suggesting a potential pro-tumorigenic role. RT-qPCR and western blotting confirmed that circARHGAP12 knockdown reduced CA9 mRNA and protein levels, whereas overexpression increased CA9 expression, consistent with RNA-seq results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). Immunohistochemistry of subcutaneous tumors in nude mice further demonstrated that circARHGAP12 overexpression promoted CA9 expression, while cisplatin treatment reduced it. Semi-quantitative scoring indicated a positive correlation between circARHGAP12 and CA9 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). As a member of the carbonic anhydrase family, CA9 regulates cellular acid\u0026ndash;base balance by catalyzing the reversible reaction CO₂ + H₂O ⇌ H⁺ + HCO₃⁻, thereby suppressing alkaliptosis induced by intracellular and extracellular pH dysregulation\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. BCECF, a neutral lipophilic bis-carboxyfluorescein derivative, freely diffuses across the plasma membrane and is hydrolyzed by intracellular esterases to retain fluorescence within the cytoplasm. Its fluorescence intensity is inversely correlated with pH\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Using both a pH meter and BCECF-AM probe, we measured intracellular and extracellular pH. CircARHGAP12 knockdown increased intracellular pH and decreased extracellular pH, whereas overexpression had the opposite effect, decreasing intracellular pH and increasing extracellular pH (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. \u003cb\u003eE-F\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTo determine whether circARHGAP12 suppresses cisplatin-induced alkaliptosis in NPC cells via CA9, we performed colony formation assays to assess cell proliferation. Compared to the control group (siNC\u0026thinsp;+\u0026thinsp;Vector), CA9 knockdown significantly reduced the clonogenic ability of NPC cells, whereas circARHGAP12 overexpression enhanced it. Notably, simultaneous circARHGAP12 overexpression and CA9 knockdown restored clonogenic capacity to levels comparable with the control group (siNC\u0026thinsp;+\u0026thinsp;Vector) (\u003cb\u003eFig. S2A\u003c/b\u003e). MTT assay yielded consistent results. (\u003cb\u003eFig. S2B\u003c/b\u003e). These findings indicate that circARHGAP12 inhibits cisplatin-induced alkaliptosis in NPC cells by upregulating CA9 expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eCircARHGAP12 upregulates CA9 through binding to G3BP1\u003c/h3\u003e\n\u003cp\u003eThe most well-established function of circRNAs is acting as competing endogenous RNAs (ceRNAs) that sequester miRNAs to regulate target gene expression\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. To explore the mechanism by which circARHGAP12 regulates CA9 mRNA, we used the online databases circinteractome and miRcode to predict miRNAs that could bind both circARHGAP12 and the CA9 3\u0026prime;UTR. No shared miRNAs were identified (\u003cb\u003eFig. S3A\u003c/b\u003e), suggesting that circARHGAP12 likely regulates CA9 through alternative mechanisms, such as interactions with RNA-binding proteins (RBPs)\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. We next performed RNA pulldown coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify potential circARHGAP12-binding proteins (\u003cb\u003eFig. S3B-C\u003c/b\u003e). Among the top candidates, G3BP1, NONO, and PTBP1 were confirmed to bind biotin-labeled circARHGAP12 by RNA pulldown validation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Western blotting indicated that circARHGAP12 did not affect the expression levels of these RBPs (\u003cb\u003eFig. S3D\u003c/b\u003e), and RT-qPCR showed that knockdown of G3BP1, NONO, or PTBP1 did not alter circARHGAP12 expression (\u003cb\u003eFig. S3E\u003c/b\u003e), suggesting that these proteins do not influence circARHGAP12 biogenesis. Collectively, these findings indicate that circARHGAP12 and its binding proteins do not directly regulate each other.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate whether G3BP1, NONO, or PTBP1 might serve as molecular scaffolds through which circARHGAP12 regulates CA9 mRNA, we first assessed CA9 expression following modulation of these RBPs. RT-qPCR and western blotting showed that G3BP1 knockdown reduced both CA9 mRNA and protein levels, whereas G3BP1 overexpression increased CA9 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). In contrast, knockdown of NONO or PTBP1 had no effect on CA9 expression (\u003cb\u003eFig. S3F-G\u003c/b\u003e), indicating G3BP1 as the primary downstream candidate. Nuclear-cytoplasmic fractionation assays revealed that circARHGAP12 overexpression or knockdown did not alter G3BP1 subcellular localization (\u003cb\u003eFig. S3H\u003c/b\u003e), supporting its potential role as a scaffold for CA9 regulation. RIP assays further demonstrated that G3BP1 antibody enriched significantly more circARHGAP12 than the IgG control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), consistent with the RNA pulldown results.\u003c/p\u003e\u003cp\u003eRT-qPCR and western blotting showed that, compared with the control group (siNC\u0026thinsp;+\u0026thinsp;Vector), G3BP1 knockdown reduced CA9 mRNA and protein levels, whereas circARHGAP12 overexpression increased them. Notably, simultaneous circARHGAP12 overexpression and G3BP1 knockdown restored CA9 expression to levels comparable with the control group (siNC\u0026thinsp;+\u0026thinsp;Vector) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cb\u003eFig. S3I\u003c/b\u003e). To assess whether circARHGAP12 suppresses cisplatin-induced alkaliptosis in NPC cells via G3BP1, cell viability was measured by MTT assay. G3BP1 knockdown decreased the IC₅₀ of NPC cells, while circARHGAP12 overexpression increased it. Concurrent circARHGAP12 overexpression and G3BP1 knockdown restored the IC₅₀ to levels similarly to the control group (siNC\u0026thinsp;+\u0026thinsp;Vector) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Colony formation assays yielded consistent results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). These findings indicate that circARHGAP12 inhibits cisplatin-induced alkaliptosis in NPC cells by binding to G3BP1 and upregulating CA9 expression.\u003c/p\u003e\n\u003ch3\u003eG3BP1 binds to and stabilizes CA9 mRNA in an rG4-dependent manner\u003c/h3\u003e\n\u003cp\u003eG-quadruplexes are three-dimensional structures formed by guanine-rich sequences, present in both DNA and RNA, collectively referred to as G4 structures. Those specifically in RNA are termed rG4s\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. G3BP1 exhibits high selectivity and affinity for rG4s, enabling it to recognize and bind rG4 motifs in the 3\u0026prime;UTR of target mRNAs, thereby preventing their degradation\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Based on these properties, we used the online tool QGRS to predict potential rG4 structures in the CA9 3\u0026prime;UTR (\u003cb\u003eFig. S4A-B\u003c/b\u003e). Pyridostatin (PDS) is a small-molecule ligand that binds and stabilizes G4 structures\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. PDS can disrupt the interaction between G3BP1 and rG4, displacing G3BP1 from target mRNAs and promoting mRNA degradation due to loss of G3BP1-mediated protection\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. To determine whether G3BP1 regulates CA9 mRNA via rG4s, we performed actinomycin D-based RNA stability assays. G3BP1 overexpression enhanced CA9 mRNA stability, whereas PDS treatment reduced it. Importantly, CA9 mRNA stability was restored to control levels when G3BP1 was overexpressed following PDS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To confirm rG4 dependence, we designed a mutant CA9 3\u0026prime;UTR in which guanine-to-adenine substitutions (GG \u0026rarr; AA) disrupted the rG4-forming region (\u003cb\u003eFig. S4C\u003c/b\u003e) and cloned it into a dual-luciferase reporter system. Dual-luciferase assays showed that G3BP1\u0026rsquo;s regulatory effect on CA9 mRNA was abolished with the mutant 3\u0026prime;UTR, indicating that G3BP1 stabilizes CA9 mRNA in an rG4-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Furthermore, RT-qPCR-based actinomycin D assays under cisplatin treatment revealed that circARHGAP12 enhanced CA9 mRNA stability, whereas circARHGAP12 knockdown decreased it (\u003cb\u003eFig. S4D\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether circARHGAP12 influences the binding of G3BP1 to CA9 mRNA, we assessed G3BP1\u0026ndash;CA9 mRNA interactions following circARHGAP12 knockdown or overexpression in NPC cells. RIP assays revealed that circARHGAP12 enhanced the association between G3BP1 and CA9 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Additionally, FISH analysis showed clear colocalization of G3BP1, circARHGAP12, and CA9 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results indicate that G3BP1 binds to the CA9 3\u0026prime;UTR in an rG4-dependent manner to stabilize CA9 expression, and that circARHGAP12 facilitates this interaction.\u003c/p\u003e\n\u003ch3\u003eCircARHGAP12 promotes cisplatin-induced Stress Granule (SG) assembly and stabilizes rG4 structures\u003c/h3\u003e\n\u003cp\u003eG3BP1 is the core assembly component of SGs\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. SGs are dynamic, membraneless organelles formed through liquid-liquid phase separation (LLPS) in response to various cellular stresses. The components within SGs are transiently segregated from the cytoplasm and can be re-released once the stress subsides\u003csup\u003e[\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown that SGs play a crucial role in helping tumor cells adapt to diverse stress conditions\u003csup\u003e[\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. To investigate whether circARHGAP12 influences SG assembly by binding to G3BP1, FISH analysis revealed that in the absence of cisplatin, knockdown or overexpression of circARHGAP12 did not alter the diffuse cytoplasmic distribution of G3BP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cb\u003eFig. S5A\u003c/b\u003e). However, upon cisplatin treatment, SGs\u0026mdash;characterized by G3BP1-positive LLPS condensates\u0026mdash;were assembled within cells, where circARHGAP12 exhibited strong colocalization with these granules and promoted their formation. Using sodium arsenite-induced SGs as a positive control\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e, we further confirmed that circARHGAP12 colocalized with G3BP1 within SGs and enhanced their assembly (\u003cb\u003eFig. S5B\u003c/b\u003e). As reported, RNA G-quadruplex (rG4) structures are often enriched within SGs, and RBP\u0026ndash;rG4 interactions are essential for SG formation\u003csup\u003e[\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. BG4, a specific antibody recognizing rG4 structures, can be used to visualize rG4-containing mRNAs in cells. To explore the relationship between rG4 and cisplatin-induced SG assembly, FISH assays demonstrated that CA9 mRNA and its associated rG4 structures showed marked colocalization with G3BP1 in cisplatin-induced SGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These findings suggest that SGs may play a critical role in sequestering and stabilizing CA9 mRNA under cisplatin stress.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs SGs are membraneless organelles formed in the cytoplasm through LLPS under stress conditions, they can transiently sequester mRNAs and proteins\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. To visualize SG dynamics, we constructed an EGFP-G3BP1 vector for live-cell imaging. Under cisplatin treatment, EGFP-G3BP1 formed distinct condensates in NPC cells. Treatment with 1,6-hexanediol, a chemical disruptor of phase separation, led to the gradual dissolution of these EGFP-G3BP1 foci, confirming that cisplatin induces G3BP1-dependent phase separation (\u003cb\u003eFig. S5C\u003c/b\u003e). FRAP assays further revealed that the fluorescence recovery rate of EGFP-G3BP1 foci was significantly faster in cisplatin-treated circARHGAP12-overexpressing cells compared with controls, indicating enhanced SG dynamics (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cb\u003eFig. S5D\u003c/b\u003e). Moreover, FISH analysis showed that circARHGAP12 promoted the assembly of G3BP1-marked SGs upon cisplatin induction, whereas this effect was reversed by PDS\u0026mdash;a small molecule that competitively binds rG4 structures and disrupts their interaction with G3BP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cb\u003eFig. S5E\u003c/b\u003e). Under cisplatin treatment, G3BP1, rG4 structures, and CA9 mRNA exhibited strong colocalization within SGs; this colocalization was markedly reduced following G3BP1 knockdown, which also impaired SG formation (\u003cb\u003eFig. S5F\u003c/b\u003e). Together, these results indicate that under cisplatin-induced stress, circARHGAP12 promotes the assembly and phase separation of G3BP1-marked SGs, thereby protecting and stabilizing CA9 mRNA within SGs in an rG4-dependent manner through G3BP1.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe treatment of NPC, particularly in patients with advanced disease, relies heavily on cisplatin-based chemotherapy regimens\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. However, the development of cisplatin resistance frequently results in treatment failure and tumor recurrence, posing a major clinical challenge\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Therefore, elucidating the molecular mechanisms underlying cisplatin resistance is of great importance for overcoming this obstacle. Traditionally, the antitumor effects of cisplatin have been attributed to its ability to induce DNA damage and trigger apoptosis\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. Recent studies, however, have demonstrated that cisplatin can also exert cytotoxicity through alternative cell death pathways such as ferroptosis and autophagy\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. In this study, we provide the first evidence that cisplatin can induce alkaliptosis in NPC cells. Furthermore, we identify circARHGAP12 as a novel and essential driver of cisplatin resistance in NPC, acting through the upregulation of CA9 to suppress alkaliptosis.\u003c/p\u003e\u003cp\u003eAlkaliptosis is a recently identified form of regulated cell death characterized by an increase in intracellular pH\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Unlike other programmed cell death pathways, alkaliptosis induced by elevated intracellular pH cannot be prevented by existing cell death inhibitors. The compound JTC801 triggers alkaliptosis by suppressing CA9 expression, thereby disrupting intracellular and extracellular pH homeostasis. CA9 functions as a pH regulator by catalyzing the reversible hydration of carbon dioxide to form bicarbonate and protons, helping to maintain acid-base balance and prevent alkaliptosis. In this study, we found that cisplatin treatment induces alkaliptosis in NPC cells. Moreover, we demonstrated that circARHGAP12 upregulates CA9 expression to suppress cisplatin-induced alkaliptosis. As a newly defined form of cell death, the relationship between alkaliptosis and chemotherapy remains poorly understood\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Our findings suggest that pharmacological induction of alkaliptosis\u0026mdash;for instance, through JTC801\u0026mdash;may enhance the antitumor efficacy of cisplatin, providing a potential therapeutic strategy for NPC. However, whether alkaliptosis represents a direct target of cisplatin or interacts with other cell death pathways warrants further investigation. Mechanistically, we identified a novel regulatory axis in which circARHGAP12 cooperates with G3BP1 under cisplatin stress to promote the assembly of SGs. These granules create a protective microenvironment that enriches rG4 structures within the CA9 mRNA 3\u0026prime;UTR. Acting as a molecular scaffold, G3BP1 stabilizes CA9 mRNA within SGs, leading to elevated CA9 expression, suppression of alkaliptosis, and ultimately enhanced cisplatin resistance.\u003c/p\u003e\u003cp\u003eStress granules (SGs) serve as a crucial adaptive mechanism that enables cells to withstand environmental stress, and their emerging link to chemotherapy resistance has garnered increasing attention. SGs are dynamic, membrane-less condensates that transiently assemble through LLPS in response to stress and are primarily composed of untranslated mRNAs and RBPs\u003csup\u003e[\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. By transiently inhibiting translation and preventing mRNA degradation, SGs facilitate cellular adaptation to adverse conditions\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. Dysregulation of G3BP1, a core SG assembly factor, has been implicated in tumor initiation, progression, and therapeutic resistance\u003csup\u003e[\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. In this study, we found that circARHGAP12 binds to G3BP1 and cooperates with cisplatin to promote SG assembly, revealing a novel role for circARHGAP12 in cisplatin-induced SG formation. Moreover, we observed enrichment of G3BP1-bound CA9 3\u0026prime;UTR rG4 structures within SGs. This finding suggests that, beyond their classical role in translational repression, SGs may contribute to chemotherapy resistance in NPC by sequestering RNA\u0026ndash;protein complexes. Such sequestration could protect key cisplatin resistance\u0026ndash;related mRNAs, such as CA9, from degradation during stress, thereby maintaining CA9 expression and suppressing alkaliptosis. Notably, cisplatin-induced SGs exhibited slower disassembly kinetics compared with classical SGs (e.g., those formed under oxidative stress)\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e. Consequently, the fate of these SGs\u0026mdash;and how their delayed disassembly influences the reinitiation of translation once cisplatin stress subsides\u0026mdash;warrants further investigation.\u003c/p\u003e\u003cp\u003eRNA G-quadruplexes (rG4s) are unique secondary structures formed by the stacking of guanine tetrads within guanine-rich RNA sequences. These structures can form in various regions of mRNAs, including the 5\u0026prime;UTR, 3\u0026prime;UTR, and coding sequences, where they play pivotal roles in regulating gene expression\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. The formation and function of rG4s are typically modulated by rG4-binding proteins (G4BPs), such as TMPRSS2 and G3BP1\u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. Notably, G3BP1 exhibits high affinity and specificity for rG4 structures located in mRNA 3\u0026prime;UTRs. This interaction is mediated by its C-terminal RGG domain and further stabilized by the RNA recognition motif (RRM) domain\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. In this study, we identified the rG4 structure within the CA9 mRNA 3\u0026prime;UTR as a critical site for G3BP1 binding and for maintaining CA9 mRNA stability\u0026mdash;a mechanism reminiscent of the rG4-dependent regulation of PITX1 mRNA by G3BP1\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Importantly, our findings reveal for the first time that circRNAs can enhance the recognition and binding of rG4 structures by RBPs, thereby stabilizing downstream target mRNAs. This discovery expands the biological significance of rG4s: beyond serving as independent regulatory elements, rG4s may act as scaffolding platforms for circRNA\u0026ndash;protein\u0026ndash;mRNA ternary complex formation. Such a mechanism suggests that rG4-dependent G3BP1-mediated transcriptomic regulation may be far more extensive than previously recognized. Furthermore, small-molecule compounds targeting rG4 structures\u0026mdash;such as 2a and 2b, which suppress KRAS translation and inhibit tumor progression in pancreatic cancer\u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e\u0026mdash;highlight the therapeutic potential of this regulatory mechanism. Analogously, small molecules that specifically target the CA9 3\u0026prime;UTR rG4 structure (e.g., PDS) may offer promising strategies to overcome cisplatin resistance in NPC.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study demonstrates that circARHGAP12 promotes cisplatin resistance in NPC. Mechanistically, circARHGAP12 directly binds to G3BP1 and, in an rG4-dependent manner, interacts with the 3\u0026prime;UTR of CA9 mRNA to enhance its stability. Further investigations revealed that circARHGAP12 facilitates cisplatin-induced stress granule (SG) assembly through G3BP1, resulting in the colocalization of circARHGAP12, G3BP1, and CA9 mRNA within SGs. This spatial organization protects CA9 mRNA from degradation under stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Collectively, these findings uncover a novel regulatory axis\u0026mdash;circARHGAP12\u0026ndash;G3BP1\u0026ndash;CA9\u0026mdash;that contributes to cisplatin resistance in NPC, and suggest that targeting this pathway to modulate SG dynamics may represent a promising therapeutic strategy for overcoming chemoresistance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCircular RNA (circRNA)\u003c/p\u003e\u003cp\u003eCarbonic Anhydrase 9 (CA9)\u003c/p\u003e\u003cp\u003eCis-diamminedichloroplatinum (II) (DDP)\u003c/p\u003e\u003cp\u003eFluorescence recovery after photobleaching (FRAP)\u003c/p\u003e\u003cp\u003eG3BP Stress Granule Assembly Factor 1 (G3BP1)\u003c/p\u003e\u003cp\u003eLiquid-Liquid Phase Separation (LLPS)\u003c/p\u003e\u003cp\u003eNasopharyngeal carcinoma (NPC)\u003c/p\u003e\u003cp\u003eRNA immunoprecipitation (RIP)\u003c/p\u003e\u003cp\u003eRNA binding proteins (RBPs)\u003c/p\u003e\u003cp\u003eRNA G-quadruplex (rG4)\u003c/p\u003e\u003cp\u003ePyridostatin (PDS)\u003c/p\u003e\u003cp\u003eStress Granules (SGs)\u003c/p\u003e\u003cp\u003eFluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH)\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn situ\u003c/em\u003e hybridization (ISH)\u003c/p\u003e\u003cp\u003eImmunohistochemistry (IHC)\u003c/p\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003e This study was approved by the Ethics Committee of Central South University (CSU-2024-0335).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003e All authors have critically reviewed the manuscript and approved its submission.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work has been supported by the National Natural Science Foundation of China (82203163), the Natural Science Foundation of Hunan Province (2025JJ50544), Young Elite Scientists Sponsorship Program by CAST (2022QNRC001), and grant from The Scientific Research Program of FuRong Laboratory (No. 2024PT5102).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eT.L. and Y.L. contributed to experiment execution, data analysis, and manuscript drafting. H.Q., D.W., J.G. performed some of the experiments. L.S., Q.Y. and W.X. participated in project design. W.X., C.F. and Z.Z. revised the manuscript. C.F. and Z.Z. were responsible for overall study design and manuscript revision.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCHEN LL. The expanding regulatory mechanisms and cellular functions of circular RNAs [J]. Nat Rev Mol Cell Biol. 2020;21(8):475\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHOU W Y, CAI Z R, LIU J, et al. Circular RNA: metabolism, functions and interactions with proteins [J]. Mol Cancer. 2020;19(1):172.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFAN C, QU H, XIONG F, et al. CircARHGAP12 promotes nasopharyngeal carcinoma migration and invasion via ezrin-mediated cytoskeletal remodeling [J]. Cancer Lett. 2021;496:41\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLI Q, ZHAO Y H, XU C, et al. Chemotherapy-Induced Senescence Reprogramming Promotes Nasopharyngeal Carcinoma Metastasis by circRNA-Mediated PKR Activation [J]. Adv Sci (Weinh). 2023;10(8):e2205668.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHONG X, LI Q, LI J, et al. CircIPO7 Promotes Nasopharyngeal Carcinoma Metastasis and Cisplatin Chemoresistance by Facilitating YBX1 Nuclear Localization [J]. Clin Cancer Res. 2022;28(20):4521\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMO Y, WANG Y, ZHANG S, et al. Circular RNA circRNF13 inhibits proliferation and metastasis of nasopharyngeal carcinoma via SUMO2 [J]. Mol Cancer. 2021;20(1):112.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGE J, WANG J, XIONG F, et al. Epstein-Barr Virus-Encoded Circular RNA CircBART2.2 Promotes Immune Escape of Nasopharyngeal Carcinoma by Regulating PD-L1 [J]. Cancer Res. 2021;81(19):5074\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCHEN Y P, CHAN A T C, LE Q T, et al. Nasopharyngeal carcinoma [J]. Lancet. 2019;394(10192):64\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLIANG Y-L, LIU X, SHEN L-F, et al. Adjuvant PD-1 Blockade With Camrelizumab for Nasopharyngeal Carcinoma: The DIPPER Randomized Clinical Trial [J]. JAMA. 2025;333(18):1589\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDAI J, ZHANG B, SU Y, et al. Induction Chemotherapy Followed by Radiotherapy vs Chemoradiotherapy in Nasopharyngeal Carcinoma: A Randomized Clinical Trial [J]. JAMA Oncol. 2024;10(4):456\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTANG L L, CHEN Y P, CHEN C B, et al. The Chinese Society of Clinical Oncology (CSCO) clinical guidelines for the diagnosis and treatment of nasopharyngeal carcinoma [J]. Cancer Commun (Lond). 2021;41(11):1195\u0026ndash;227.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGUO TANGLL, ZHANG R. Effect of Radiotherapy Alone vs Radiotherapy With Concurrent Chemoradiotherapy on Survival Without Disease Relapse in Patients With Low-risk Nasopharyngeal Carcinoma: A Randomized Clinical Trial [J]. JAMA. 2022;328(8):728\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHANG Y, CHEN L, HU G Q, et al. Gemcitabine and Cisplatin Induction Chemotherapy in Nasopharyngeal Carcinoma [J]. N Engl J Med. 2019;381(12):1124\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHANG Y, SUN Y. Induction gemcitabine and cisplatin in locoregionally advanced nasopharyngeal carcinoma [J]. Cancer Commun (Lond). 2019;39(1):39.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTANG L-L, GUO R, ZHANG N, et al. Effect of Radiotherapy Alone vs Radiotherapy With Concurrent Chemoradiotherapy on Survival Without Disease Relapse in Patients With Low-risk Nasopharyngeal Carcinoma: A Randomized Clinical Trial [J]. JAMA. 2022;328(8):728\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJI F, LU Y, CHEN S, et al. IGF2BP2-modified circular RNA circARHGAP12 promotes cervical cancer progression by interacting m6A/FOXM1 manner [J]. Cell Death Discovery. 2021;7(1):215.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHANG D, GUO Q, YOU K, et al. m6A-modified circARHGAP12 promotes the aerobic glycolysis of doxorubicin-resistance osteosarcoma by targeting c-Myc [J]. J Orthop Surg Res. 2024;19(1):33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMAO C, WANG M. ZHUANG L, et al. Metabolic cell death in cancer: ferroptosis, cuproptosis, disulfidptosis, and beyond [J]. Volume 15. Protein \u0026amp; Cell; 2024. pp. 642\u0026ndash;60. 9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSONG X, ZHU S, XIE Y, et al. JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice [J]. Gastroenterology. 2018;154(5):1480\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMCDONALD P C, CHAFE S C, BROWN W S, et al. Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia [J]. Gastroenterology. 2019;157(3):823\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLI Z, JIANG L, CHEW SH et al. Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia [J]. Redox Biol, 2019, 26(101297.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKOUKOURAKIS M I, GIATROMANOLAKI A, SIVRIDIS E, et al. Hypoxia-regulated carbonic anhydrase-9 (CA9) relates to poor vascularization and resistance of squamous cell head and neck cancer to chemoradiotherapy [J]. Clin Cancer Res. 2001;7(11):3399\u0026ndash;403.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMACKAY C, CARROLL E, IBRAHIM A F M, et al. E3 ubiquitin ligase HOIP attenuates apoptotic cell death induced by cisplatin [J]. Cancer Res. 2014;74(8):2246\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYANG Y, LIU L, TIAN Y et al. Autophagy-driven regulation of cisplatin response in human cancers: Exploring molecular and cell death dynamics [J]. Cancer Lett, 2024, 587(216659.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGUO J, XU B, HAN Q, et al. Ferroptosis: A Novel Anti-tumor Action for Cisplatin [J]. Cancer Res Treat. 2017;50(2):445\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXU Y, MA H, SHAO J, et al. A Role for Tubular Necroptosis in Cisplatin-Induced AKI [J]. J Am Soc Nephrol. 2015;26(11):2647\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLIU C, LI D, WANG J, et al. Arenobufagin increases the sensitivity of gastric cancer to cisplatin via alkaliptosis [J]. Heliyon. 2023;9(11):e21110.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTANG D, KANG R, BERGHE T V, et al. The molecular machinery of regulated cell death [J]. Cell Res. 2019;29(5):347\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMAO C, WANG M. Metabolic cell death in cancer: ferroptosis, cuproptosis, disulfidptosis, and beyond [J]. Protein Cell. 2024;15(9):642\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHENG G, PENG C, JIA X et al. ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH [J]. Molecular cancer, 2015, 14(84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCONN V M, CHINNAIYAN A M, CONN S J. Circular RNA in cancer [J]. Nat Rev Cancer. 2024;24(9):597\u0026ndash;613.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLI J, SUN D, PU W, et al. Circular RNAs in Cancer: Biogenesis, Function, and Clinical Significance [J]. Trends Cancer. 2020;6(4):319\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLYU K, CHOW E Y, MOU X, et al. RNA G-quadruplexes (rG4s): genomics and biological functions [J]. Nucleic Acids Res. 2021;49(10):5426\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHE X, YUAN J, WANG Y. G3BP1 binds to guanine quadruplexes in mRNAs to modulate their stabilities [J]. Nucleic Acids Res. 2021;49(19):11323\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTURNER M, DANINO Y M, BARSHAI M, et al. rG4detector, a novel RNA G-quadruplex predictor, uncovers their impact on stress granule formation [J]. Nucleic Acids Res. 2022;50(20):11426\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYANG P, MATHIEU C, KOLAITIS R M et al. G3BP1 Is a Tunable Switch that Triggers Phase Separation to Assemble Stress Granules [J]. Cell, 2020, 181(2): 325\u0026thinsp;\u0026ndash;\u0026thinsp;45.e28.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYOUN J Y, DYAKOV B J A, ZHANG J, et al. Properties of Stress Granule and P-Body Proteomes [J]. Mol Cell. 2019;76(2):286\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePROTTER D S W PARKERR. Principles and Properties of Stress Granules [J]. Trends Cell Biol. 2016;26(9):668\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKEDERSHA N, IVANOV P, ANDERSON P. Stress granules and cell signaling: more than just a passing phase? [J]. Trends Biochem Sci. 2013;38(10):494\u0026ndash;506.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWHEELER J R JAINS, WALTERS R W, et al. ATPase-Modulated Stress Granules Contain a Diverse Proteome and Substructure [J]. Cell. 2016;164(3):487\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eREDDING A, GRABOCKA E. Stress granules and hormetic adaptation of cancer [J]. Trends Cancer. 2023;9(12):995\u0026ndash;1005.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLI T, ZENG Z, FAN C, et al. Role of stress granules in tumorigenesis and cancer therapy [J]. Biochim Biophys Acta Rev Cancer. 2023;1878(6):189006.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXING F, QIN Y, XU J, et al. Stress granules dynamics and promising functions in pancreatic cancer [J]. Biochim Biophys Acta Rev Cancer. 2023;1878(3):188885.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHAO T, XIONG W, CAI J et al. YTHDF2 phase separation promotes arsenic-induced keratinocyte transformation in a poly-m6A-dependent manner by inhibiting translational initiation of the key tumor suppressor PTEN [J]. J Hazard Mater, 2024, 480(136243.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLI Q, LIU J, GUO L, et al. Decoding the interplay between m6A modification and stress granule stability by live-cell imaging [J]. Sci Adv. 2024;10(46):eadp5689.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eASAMITSU S, YABUKI Y, MATSUO K et al. RNA G-quadruplex organizes stress granule assembly through DNAPTP6 in neurons [J]. Sci Adv, 2023, 9(8): eade2035.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKHAREL P. RNA G-quadruplexes and stress: emerging mechanisms and functions [J]. Trends Cell Biol. 2024;34(9):771\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTURNER M, DANINO Y M, BARSHAI M, et al. rG4detector, a novel RNA G-quadruplex predictor, uncovers their impact on stress granule formation [J]. Nucleic Acids Res. 2022;50(20):11426\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDANINO YM, ROSENBAUM-COHEN T MOLITORL, et al. BLM helicase protein negatively regulates stress granule formation through unwinding RNA G-quadruplex structures [J]. Nucleic Acids Res. 2023;51(17):9369\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMAO L-H YUANL, HUANG Y-Y, et al. Stress granules: emerging players in neurodegenerative diseases [J]. Transl Neurodegener. 2025;14(1):22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFANG M, LUO L, CHEN Y et al. Perillaldehyde Improves Parkinson-Like Deficits by Targeting G3BP Mediated Stress Granule Assembly in Preclinical Models [J]. Advanced Science (Weinheim, Baden-Wurttemberg, Germany), 2025, 12(14): e2412152.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLIU C, ZHOU L, CHEN J et al. Galectin-7 promotes cisplatin efficacy by facilitating apoptosis and G3BP1 degradation in cervical cancer [J]. Biochem Pharmacol, 2023, 217(115834.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTUFO G, JONES A W, WANG Z, et al. The protein disulfide isomerases PDIA4 and PDIA6 mediate resistance to cisplatin-induced cell death in lung adenocarcinoma [J]. Cell Death Differ. 2014;21(5):685\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLEE C Y SEYDOUXG. Dynamics of mRNA entry into stress granules [J]. Nat Cell Biol. 2019;21(2):116\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHAO Z, QING Y. QKI shuttles internal m(7)G-modified transcripts into stress granules and modulates mRNA metabolism [J]. Cell. 2023;186(15):3208\u0026ndash;e2627.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePIETRAS P, AULAS A, FAY M M et al. Translation inhibition and suppression of stress granules formation by cisplatin [J]. Biomed Pharmacother, 2022, 145(112382.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLIU G, DU W. RNA G-quadruplex in TMPRSS2 reduces SARS-CoV-2 infection [J]. Nat Commun. 2022;13(1):1444.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMIGLIETTA G, COGOI S, MARINELLO J, et al. RNA G-Quadruplexes in Kirsten Ras (KRAS) Oncogene as Targets for Small Molecules Inhibiting Translation [J]. J Med Chem. 2017;60(23):9448\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nasopharyngeal carcinoma, CircARHGAP12, Alkaliptosis, RNA G-quadruplex, Stress granule, Cisplatin resistance, Phase separation","lastPublishedDoi":"10.21203/rs.3.rs-7984659/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7984659/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eNasopharyngeal carcinoma (NPC) is a malignant epithelial tumor with a high prevalence in Southern China, and cisplatin remains a commonly used first-line chemotherapeutic agent. However, the effectiveness of cisplatin-based therapy is often compromised by the development of drug resistance, posing a major clinical challenge. Our previous studies demonstrated that circular RNA circARHGAP12 is highly expressed in NPC and promotes tumor cell migration and invasion, although its other potential functions remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eTo evaluate the effect of circARHGAP12 on cisplatin resistance, MTT assays, colony formation assays, and nude mouse xenograft models were utilized in vitro and in vivo. Targeted genes of circARHGAP 12 were screened by RNA sequencing, combined with RT-qPCR, western blotting, and rescue experiments to validate their functions. RNA pulldown, RNA immunoprecipitation (RIP), dual-luciferase reporter assays, and actinomycin D experiments were used to elucidate the circARHGAP12/G3BP1/CA9 regulatory axis. Analysis of stress granule dynamics using immunofluorescence, fluorescence in situ hybridization, and fluorescence recovery after photobleaching (FRAP).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn this study, we found that circARHGAP12 confers cisplatin resistance in NPC by suppressing cisplatin-induced alkaliptosis, a process mediated by the upregulation of carbonic anhydrase 9 (CA9) and reversed upon CA9 knockdown. Mechanistically, circARHGAP12 stabilizes CA9 mRNA in an RNA G-quadruplex (rG4)-dependent manner through interaction with the RNA-binding protein G3BP1. In addition, circARHGAP12 enhances the assembly of cisplatin-induced stress granules, an effect abolished by either G3BP1 silencing or treatment with pyridostatin (PDS), which disrupts the G3BP1-rG4 interaction.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eCollectively, our findings reveal a novel role of circARHGAP12 in mediating cisplatin resistance in NPC through G3BP1-dependent stabilization of CA9 mRNA and modulation of stress granule dynamics. Targeting the circARHGAP12\u0026ndash;G3BP1\u0026ndash;CA9 axis may therefore represent a promising therapeutic strategy to overcome chemotherapy resistance in NPC.\u003c/p\u003e","manuscriptTitle":"CircARHGAP12 suppresses cisplatin-induced alkaliptosis in nasopharyngeal carcinoma through G3BP1-mediated upregulation of CA9 in an RNA G-quadruplex-dependent manner","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 16:46:28","doi":"10.21203/rs.3.rs-7984659/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bd9e5a8a-a1f2-452c-9227-9bf71d691c4f","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-06T01:09:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-05 16:46:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7984659","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7984659","identity":"rs-7984659","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}