LncRNA NORAD Promotes Oxaliplatin Resistance in Adenocarcinoma of Esophagogastric Junction via Sponging miR-433-3p and Activating Autophagy

LncRNA NORAD Promotes Oxaliplatin Resistance in Adenocarcinoma of Esophagogastric Junction via Sponging miR-433-3p and Activating Autophagy | 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 LncRNA NORAD Promotes Oxaliplatin Resistance in Adenocarcinoma of Esophagogastric Junction via Sponging miR-433-3p and Activating Autophagy Huabing Ma, Jing Wang, Jiaxin Liu, Hui Li, Xiaomin Zhang, Yanchao Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7500532/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Objective Adenocarcinoma of esophagogastric junction (AEG) is a clinically aggressive malignancy with an increasing incidence worldwide. Although oxaliplatin-based chemotherapy represents a cornerstone of treatment, the development of drug resistance remains a significant clinical challenge. This study aims to explore the role and underlying mechanism of the long non-coding RNA NORAD in oxaliplatin resistance in AEG, focusing on the NORAD/miR-433-3p/autophagy regulatory axis, as well as to evaluate the potential of serum exosomal NORAD as a novel biomarker. Methods Paired tumor and adjacent normal tissues were obtained from 56 patients with AEG and analyzed. Differentially expressed lncRNAs were identified using lncRNA microarray profiling and validated by qRT-PCR. Oxaliplatin-resistant cell lines (PDC-R and OE19-R) were established through long-term drug exposure. Serum-derived exosomes were isolated and characterized in terms of morphology and marker expression. Bioinformatics tools predicted potential interactions between NORAD and miR-433-3p, which were subsequently confirmed using dual-luciferase reporter assays. NORAD knockdown was effectively achieved via lentiviral transduction with shRNA targeting NORAD. Cell viability was evaluated using the CCK-8 assay. The levels of autophagy-related proteins, including LC3B-II and p62, were assessed by western blot analysis. Results NORAD was markedly upregulated in AEG tissues and further increased in oxaliplatin-resistant cells, whereas miR-433-3p expression was downregulated. A strong inverse correlation was observed between NORAD and miR-433-3p levels ( r = -0.864, p < 0.001). Luciferase assays confirmed that NORAD directly interacts with miR-433-3p. Serum exosomal NORAD levels were significantly elevated in AEG patients compared to those in healthy controls and showed a positive correlation with NORAD expression in tumor tissues ( r = 0.8858, p < 0.001). Knockdown of NORAD enhanced the sensitivity of AEG cells to oxaliplatin, as indicated by reduced IC₅₀ values and resistance indices. Furthermore, NORAD silencing impaired autophagic flux, as demonstrated by decreased LC3B-II levels and increased p62 accumulation. Conclusions LncRNA NORAD contributes to oxaliplatin resistance in AEG by acting as a sponge for miR-433-3p and subsequently promoting autophagy. Serum-derived exosomal NORAD holds promise as a non-invasive biomarker for AEG. Targeting the NORAD/miR-433-3p/autophagy axis may offer a novel therapeutic approach to counteract chemoresistance. Adenocarcinoma of esophagogastric junction Oxaliplatin resistance Long non-coding RNA NORAD miR-433-3p Autophagy Exosomes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Adenocarcinoma of the esophagogastric junction (AEG) is a clinically aggressive malignancy that exhibits distinct clinicopathological characteristics compared to distal gastric cancers [ 1 ]. The incidence of AEG has increased rapidly in Western countries and certain regions of Asia over the past few decades [ 2 ]. Studies have shown that the combination of perioperative chemotherapy and surgical resection provides significant survival advantages over surgery alone in patients with advanced AEG [ 3 , 4 ]. Currently, oxaliplatin-based regimens remain a key component of systemic therapy for advanced stages of this disease [ 5 ]. However, the emergence of oxaliplatin resistance has become a major challenge, often leading to treatment failure and disease progression [ 6 ], underscoring the urgent need to investigate the underlying molecular mechanisms. Autophagy, an evolutionarily conserved lysosomal degradation pathway, plays a critical role in removing damaged organelles and proteins, thereby maintaining cellular homeostasis. Increasing evidence suggests that excessive activation of autophagy enhances chemoresistance in multiple types of malignancies, such as gastric [ 7 ], hepatocellular [ 8 ], and colorectal cancers [ 9 ]. This adaptive response allows cancer cells to counteract the detrimental effects of chemotherapy and avoid apoptosis, which ultimately leads to therapeutic failure [ 10 ]. Long non-coding RNA activated by DNA damage (NORAD, also known as LINC00657) has been increasingly recognized as a key oncogenic regulator across multiple cancer types [ 11 ]. It promotes tumor progression by enhancing cell migration, metastatic capacity, and resistance to apoptosis [ 12 ]. Notably, NORAD contributes to chemotherapy resistance in various malignancies, including non-small cell lung cancer [ 12 ], neuroblastoma [ 13 ], osteosarcoma [ 14 ], bladder cancer [ 15 ], colorectal cancer [ 16 ], and esophageal squamous cell carcinoma [ 17 ], through diverse molecular mechanisms. Furthermore, quantitative proteomic studies using TMT (Tandem Mass Tag) labeling have indicated that NORAD modulates autophagic flux in gastric cancer [ 18 ], underscoring its potential involvement in cellular stress responses. A significant proportion of long non-coding RNAs (lncRNAs) act as competitive endogenous RNAs (ceRNAs) by binding to microRNAs (miRNAs), thereby relieving the repression of downstream target genes [ 19 ]. MiRNAs are small non-coding RNAs that typically bind to the 3′-untranslated regions (3′-UTRs) of target messenger RNAs, leading to translational inhibition or mRNA degradation [ 20 ]. Numerous lncRNAs display co-expression patterns or functional associations with adjacent miRNAs [ 21 ]. Notably, microRNA-433-3p (miR-433-3p) has been demonstrated to regulate chemosensitivity. For instance, it enhances cisplatin sensitivity in glioma by targeting NR5A2 [ 22 ], and the knockdown of circ_0011292 improves paclitaxel sensitivity in non-small-cell lung cancer via the miR-433-3p/CHEK1 pathway [ 23 ]. Using bioinformatic tools such as StarBase V3.0 and DIANA-LncBase V3.0, we identified a potential binding site between NORAD and miR-433-3p (chr20:36045805–36045826). Structural predictions based on RNAhybrid, along with fluorescence in situ hybridization, confirmed their cytoplasmic co-localization and interaction, indicating a possible regulatory mechanism. Exosomes (30–200 nm extracellular vesicles) serve as critical mediators of intercellular communication by transporting bioactive molecules, including lncRNAs [ 24 ]. Accumulating evidence suggests that exosomal lncRNAs play a crucial role in the development of chemotherapy resistance through intercellular horizontal transfer [ 25 ]. For instance, exosomal lincROR derived from prostate cancer cells enhances docetaxel resistance via a β-catenin/HIF1α feedback mechanism [ 25 ]; exosomal CRNDE secreted by tumor-associated macrophages promotes cisplatin resistance in gastric cancer [ 26 ]; exosomal DACT3-AS1 from cancer-associated fibroblasts reduces oxaliplatin resistance in gastric cancer [ 27 ]; and exosomal LOC85009 suppresses docetaxel resistance in lung adenocarcinoma by modulating ATG5-mediated autophagy [ 28 ]. However, the expression pattern and biological function of exosomal lncRNA NORAD in AEG remain poorly understood. Whether exosomal NORAD contributes to oxaliplatin resistance in AEG remains to be elucidated. AEG exhibits a distinct natural history, histogenesis, and molecular profile compared to middle and distal gastric cancers [ 29 ]. In gastric cancer, Wang et al . reported that NORAD enhances autophagic flux and confers resistance to oxaliplatin by stabilizing the ATG5-ATG12 complex through miR-433-3p sponging [ 18 ]. Based on these findings, we propose that NORAD may similarly contribute to oxaliplatin resistance in AEG by promoting autophagy via miR-433-3p sequestration. This study aims to investigate the role and underlying mechanisms of the NORAD/miR-433-3p/autophagy axis in oxaliplatin resistance in AEG and to evaluate the potential of serum exosomal NORAD as a non-invasive biomarker. 2 Materials and Methods 2.1 Patients and Tissue Samples A total of 56 patients with locally advanced AEG and 56 age- and sex-matched healthy volunteers were enrolled in this study. From each AEG patient, paired samples of primary tumor tissue and adjacent normal tissue (ANT), collected from a distance of at least 5 cm from the tumor margin, were obtained during curative resection at Anyang Tumor Hospital between January 2024 and September 2024. All tumors were histopathologically confirmed as adenocarcinoma according to the WHO classification system. None of the patients had received neoadjuvant chemotherapy or radiotherapy prior to surgery. The study was approved by the Ethics Committee of Anyang Tumor Hospital (AZLL0220240103008) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. 2.2 Blood Collection and Serum Isolation Peripheral blood (5 mL) was collected from each participant using vacuum tubes containing EDTA (BD Biosciences, USA). The blood samples were kept on ice and centrifuged within one hour at 1000 × g for 20 minutes at 4°C to separate the cellular components. The serum was then carefully aliquoted and stored at -80°C until further analysis to ensure the integrity of exosomes and the stability of RNA. 2.3 Exosome Isolation and Characterization Serum exosomes were isolated using the exoRNeasy Serum Midi Kit (Qiagen, Germany) according to the manufacturer's instructions. The size distribution and concentration of the exosomes were analyzed by nanoparticle tracking analysis (NTA; Malvern NanoSight LM10), and their morphology was examined using transmission electron microscopy (TEM; Philips Tecnai 12). To confirm exosomal purity, standard markers (CD63, CD81, and TSG101) were detected by western blotting. Total RNA was extracted from the isolated exosomes using the same kit and subsequently reverse-transcribed using the miScript II RT Kit (Qiagen) for further analysis [ 30 ]. 2.4 RNA Extraction and lncRNA Microarray Total RNA was extracted from tissue samples using TRIzol® Reagent (Invitrogen, USA) in accordance with the manufacturer's instructions. RNA quality and concentration were evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). LncRNA and mRNA expression profiles were generated using the ArrayStar Human LncRNA/mRNA Expression Microarray V3.0 (Arraystar, USA), following the manufacturer's recommended protocols. The microarray annotations were based on databases including NCBI RefSeq, UCSC, RNAdb, and literature-validated lncRNAs [ 31 ]. Microarray hybridization and preliminary data processing were carried out by KangChen Biotech (Shanghai, China). Differentially expressed transcripts were identified using a threshold of fold change ≥ 2.0 and an adjusted p-value < 0.05. 2.5 Cell Lines and Culture Conditions The human gastric mucosal epithelial cell line GES-1 was purchased from the American Type Culture Collection (ATCC, USA) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Thermo Scientific, USA) and 1% penicillin/streptomycin (HyClone, USA) at 37°C in a humidified atmosphere containing 5% CO₂. The OE19 cell line, derived from Siewert type II AEG, was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (China) and maintained under identical culture conditions. Cells were subcultured every 3–5 days using 0.25% trypsin-EDTA (Sigma-Aldrich, USA) and were routinely tested for mycoplasma contamination. 2.6 Patient-Derived Cell (PDC) Culture Fresh primary tumor tissues from advanced AEG patients were rinsed with phosphate-buffered saline (PBS), cut into small fragments (~ 1 mm³), and digested with EZ Enzyme (Kurabo, Japan) for 2 hours at 37°C with gentle agitation. The resulting cell pellets were resuspended and cultured in either PCM-2 or ACL4 medium (Kurabo, Japan) until stromal cell contamination was effectively minimized [ 32 ]. Passage 3 to 8 PDCs were selected for experimental use to ensure biological consistency and relevance. 2.7 Generation of Oxaliplatin-Resistant Cells Oxaliplatin-resistant sublines (PDC-R and OE19-R) were developed through stepwise exposure to gradually increasing concentrations of oxaliplatin (2–10 µg/mL; Sigma-Aldrich, USA) over multiple passages (approximately six months). Resistance was verified by continuous proliferation under oxaliplatin treatment and through calculated resistance indices (RI). To maintain the resistant phenotype, all resistant cell lines were cultured in complete medium supplemented with 2 µg/mL oxaliplatin. 2.8 Bioinformatic Analysis Putative interactions between NORAD and miR-433-3p were predicted using StarBase v3.0 ( http://starbase.sysu.edu.cn ) and DIANA-LncBase v3.0 ( http://diana.e-ce.uth.gr/lncbasev3 ) [ 32 ]. The binding sites were visualized using RNAhybrid ( http://bibiserv.cebitec.uni-bielefeld.de/rnahybrid ). Potential targets of miR-433-3p were identified using miRDB ( http://mirdb.org ) and TargetScan ( http://targetscan.org ). 2.9 Dual-Luciferase Reporter Assay Fragments of NORAD containing either wild-type (WT) or mutated (Mut) miR-433-3p binding sites were cloned into the GV272 vector (GeneChem, China) [ 18 ]. The cells were co-transfected with the reporter constructs and either a miR-433-3p mimic or a negative control (NC) using Lipofectamine 3000 (Invitrogen, USA). Luciferase activity was assessed 48 hours after transfection using a dual-luciferase assay system (Promega, USA) on a GloMax 20/20 Luminometer (Promega, USA). Firefly luciferase activity was normalized relative to Renilla luciferase activity in each sample. 2.10 Lentiviral Transduction and Knockdown Lentiviral particles carrying short hairpin RNA (shRNA) targeting NORAD or a non-targeting control (NC) were synthesized by GeneChem (China). Cells were transduced at a multiplicity of infection (MOI) of 20 in the presence of Polybrene (8 µg/mL; Sigma-Aldrich, USA). Stably transduced cell pools were selected using puromycin (2 µg/mL; Sigma-Aldrich, USA) over a period of two weeks. Knockdown efficiency was confirmed by qRT-PCR. 2.11 Cell Viability and IC₅₀ Assay Cells were seeded in 96-well plates at a density of 3×10³ cells/well and exposed to varying concentrations of oxaliplatin (1–64 µg/mL) for 48 hours. Cell viability was evaluated using the CCK-8 kit (ApexBio, USA) according to the manufacturer's protocol [ 15 ]. Absorbance was then measured at 490 nm using a microplate reader (BioTek, USA). IC₅₀ values (half-maximal inhibitory concentration) were determined through nonlinear regression analysis with GraphPad Prism v 9.0 (GraphPad Software, USA). The resistance index (RI) was defined as the ratio of IC₅₀ values between resistant and parental cell lines. 2.12 Quantitative Real-Time PCR (qRT-PCR) Total RNA was reverse-transcribed using the PrimeScript RT Reagent (Takara, Japan). Quantitative PCR (qPCR) was carried out using SYBR Premix Ex Taq II (Takara, Japan) on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, USA). miRNA expression was analyzed using the Mir-X miRNA RT-PCR Kit (Takara, Japan). GAPDH and U6 small nuclear RNA were used as endogenous controls for NORAD and miR-433-3p, respectively. Relative gene expression was calculated using the 2 −ΔΔCt method. The primer sequences used were as follows: NORAD forward, 5'-ACAGCCAAATCACAGCCACA-3'; reverse, 5'-TGGGCTCTGTGTTTGCATCC-3'; miR-433-3p forward, 5'-ATCACAUUGGGCUCCUCGGUGU-3'; reverse: universal primer provided in the kit; GAPDH forward, 5'-GGAGCGAGATCCCTCCAAAAT-3'; reverse, 5'-GGCTGTTGTCATACTTCTCATGG-3'; U6 forward, 5'-CTCGCTTCGGCAGCACA-3'; reverse, 5'-AACGCTTCACGAATTTGCGT-3'. 2.13 Protein extraction and Western Blotting GO pathway analysis revealed the potential involvement of NORAD in the assembly process of autophagosomes [ 17 ]. The conversion of the cytosolic LC3B-I protein to the membrane-bound LC3B-II isoform on the phagophore is a key hallmark of autophagy, and the quantification of LC3B-II levels represents the most reliable method for assessing autophagic flux in tumor cells [ 33 , 34 ]. Moreover, a negative correlation exists between p62 protein abundance and the strength of autophagic flux. Proteins were extracted using RIPA buffer (Beyotime, China), supplemented with protease inhibitors (Roche, Switzerland). Protein concentrations in the lysates were determined using a bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich, USA). Equal amounts of protein (20–30 µg) were separated by 10% SDS-PAGE and then transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 5% non-fat milk to minimize nonspecific binding. Primary antibodies against LC3B (1:1000; Novus Biologicals, USA), p62 (1:800; Santa Cruz Biotechnology, USA), and GAPDH (1:5000; Proteintech, USA) were incubated overnight at 4°C. After incubation with primary antibodies, the membranes were treated with HRP-conjugated secondary antibodies (1:5000; Cell Signaling Technology, USA), and the protein bands were visualized using ECL reagent (Bio-Rad, USA). Band intensities were analyzed and quantified using ImageJ software (NIH, USA). 2.14 Statistical Analysis Data are expressed as mean ± standard deviation (SD). Comparisons between two groups were made using two-tailed Student's t-tests , while one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used for comparisons among multiple groups. Pearson's correlation coefficient was employed to assess correlations. Statistical analyses were performed using SPSS V 22.0 (IBM, USA) and GraphPad Prism V 9.0 . A p-value less than 0.05 was considered statistically significant. All experiments were independently repeated at least three times. 3 Results 3.1 Identification of Differentially Expressed lncRNAs in AEG We analyzed lncRNA expression profiles in primary tumor tissues and paired ANTs from four treatment-naïve patients with locally advanced AEG using the ArrayStar Human LncRNA/mRNA Microarray V3.0. Among the 503,687 lncRNAs profiled, several differentially expressed transcripts were identified. Notably, the lncRNA NORAD was significantly upregulated in AEG tissues compared to ANTs (fold change ≥ 2.0, p < 0.05; Fig. 1 A). This result was further validated by qRT-PCR in an expanded cohort of 56 AEG patients, which confirmed the overexpression of NORAD in tumor tissues ( t = 21.213, p < 0.001; Fig. 1 B). 3.2 NORAD and miR-433-3p Are Differentially Expressed in AEG Tissues and Show Inverse Correlation Bioinformatic analysis using StarBase v3.0 and DIANA-LncBase v3.0 identified miR-433-3p as a direct target of NORAD, suggesting a potential competing endogenous RNA (ceRNA) mechanism in oxaliplatin resistance. Quantitative RT-PCR validation in 56 AEG patients confirmed that NORAD was significantly upregulated ( t = 21.213, p < 0.001), while miR-433-3p was significantly downregulated ( t = 19.463, p < 0.001) in tumor tissues compared with ANTs (Table 1 ). A significant inverse correlation was observed between NORAD and miR-433-3p expression levels in AEG tissues ( r = -0.864, p < 0.001; Fig. 2 ). Table 1 Expression of NORAD and miR-433-3p in 56 AEG patients compared with ANT. Group NORAD miR-433-3p mean ± standard deviation P -value mean ± standard deviation P -value AEG 2.245 ± 0.376 P < 0.000 0.444 ± 0.143 P < 0.000 ANT 1.005 ± 0.265 1.039 ± 0.161 3.3 NORAD Directly Binds to miR-433-3p Dual-luciferase reporter assays further confirmed that miR-433-3p directly targets NORAD. As shown by the significantly reduced luciferase activity in PDCs and PDC-Rs co-transfected with wild-type NORAD and miR-433-3p mimics (approximately 60% reduction compared to the control ( p < 0.001; Fig. 3 ), this interaction is functionally relevant. Mutation of the predicted miR-433-3p binding site within NORAD completely restored luciferase activity, thereby validating the specificity of the interaction. 3.4 Expression of NORAD and miR-433-3p in AEG Cell Lines Oxaliplatin-resistant sublines (PDC-R and OE19-R) were derived from their parental PDC and OE19 cells through prolonged stepwise exposure to oxaliplatin. NORAD-knockdown cells (shNORAD) were successfully established via lentiviral transduction, achieving a knockdown efficiency exceeding 80% across all cell types ( p < 0.001). qRT-PCR analysis demonstrated that NORAD expression was significantly upregulated in both PDC and PDC-R cells compared to the non-tumoral GES-1 cells (both p < 0.001), with a further significant increase observed in PDC-R cells relative to PDC cells ( t = 19.432, p < 0.001; Fig. 4 A). In contrast, miR-433-3p expression was markedly downregulated in all AEG-derived cell lines compared to GES-1 cells ( F = 147.567, p < 0.001). Knockdown of NORAD significantly elevated miR-433-3p levels in both PDC and PDC-R cells (Fig. 4 A). Consistent trends were observed in the OE19-based cell lines (Fig. 4 B). 3.5 Serum Exosomal NORAD Is Elevated in AEG and Correlates with Tumor Expression Serum exosomes were isolated from patients with AEG and healthy volunteers and were characterized using NTA and TEM. The exosomes exhibited the typical cup-shaped morphology with diameters ranging from 50 to 150 nm. Western blot analysis confirmed the enrichment of exosomal markers, including CD63, CD81, and TSG101, as well as the absence of the negative marker calnexin. Notably, exosomal NORAD levels were significantly elevated in AEG patients compared to those in healthy volunteers ( t = 35.646, p < 0.001; Fig. 5 A). Furthermore, a strong positive correlation was observed between exosomal NORAD expression and NORAD levels in matched tumor tissues ( r = 0.8858, p < 0.001; Fig. 5 B), indicating its potential as a non-invasive biomarker for clinical applications. 3.6 NORAD Knockdown Sensitizes AEG Cells to Oxaliplatin CCK-8 assays demonstrated that oxaliplatin-resistant cell lines (PDC-R and OE19-R) exhibited significantly higher IC₅₀ values compared to their parental counterparts (PDC-R: 22.27 µg/mL vs . PDC: 14.28 µg/mL; resistance index = 1.559; OE19-R: 25.43 µg/mL vs . OE19: 15.16 µg/mL; resistance index = 1.677). NORAD knockdown substantially decreased oxaliplatin resistance, resulting in IC₅₀ values of 2.00 µg/mL in shNORAD-transduced PDC cells and 2.51 µg/mL in shNORAD-transduced PDC-R cells, corresponding to resistance indices of 0.140 and 0.113, respectively (Fig. 6 A). A similar resensitization effect was observed in the OE19-derived cell lines (Fig. 6 B). 3.7 NORAD Regulates Autophagic Activity in AEG Cells Western blot analysis demonstrated that oxaliplatin-resistant cells (PDC-R and OE19-R) displayed elevated levels of LC3B-II and reduced expression of p62 compared to their parental counterparts, suggesting an enhancement in autophagic flux (Fig. 7 ). NORAD knockdown reversed this effect, leading to decreased LC3B-II levels and increased p62 expression in the resistant cells (Fig. 7 ). These findings indicate that NORAD contributes to oxaliplatin resistance by promoting autophagy in AEG cells. 3.8 NORAD was implicated in the regulation of autophagy in AEG cells We employed Western blotting to detect and compare the relative expression levels of LC3B-I, LC3B-II, and p62 in PDC, PDC-R, and shNORAD PDC-R cell lines. In parallel, we assessed these markers in OE19, OE19-R, and shNORAD OE19-R cell lines. Our results revealed a significant increase in LC3B-II levels in PDC-R cells compared to PDC cells. Knockdown of NORAD in PDC-R cells led to a decrease in LC3B-II expression, suggesting a reduction in autophagic flux. In contrast, p62 protein levels were lower in PDC-R cells than in PDC cells; however, NORAD knockdown mediated by shRNA resulted in an accumulation of p62 (Fig. 5 A). Consistent results were observed in the AEG cell line OE19 (Fig. 5 B). 4 Discussion This study demonstrates that the long non-coding RNA (lncRNA) NORAD is significantly overexpressed in both tumor tissues and serum exosomes obtained from patients with AEG. We present mechanistic evidence indicating that NORAD promotes oxaliplatin resistance by enhancing autophagy through a ceRNA mechanism, which involves sponging miR-433-3p and subsequently derepressing ATG5. Consistent with its previously reported oncogenic roles in other malignancies [ 18 ], NORAD expression is markedly upregulated in AEG tissues compared to adjacent normal tissues. Importantly, we report for the first time that serum exosomal levels of NORAD are significantly elevated in AEG patients and show a strong correlation with NORAD expression in matched tumor tissues. These findings suggest that exosomal NORAD may serve as a non-invasive biomarker for AEG diagnosis and disease monitoring, supporting the growing recognition of exosomal lncRNAs as mediators of intercellular communication and contributors to chemotherapy resistance [ 35 ]. Our findings corroborate recent studies highlighting exosomal lncRNAs as key regulators of drug resistance in gastric cancer [ 27 , 36 ] and extend this concept to AEG, a distinct clinical entity with unique molecular features [ 37 ]. Through bioinformatic prediction and experimental validation, we identified miR-433-3p as a direct target of NORAD. The inverse correlation between NORAD and miR-433-3p expression across clinical samples and cell models supports a ceRNA mechanism, whereby NORAD functions as a molecular sponge to sequester miR-433-3p, thereby attenuating its tumor-suppressive effects. This regulatory axis aligns with previous findings in gastric cancer, where NORAD modulates autophagy and chemoresistance through miR-433-3p [ 18 ]. Our study further demonstrates that this mechanism is present in AEG and is particularly relevant to oxaliplatin resistance. Functional assays confirmed that NORAD knockdown significantly enhanced the sensitivity of both parental and oxaliplatin-resistant AEG cells to oxaliplatin, highlighting its essential role in mediating drug resistance. Western blot analysis further demonstrated that NORAD upregulation promotes autophagic flux, as indicated by increased levels of LC3B-II and decreased expression of p62—effects that were reversed upon NORAD silencing. These findings suggest that NORAD contributes to oxaliplatin resistance by activating autophagy, potentially through the derepression of the ATG5-ATG12 complex formation via sponging miR-433-3p. This mechanism is particularly relevant given the well-established role of autophagy in chemoresistance across multiple cancer types [ 7 , 38 ]. Our study also highlights the therapeutic potential of targeting the NORAD/miR-433-3p/autophagy axis. The marked reduction in oxaliplatin resistance following NORAD knockdown suggests that pharmacological inhibition of NORAD may represent a promising strategy to overcome chemoresistance in AEG patients. Several approaches for targeting lncRNAs are currently under investigation, including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and CRISPR-based technologies [ 39 ]. Furthermore, the strong correlation between serum exosomal NORAD levels and tumor expression suggests that NORAD may serve as a non-invasive biomarker for predicting treatment response and monitoring disease progression. Several limitations of our study should be acknowledged. First, the sample size was relatively small and derived from a single institution, underscoring the necessity for future multi-center studies with larger cohorts to validate the clinical utility of serum exosomal NORAD. Second, although we demonstrated that NORAD contributes to oxaliplatin resistance by modulating autophagy via miR-433-3p sponging, we cannot exclude the possibility that additional mechanisms may also be involved. Third, the mechanisms responsible for the selective packaging of NORAD into exosomes remain unclear and require further investigation. Finally, in vivo studies are needed to confirm the therapeutic potential of targeting NORAD in AEG and to assess any potential off-target effects. In conclusion, our findings establish NORAD as a critical player in oxaliplatin resistance in AEG by regulating the miR-433-3p/autophagy axis. The strong correlation between serum exosomal NORAD levels and tumor expression suggests its potential as a non-invasive biomarker. Targeting the NORAD/miR-433-3p/autophagy pathway may represent a promising therapeutic strategy for overcoming oxaliplatin resistance in AEG patients. 5 Conclusion This study reveals that the lncRNA NORAD is markedly upregulated in both AEG tissues and serum exosomes, where it contributes to oxaliplatin resistance by sequestering miR-433-3p and inducing autophagy. Knockdown of NORAD enhances the sensitivity of AEG cells to oxaliplatin, suppresses autophagic flux, and promotes apoptosis. Notably, serum exosomal levels of NORAD show a strong correlation with its expression in tumor tissues, underscoring its potential as a non-invasive biomarker. Targeting the NORAD/miR-433-3p/autophagy pathway may represent a novel therapeutic approach to reverse chemoresistance in patients with AEG. Declarations ACKNOWLEDGEMENTS The authors would like to express their sincere gratitude to Professor Baozhong Li, Professor Zhiqiang Liu, Professor Wei Zhang, Miss Gongjing Lu, and Anyang Tumor Hospital for their invaluable support. We are grateful to Editage (www.editage.cn) for providing English language editing services. Institutional review board statement : Informed written consent was obtained from all patients. The present study was approved by the Ethics Committee of the Anyang Tumor Hospital (Anyang, China). Conflict-of-interest statement : All authors declare that they have no competing interests. The authors declare that they have no conflicts of interest. All the authors agree to share the data and materials used in this study. All authors declare their consent to publish this study. Funding : This research was funded by Henan Province Science and Technology Tackling Key Project (232102310090) and (No. 242102310125). Ethics approval and consent to participate: This study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Anyang Tumor Hospital. The patients informed consent was obtained according to regional regulations. Data Availability Statement: All data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding author, Dr. Shoumiao Li ( E-Mail: [email protected] ). All authors agree to share the data and materials used in this study. For more detailed information, please contact Dr. Shoumiao Li, who will be pleased to provide further assistance. Author contribution statement: The final manuscript has undergone thorough review and approval by all authors, who disclose no conflicts of interest. 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Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 28 Oct, 2025 Reviews received at journal 28 Oct, 2025 Reviewers agreed at journal 28 Oct, 2025 Reviews received at journal 09 Oct, 2025 Reviewers agreed at journal 20 Sep, 2025 Reviewers agreed at journal 19 Sep, 2025 Reviewers invited by journal 14 Sep, 2025 Editor assigned by journal 03 Sep, 2025 Submission checks completed at journal 03 Sep, 2025 First submitted to journal 31 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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05:05:38","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1123894,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFIGURE6.png","url":"https://assets-eu.researchsquare.com/files/rs-7500532/v1/1327f81b6e6b4a8f5980b52c.png"},{"id":92047006,"identity":"7cf07a22-a8d4-4eb7-93a3-b274ec2ce1f4","added_by":"auto","created_at":"2025-09-24 05:05:39","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3788572,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFIGURE7.png","url":"https://assets-eu.researchsquare.com/files/rs-7500532/v1/2df71a79146c5710be6a52e6.png"},{"id":92048340,"identity":"95bf2208-f16b-47d7-ac99-0b73745f96bd","added_by":"auto","created_at":"2025-09-24 05:13:38","extension":"xml","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102863,"visible":true,"origin":"","legend":"","description":"","filename":"831c8476c3964f14abce77ad89b99e2b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7500532/v1/341782875db4aef4ff87c2c9.xml"},{"id":92046995,"identity":"99cec8c9-3088-4476-9bbe-604d2a88c370","added_by":"auto","created_at":"2025-09-24 05:05:38","extension":"html","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115323,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7500532/v1/63895a7d31f91f6cc5012f9f.html"},{"id":92046973,"identity":"ab401f9c-cac4-413b-a523-eaa2f9ed7715","added_by":"auto","created_at":"2025-09-24 05:05:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1301831,"visible":true,"origin":"","legend":"\u003cp\u003eNORAD is overexpressed in AEG tissues. (A)\u003cstrong\u003e \u003c/strong\u003eMicroarray analysis showing differentially expressed lncRNAs in AEG tissues compared to adjacent normal tissues (ANT). (B) qRT-PCR validation of NORAD overexpression in 56 AEG patients ( \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"FIGURE1.png","url":"https://assets-eu.researchsquare.com/files/rs-7500532/v1/95d746ce75ca387a493b746c.png"},{"id":92063253,"identity":"e119cd9a-6a27-4cf0-944b-5fa1af51ee7e","added_by":"auto","created_at":"2025-09-24 08:37:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":978879,"visible":true,"origin":"","legend":"\u003cp\u003eNORAD and miR-433-3p expression levels in AEG tissues.\u003c/p\u003e","description":"","filename":"FIGURE2.png","url":"https://assets-eu.researchsquare.com/files/rs-7500532/v1/d7a3bcf687269328f584fa4c.png"},{"id":92046974,"identity":"7a3e9423-1269-42f2-9a1d-3706d1692dc3","added_by":"auto","created_at":"2025-09-24 05:05:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1512971,"visible":true,"origin":"","legend":"\u003cp\u003eDual-luciferase reporter assays confirmed that miR-433-3p directly targets NORAD. (A) The luciferase activity was significantly reduced in PDCs co-transfected with wild-type NORAD and miR-433-3p mimics. (B) The luciferase activity was significantly reduced in PDC-Rs co-transfected with wild-type NORAD and miR-433-3p mimics.\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-7500532/v1/3dfc552f87aee93b73d4f213.png"},{"id":92048330,"identity":"09b42754-eed5-4a91-9d5a-f5d8b55ffc8a","added_by":"auto","created_at":"2025-09-24 05:13:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1607619,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of NORAD and miR-433-3p in AEG cell lines. (A) qRT-PCR analysis of NORAD and miR-433-3p expression in patient-derived cells (PDC) and oxaliplatin-resistant sublines (PDC-R) with or without NORAD knockdown. (B) Similar trends observed in OE19 and OE19-R cells.\u003c/p\u003e","description":"","filename":"FIGURE4.png","url":"https://assets-eu.researchsquare.com/files/rs-7500532/v1/791522eebeec1d506f5d03cf.png"},{"id":92063194,"identity":"4b5767c8-7e6f-4856-845d-6e8f01ef070e","added_by":"auto","created_at":"2025-09-24 08:37:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1339131,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of serum exosomes and exosomal NORAD expression.\u003c/p\u003e\n\u003cp\u003e(A) Exosomal NORAD levels are significantly higher in AEG patients than healthy controls. (B) Strong correlation between exosomal NORAD and tumor NORAD expression.\u003c/p\u003e","description":"","filename":"FIGURE5.png","url":"https://assets-eu.researchsquare.com/files/rs-7500532/v1/0ded66e3ad52a32d645d27b0.png"},{"id":92048333,"identity":"d23eb21b-e074-4d93-a304-9131d952ed0b","added_by":"auto","created_at":"2025-09-24 05:13:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":973830,"visible":true,"origin":"","legend":"\u003cp\u003eNORAD knockdown sensitizes AEG cells to oxaliplatin. (A) IC₅₀ values and resistance indices (RI) in PDC and PDC-R cells with or without NORAD knockdown. (B) Similar effects observed in OE19 and OE19-R cells.\u003c/p\u003e","description":"","filename":"FIGURE6.png","url":"https://assets-eu.researchsquare.com/files/rs-7500532/v1/2d40527190f4dd1f677a6ea5.png"},{"id":92048339,"identity":"95bb5757-475a-43da-ae43-5e7b2e139926","added_by":"auto","created_at":"2025-09-24 05:13:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2163853,"visible":true,"origin":"","legend":"\u003cp\u003eNORAD regulates autophagic activity in AEG cells. (A) Western blot analysis of LC3B-I/II and p62 in PDC and PDC-R cells with or without NORAD knockdown. (B) Similar results in OE19 and OE19-R cells.\u003c/p\u003e","description":"","filename":"FIGURE7.png","url":"https://assets-eu.researchsquare.com/files/rs-7500532/v1/f5e4a3f24d82d140e17a4914.png"},{"id":92063562,"identity":"83615654-bced-456f-bfad-eb730612dbdb","added_by":"auto","created_at":"2025-09-24 08:38:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10155936,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7500532/v1/6c19a59b-7c94-4414-99d7-9ca74b5a2b30.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"LncRNA NORAD Promotes Oxaliplatin Resistance in Adenocarcinoma of Esophagogastric Junction via Sponging miR-433-3p and Activating Autophagy","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAdenocarcinoma of the esophagogastric junction (AEG) is a clinically aggressive malignancy that exhibits distinct clinicopathological characteristics compared to distal gastric cancers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The incidence of AEG has increased rapidly in Western countries and certain regions of Asia over the past few decades [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Studies have shown that the combination of perioperative chemotherapy and surgical resection provides significant survival advantages over surgery alone in patients with advanced AEG [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Currently, oxaliplatin-based regimens remain a key component of systemic therapy for advanced stages of this disease [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the emergence of oxaliplatin resistance has become a major challenge, often leading to treatment failure and disease progression [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], underscoring the urgent need to investigate the underlying molecular mechanisms.\u003c/p\u003e\u003cp\u003eAutophagy, an evolutionarily conserved lysosomal degradation pathway, plays a critical role in removing damaged organelles and proteins, thereby maintaining cellular homeostasis. Increasing evidence suggests that excessive activation of autophagy enhances chemoresistance in multiple types of malignancies, such as gastric [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], hepatocellular [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and colorectal cancers [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This adaptive response allows cancer cells to counteract the detrimental effects of chemotherapy and avoid apoptosis, which ultimately leads to therapeutic failure [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLong non-coding RNA activated by DNA damage (NORAD, also known as LINC00657) has been increasingly recognized as a key oncogenic regulator across multiple cancer types [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It promotes tumor progression by enhancing cell migration, metastatic capacity, and resistance to apoptosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Notably, NORAD contributes to chemotherapy resistance in various malignancies, including non-small cell lung cancer [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], neuroblastoma [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], osteosarcoma [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], bladder cancer [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], colorectal cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and esophageal squamous cell carcinoma [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], through diverse molecular mechanisms. Furthermore, quantitative proteomic studies using TMT (Tandem Mass Tag) labeling have indicated that NORAD modulates autophagic flux in gastric cancer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], underscoring its potential involvement in cellular stress responses.\u003c/p\u003e\u003cp\u003eA significant proportion of long non-coding RNAs (lncRNAs) act as competitive endogenous RNAs (ceRNAs) by binding to microRNAs (miRNAs), thereby relieving the repression of downstream target genes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. MiRNAs are small non-coding RNAs that typically bind to the 3\u0026prime;-untranslated regions (3\u0026prime;-UTRs) of target messenger RNAs, leading to translational inhibition or mRNA degradation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Numerous lncRNAs display co-expression patterns or functional associations with adjacent miRNAs [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Notably, microRNA-433-3p (miR-433-3p) has been demonstrated to regulate chemosensitivity. For instance, it enhances cisplatin sensitivity in glioma by targeting NR5A2 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and the knockdown of circ_0011292 improves paclitaxel sensitivity in non-small-cell lung cancer via the miR-433-3p/CHEK1 pathway [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Using bioinformatic tools such as StarBase V3.0 and DIANA-LncBase V3.0, we identified a potential binding site between NORAD and miR-433-3p (chr20:36045805\u0026ndash;36045826). Structural predictions based on RNAhybrid, along with fluorescence in situ hybridization, confirmed their cytoplasmic co-localization and interaction, indicating a possible regulatory mechanism.\u003c/p\u003e\u003cp\u003eExosomes (30\u0026ndash;200 nm extracellular vesicles) serve as critical mediators of intercellular communication by transporting bioactive molecules, including lncRNAs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Accumulating evidence suggests that exosomal lncRNAs play a crucial role in the development of chemotherapy resistance through intercellular horizontal transfer [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For instance, exosomal lincROR derived from prostate cancer cells enhances docetaxel resistance via a β-catenin/HIF1α feedback mechanism [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]; exosomal CRNDE secreted by tumor-associated macrophages promotes cisplatin resistance in gastric cancer [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]; exosomal DACT3-AS1 from cancer-associated fibroblasts reduces oxaliplatin resistance in gastric cancer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]; and exosomal LOC85009 suppresses docetaxel resistance in lung adenocarcinoma by modulating ATG5-mediated autophagy [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the expression pattern and biological function of exosomal lncRNA NORAD in AEG remain poorly understood. Whether exosomal NORAD contributes to oxaliplatin resistance in AEG remains to be elucidated. AEG exhibits a distinct natural history, histogenesis, and molecular profile compared to middle and distal gastric cancers [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In gastric cancer, \u003cem\u003eWang et al\u003c/em\u003e. reported that NORAD enhances autophagic flux and confers resistance to oxaliplatin by stabilizing the ATG5-ATG12 complex through miR-433-3p sponging [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Based on these findings, we propose that NORAD may similarly contribute to oxaliplatin resistance in AEG by promoting autophagy via miR-433-3p sequestration. This study aims to investigate the role and underlying mechanisms of the NORAD/miR-433-3p/autophagy axis in oxaliplatin resistance in AEG and to evaluate the potential of serum exosomal NORAD as a non-invasive biomarker.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Patients and Tissue Samples\u003c/h2\u003e\u003cp\u003eA total of 56 patients with locally advanced AEG and 56 age- and sex-matched healthy volunteers were enrolled in this study. From each AEG patient, paired samples of primary tumor tissue and adjacent normal tissue (ANT), collected from a distance of at least 5 cm from the tumor margin, were obtained during curative resection at Anyang Tumor Hospital between January 2024 and September 2024. All tumors were histopathologically confirmed as adenocarcinoma according to the WHO classification system. None of the patients had received neoadjuvant chemotherapy or radiotherapy prior to surgery. The study was approved by the Ethics Committee of Anyang Tumor Hospital (AZLL0220240103008) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Blood Collection and Serum Isolation\u003c/h2\u003e\u003cp\u003ePeripheral blood (5 mL) was collected from each participant using vacuum tubes containing EDTA (BD Biosciences, USA). The blood samples were kept on ice and centrifuged within one hour at 1000 \u0026times; g for 20 minutes at 4\u0026deg;C to separate the cellular components. The serum was then carefully aliquoted and stored at -80\u0026deg;C until further analysis to ensure the integrity of exosomes and the stability of RNA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Exosome Isolation and Characterization\u003c/h2\u003e\u003cp\u003eSerum exosomes were isolated using the exoRNeasy Serum Midi Kit (Qiagen, Germany) according to the manufacturer's instructions. The size distribution and concentration of the exosomes were analyzed by nanoparticle tracking analysis (NTA; Malvern NanoSight LM10), and their morphology was examined using transmission electron microscopy (TEM; Philips Tecnai 12). To confirm exosomal purity, standard markers (CD63, CD81, and TSG101) were detected by western blotting. Total RNA was extracted from the isolated exosomes using the same kit and subsequently reverse-transcribed using the miScript II RT Kit (Qiagen) for further analysis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 RNA Extraction and lncRNA Microarray\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from tissue samples using TRIzol\u0026reg; Reagent (Invitrogen, USA) in accordance with the manufacturer's instructions. RNA quality and concentration were evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). LncRNA and mRNA expression profiles were generated using the ArrayStar Human LncRNA/mRNA Expression Microarray V3.0 (Arraystar, USA), following the manufacturer's recommended protocols. The microarray annotations were based on databases including NCBI RefSeq, UCSC, RNAdb, and literature-validated lncRNAs [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Microarray hybridization and preliminary data processing were carried out by KangChen Biotech (Shanghai, China). Differentially expressed transcripts were identified using a threshold of fold change\u0026thinsp;\u0026ge;\u0026thinsp;2.0 and an adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Cell Lines and Culture Conditions\u003c/h2\u003e\u003cp\u003eThe human gastric mucosal epithelial cell line GES-1 was purchased from the American Type Culture Collection (ATCC, USA) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Thermo Scientific, USA) and 1% penicillin/streptomycin (HyClone, USA) at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. The OE19 cell line, derived from Siewert type II AEG, was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (China) and maintained under identical culture conditions. Cells were subcultured every 3\u0026ndash;5 days using 0.25% trypsin-EDTA (Sigma-Aldrich, USA) and were routinely tested for mycoplasma contamination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Patient-Derived Cell (PDC) Culture\u003c/h2\u003e\u003cp\u003eFresh primary tumor tissues from advanced AEG patients were rinsed with phosphate-buffered saline (PBS), cut into small fragments (~\u0026thinsp;1 mm\u0026sup3;), and digested with EZ Enzyme (Kurabo, Japan) for 2 hours at 37\u0026deg;C with gentle agitation. The resulting cell pellets were resuspended and cultured in either PCM-2 or ACL4 medium (Kurabo, Japan) until stromal cell contamination was effectively minimized [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Passage 3 to 8 PDCs were selected for experimental use to ensure biological consistency and relevance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Generation of Oxaliplatin-Resistant Cells\u003c/h2\u003e\u003cp\u003eOxaliplatin-resistant sublines (PDC-R and OE19-R) were developed through stepwise exposure to gradually increasing concentrations of oxaliplatin (2\u0026ndash;10 \u0026micro;g/mL; Sigma-Aldrich, USA) over multiple passages (approximately six months). Resistance was verified by continuous proliferation under oxaliplatin treatment and through calculated resistance indices (RI). To maintain the resistant phenotype, all resistant cell lines were cultured in complete medium supplemented with 2 \u0026micro;g/mL oxaliplatin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Bioinformatic Analysis\u003c/h2\u003e\u003cp\u003ePutative interactions between NORAD and miR-433-3p were predicted using StarBase v3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://starbase.sysu.edu.cn\u003c/span\u003e\u003cspan address=\"http://starbase.sysu.edu.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and DIANA-LncBase v3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://diana.e-ce.uth.gr/lncbasev3\u003c/span\u003e\u003cspan address=\"http://diana.e-ce.uth.gr/lncbasev3\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The binding sites were visualized using RNAhybrid (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bibiserv.cebitec.uni-bielefeld.de/rnahybrid\u003c/span\u003e\u003cspan address=\"http://bibiserv.cebitec.uni-bielefeld.de/rnahybrid\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Potential targets of miR-433-3p were identified using miRDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mirdb.org\u003c/span\u003e\u003cspan address=\"http://mirdb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and TargetScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://targetscan.org\u003c/span\u003e\u003cspan address=\"http://targetscan.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Dual-Luciferase Reporter Assay\u003c/h2\u003e\u003cp\u003eFragments of NORAD containing either wild-type (WT) or mutated (Mut) miR-433-3p binding sites were cloned into the GV272 vector (GeneChem, China) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The cells were co-transfected with the reporter constructs and either a miR-433-3p mimic or a negative control (NC) using Lipofectamine 3000 (Invitrogen, USA). Luciferase activity was assessed 48 hours after transfection using a dual-luciferase assay system (Promega, USA) on a GloMax 20/20 Luminometer (Promega, USA). Firefly luciferase activity was normalized relative to Renilla luciferase activity in each sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Lentiviral Transduction and Knockdown\u003c/h2\u003e\u003cp\u003eLentiviral particles carrying short hairpin RNA (shRNA) targeting NORAD or a non-targeting control (NC) were synthesized by GeneChem (China). Cells were transduced at a multiplicity of infection (MOI) of 20 in the presence of Polybrene (8 \u0026micro;g/mL; Sigma-Aldrich, USA). Stably transduced cell pools were selected using puromycin (2 \u0026micro;g/mL; Sigma-Aldrich, USA) over a period of two weeks. Knockdown efficiency was confirmed by qRT-PCR.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Cell Viability and IC₅₀ Assay\u003c/h2\u003e\u003cp\u003eCells were seeded in 96-well plates at a density of 3\u0026times;10\u0026sup3; cells/well and exposed to varying concentrations of oxaliplatin (1\u0026ndash;64 \u0026micro;g/mL) for 48 hours. Cell viability was evaluated using the CCK-8 kit (ApexBio, USA) according to the manufacturer's protocol [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Absorbance was then measured at 490 nm using a microplate reader (BioTek, USA). IC₅₀ values (half-maximal inhibitory concentration) were determined through nonlinear regression analysis with GraphPad Prism v 9.0 (GraphPad Software, USA). The resistance index (RI) was defined as the ratio of IC₅₀ values between resistant and parental cell lines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.12 Quantitative Real-Time PCR (qRT-PCR)\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eTotal RNA was reverse-transcribed using the PrimeScript RT Reagent (Takara, Japan). Quantitative PCR (qPCR) was carried out using SYBR Premix Ex Taq II (Takara, Japan) on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, USA). miRNA expression was analyzed using the Mir-X miRNA RT-PCR Kit (Takara, Japan). GAPDH and U6 small nuclear RNA were used as endogenous controls for NORAD and miR-433-3p, respectively. Relative gene expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. The primer sequences used were as follows: NORAD forward, 5'-ACAGCCAAATCACAGCCACA-3'; reverse, 5'-TGGGCTCTGTGTTTGCATCC-3'; miR-433-3p forward, 5'-ATCACAUUGGGCUCCUCGGUGU-3'; reverse: universal primer provided in the kit; GAPDH forward, 5'-GGAGCGAGATCCCTCCAAAAT-3'; reverse, 5'-GGCTGTTGTCATACTTCTCATGG-3'; U6 forward, 5'-CTCGCTTCGGCAGCACA-3'; reverse, 5'-AACGCTTCACGAATTTGCGT-3'.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Protein extraction and Western Blotting\u003c/h2\u003e\u003cp\u003eGO pathway analysis revealed the potential involvement of NORAD in the assembly process of autophagosomes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The conversion of the cytosolic LC3B-I protein to the membrane-bound LC3B-II isoform on the phagophore is a key hallmark of autophagy, and the quantification of LC3B-II levels represents the most reliable method for assessing autophagic flux in tumor cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Moreover, a negative correlation exists between p62 protein abundance and the strength of autophagic flux. Proteins were extracted using RIPA buffer (Beyotime, China), supplemented with protease inhibitors (Roche, Switzerland). Protein concentrations in the lysates were determined using a bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich, USA). Equal amounts of protein (20\u0026ndash;30 \u0026micro;g) were separated by 10% SDS-PAGE and then transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 5% non-fat milk to minimize nonspecific binding. Primary antibodies against LC3B (1:1000; Novus Biologicals, USA), p62 (1:800; Santa Cruz Biotechnology, USA), and GAPDH (1:5000; Proteintech, USA) were incubated overnight at 4\u0026deg;C. After incubation with primary antibodies, the membranes were treated with HRP-conjugated secondary antibodies (1:5000; Cell Signaling Technology, USA), and the protein bands were visualized using ECL reagent (Bio-Rad, USA). Band intensities were analyzed and quantified using ImageJ software (NIH, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Statistical Analysis\u003c/h2\u003e\u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Comparisons between two groups were made using two-tailed \u003cem\u003eStudent's t-tests\u003c/em\u003e, while \u003cem\u003eone-way analysis of variance (ANOVA)\u003c/em\u003e followed by Tukey's post hoc test was used for comparisons among multiple groups. Pearson's correlation coefficient was employed to assess correlations. Statistical analyses were performed using \u003cem\u003eSPSS V 22.0\u003c/em\u003e (IBM, USA) and \u003cem\u003eGraphPad Prism V 9.0\u003c/em\u003e. A p-value less than 0.05 was considered statistically significant. All experiments were independently repeated at least three times.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Identification of Differentially Expressed lncRNAs in AEG\u003c/h2\u003e\u003cp\u003eWe analyzed lncRNA expression profiles in primary tumor tissues and paired ANTs from four treatment-na\u0026iuml;ve patients with locally advanced AEG using the ArrayStar Human LncRNA/mRNA Microarray V3.0. Among the 503,687 lncRNAs profiled, several differentially expressed transcripts were identified. Notably, the lncRNA NORAD was significantly upregulated in AEG tissues compared to ANTs (fold change\u0026thinsp;\u0026ge;\u0026thinsp;2.0, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This result was further validated by qRT-PCR in an expanded cohort of 56 AEG patients, which confirmed the overexpression of NORAD in tumor tissues (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21.213, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.2 NORAD and miR-433-3p Are Differentially Expressed in AEG Tissues and Show Inverse Correlation\u003c/h2\u003e\u003cp\u003eBioinformatic analysis using StarBase v3.0 and DIANA-LncBase v3.0 identified miR-433-3p as a direct target of NORAD, suggesting a potential competing endogenous RNA (ceRNA) mechanism in oxaliplatin resistance. Quantitative RT-PCR validation in 56 AEG patients confirmed that NORAD was significantly upregulated (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21.213, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while miR-433-3p was significantly downregulated (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;19.463, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in tumor tissues compared with ANTs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A significant inverse correlation was observed between NORAD and miR-433-3p expression levels in AEG tissues (\u003cem\u003er\u003c/em\u003e = -0.864, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExpression of NORAD and miR-433-3p in 56 AEG patients compared with ANT.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eNORAD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003emiR-433-3p\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003emean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAEG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.245\u0026thinsp;\u0026plusmn;\u0026thinsp;0.376\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.000\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.444\u0026thinsp;\u0026plusmn;\u0026thinsp;0.143\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.000\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eANT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.265\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.039\u0026thinsp;\u0026plusmn;\u0026thinsp;0.161\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.3 NORAD Directly Binds to miR-433-3p\u003c/h2\u003e\u003cp\u003eDual-luciferase reporter assays further confirmed that miR-433-3p directly targets NORAD. As shown by the significantly reduced luciferase activity in PDCs and PDC-Rs co-transfected with wild-type NORAD and miR-433-3p mimics (approximately 60% reduction compared to the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), this interaction is functionally relevant. Mutation of the predicted miR-433-3p binding site within NORAD completely restored luciferase activity, thereby validating the specificity of the interaction.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.4 Expression of NORAD and miR-433-3p in AEG Cell Lines\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eOxaliplatin-resistant sublines (PDC-R and OE19-R) were derived from their parental PDC and OE19 cells through prolonged stepwise exposure to oxaliplatin. NORAD-knockdown cells (shNORAD) were successfully established via lentiviral transduction, achieving a knockdown efficiency exceeding 80% across all cell types (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). qRT-PCR analysis demonstrated that NORAD expression was significantly upregulated in both PDC and PDC-R cells compared to the non-tumoral GES-1 cells (both \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with a further significant increase observed in PDC-R cells relative to PDC cells (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;19.432, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, miR-433-3p expression was markedly downregulated in all AEG-derived cell lines compared to GES-1 cells (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;147.567, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Knockdown of NORAD significantly elevated miR-433-3p levels in both PDC and PDC-R cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Consistent trends were observed in the OE19-based cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Serum Exosomal NORAD Is Elevated in AEG and Correlates with Tumor Expression\u003c/h2\u003e\u003cp\u003eSerum exosomes were isolated from patients with AEG and healthy volunteers and were characterized using NTA and TEM. The exosomes exhibited the typical cup-shaped morphology with diameters ranging from 50 to 150 nm. Western blot analysis confirmed the enrichment of exosomal markers, including CD63, CD81, and TSG101, as well as the absence of the negative marker calnexin. Notably, exosomal NORAD levels were significantly elevated in AEG patients compared to those in healthy volunteers (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;35.646, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Furthermore, a strong positive correlation was observed between exosomal NORAD expression and NORAD levels in matched tumor tissues (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8858, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating its potential as a non-invasive biomarker for clinical applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.6 NORAD Knockdown Sensitizes AEG Cells to Oxaliplatin\u003c/h2\u003e\u003cp\u003eCCK-8 assays demonstrated that oxaliplatin-resistant cell lines (PDC-R and OE19-R) exhibited significantly higher IC₅₀ values compared to their parental counterparts (PDC-R: 22.27 \u0026micro;g/mL \u003cem\u003evs\u003c/em\u003e. PDC: 14.28 \u0026micro;g/mL; resistance index\u0026thinsp;=\u0026thinsp;1.559; OE19-R: 25.43 \u0026micro;g/mL \u003cem\u003evs\u003c/em\u003e. OE19: 15.16 \u0026micro;g/mL; resistance index\u0026thinsp;=\u0026thinsp;1.677). NORAD knockdown substantially decreased oxaliplatin resistance, resulting in IC₅₀ values of 2.00 \u0026micro;g/mL in shNORAD-transduced PDC cells and 2.51 \u0026micro;g/mL in shNORAD-transduced PDC-R cells, corresponding to resistance indices of 0.140 and 0.113, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). A similar resensitization effect was observed in the OE19-derived cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.7 NORAD Regulates Autophagic Activity in AEG Cells\u003c/h2\u003e\u003cp\u003eWestern blot analysis demonstrated that oxaliplatin-resistant cells (PDC-R and OE19-R) displayed elevated levels of LC3B-II and reduced expression of p62 compared to their parental counterparts, suggesting an enhancement in autophagic flux (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). NORAD knockdown reversed this effect, leading to decreased LC3B-II levels and increased p62 expression in the resistant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings indicate that NORAD contributes to oxaliplatin resistance by promoting autophagy in AEG cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.8 NORAD was implicated in the regulation of autophagy in AEG cells\u003c/h2\u003e\u003cp\u003eWe employed Western blotting to detect and compare the relative expression levels of LC3B-I, LC3B-II, and p62 in PDC, PDC-R, and shNORAD PDC-R cell lines. In parallel, we assessed these markers in OE19, OE19-R, and shNORAD OE19-R cell lines. Our results revealed a significant increase in LC3B-II levels in PDC-R cells compared to PDC cells. Knockdown of NORAD in PDC-R cells led to a decrease in LC3B-II expression, suggesting a reduction in autophagic flux. In contrast, p62 protein levels were lower in PDC-R cells than in PDC cells; however, NORAD knockdown mediated by shRNA resulted in an accumulation of p62 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Consistent results were observed in the AEG cell line OE19 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThis study demonstrates that the long non-coding RNA (lncRNA) NORAD is significantly overexpressed in both tumor tissues and serum exosomes obtained from patients with AEG. We present mechanistic evidence indicating that NORAD promotes oxaliplatin resistance by enhancing autophagy through a ceRNA mechanism, which involves sponging miR-433-3p and subsequently derepressing ATG5. Consistent with its previously reported oncogenic roles in other malignancies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], NORAD expression is markedly upregulated in AEG tissues compared to adjacent normal tissues. Importantly, we report for the first time that serum exosomal levels of NORAD are significantly elevated in AEG patients and show a strong correlation with NORAD expression in matched tumor tissues. These findings suggest that exosomal NORAD may serve as a non-invasive biomarker for AEG diagnosis and disease monitoring, supporting the growing recognition of exosomal lncRNAs as mediators of intercellular communication and contributors to chemotherapy resistance [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our findings corroborate recent studies highlighting exosomal lncRNAs as key regulators of drug resistance in gastric cancer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and extend this concept to AEG, a distinct clinical entity with unique molecular features [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThrough bioinformatic prediction and experimental validation, we identified miR-433-3p as a direct target of NORAD. The inverse correlation between NORAD and miR-433-3p expression across clinical samples and cell models supports a ceRNA mechanism, whereby NORAD functions as a molecular sponge to sequester miR-433-3p, thereby attenuating its tumor-suppressive effects. This regulatory axis aligns with previous findings in gastric cancer, where NORAD modulates autophagy and chemoresistance through miR-433-3p [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our study further demonstrates that this mechanism is present in AEG and is particularly relevant to oxaliplatin resistance. Functional assays confirmed that NORAD knockdown significantly enhanced the sensitivity of both parental and oxaliplatin-resistant AEG cells to oxaliplatin, highlighting its essential role in mediating drug resistance. Western blot analysis further demonstrated that NORAD upregulation promotes autophagic flux, as indicated by increased levels of LC3B-II and decreased expression of p62\u0026mdash;effects that were reversed upon NORAD silencing. These findings suggest that NORAD contributes to oxaliplatin resistance by activating autophagy, potentially through the derepression of the ATG5-ATG12 complex formation via sponging miR-433-3p. This mechanism is particularly relevant given the well-established role of autophagy in chemoresistance across multiple cancer types [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our study also highlights the therapeutic potential of targeting the NORAD/miR-433-3p/autophagy axis. The marked reduction in oxaliplatin resistance following NORAD knockdown suggests that pharmacological inhibition of NORAD may represent a promising strategy to overcome chemoresistance in AEG patients. Several approaches for targeting lncRNAs are currently under investigation, including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and CRISPR-based technologies [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Furthermore, the strong correlation between serum exosomal NORAD levels and tumor expression suggests that NORAD may serve as a non-invasive biomarker for predicting treatment response and monitoring disease progression.\u003c/p\u003e\u003cp\u003eSeveral limitations of our study should be acknowledged. First, the sample size was relatively small and derived from a single institution, underscoring the necessity for future multi-center studies with larger cohorts to validate the clinical utility of serum exosomal NORAD. Second, although we demonstrated that NORAD contributes to oxaliplatin resistance by modulating autophagy via miR-433-3p sponging, we cannot exclude the possibility that additional mechanisms may also be involved. Third, the mechanisms responsible for the selective packaging of NORAD into exosomes remain unclear and require further investigation. Finally, in vivo studies are needed to confirm the therapeutic potential of targeting NORAD in AEG and to assess any potential off-target effects. In conclusion, our findings establish NORAD as a critical player in oxaliplatin resistance in AEG by regulating the miR-433-3p/autophagy axis. The strong correlation between serum exosomal NORAD levels and tumor expression suggests its potential as a non-invasive biomarker. Targeting the NORAD/miR-433-3p/autophagy pathway may represent a promising therapeutic strategy for overcoming oxaliplatin resistance in AEG patients.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThis study reveals that the lncRNA NORAD is markedly upregulated in both AEG tissues and serum exosomes, where it contributes to oxaliplatin resistance by sequestering miR-433-3p and inducing autophagy. Knockdown of NORAD enhances the sensitivity of AEG cells to oxaliplatin, suppresses autophagic flux, and promotes apoptosis. Notably, serum exosomal levels of NORAD show a strong correlation with its expression in tumor tissues, underscoring its potential as a non-invasive biomarker. Targeting the NORAD/miR-433-3p/autophagy pathway may represent a novel therapeutic approach to reverse chemoresistance in patients with AEG.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their sincere gratitude to Professor Baozhong Li, Professor Zhiqiang Liu, Professor Wei Zhang, Miss Gongjing Lu, and Anyang Tumor Hospital for their invaluable support. We are grateful to Editage (www.editage.cn) for providing English language editing services.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional review board statement\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInformed written consent was obtained from all patients. The present study was approved by the Ethics Committee of the Anyang Tumor Hospital (Anyang, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict-of-interest statement\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no competing interests. The authors declare that they have no conflicts of interest. All the authors agree to share the data and materials used in this study. All authors declare their consent to publish this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Henan Province Science and Technology Tackling Key Project (232102310090) and (No. 242102310125).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Anyang Tumor Hospital. The patients informed consent was obtained according to regional regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding author, Dr. Shoumiao Li ( E-Mail: [email protected] ). All authors agree to share the data and materials used in this study. For more detailed information, please contact Dr. Shoumiao Li, who will be pleased to provide further assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe final manuscript has undergone thorough review and approval by all authors, who disclose no conflicts of interest. The authors unanimously consent to the sharing of data and materials utilized in this study, as well as granting permission for publication of this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDemicco EG, Farris AB 3rd, Baba Y, et al. The dichotomy in carcinogenesis of the distal esophagus and esophagogastric junction: intestinal - type vs cardiac - type mucosa - associated adenocarcinoma. 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Exosomal PD-L1 and lactate versus tissue PD-L1 as biomarkers for clinical outcomes of PD-1 Blockade plus chemotherapy in metastatic esophagogastric signet ring cell carcinoma. Exp Hematol Oncol. 2025;14:34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu S, Mao J, Zhang X, et al. CAF-derived exosomal lncRNA FAL1 promotes chemoresistance to oxaliplatin by regulating autophagy in colorectal cancer. Dig Liver Dis. 2024;56:330\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlnefaie GO. A review of the complex interplay between chemoresistance and lncRNAs in lung cancer. J Transl Med. 2024;22:1109.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Adenocarcinoma of esophagogastric junction, Oxaliplatin resistance, Long non-coding RNA NORAD, miR-433-3p, Autophagy, Exosomes","lastPublishedDoi":"10.21203/rs.3.rs-7500532/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7500532/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eAdenocarcinoma of esophagogastric junction (AEG) is a clinically aggressive malignancy with an increasing incidence worldwide. Although oxaliplatin-based chemotherapy represents a cornerstone of treatment, the development of drug resistance remains a significant clinical challenge. This study aims to explore the role and underlying mechanism of the long non-coding RNA NORAD in oxaliplatin resistance in AEG, focusing on the NORAD/miR-433-3p/autophagy regulatory axis, as well as to evaluate the potential of serum exosomal NORAD as a novel biomarker.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003ePaired tumor and adjacent normal tissues were obtained from 56 patients with AEG and analyzed. Differentially expressed lncRNAs were identified using lncRNA microarray profiling and validated by qRT-PCR. Oxaliplatin-resistant cell lines (PDC-R and OE19-R) were established through long-term drug exposure. Serum-derived exosomes were isolated and characterized in terms of morphology and marker expression. Bioinformatics tools predicted potential interactions between NORAD and miR-433-3p, which were subsequently confirmed using dual-luciferase reporter assays. NORAD knockdown was effectively achieved via lentiviral transduction with shRNA targeting NORAD. Cell viability was evaluated using the CCK-8 assay. The levels of autophagy-related proteins, including LC3B-II and p62, were assessed by western blot analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eNORAD was markedly upregulated in AEG tissues and further increased in oxaliplatin-resistant cells, whereas miR-433-3p expression was downregulated. A strong inverse correlation was observed between NORAD and miR-433-3p levels (\u003cem\u003er\u003c/em\u003e = -0.864, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Luciferase assays confirmed that NORAD directly interacts with miR-433-3p. Serum exosomal NORAD levels were significantly elevated in AEG patients compared to those in healthy controls and showed a positive correlation with NORAD expression in tumor tissues (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8858, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Knockdown of NORAD enhanced the sensitivity of AEG cells to oxaliplatin, as indicated by reduced IC₅₀ values and resistance indices. Furthermore, NORAD silencing impaired autophagic flux, as demonstrated by decreased LC3B-II levels and increased p62 accumulation.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eLncRNA NORAD contributes to oxaliplatin resistance in AEG by acting as a sponge for miR-433-3p and subsequently promoting autophagy. Serum-derived exosomal NORAD holds promise as a non-invasive biomarker for AEG. Targeting the NORAD/miR-433-3p/autophagy axis may offer a novel therapeutic approach to counteract chemoresistance.\u003c/p\u003e","manuscriptTitle":"LncRNA NORAD Promotes Oxaliplatin Resistance in Adenocarcinoma of Esophagogastric Junction via Sponging miR-433-3p and Activating Autophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 05:05:33","doi":"10.21203/rs.3.rs-7500532/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-29T03:26:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T07:58:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253557524949498519343846753628713739905","date":"2025-10-28T06:58:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-09T17:11:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150332220259364295706894061231040722588","date":"2025-09-20T09:12:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54077920669351808652917811517211105561","date":"2025-09-19T11:59:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-15T01:29:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-03T09:00:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-03T09:00:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Cell International","date":"2025-08-31T12:10:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f2d24a0-758a-4fc1-a585-6a71f3142539","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T09:26:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-24 05:05:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7500532","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7500532","identity":"rs-7500532","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}