A novel antisense lncRNA, LPCRL, functions as a molecular scaffold for the USP15/MIB1 complex to promote primary cisplatin resistance and tumor progression in lung squamous cell carcinoma

A novel antisense lncRNA, LPCRL, functions as a molecular scaffold for the USP15/MIB1 complex to promote primary cisplatin resistance and tumor progression in lung squamous cell carcinoma | 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 A novel antisense lncRNA, LPCRL, functions as a molecular scaffold for the USP15/MIB1 complex to promote primary cisplatin resistance and tumor progression in lung squamous cell carcinoma Peng Luo, Dapeng Lu, Shuang Zhang, Wenqian Dong, Kai Fang, Shihao Yu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8106822/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 May, 2026 Read the published version in Journal of Experimental & Clinical Cancer Research → Version 1 posted 8 You are reading this latest preprint version Abstract Background Platinum-based chemotherapy remains the first-line treatment for advanced lung squamous cell carcinoma (LUSC), but its efficacy is often hindered by the development of chemoresistance. Although long noncoding RNAs (lncRNAs) are recognized as regulators of tumor progression and drug resistance, the functional contribution of natural antisense transcripts (NATs), a major subclass of lncRNAs involved in cisplatin resistance in LUSC, remains poorly understood. Methods Patient-derived xenograft (PDX) models of LUSC were established and treated with cisplatin to identify cisplatin-resistant and cisplatin-sensitive tumor tissues. LncRNA microarray profiling was used to identify transcripts associated with cisplatin resistance. The functional role of a candidate lncRNA, termed LPCRL (LUSC primary cisplatin resistance-associated LncRNA), was assessed in vitro via MTT, flow cytometry, colony formation, and Transwell migration assays. Its effects on tumor growth and metastasis were further validated in vivo. Mechanistic insights were gained through RNA pull-down, silver staining, RNA immunoprecipitation (RIP), coimmunoprecipitation (Co-IP), and Western blot analyses. Finally, the therapeutic potential of LPCRL-targeting siRNA was assessed in a LUSC PDX model. Results We found that LPCRL was significantly upregulated in primary cisplatin-resistant PDX tissues. Functionally, LPCRL promoted primary cisplatin resistance and enhanced the proliferation and migration of LUSC cells both in vitro and in vivo. Mechanistically, LPCRL functions as a molecular scaffold to facilitate the interaction between MIB1 and USP15. This complex enables USP15 to deubiquitinate MIB1, thereby increasing MIB1 stability and promoting its nuclear export. The subsequent cytoplasmic accumulation of MIB1 enhances the ubiquitination of DLL4, leading to Notch pathway activation and upregulation of the downstream effector HES1. Importantly, intratumoral administration of LPCRL-targeting siRNA in PDX models suppressed tumor growth and sensitized tumors to cisplatin in vivo. Conclusions Our study revealed that LPCRL promotes LUSC malignancy and cisplatin resistance via the USP15/MIB1/Notch axis, highlighting LPCRL as a promising therapeutic target. Lung squamous cell carcinoma Cisplatin resistance Antisense long noncoding RNA LPCRL USP15/MIB1 complex Molecular scaffold Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Non-small cell lung cancer (NSCLC) accounts for 80%-85% of all lung cancer cases, with the squamous cell subtype comprising 20%-30% of NSCLCs [ 1 ]. Unfortunately, most LUSC patients are diagnosed at advanced stages, resulting in high mortality rates [ 2 ]. Although targeted therapies and immunotherapies have transformed the treatment landscape for LUSC [ 3 ], platinum-based chemotherapy remains the cornerstone of first-line treatment for advanced cases [ 4 , 5 ]. However, its efficacy is often limited, with objective response rates of only 30%-40%, primarily due to intrinsic resistance [ 6 , 7 ]. Therefore, elucidating the molecular mechanisms underlying primary platinum resistance in LUSC is critical for developing novel therapeutic strategies aimed at improving patient outcomes. Long noncoding RNAs (lncRNAs) are defined as RNA transcripts longer than 200 nucleotides that lack protein-coding capacity but are critically involved in regulating diverse biological processes in lung cancer [ 8 – 11 ]. Antisense lncRNAs, a major subclass of lncRNAs, are transcribed from the complementary strand of protein-coding or noncoding genes. They account for approximately 50%-70% of all annotated lncRNAs and are widely distributed across both eukaryotic and prokaryotic genomes. Notably, antisense lncRNAs can regulate gene expression at multiple levels, including the pretranscriptional, transcriptional, and posttranscriptional levels, through interactions with DNA, RNA, or proteins [ 12 ]. Emerging evidence has linked antisense lncRNAs to various aspects of cancer biology, including drug resistance [ 10 ], metabolic reprogramming [ 11 ], phase separation [ 13 ], cell proliferation, and metastasis [ 14 ]. Nevertheless, the specific roles and mechanisms of antisense lncRNAs in driving primary cisplatin resistance in LUSC remain largely unexplored. Patient-derived xenograft (PDX) models are widely recognized as robust preclinical tools that recapitulate intratumoral heterogeneity, preserve native tumor architecture, and reliably reflect drug responses and resistance mechanisms [ 15 ]. After serial passaging to the third generation (P3), these models reach a biological "plateau" with stabilized characteristics, enhancing their reproducibility for drug studies [ 16 ]. In this study, microarray analysis of the lncRNA expression profiles of third-generation xenograft tumors revealed a clear transcriptional distinction between the cisplatin-sensitive and cisplatin-resistant groups via principal component analysis. We identified a markedly upregulated antisense transcript, uc002ktr.3, hereafter referred to as LPCRL (LUSC primary cisplatin resistance-associated LncRNA), in the cisplatin-resistant cohort. This transcript is derived from the MIR133A1HG gene locus, which overlaps the antisense strand of the twelfth intron of MIB1 . Functional studies demonstrated that LPCRL enhance cisplatin resistance, promote proliferation, and facilitate metastasis in LUSC cells. Mechanistically, LPCRL functions as a molecular scaffold that directly mediates the interaction between MIB1 and USP15. This promotes USP15-mediated deubiquitination of nuclear MIB1. This posttranslational modification stabilizes MIB1 and promotes its nuclear export. The resulting cytoplasmic accumulation of MIB1 activates the Notch signaling pathway. Importantly, the siRNA-mediated silencing of LPCRL in vivo significantly suppressed tumor growth and migration while simultaneously enhancing cisplatin sensitivity. In summary, our study reveals a pivotal role for the LPCRL/USP15/MIB1/Notch signaling axis in promoting cisplatin resistance and tumor progression in LUSC, identifying LPCRL as a promising molecular target for therapeutic intervention. Materials and methods Clinical specimens Patient-derived xenograft (PDX) models were established using tumor tissues from patients diagnosed with LUSC at The First Affiliated Hospital of the University of Science and Technology of China. All tumor samples were collected with approval from the Ethics Committee of the University of Science and Technology of China (Approval No. 2019-N(H)-128), and written informed consent was obtained from all participants. Establishment of PDX models and cisplatin chemosensitivity testing All animal procedures were conducted in accordance with the Declaration of Helsinki and were approved by the Institutional Animal Care and Use Committee of the University of Science and Technology of China (Approval No. 2019-N (A)-179). PDX models were generated as previously described [ 17 ]. Briefly, fresh tumor tissues were sectioned into ~ 3 mm³ fragments and implanted subcutaneously into the flanks of BALB/c nude mice. Following the same method, the transplanted tumors were serially passaged to the third generation. Once the third-generation xenograft tumor volume reached 50–200 mm³, 3–5 mice per model were randomized into treatment or control groups. The tumor volume was calculated as (length × width^2)/2. Cisplatin (20 mg/mL; Jiangsu Hansoh Pharmaceutical Co., Ltd.) was diluted to 0.5 mg/mL in saline and administered intraperitoneally at 5 mg/kg once weekly for 3 consecutive weeks (treatment group). The control mice received an equivalent volume of saline. The injection volume was standardized at 0.2 mL per 20 g body weight. A tumor growth inhibition rate (TIR = 1 - (average tumor volume in the cisplatin group/average tumor volume in the PBS group)×100%) ≥ 50% was considered cisplatin sensitive; <50% was considered resistant. Establishment of primary LUSC cells Primary LUSC cells were established as follows. Briefly, fresh LUSC tumor tissue from surgical resection was placed in cold preservation medium (DMEM with antibiotics/antimycotics) on ice. The tissue was minced into 1–2 mm³ fragments and digested in a collagenase solution (200 U/mL, Sigma Aldrich, Saint Louis, MO, USA) at 37°C with periodic shaking or resuspension. The resulting cell suspension was filtered through a 70 µm cell strainer, and the filtrate was centrifuged. The cell pellet was washed with PBS, resuspended in culture medium (RPMI 1640 medium (HyClone) with 10% FBS), and seeded. Cultures were maintained in a humidified incubator at 37°C with 5% CO₂, with regular medium changes to remove nonadherent cells and debris. Once the cells reached 80%-90% confluence, they were passaged with trypsin-EDTA (Beyotime). LncRNA microarray and bioinformatic analysis Total RNA was extracted from cisplatin-resistant and cisplatin-sensitive PDX samples for microarray analysis via the Arraystar Human LncRNA Microarray V3.0 (Agilent Technologies). Feature extraction was performed with Agilent Feature Extraction software (v11.0.1.1), and quantile normalization and data processing were conducted via GeneSpring GX v12.1 (Agilent Technologies). Differentially expressed lncRNAs were identified by a fold change > 1.5 and a p value < 0.05. Principal component analysis and volcano plots were generated via in-house scripts. Cell culture SK-MES-1 and HEK293T cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The NCI-H520 cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). LUSC primary cells were established in our laboratory. NCI-H520 and primary cells were cultured in RPMI-1640 medium (HyClone) supplemented with 10% fetal bovine serum (FBS; Proteintech). SK-MES-1 and HEK293T cells were cultured in DMEM/high-glucose medium (HyClone) supplemented with 10% FBS. All the cells were maintained at 37°C in a humidified incubator with 5% CO₂ and passaged using 0.25% trypsin-EDTA (Beyotime). RNA extraction, reverse transcription and quantitative PCR ( RT‒qPCR) Total RNA was extracted via TRIzol Reagent (Life Technologies, CA, USA). Nuclear and cytoplasmic RNA fractions were isolated via nuclear and cytoplasmic extraction reagents (Invitrogen, NY, USA). Complementary DNA (cDNA) was synthesized with the PrimeScript™ RT‒PCR Kit (Takara Bio, Otsu, Shiga, Japan), and quantitative PCR was performed via SYBR® Select Master Mix (Vazyme, Nanjing, China) on an ABI 7500 real-time PCR system (Thermo Fisher Scientific, USA). The primer sequences are listed in Supplementary Table S1 . Small interfering RNA (siRNA), antisense oligonucleotides, plasmid construction and cell transfection Small interfering RNAs (siRNAs) were obtained from Generalbiol (Shanghai, China). Antisense oligonucleotides (ASOs) were obtained from RiboBio (Guangzhou, China). Plasmid vectors (pCMV-LPCRL, pCMV-LPCRL-M1, pCMV-LPCRL-M2, pCMV-LPCRL-M3, pCMV-Myc-MIB1, pCMV-HA-USP15, and the empty pCMV vector) were obtained from the Miaoling Plasmid Platform (Wuhan, China). siRNAs and ASOs were transfected via a Lipofectamine™ 2000 kit (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol. Plasmid transfection was performed using a PEI MW40000 (Yeasen Biotechnology, Shanghai, China). All the siRNA sequences used are listed in Supplementary Table S2 . Lentiviral transfection Lentiviral-shLPCRL and Lentiviral-LPCRL were obtained from Hanheng Biological Company (Shanghai, China). Lentiviral (LV)-short hairpin negative control (shNC) and LV-sh LPCRL (sh LPCRL-1, sh LPCRL-2) were used to transfect SK-MES-1 cells. The short hairpin RNA sequences used to silence the LPCRL are listed in Supplementary Table S1 . Lentiviral (LV)-LPCRL (oeLPCRL) was used to transfect NCI-H520 cells. Inducible cell sublines were established via puromycin selection and validated via RT‒qPCR. MTT and colony formation assays For the MTT assays, 96-well plates were seeded with cells and incubated with MTT (5 mg/mL; Biosharp, Anhui, China) for 4 hours. The absorbance was measured at 490 nm. For the colony formation assays, 1,000 cells/well were seeded in 6-well plates and cultured for 1–2 weeks. Colonies were fixed in 4% paraformaldehyde, stained with crystal violet (Sangon Biotech, Shanghai, China), and counted manually. Transwell migration assay For the migration assays, 8 × 10⁴ cells in 200 µL of serum-free DMEM were seeded into the upper chambers of Transwell plates (8-µm pores; Corning, NY, USA). The lower chambers contained 800 µL of medium supplemented with 10% FBS as a chemoattractant. After 24–48 hours, the migrated cells were fixed, stained with crystal violet, and quantified under a microscope. IC50 assay The cells (8 × 10⁴ cells/well) were seeded in 96-well plates, allowed to adhere, and then treated with various concentrations of cisplatin for 48 hours. IC₅₀ values were calculated via GraphPad Prism v9.5.0 on the basis of the logarithmic relationship between drug concentration and the cellular response. Flow cytometry apoptosis assay Apoptosis was assessed via an Annexin V-FITC/PI Apoptosis Detection Kit (Keygen Biotech, Nanjing, China). After 24 hours of cisplatin treatment, the cells were washed with PBS, resuspended in 500 µL of binding buffer, and stained with 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) for 10 minutes in the dark. Apoptosis was analyzed via a CytoFLEX flow cytometer and CytExpert software (Beckman Coulter). The cells were categorized as viable (FITC⁻/PI⁻), early apoptotic (FITC⁺/PI⁻), or late apoptotic/dead (FITC⁺/PI⁺). Fluorescence in situ hybridization (FISH) Biotin-labeled LPCRL probes (Ribobio, Guangzhou, China) were used to detect LPCRL localization with a FISH detection kit (Ribobio). Fixed cells were hybridized with probes overnight, washed, stained with DAPI, and visualized via fluorescence microscopy. The tissue samples were cryosectioned at 4 µm after fixation in 4% paraformaldehyde for 1 hour and processed similarly. RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assays The RNA pull-down assays used biotin-labeled LPCRL probes (RiboBio) and their antisense controls. SK-MES-1 cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 5 mM EDTA; 1% NP-40; 0.1% SDS; 1 mM DTT; 1× protease inhibitor cocktail; 0.1 U/µL RNase inhibitor) for 15 minutes on ice. The lysates were centrifuged at 12,000 × g for 15 minutes, and the supernatants were incubated with 100 pmol of biotinylated oligonucleotides or 2 µg of antibodies (MIB1, USP15 for RIP) overnight at 4°C. M-280 streptavidin Dynabeads (Invitrogen, 11206D, for RNA pull-down) or Protein G Dynabeads (Invitrogen, 10004D, for RIP) preblocked with 500 ng/µL yeast total RNA and 5% BSA were added for 2 hours at room temperature. The beads were washed with lysis buffer and high-salt lysis buffer (500 mM NaCl). The purified RNAs were analyzed via RT‒qPCR, and the proteins were analyzed via western blotting after silver staining. 5′ and 3′ rapid amplification of cDNA ends (RACE) RACE was performed via the SMARTer RACE 5′/3′ Kit (Takara). Total RNA from SK-MES-1 cells was used to synthesize 5′- and 3′-RACE-ready cDNA, which was amplified via nested PCR with universal primers and gene-specific primers ( Supplementary Table S2 ). Western blot Total protein was extracted via a Total Protein Extraction Kit (Bestbio, Shanghai, China) with protease and phosphatase inhibitors (Epizyme, Shanghai, China). Proteins were quantified and separated by SDS‒PAGE, transferred to PVDF membranes (Merck Millipore), blocked with 5% skim milk, and incubated with primary antibodies overnight, followed by incubation with secondary antibodies (Proteintech). Bands were detected via a Tanon multigel imaging system. The following antibodies were used: MIB1 (sc-393551, Santa Cruz, 1:500), USP15 (66310s, CST, 1:1000), c-PARP (ab32561, Abcam, 1:1000), γ-H2AX (CY6572, Abways, 1:1000), β-Actin (66009-1-Ig, Proteintech, 1:10000), GAPDH (60004-1-Ig, Proteintech, 1:10000), Ub (#20326, CST, 1:1000), Myc (60003-2-Ig, Proteintech, 1:2000), HA (51064-2-AP, Proteintech, 1:2000), DLL4 (21584-1-AP, Proteintech, 1:2000), LaminB1 (12987-1-AP, Proteintech, 1:2000), NICD (10062-2-AP, Proteintech, 1:2000), N-cadherin (22018-1-AP, Proteintech, 1:2000), PCNA (60097-1-Ig, Proteintech, 1:5000), Vimentin (10366-1-AP, Proteintech, 1:5000), and c-Myc (10828-1-AP, Proteintech, 1:5000). Immunoprecipitation (IP) The cells were lysed in IP buffer supplemented with protease and phosphatase inhibitors, and the lysates were subsequently centrifuged. The supernatants were immunoprecipitated with antibodies against MIB1 (Santa Cruz), USP15 (CST), DLL4 (Proteintech), Myc (Proteintech), or HA (Proteintech) overnight at 4°C. Immune complexes were captured with protein A/G magnetic beads (Biolinkedin, Shanghai, China), washed with ice-cold PBS, 20 µL of 1× loading buffer was added, the mixture was heated at 95°C for 10 minutes, and the proteins were analyzed by western blotting. Animal experiments Female NOD-SCID mice (4–6 weeks old; n = 6, 3 per group) and BALB/c nude mice (4–6 weeks old; n = 18, 9 per group) were obtained from Charles River Laboratories (Zhejiang, China). SK-MES-1 cells (5 × 10⁶ cells/mouse) transfected with si-LPCRL or si-NC were injected subcutaneously into BALB/c nude mice. Tumor growth was monitored every 5 days. After 4 weeks, the mice were euthanized, and the tumors were fixed in 10% formalin for pathological analysis. For the metastasis assays, luciferase-labeled SK-MES-1 cells (1 × 10⁶) transfected with si-LPCRL or si-NC were injected via the tail vein into NOD-SCID mice. Lung metastases were monitored by bioluminescence imaging (IVIS Spectrum). After 3 weeks, the mice were euthanized, and the lungs were fixed and stained with H&E for histological confirmation. In vivo PDX model-based therapeutic study When the PDX tumor volume reached 50–100 mm³, the mice were randomized into 5 groups (n = 8/group): (a) blank control (5% glucose); (b) si-NC; (c) si-LPCRL; (d) si-NC + cisplatin; and (e) si-LPCRL + cisplatin. Si-LPCRL or si-NC (10 µg/tumor/dose) in 5% glucose was administered intratumorally via in vivo jetPEI® (Polyplus, France) every 5 days for 4 doses. Cisplatin (2.5 mg/kg) was administered intraperitoneally every 5 days for 3 doses in the combination groups. The mice were euthanized, and the tumors were excised, weighed, measured, and paraffin-embedded for IHC. Immunohistochemistry (IHC) The tumor sections were deparaffinized, rehydrated, and subjected to antigen retrieval with sodium citrate (Beyotime). After blocking, the sections were incubated with primary antibodies (MIB1, Proteintech, 1:200; HES1, Bioss, Beijing, 1:200; γ-H2AX, Bioss, 1:200; Ki-67, Abcam, 1:200) overnight at 4°C and then with HRP-labeled secondary antibodies (Boster). Staining was performed with DAB (Beyotime), and the sections were counterstained with hematoxylin. Images were captured with a Nikon microscope. Expression was quantified by the H score (staining intensity × percentage of positive cells). Intensities: 0 (none), 1 (light brown), 2 (brown), and 3 (dark brown). Positive cells: 0 (< 5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). Statistical analysis All the experiments were performed in triplicate. The data are presented as the means ± standard deviations (SDs). Comparisons between two groups were performed via two-tailed Student’s t tests. Paired samples were analyzed with paired t tests. A p value of less than 0.05 was considered statistically significant. Statistical analyses were conducted via SPSS (version 17.0). RESULTS LPCRL is upregulated in primary cisplatin-resistant LUSC tissues To identify key lncRNAs associated with primary cisplatin resistance in LUSC, we established six LUSC PDX models, which were serially passaged to the third generation. After three cycles of cisplatin treatment, the models were classified into cisplatin-sensitive (tumor inhibition rate, TIR > 50%; n = 2) and cisplatin-resistant (TIR < 50%; n = 4) groups (Fig. 1 A; Supplementary Fig. S1 A, S1B). Microarray analysis of the lncRNA expression profiles of third-generation xenograft tumors revealed a clear transcriptional distinction between the cisplatin-sensitive and cisplatin-resistant groups via principal component analysis (Fig. 1 B). Volcano plot analysis identified 847 differentially expressed lncRNAs (|fold change| >1.5, p < 0.05). Among these, uc002ktr.3, which we designated LPCRL (lung squamous cell carcinoma primary cisplatin resistance-associated long noncoding RNA), was the most significantly upregulated antisense lncRNA in cisplatin-resistant tissues (Fig. 1 C). RT‒qPCR analysis confirmed significantly increased LPCRL expression in cisplatin-resistant tissues (n = 3) from eight additional PDX models, which were established and classified on the basis of cisplatin screening (Fig. 1 D). Bioinformatic analysis revealed that the LPCRL is located on chromosome 18 and is transcribed from the MIR133A1HG gene, which overlaps with the antisense strand of the twelfth intron of MIB1 (Fig. 1 E). Kaplan‒Meier analysis revealed that high MIR133A1HG expression was correlated with poorer overall survival in LUSC patients [ 19 ] (Fig. 1 F). Analysis of the GDC TCGA LUSC cohort revealed that 42.6% (207/486) of patients harbored MIR133A1HG copy number gains (Fig. 1 G). Given the role of copy number variations (CNVs) in driving gene expression and tumorigenesis [ 20 , 21 ], we hypothesized that LPCRL expression would be elevated in LUSC. Consistent with these findings, RT‒qPCR confirmed significantly higher LPCRL levels in LUSC tissues than in paired normal adjacent tissues (Fig. 1 H). This tumor-specific overexpression was further corroborated by fluorescence in situ hybridization (FISH), which revealed markedly stronger LPCRL signals in tumor cells than in stromal cells within the same tissue (Supplementary Fig. S1 C). In summary, these data establish LPCRL as an antisense lncRNA that not only is highly expressed in LUSC but is also further upregulated in a cisplatin-resistant context, positioning it as a key mediator of LUSC resistance to cisplatin. To molecularly characterize LPCRL, we performed 5' and 3' RACE in LUSC cells. Using a 5,912-nt reference sequence from the UCSC Genome Browser (which annotates four MIR133A1HG transcripts; Supplementary Fig. S1 D), we identified a single 688-nt, single-exon transcript in SK-MES-1 cells. This variant was confirmed by ORF Finder to lack protein-coding potential (Supplementary Fig. S1 E) and constitutes a partial segment of the MIR133A1HG-204 isoform (Supplementary Fig. S1 F). RNA FISH and subcellular fractionation assays revealed that LPCRL is predominantly localized in the nucleus of both SK-MES-1 and NCI-H520 cells (Fig. 1 I, 1 J; Supplementary Fig. S1 G, S1H), two widely used cell lines in LUSC research. Given the high expression of LPCRL in LUSC, we performed siRNA-mediated knockdown in primary LUSC cells established in our laboratory (Supplementary Fig. S1 I). LPCRL knockdown significantly increased cisplatin sensitivity and reduced cell proliferation (Fig. 1 K, 1 L). Taken together, our results define LPCRL as a nuclear-enriched antisense lncRNA associated with cisplatin resistance and cell proliferation in LUSC. LPCRL promotes cisplatin resistance, proliferation and migration in LUSC To further investigate the role of LPCRL in primary cisplatin resistance in LUSC, we first assessed its expression in SK-MES-1 and NCI-H520 cells. RT‒qPCR analysis revealed significantly higher LPCRL expression in SK-MES-1 cells than in NCI-H520 cells (Fig. 2 A). Consistent with these findings, SK-MES-1 cells exhibited greater intrinsic cisplatin resistance, as indicated by a higher IC 50 in the MTT assays (Fig. 2 B). To silence the nucleus-localized LPCRL, we designed a panel of siRNAs, shRNAs, and ASOs and evaluated their silencing efficacy (Supplementary Fig. S2A-S2C). Among these, only one siRNA demonstrated high interference efficiency. This siRNA, which targets the stem region of a predicted stem loop in the LPCRL secondary structure (modeled via RNAstructure software via the minimum free energy method), was selected for subsequent experiments (Supplementary Fig. S2D). We also stably overexpressed exogenous LPCRL in NCI-H520 cells, which presented relatively low endogenous LPCRL levels (Supplementary Fig. S2E). Functionally, LPCRL silencing significantly decreased the IC 50 of cisplatin in SK-MES-1 cells (Fig. 2 C), whereas its overexpression increased the IC 50 of cisplatin in NCI-H520 cells (Supplementary Fig. S2F). Given that cisplatin exerts its antitumor effects primarily by inducing DNA damage and subsequent apoptosis, we next examined these processes. Flow cytometry analysis revealed that LPCRL silencing enhanced cisplatin-induced apoptosis in SK-MES-1 cells (Fig. 2 D), which was supported by elevated levels of γ-H2AX (a DNA damage marker) and cleaved PARP (an apoptosis marker; Fig. 2 E). Conversely, LPCRL overexpression in NCI-H520 cells attenuated cisplatin-induced apoptosis and reduced the expression of these markers (Supplementary Fig. S2G, S2H). To investigate the impact of LPCRL on LUSC cell proliferation and migration in vitro, we performed colony formation and Transwell migration assays. LPCRL knockdown significantly suppressed the proliferation and migration of SK-MES-1 cells (Fig. 2 F, 2 G), whereas its ectopic overexpression enhanced these abilities in NCI-H520 cells (Supplementary Fig. S2I, S2J). Consistent with these phenotypic alterations, the expression of proliferation markers (PCNA) and epithelial‒mesenchymal transition (EMT)-related migration markers (N-cadherin and vimentin) was downregulated following LPCRL knockdown in SK-MES-1 cells (Fig. 2 H) but upregulated upon LPCRL overexpression in NCI-H520 cells (Supplementary Fig. S2K). To further validate the role of LPCRL in promoting proliferation in vivo, we subcutaneously injected BALB/c nude mice with SK-MES-1 cells transfected with LPCRL-targeting siRNA (si-LPCRL) or control siRNA (si-NC) (Supplementary Fig. S2L). Compared with the si-NC, si-LPCRL not only reduced the tumor incidence but also suppressed the tumor growth rate (Fig. 2 I, 2 J). Consistently, immunohistochemical (IHC) analysis revealed that LPCRL knockdown reduced Ki-67 positivity (a marker of cell proliferation; Fig. 2 K). To further assess the role of LPCRL in metastasis in vivo, we established an experimental metastasis model by intravenously injecting NOD/SCID mice with luciferase-labeled SK-MES-1 cells transfected with si-NC or si-LPCRL (Supplementary Fig. S2M). si-LPCRL attenuated the metastatic burden, as shown by a reduced bioluminescence signal in the lungs (Fig. 2 L, 2 M). Histological examination of lung tissues confirmed a reduction in both the size and number of metastatic lesions in the si-LPCRL group (Fig. 2 N, 2 O). Collectively, these findings indicate that LPCRL contribute to cisplatin resistance and promote tumor proliferation and metastasis in LUSC. LPCRL binds to MIB1 and inhibits ubiquitin-mediated degradation Antisense lncRNAs regulate the expression of their corresponding sense genes at the transcriptional or posttranscriptional level through interactions with DNA, RNA, or proteins [ 12 ]. Notably, LPCRL is transcribed from an antisense intronic region within the MIB1 gene. We therefore hypothesized that LPCRL may promote LUSC progression by regulating MIB1, an E3 ubiquitin ligase known to be overexpressed in lung cancer and associated with poor prognosis [ 22 ]. Consistent with this hypothesis, CPTAC data and our IHC analyses confirmed significantly elevated MIB1 protein levels in LUSC tissues relative to normal tissues (Supplementary Fig. S3A, S3B). Surprisingly, neither LPCRL knockdown nor LPCRL overexpression significantly altered MIB1 mRNA expression (Supplementary Fig. S3C, S3D); likewise, modulating MIB1 expression did not affect the abundance of LPCRL transcripts (Supplementary Fig. S3E, S3F). However, LPCRL knockdown markedly reduced MIB1 protein levels, whereas LPCRL overexpression significantly increased them (Fig. 3 A), indicating a posttranscriptional regulatory mechanism. Previous studies have shown that lncRNAs can stabilize their interacting proteins by inhibiting ubiquitin-proteasome-mediated degradation [ 23 – 26 ]. To determine if LPCRL physically interacts with MIB1, we first employed RPISeq, which predicts a high-confidence interaction (RF/SVM scores: 0.65/0.81). We next performed RNA pull-down assays in SK-MES-1 cells using a biotin-labeled LPCRL probe. Following efficient probe enrichment (Fig. 3 B), silver staining revealed a specific ~ 110 kDa band (Fig. 3 C), which was confirmed via western blot analysis as MIB1 (Fig. 3 D). RNA immunoprecipitation (RIP) assays independently validated this interaction (Fig. 3 E). To map the binding domain, we analyzed the secondary structure of LPCRL, identifying three major loops: Loop1 (1–252 nt), Loop2 (253–476 nt), and Loop3 (477–688 nt) (Fig. 3 F). RIP assays with full-length and truncated LPCRL constructs revealed significant enrichment only for the full-length and Loop2 fragments (Fig. 3 G), thereby mapping the core MIB1-binding region to nucleotides 253–476 (Fig. 3 H). To assess whether LPCRL stabilizes MIB1 via the ubiquitin‒proteasome pathway, LUSC cells were treated with the proteasome inhibitor MG132 or the protein synthesis inhibitor cycloheximide (CHX). MG132 treatment significantly increased MIB1 protein levels in both SK-MES-1 and NCI-H520 cells (Fig. 3 I), confirming the proteasomal degradation of MIB1. Consistent with these findings, LPCRL knockdown increased MIB1 ubiquitination, whereas LPCRL overexpression reduced MIB1 ubiquitination (Fig. 3 J). In CHX chase assays, LPCRL silencing accelerated MIB1 degradation, whereas LPCRL overexpression extended its half-life (Fig. 3 K). These results demonstrate that LPCRL stabilizes MIB1 by inhibiting its ubiquitin-mediated proteasomal degradation. To investigate whether LPCRL promotes LUSC progression via MIB1, we first examined the role of MIB1 in LUSC cells. In SK-MES-1 cells, MIB1 knockdown significantly downregulated key markers of cell proliferation and migration, including PCNA, N-cadherin, and vimentin (Supplementary Fig. S3G). Furthermore, MIB1 silencing followed by cisplatin treatment markedly increased the levels of γ-H2AX and cleaved PARP, indicating enhanced DNA damage and apoptosis (Supplementary Fig. S3H). To confirm that LPCRL functions through MIB1, we silenced MIB1 in NCI-H520 cells stably overexpressing LPCRL. MTT assays revealed that MIB1 knockdown abolished the LPCRL-induced increase in the IC 50 of cisplatin (Fig. 3 L). Furthermore, colony formation and Transwell assays revealed that MIB1 silencing attenuated the enhanced proliferative and migratory capacities conferred by LPCRL overexpression (Fig. 3 M, 3 N), which was consistent with decreased PCNA and vimentin protein levels (Fig. 3 O). Upon cisplatin exposure, MIB1 silencing abrogated the LPCRL-mediated suppression of γ-H2AX and cleaved PARP (Fig. 3 P). These findings collectively support that MIB1 is a key mediator of the oncogenic effects of LPCRL in regulating proliferation, migration, and cisplatin resistance in LUSC. LPCRL stabilizes MIB1 via USP15-mediated deubiquitination LPCRL lacks protein-coding capacity; therefore, the precise mechanism by which it interacts with MIB1 to regulate MIB1 ubiquitination remains incompletely understood. Accumulating evidence indicates that lncRNAs function as molecular scaffolds to facilitate protein‒protein interactions [ 27 – 29 ]. On the basis of these findings, we hypothesized that LPCRL may act as a scaffold to recruit a deubiquitinating enzyme that interacts with MIB1, thereby inhibiting MIB1 ubiquitination and subsequent proteasomal degradation. To identify MIB1-associated deubiquitinating enzymes, analysis of BioID proteomic datasets and BioGRID interactors identified USP9X, CYLD, and USP15 as potential MIB1-interacting candidates [ 30 ]. While interactions between MIB1 and USP9X or CYLD have been reported [ 31 – 32 ], the MIB1-USP15 interaction remains uncharacterized. USP15 is a critical deubiquitinating enzyme implicated in multiple malignancies and has been reported to promote lung cancer cell proliferation [ 33 ]. Moreover, RPISeq analysis predicted a high-probability interaction between LPCRL and USP15 (RF/SVM scores: 0.75 and 0.97). On the basis of these findings, we hypothesized that LPCRL serves as a molecular scaffold, facilitating the interaction of MIB1 with USP15 in LUSC cells. Western blot analysis confirmed the specific enrichment of USP15 by the biotin-labeled LPCRL probe (Fig. 4 A). Furthermore, RIP assays revealed significant enrichment of LPCRL in USP15-immunoprecipitated complexes (Fig. 4 B). Similarly, using full-length and truncated LPCRL constructs transfected into SK-MES-1 cells, RIP assays revealed significant enrichment of the Loop1 region (nucleotides 1–252) in USP15-immunoprecipitated complexes (Fig. 4 C). Collectively, these results indicate that MIB1 primarily binds to Loop2 of LPCRL, whereas USP15 preferentially associates with Loop1, supporting the role of LPCRL as a molecular scaffold (Fig. 4 D). These results indicate that LPCRL binds independently to MIB1 and USP15. We therefore hypothesized that LPCRL serves as a molecular scaffold to promote the MIB1-USP15 interaction. To this end, we first assessed whether MIB1 and USP15 physically interact. Molecular docking analysis predicted multiple potential binding modes between MIB1 and USP15 (Fig. 4 E). Co-IP assays confirmed the endogenous interaction between MIB1 and USP15 in both SK-MES-1 and NCI-H520 cells (Fig. 4 F, Supplementary Fig. S4A). Furthermore, immunofluorescence (IF) staining demonstrated their nuclear colocalization in these cell lines (Fig. 4 G, Supplementary Fig. S4B), which is consistent with the established nuclear localization of LPCRL. To further validate the scaffolding role of LPCRL, we initially conducted co-IP assays to assess exogenous MIB1-USP15 interactions in HEK293T cells with or without RNase A treatment. The results showed that MIB1 interacted with USP15 in the absence of RNase A; however, this interaction was markedly diminished upon RNase A treatment (Fig. 4 H). Additionally, LPCRL knockdown weakened the interaction between MIB1 and USP15 (Fig. 4 I), whereas LPCRL overexpression increased this interaction (Fig. 4 J), thus supporting the hypothesis that LPCRL is essential for mediating this interaction. Taken together, these data indicate that LPCRL functions as a molecular scaffold to facilitate the formation of the MIB1-USP15 complex. Functionally, USP15 knockdown reduced MIB1 protein levels and increased MIB1 ubiquitination (Fig. 4 K, 4 L; Supplementary Fig. S4C, S4D). Conversely, USP15 overexpression had the opposite effect (Fig. 4 M, 4 N; Supplementary Fig. S4E, S4F). Moreover, LPCRL knockdown decreased the USP15 protein level without affecting its mRNA level, whereas LPCRL overexpression increased it (Supplementary Fig. S4G-S4J). This finding suggests that LPCRL posttranslationally stabilizes both MIB1 and USP15. Collectively, these findings demonstrate that LPCRL promotes the assembly of the USP15-MIB1-LPCRL ternary complex, which enables USP15-mediated deubiquitination of MIB1, thereby shielding it from proteasomal degradation and enhancing its protein stability (Fig. 4 O). The LPCRL-MIB1-USP15 complex activates the Notch signaling pathway Our previous studies revealed that the LPCRL-MIB1-USP15 complex enhances MIB1 stability in LUSC cells. As an E3 ubiquitin ligase, MIB1 activates the Notch signaling pathway via ubiquitination and endocytosis of the intracellular domains of its ligands (Delta/Jagged). Notably, activation of the Notch signaling pathway is critical for tumorigenesis and progression and is implicated in promoting proliferation, metastasis, and chemoresistance in various cancers [ 34 – 36 ]. We thus assessed whether LPCRL-driven malignant progression in LUSC cells is dependent on the Notch signaling pathway. Co-IP assays demonstrated that MIB1 interacts with DLL4, a Notch ligand, in both SK-MES-1 and NCI-H520 cells (Supplementary Fig. S5A, S5B). LPCRL knockdown significantly reduced DLL4 ubiquitination (Fig. 5 A), whereas LPCRL overexpression increased it (Fig. 5 B). Notably, this increase was substantially mitigated upon MIB1 knockdown (Fig. 5 C), confirming the role of MIB1 in this process. Furthermore, LPCRL knockdown decreased the nuclear accumulation of the Notch intracellular domain (NICD) (Fig. 5 D) and downregulated the mRNA and protein levels of the Notch downstream effectors HES1 and c-Myc (Fig. 5 E, 5 F). Conversely, LPCRL overexpression upregulated these effectors (Fig. 5 G- 5 I), indicating that LPCRL effectively activated the Notch signaling pathway. Our experiments demonstrated that LPCRL inhibits MIB1 degradation via the USP15-mediated ubiquitin–proteasome pathway in LUSC cell nuclei, thereby maintaining MIB1 homeostasis. However, MIB1 is known to activate the Notch signaling pathway by catalyzing the ubiquitination of its specific ligands in the cytoplasm [ 37 ]. We thus investigated whether the LPCRL-MIB1-USP15 complex facilitates MIB1 nuclear export to enable Notch signaling pathway activation. Both nuclear-cytoplasmic fractionation and IF assays revealed that the overexpression of LPCRL or USP15 increased the cytoplasmic MIB1 level (Fig. 5 J, 5 K; Supplementary Fig. S5C, S5D). Collectively, these findings indicate that the LPCRL-MIB1-USP15 complex regulates not only MIB1 stability but also its nuclear export, providing new insights into the molecular mechanism of LPCRL in LUSC. HES1 plays a pivotal role in tumorigenesis, tumor progression, and therapeutic resistance [ 37 ]. To determine whether LPCRL contributes to LUSC progression by modulating HES1, we first assessed the role of HES1 in LUSC via multiple approaches. TCGA database analysis revealed significantly elevated HES1 mRNA in LUSC tissues compared with normal lung tissues (Fig. 5 L), and Kaplan‒Meier survival analysis revealed that high HES1 expression was correlated with reduced overall survival in LUSC patients (Fig. 5 M). CPTAC database data and our IHC analysis confirmed that HES1 protein expression was increased in LUSC clinical samples compared with normal tissues (Supplementary Fig. S5E, S5F). Furthermore, HES1 silencing reduced the expression of proliferation- and migration-related proteins (PCNA, N-cadherin, and vimentin; Supplementary Fig. S5G) and elevated cisplatin-induced γ-H2AX and cleaved PARP levels (Supplementary Fig. S5H). HES1 expression was also elevated in cisplatin-resistant patient-derived xenograft (PDX) tissues (Supplementary Fig. S5I). NCI-H520 cells stably overexpressing LPCRL were subsequently transfected with HES1-targeting siRNAs. MTT assays revealed that HES1 knockdown reversed the increase in the IC 50 of cisplatin induced by LPCRL (Fig. 5 N) and abolished the suppressive effect of LPCRL on cisplatin-induced γ-H2AX and cleaved PARP (Supplementary Fig. S5J). Colony formation and Transwell migration assays further demonstrated that HES1 silencing mitigated the increase in cell proliferation and migration driven by LPCRL (Fig. 5 O, 5 P). Consistent with these functional changes, HES1 knockdown also reduced the expression of PCNA and vimentin (Supplementary Fig. S5K). Notably, treatment with tangeretin, a Notch inhibitor, also inhibited LPCRL-mediated HES1 upregulation and cisplatin resistance. This was evidenced by the reversal of the effects of LPCRL on γ-H2AX and cleaved PARP (c-PARP) levels (Fig. 5 Q, 5 R). Collectively, these findings confirm that LPCRL exerts its oncogenic functions through the MIB1-USP15-Notch-HES1 axis. LPCRL is potential therapeutic targets for the treatment of LUSC To assess the therapeutic potential of LPCRL inhibition, we knocked down LPCRL in primary LUSC cells derived from a patient. LPCRL silencing significantly suppressed colony formation, reduced the cisplatin IC 50 , and enhanced apoptosis, as evidenced by elevated γ-H2AX and c-PARP levels (Fig. 1 K, 1 L; Supplementary Fig. S6A, S6B). MIB1 protein expression also decreased without altering its mRNA levels (Supplementary Fig. S6C, S6D). The endogenous interaction between MIB1 and USP15 was confirmed by Co-IP, and manipulation of USP15 levels altered MIB1 protein expression accordingly (Supplementary Fig. S6E, S6F). Collectively, these results corroborate our previous observations in LUSC cell lines and support the therapeutic potential of targeting LPCRL in LUSC. To test its efficacy in vivo, we further employed a PDX model established from the same LUSC patient as the primary cells. When the tumor volume reached 50–100 mm³, the mice were intratumorally injected with LPCRL-targeting siRNA (si-LPCRL), si-NC, or glucose solution (Blank). Cisplatin was administered intraperitoneally every five days (Fig. 6 A). Consistent with the in vitro findings, si-LPCRL significantly inhibited tumor growth and reduced tumor weight compared with those in the si-NC and blank groups. Importantly, the combination of cisplatin and si-LPCRL enhanced the inhibitory effect compared with that of cisplatin plus si-NC (Fig. 6 B- 6 D). No significant body weight changes were observed across groups (Fig. 6 E), indicating that si-LPCRL inhibits tumor growth and enhances cisplatin sensitivity without overt systemic toxicity in LUSC. The knockdown efficiency was confirmed by RT‒qPCR (Fig. 6 F). Compared with those in the si-NC or blank groups, IHC staining revealed markedly lower Ki67, MIB1, and HES1 expression in the si-LPCRL-treated tumors (Fig. 6 G), and the si-LPCRL plus cisplatin combination notably increased DNA damage (elevated γ-H2AX) and inhibited proliferation (decreased Ki67) compared with those in the cisplatin plus si-NC group (Fig. 6 H). These results demonstrate that targeting LPCRL suppresses cancer cell proliferation, reduces MIB1 and its downstream HES1 expression, and enhances cisplatin sensitivity in vivo, supporting its potential as a therapeutic target. Discussion Platinum-based chemotherapy, particularly cisplatin, is a cornerstone of LUSC treatment; however, its efficacy is severely hindered by the emergence of drug resistance [ 38 ]. These challenges underscore the urgent need to elucidate the molecular mechanisms underlying cisplatin resistance and identify novel therapeutic strategies. In this study, we identified a novel antisense lncRNA, designated LPCRL, which is highly expressed in cisplatin-resistant LUSC xenograft tumors. Bioinformatic analysis revealed that LPCRL is transcribed from the MIR133A1HG locus and is located on the antisense strand within the twelfth intron of its homologous gene, MIB1, thus constituting a natural antisense transcript (NAT). While NATs typically regulate their sense counterparts through interactions with DNA or RNA [ 12 ], we uncovered a distinct mechanism: LPCRL promotes cisplatin resistance in LUSC by functioning as a molecular scaffold that directly mediates the protein interaction between MIB1 and USP15, thereby activating the Notch signaling pathway. Our data demonstrate that the LPCRL/USP15/MIB1/Notch axis is a hitherto unidentified mechanism driving cisplatin resistance. This regulatory mode is particularly intriguing given the genomic context of LPCRL. The MIR133A1HG locus is well established as a precursor for the tumor-suppressive microRNAs miR-133a and miR-1 [ 39 , 40 ]. In addition to its role in solid tumors, MIR133A1HG has been implicated in hematological malignancies; for example, MIR133A1HG was identified among a set of autophagy-related lncRNAs that improve the prognosis of acute myeloid leukemia (AML) patients, with higher expression correlating with better outcomes [ 41 ]. However, its potential functions in AML remain largely unexplored. In contrast, our work revealed that LPCRL, a lncRNA isoform from the same locus, functions as an oncogene in LUSC. This functional divergence underscores the remarkable transcriptional complexity of a single genomic locus and highlights the critical importance of isoform-specific investigations in cancer research. We further elucidated the spatial dynamics of this axis. Although MIB1, an E3 ubiquitin ligase, is known to increase Notch signaling in the cytoplasm, we found that LPCRL, MIB1, and USP15 are predominantly colocalized within the nucleus and activate Notch signaling. This observation prompted us to investigate their role in regulating the nucleocytoplasmic shuttling of MIB1. Nuclear‒cytoplasmic fractionation and IF analyses demonstrated that cytoplasmic MIB1 was significantly increased upon overexpression of LPCRL or USP15. This finding reveals a regulatory mechanism whereby LPCRL and USP15 modulate the nuclear stability and subsequent cytoplasmic translocation of MIB1, which is crucial for its oncogenic function. This axis is analogous to the USP7-mediated regulation of PTEN, suggesting that nuclear stabilization followed by cytoplasmic translocation may be a common strategy for regulating key signaling proteins [ 42 ]. From a translational perspective, we successfully identified a specific and efficient siRNA against LPCRL after extensive screening. The scarcity of effective inhibitors may be attributed to the nuclear localization and complex secondary structure of LPCRL. Notably, the effective siRNA targeted the stem region within LPCRL-Loop2 (Supplementary Fig. S2D), underscoring the importance of considering the RNA secondary structure in therapeutic design. Recent advancements in structural probing techniques have revealed critical structural motifs and RNA‒protein interfaces that contribute to lncRNA dysfunction. Furthermore, developing small molecules, antisense oligonucleotides, and peptidomimetic-based therapeutic agents that target these motifs and interfaces presents promising strategies for treating lncRNA-mediated diseases [ 43 ]. In PDX models, intratumoral administration of LPCRL-siRNA significantly suppressed tumor growth, and compared with monotherapy, its combination with cisplatin achieved superior efficacy. Moreover, with advances in drug delivery systems, such as inhalable lipid nanoparticles that enable targeted lung delivery with increased local accumulation and reduced systemic exposure [ 44 ], LPCRL-siRNA holds significant promise for clinical translation as a novel therapeutic strategy for LUSC. Despite these significant findings, our study has several limitations. First, large-scale, multicenter clinical studies are necessary to validate the association between LPCRL expression and cisplatin response, as well as its prognostic value in patients with LUSC. Second, the functional role of nuclear MIB1 remains incompletely understood; it may have nuclear substrates other than Notch ligands, and proximity-labeling techniques such as BioID may help delineate the nuclear interactome of MIB1. Third, the dynamics of MIB1 nucleocytoplasmic shuttling and the precise mechanisms governing its translocation require further elucidation, potentially through live-cell imaging at the single-cell level. Together, these future directions will not only elucidate the noncanonical roles of nuclear MIB1 and the precise mechanisms of MIB1 nucleocytoplasmic shuttling but also pave the way for rationally designed LPCRL-targeted therapies. Conclusions Our study identified LPCRL as a novel NAT in LUSC. It functions as a molecular scaffold that facilitates the MIB1-USP15 interaction and promotes the nuclear export of MIB1, which in turn activates the Notch signaling pathway. This activation protects cells from DNA damage and cisplatin-induced cell death. Silencing LPCRL (si-LPCRL) disrupts signaling through the LPCRL/USP15/MIB1 axis and enhances the efficacy of cisplatin-based chemotherapy. Abbreviations LUSClung squamous cell carcinoma long noncoding RNAs lncRNAs natural antisense transcripts NATs patient-derived xenograft PDX LPCRL LUSC primary cisplatin resistance-associated lncRNA Copy number variations CNVs Co-IP Coimmunoprecipitation FISH Fluorescence in situ hybridization RIP RNA immunoprecipitation IHC Immunohistochemistry Declarations Ethics approval and consent to participate All the mouse experiments in this study were approved by the Institutional Animal Care and Use Committee of the University of Science and Technology of China (Approval No. 2019-N(A)-179). The collection of human samples and research were approved by the Ethics Committee of the University of Science and Technology of China (Approval No. 2019-N(H)-128), and written informed consent was obtained from all participants. Consent for publication Not applicable. Availability of data and materials All raw lncRNA microarray data are available upon request from the corresponding author. Competing interests The authors declare that they have no competing interests. Funding This study was supported by funding from the National Natural Science Foundation of China (81972006) and Fundamental Research Funds for the Central Universities (WK9110000101). Authors’ contributions BLW conceived and designed the research. PL carried out the molecular genetic studies and drafted the manuscript. DPL performed cell experiments. SZ analyzed the data and drafted the manuscript. KF performed the statistical analysis. WQD helped to draft the manuscript. 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1","display":"","copyAsset":false,"role":"figure","size":869580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLPCRL is upregulated in primary cisplatin-resistant LUSC tissues. (A) \u003c/strong\u003eSchematic illustration showing cisplatin or PBS treatment in patient-derived xenografts (PDXs) of lung squamous cell carcinomas (LUSC) and the establishment of primary cells. \u003cstrong\u003e(B)\u003c/strong\u003ePrincipal component analysis of long noncoding RNA (lncRNA) data from third-generation xenograft tumors. Each dot represents a sample, colored according to its response to cisplatin (blue, sensitive; red, resistant). \u003cstrong\u003e(C)\u003c/strong\u003eVolcano plots showing differentially expressed lncRNAs in the cisplatin-resistant group versus the cisplatin-sensitive group (fold change \u0026gt;1.5; adjusted p value \u0026lt;0.05). \u003cstrong\u003e(D)\u003c/strong\u003e RT‒qPCR analysis showing relative LPCRL expression in the cisplatin-resistant group (n = 3) and the cisplatin-sensitive group (n = 5). \u003cstrong\u003e(E)\u003c/strong\u003e Schematic of the genomic localization of MIR133A1HG (chr18q11.2), an antisense lncRNA hereafter referred to as LPCRL that overlaps the antisense strand of intron 12 of MIB1, on the basis of the UCSC Genome Browser. \u003cstrong\u003e(F)\u003c/strong\u003e Kaplan‒Meier analysis showing the association between MIR133A1HG expression and overall survival (OS). \u003cstrong\u003e(G)\u003c/strong\u003e Pie chart showing the absolute MIR133A1HG copy number distribution in LUSC (TCGA dataset, n = 486). \u003cstrong\u003e(H)\u003c/strong\u003e RT‒qPCR analysis showing relative LPCRL expression in LUSC tumor tissues and adjacent normal tissues (n = 14 pairs) (paired t test). \u003cstrong\u003e(I)\u003c/strong\u003e Subcellular localization of LPCRL in SK-MES-1 cells on the basis of nuclear and cytoplasmic RNA separation followed by RT‒qPCR analysis. GAPDH (cytoplasmic) and U6 (nuclear) were used as controls. \u003cstrong\u003e(J)\u003c/strong\u003e Representative fluorescence in situ hybridization (FISH) images showing LPCRL signals (red) in SK-MES-1cells. Nuclei are stained with DAPI (blue). Scale bar: 50 μm. \u003cstrong\u003e(K)\u003c/strong\u003e The viability of primary cells transfected with si-LPCRL or si-NC was evaluated via the MTT assay after treatment with various concentrations of cisplatin for 48 hours. \u003cstrong\u003e(L)\u003c/strong\u003e Colony formation assays showing the proliferative capacity of primary cells transfected with si-LPCRL or si-NC. Representative images (left panel) and quantitative analysis of colony numbers (right panel) are presented\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8106822/v1/849ddcd0ac7a3fa4311888d5.jpeg"},{"id":97114663,"identity":"11b3fdbf-96d6-4a89-aa47-f2e2d63ab1f5","added_by":"auto","created_at":"2025-12-01 07:00:41","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1030350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLPCRL promotes cisplatin resistance, cell proliferation and migrationin SK-MES-1 cells. (A) \u003c/strong\u003eRT‒qPCR analysis of endogenous LPCRL expression in SK-MES-1 and NCI-H520 cells. \u003cstrong\u003e(B-C)\u003c/strong\u003e Cell viability was evaluated by the MTT assay in SK-MES-1 cells, NCI-H520 cells (B), and SK-MES-1 cells transfected with si-LPCRL or si-NC (C), followed by treatment with various concentrations of cisplatin for 48 hours. \u003cstrong\u003e(D) \u003c/strong\u003eRepresentative Annexin V-FITC/PI staining images (left panel) showing apoptosis in si-LPCRL- or si-NC-transfected SK-MES-1 cells after 24hours of treatment with 40 μM cisplatin and quantification of apoptotic rates (right panel). \u003cstrong\u003e(E) \u003c/strong\u003eWestern blot analysis showing the protein expression of γ-H2AX (a DNA damage marker) and cleaved PARP (c-PARP, an apoptosis marker) in si-LPCRL- or si-NC-transfected SK-MES-1 cells after 48 hours oftreatment with 20 μM cisplatin. \u003cstrong\u003e(F-G)\u003c/strong\u003e Colony formation (F) and Transwell (G) assays showing the proliferative capacity and migratory capacity of si-LPCRL- or si-NC-transfected SK-MES-1 cells. Representative images (left panel) and quantitative analysis (right panel) are presented. \u003cstrong\u003e(H)\u003c/strong\u003eWestern blot analysis showing the protein expression of PCNA, N-cadherin, and vimentin in si-LPCRL- or si-NC-transfected SK-MES-1 cells. \u003cstrong\u003e(I-J) \u003c/strong\u003eBALB/c nude mice (n=9 per group) were subcutaneously injected with si-LPCRL- or si-NC-transfected SK-MES-1 cells to induce tumor formation. (I) Representative images of excised tumors; (J) tumorgrowth curves generated from measurements at the indicated time points. \u003cstrong\u003e(K) \u003c/strong\u003eRepresentative immunohistochemical (IHC) images (left panel) and Ki-67 quantification by H-scores(right panel) in the tumors from (I). Scale bars: 100 μm (upper); 50 μm (lower). \u003cstrong\u003e(L-M)\u003c/strong\u003e In vivo metastasis was assessed in NOD/SCID mice (n=3 per group) following intravenous injection of luciferase-expressing SK-MES-1 cells transfected with si-LPCRL or si-NC. Representative bioluminescence mouse images (L) and the corresponding quantification of luminescence radiance (M) are shown. \u003cstrong\u003e(N)\u003c/strong\u003e Representative H\u0026amp;E-stained images of lung metastatic lesions; red arrows indicate metastatic tumor foci. Scale bars: 500 μm (upper); 100 μm (lower). \u003cstrong\u003e(O)\u003c/strong\u003eStatistical analysis of the number of lung metastatic lesions in the indicated groups.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8106822/v1/ca28cc2491bc23570aa61be9.jpeg"},{"id":97114668,"identity":"d854ee4b-777d-4cca-81c9-c93dfc97c39b","added_by":"auto","created_at":"2025-12-01 07:00:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1101638,"visible":true,"origin":"","legend":"\u003cp\u003eLPCRL binds to MIB1 and blocks its ubiquitination. \u003cstrong\u003e(A)\u003c/strong\u003e Western blot analysis of MIB1 protein levels in in SK-MES-1 cells (si-LPCRL/si-NC) and NCI-H520 cells (oeLPCRL/vector). \u003cstrong\u003e(B)\u003c/strong\u003e RT‒qPCR validation of LPCRL enrichment in SK-MES-1 pull-down assays. \u003cstrong\u003e(C)\u003c/strong\u003e Silver staining of proteins pulled down by the biotinylated LPCRL probe in SK-MES-1 cells. \u003cstrong\u003e(D)\u003c/strong\u003e MIB1 enrichment in pull-down samples from SK-MES-1 lysates using biotinylated LPCRL RNA. \u003cstrong\u003e(E)\u003c/strong\u003e RIP assays with anti-MIB1 antibody showing LPCRL enrichment versus IgG control in SK-MES-1 cells. \u003cstrong\u003e(F)\u003c/strong\u003e Schematic representation of the full-length LPCRL and its truncated variants (M1, M2, M3) on the basis of secondary structure prediction. \u003cstrong\u003e(G)\u003c/strong\u003e RIP assays in SK-MES-1 cells expressing full-length LPCRL or truncations, showing enrichment of full-length and M2 (253–476 nt). \u003cstrong\u003e(H)\u003c/strong\u003e Schematic illustration of the interaction between LPCRL and MIB1. \u003cstrong\u003e(I)\u003c/strong\u003e Western blot analysis of MIB1 protein levels in SK-MES-1 and in NCI-H520 cells following 6hours of treatment with the proteasome inhibitor MG132 (20 μM). \u003cstrong\u003e(J)\u003c/strong\u003e Western blot analysis of MIB1 ubiquitination levels in SK-MES-1 (si-LPCRL/si-NC) and NCI-H520 (oeLPCRL/vector) cells. \u003cstrong\u003e(K)\u003c/strong\u003e Western blot analysis of MIB1 protein levels in SK-MES-1 (si-LPCRL/si-NC) and NCI-H520 (oeLPCRL/vector) cell lines after CHX (50 μg/mL) treatment at indicated times; protein levels were normalized to those of GAPDH via ImageJ. \u003cstrong\u003e(L)\u003c/strong\u003e MTT assay for cell viability in NCI-H520 cells (oeLPCRL + si-MIB1) after 48h cisplatin treatment. \u003cstrong\u003e(M-N)\u003c/strong\u003e Colony formation (M) and Transwell assays (N) assessing the proliferative and migratory capacities of NCI-H520 cells (oeLPCRL + si-MIB1). Representative images (left) and quantification of colony numbers or relative migrated cell numbers (right) are shown. \u003cstrong\u003e(O)\u003c/strong\u003e Western blot analysis of vimentin and PCNA protein levels in NCI-H520 cells (oeLPCRL + si-MIB1). \u003cstrong\u003e(P)\u003c/strong\u003e Western blot analysis of γ-H2AX and cleaved PARP (c-PARP; an apoptosis marker) protein levels in NCI-H520 cells (oeLPCRL + si-MIB1) following 48 hours of treatment with cisplatin (20 μM).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8106822/v1/57afbb3dd459a5c5d078c72f.jpeg"},{"id":97114669,"identity":"0b19b361-607c-45f5-a001-62b6c0f37329","added_by":"auto","created_at":"2025-12-01 07:00:41","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":838273,"visible":true,"origin":"","legend":"\u003cp\u003eLPCRL serves as a scaffold that promotes the interaction between MIB1 and USP15. \u003cstrong\u003e(A) Western blot analysis of USP15 enrichment in pull-down samples from SK-MES-1 cell lysates via biotinylated LPCRL RNA. (B) RIP assays with an anti-USP15 antibody in SK-MES-1 cells showing LPCRL enrichment relative to the IgG control group. (C) RIP assays with an anti-USP15 antibody in SK-MES-1 cells (transfected with plasmids expressing full-length LPCRL or the indicated truncation mutants) showing enrichment of the full-length LPCRL and the M1 truncation (1–252 nt). (D) Schematic illustration of the interaction between LPCRL, MIB1, and USP15. (E) Predicted binding mode between MIB1 (blue) and USP15 (wheat) via the protein‒protein docking program HDock. (F) Co-IP showing the endogenous interaction between MIB1 and USP15 in SK-MES-1 cells. (G) Representative immunofluorescence images of endogenous MIB1 (green) and USP15 (red) in SK-MES-1 cells; nuclei were stained with DAPI (blue). Scale bar: 20 µm. (H) Co-IP showing the interaction between Myc-MIB1 and HA-USP15 in lysates from HEK293T cells transiently cotransfected with Myc-MIB1 and HA-USP15 with or without RNase A treatment. (I–J) Co-IP showing the endogenous interaction between MIB1 and USP15 in SK-MES-1 cells with LPCRL knockdown (I) or in NCI-H520 cells with stable LPCRL overexpression (oeLPCRL) (J). (K–L) Western blot analysis of MIB1 protein levels (K) and its ubiquitination (L) in SK-MES-1 cells transfected with si-USP15 or siNC. (M–N) Western blot analysis of MIB1 protein levels (M) and its ubiquitination (N) in SK-MES-1 cells transfected with USP15-expressing plasmids. (O) Schematic illustration showing that LPCRL acts as a molecular scaffold to facilitate the interaction between MIB1 and USP15, enabling USP15-mediated deubiquitination of MIB1, thereby preventing its proteasomal degradation and enhancing protein stability.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8106822/v1/80bf605df981371a83883f89.jpeg"},{"id":97114677,"identity":"18b4518a-df79-4b82-915b-859d95e59c5e","added_by":"auto","created_at":"2025-12-01 07:00:41","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1310460,"visible":true,"origin":"","legend":"\u003cp\u003eThe LPCRL-MIB1-USP15 complex activates the Notch signaling pathway. \u003cstrong\u003e(A-B)\u003c/strong\u003e Co-IP analysis of DLL4 ubiquitination levels in SK-MES-1 cells (si-LPCRL/si-NC) (A) and in NCI-H520 cells (oeLPCRL/vector) (B). \u003cstrong\u003e(C)\u003c/strong\u003e Co-IP analysis of DLL4 ubiquitination in NCI-H520 cells (oeLPCRL+si-MIB1) . \u003cstrong\u003e(D, G)\u003c/strong\u003e Western blot analysis of nuclear NICD protein levels in SK-MES-1 cells (si-LPCRL/si-NC) (D) and in NCI-H520 cells (oeLPCRL/vector) (G). \u003cstrong\u003e(E, H)\u003c/strong\u003e RT‒qPCR analysis of Notch target genes (HES1, p21, c-Myc, and CyclinD1) in SK-MES-1 cells (si-LPCRL/si-NC) (E) and in NCI-H520 cells (oeLPCRL/vector) (H). \u003cstrong\u003e(F, I)\u003c/strong\u003eWestern blot analysis of c-Myc and HES1 protein levels in SK-MES-1 cells (si-LPCRL/si-NC) (F) and in NCI-H520 cells (oeLPCRL/vector) (I). \u003cstrong\u003e(J)\u003c/strong\u003e Representative immunofluorescence images showing the subcellular localization of MIB1 (green) in SK-MES-1 cells with or without transient LPCRL overexpression; nuclei were counterstained with DAPI (blue). Scale bar: 20 µm. \u003cstrong\u003e(K)\u003c/strong\u003e Western blot analysis of MIB1 protein levels in subcellular fractions from SK-MES-1 cells with or without transient LPCRL overexpression. C, cytosolic fraction; N, nuclear fraction. \u003cstrong\u003e(L)\u003c/strong\u003e Bar chart showing HES1 expression levels across various TCGA cancer types (tumor vs. normal samples). \u003cstrong\u003e(M)\u003c/strong\u003e Kaplan‒Meier analysis showing that LUSC patients with higher HES1 expression had worse overall survival (OS). \u003cstrong\u003e(N)\u003c/strong\u003e Cell viability was evaluated via an MTT assay in NCI-H520 cells (oeLPCRL+si-HES1) following 48 hours of treatment with the indicated concentrations of cisplatin. \u003cstrong\u003e(O-P)\u003c/strong\u003e Colony formation (O) and Transwell assays (P) assessing the proliferative and migratory capacities of NCI-H520 cells (oeLPCRL+si-HES1). Representative images (left) and quantification of colony numbersor relative migrated cell numbers (right) are shown. \u003cstrong\u003e(Q)\u003c/strong\u003e Western blot analysis of HES1 protein levels in NCI-H520 cells stably overexpressing LPCRL (oeLPCRL) with or without treatment with tangeretin (a Notch inhibitor, 20 µM). \u003cstrong\u003e(R)\u003c/strong\u003e Western blot analysis of γ-H2AX and c-PARP protein levels in LPCRL-overexpressing NCI-H520 cells treated with cisplatin (20 µM) in the presence or absence of tangeretin (20 µM).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8106822/v1/1ff963692553fd20b05a93aa.jpeg"},{"id":97114671,"identity":"e99a17f7-51af-4d68-93df-22489e97683a","added_by":"auto","created_at":"2025-12-01 07:00:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2300379,"visible":true,"origin":"","legend":"\u003cp\u003eSilencing of LPCRL inhibits tumor growth and enhances cisplatin sensitivity in PDX models. (A) Schematic illustration of PDX model establishment, followed by the administration of LPCRL-targeting siRNA (si-LPCRL, 10 μg per injection), si-NC, glucose solution (Blank), si-NC plus cisplatin (2.5 mg/kg per injection), or si-LPCRL plus cisplatin. Nude mice were euthanized after the indicated treatments were completed. (B-C) Excised tumors were imaged (B), with tumor weights measured in each group (C). (D) Growth curves of subcutaneously transplanted tumors across groups. (E) Body weights were measured in each group. (F) RT‒qPCR analysis of LPCRL expression in the blank, si-NC, and si-LPCRL groups. (G) Representative immunohistochemistry (IHC) images (left) and quantification (H-score values, right) of Ki-67, MIB1, and HES1 in tumors from the glucose solution (Blank), si-NC, and si-LPCRL groups. Scale bar: 20 μm. (H) Representative IHC images (left) and quantification (H-score values, right) of Ki-67 and γ-H2AX in tumors from the si-NC plus cisplatin and si-LPCRL plus cisplatin groups. Scale bar: 20 μm.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8106822/v1/22440bf606fd3fd7cb9ff0e8.png"},{"id":108437798,"identity":"83f30074-537b-4b39-9477-cf335705c724","added_by":"auto","created_at":"2026-05-04 16:03:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7640250,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8106822/v1/616bf77e-08f6-4165-bfe2-95f4c2b0982b.pdf"},{"id":97141519,"identity":"52e0a30d-6f59-49ad-b97e-724760bea3ec","added_by":"auto","created_at":"2025-12-01 10:06:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3916927,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-8106822/v1/b3841bbf5d8fee42029ac4a6.docx"},{"id":97114666,"identity":"9e634de7-f810-4bea-9366-1c5aaffcb44b","added_by":"auto","created_at":"2025-12-01 07:00:41","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":408812,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8106822/v1/eb6dcf8bf4dc5880f1397136.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel antisense lncRNA, LPCRL, functions as a molecular scaffold for the USP15/MIB1 complex to promote primary cisplatin resistance and tumor progression in lung squamous cell carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNon-small cell lung cancer (NSCLC) accounts for 80%-85% of all lung cancer cases, with the squamous cell subtype comprising 20%-30% of NSCLCs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Unfortunately, most LUSC patients are diagnosed at advanced stages, resulting in high mortality rates [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although targeted therapies and immunotherapies have transformed the treatment landscape for LUSC [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], platinum-based chemotherapy remains the cornerstone of first-line treatment for advanced cases [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, its efficacy is often limited, with objective response rates of only 30%-40%, primarily due to intrinsic resistance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, elucidating the molecular mechanisms underlying primary platinum resistance in LUSC is critical for developing novel therapeutic strategies aimed at improving patient outcomes.\u003c/p\u003e\u003cp\u003eLong noncoding RNAs (lncRNAs) are defined as RNA transcripts longer than 200 nucleotides that lack protein-coding capacity but are critically involved in regulating diverse biological processes in lung cancer [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Antisense lncRNAs, a major subclass of lncRNAs, are transcribed from the complementary strand of protein-coding or noncoding genes. They account for approximately 50%-70% of all annotated lncRNAs and are widely distributed across both eukaryotic and prokaryotic genomes. Notably, antisense lncRNAs can regulate gene expression at multiple levels, including the pretranscriptional, transcriptional, and posttranscriptional levels, through interactions with DNA, RNA, or proteins [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Emerging evidence has linked antisense lncRNAs to various aspects of cancer biology, including drug resistance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], metabolic reprogramming [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], phase separation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], cell proliferation, and metastasis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Nevertheless, the specific roles and mechanisms of antisense lncRNAs in driving primary cisplatin resistance in LUSC remain largely unexplored.\u003c/p\u003e\u003cp\u003ePatient-derived xenograft (PDX) models are widely recognized as robust preclinical tools that recapitulate intratumoral heterogeneity, preserve native tumor architecture, and reliably reflect drug responses and resistance mechanisms [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. After serial passaging to the third generation (P3), these models reach a biological \"plateau\" with stabilized characteristics, enhancing their reproducibility for drug studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, microarray analysis of the lncRNA expression profiles of third-generation xenograft tumors revealed a clear transcriptional distinction between the cisplatin-sensitive and cisplatin-resistant groups via principal component analysis. We identified a markedly upregulated antisense transcript, uc002ktr.3, hereafter referred to as LPCRL (LUSC primary cisplatin resistance-associated LncRNA), in the cisplatin-resistant cohort. This transcript is derived from the \u003cem\u003eMIR133A1HG\u003c/em\u003e gene locus, which overlaps the antisense strand of the twelfth intron of \u003cem\u003eMIB1\u003c/em\u003e. Functional studies demonstrated that LPCRL enhance cisplatin resistance, promote proliferation, and facilitate metastasis in LUSC cells. Mechanistically, LPCRL functions as a molecular scaffold that directly mediates the interaction between MIB1 and USP15. This promotes USP15-mediated deubiquitination of nuclear MIB1. This posttranslational modification stabilizes MIB1 and promotes its nuclear export. The resulting cytoplasmic accumulation of MIB1 activates the Notch signaling pathway. Importantly, the siRNA-mediated silencing of LPCRL in vivo significantly suppressed tumor growth and migration while simultaneously enhancing cisplatin sensitivity. In summary, our study reveals a pivotal role for the LPCRL/USP15/MIB1/Notch signaling axis in promoting cisplatin resistance and tumor progression in LUSC, identifying LPCRL as a promising molecular target for therapeutic intervention.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eClinical specimens\u003c/h2\u003e\u003cp\u003ePatient-derived xenograft (PDX) models were established using tumor tissues from patients diagnosed with LUSC at The First Affiliated Hospital of the University of Science and Technology of China. All tumor samples were collected with approval from the Ethics Committee of the University of Science and Technology of China (Approval No. 2019-N(H)-128), and written informed consent was obtained from all participants.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEstablishment of PDX models and cisplatin chemosensitivity testing\u003c/h3\u003e\n\u003cp\u003e All animal procedures were conducted in accordance with the Declaration of Helsinki and were approved by the Institutional Animal Care and Use Committee of the University of Science and Technology of China (Approval No. 2019-N (A)-179). PDX models were generated as previously described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, fresh tumor tissues were sectioned into ~\u0026thinsp;3 mm\u0026sup3; fragments and implanted subcutaneously into the flanks of BALB/c nude mice. Following the same method, the transplanted tumors were serially passaged to the third generation. Once the third-generation xenograft tumor volume reached 50\u0026ndash;200 mm\u0026sup3;, 3\u0026ndash;5 mice per model were randomized into treatment or control groups. The tumor volume was calculated as (length \u0026times; width^2)/2.\u003c/p\u003e\u003cp\u003eCisplatin (20 mg/mL; Jiangsu Hansoh Pharmaceutical Co., Ltd.) was diluted to 0.5 mg/mL in saline and administered intraperitoneally at 5 mg/kg once weekly for 3 consecutive weeks (treatment group). The control mice received an equivalent volume of saline. The injection volume was standardized at 0.2 mL per 20 g body weight. A tumor growth inhibition rate (TIR\u0026thinsp;=\u0026thinsp;1 - (average tumor volume in the cisplatin group/average tumor volume in the PBS group)\u0026times;100%)\u0026thinsp;\u0026ge;\u0026thinsp;50% was considered cisplatin sensitive; \u0026lt;50% was considered resistant.\u003c/p\u003e\n\u003ch3\u003eEstablishment of primary LUSC cells\u003c/h3\u003e\n\u003cp\u003ePrimary LUSC cells were established as follows. Briefly, fresh LUSC tumor tissue from surgical resection was placed in cold preservation medium (DMEM with antibiotics/antimycotics) on ice. The tissue was minced into 1\u0026ndash;2 mm\u0026sup3; fragments and digested in a collagenase solution (200 U/mL, Sigma Aldrich, Saint Louis, MO, USA) at 37\u0026deg;C with periodic shaking or resuspension. The resulting cell suspension was filtered through a 70 \u0026micro;m cell strainer, and the filtrate was centrifuged. The cell pellet was washed with PBS, resuspended in culture medium (RPMI 1640 medium (HyClone) with 10% FBS), and seeded. Cultures were maintained in a humidified incubator at 37\u0026deg;C with 5% CO₂, with regular medium changes to remove nonadherent cells and debris. Once the cells reached 80%-90% confluence, they were passaged with trypsin-EDTA (Beyotime).\u003c/p\u003e\n\u003ch3\u003eLncRNA microarray and bioinformatic analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from cisplatin-resistant and cisplatin-sensitive PDX samples for microarray analysis via the Arraystar Human LncRNA Microarray V3.0 (Agilent Technologies). Feature extraction was performed with Agilent Feature Extraction software (v11.0.1.1), and quantile normalization and data processing were conducted via GeneSpring GX v12.1 (Agilent Technologies). Differentially expressed lncRNAs were identified by a fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5 and a \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Principal component analysis and volcano plots were generated via in-house scripts.\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eSK-MES-1 and HEK293T cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The NCI-H520 cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). LUSC primary cells were established in our laboratory. NCI-H520 and primary cells were cultured in RPMI-1640 medium (HyClone) supplemented with 10% fetal bovine serum (FBS; Proteintech). SK-MES-1 and HEK293T cells were cultured in DMEM/high-glucose medium (HyClone) supplemented with 10% FBS. All the cells were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂ and passaged using 0.25% trypsin-EDTA (Beyotime).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA extraction, reverse transcription and quantitative PCR (\u003c/b\u003eRT‒qPCR)\u003c/p\u003e\u003cp\u003eTotal RNA was extracted via TRIzol Reagent (Life Technologies, CA, USA). Nuclear and cytoplasmic RNA fractions were isolated via nuclear and cytoplasmic extraction reagents (Invitrogen, NY, USA). Complementary DNA (cDNA) was synthesized with the PrimeScript\u0026trade; RT‒PCR Kit (Takara Bio, Otsu, Shiga, Japan), and quantitative PCR was performed via SYBR\u0026reg; Select Master Mix (Vazyme, Nanjing, China) on an ABI 7500 real-time PCR system (Thermo Fisher Scientific, USA). The primer sequences are listed in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSmall interfering RNA (siRNA), antisense oligonucleotides, plasmid construction and cell transfection\u003c/h2\u003e\u003cp\u003eSmall interfering RNAs (siRNAs) were obtained from Generalbiol (Shanghai, China). Antisense oligonucleotides (ASOs) were obtained from RiboBio (Guangzhou, China). Plasmid vectors (pCMV-LPCRL, pCMV-LPCRL-M1, pCMV-LPCRL-M2, pCMV-LPCRL-M3, pCMV-Myc-MIB1, pCMV-HA-USP15, and the empty pCMV vector) were obtained from the Miaoling Plasmid Platform (Wuhan, China). siRNAs and ASOs were transfected via a Lipofectamine\u0026trade; 2000 kit (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer\u0026rsquo;s protocol. Plasmid transfection was performed using a PEI MW40000 (Yeasen Biotechnology, Shanghai, China). All the siRNA sequences used are listed in \u003cb\u003eSupplementary Table S2\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLentiviral transfection\u003c/h3\u003e\n\u003cp\u003eLentiviral-shLPCRL and Lentiviral-LPCRL were obtained from Hanheng Biological Company (Shanghai, China). Lentiviral (LV)-short hairpin negative control (shNC) and LV-sh LPCRL (sh LPCRL-1, sh LPCRL-2) were used to transfect SK-MES-1 cells. The short hairpin RNA sequences used to silence the LPCRL are listed in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. Lentiviral (LV)-LPCRL (oeLPCRL) was used to transfect NCI-H520 cells. Inducible cell sublines were established via puromycin selection and validated via RT‒qPCR.\u003c/p\u003e\n\u003ch3\u003eMTT and colony formation assays\u003c/h3\u003e\n\u003cp\u003eFor the MTT assays, 96-well plates were seeded with cells and incubated with MTT (5 mg/mL; Biosharp, Anhui, China) for 4 hours. The absorbance was measured at 490 nm. For the colony formation assays, 1,000 cells/well were seeded in 6-well plates and cultured for 1\u0026ndash;2 weeks. Colonies were fixed in 4% paraformaldehyde, stained with crystal violet (Sangon Biotech, Shanghai, China), and counted manually.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eTranswell migration assay\u003c/h2\u003e\u003cp\u003eFor the migration assays, 8 \u0026times; 10⁴ cells in 200 \u0026micro;L of serum-free DMEM were seeded into the upper chambers of Transwell plates (8-\u0026micro;m pores; Corning, NY, USA). The lower chambers contained 800 \u0026micro;L of medium supplemented with 10% FBS as a chemoattractant. After 24\u0026ndash;48 hours, the migrated cells were fixed, stained with crystal violet, and quantified under a microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eIC50 assay\u003c/h2\u003e\u003cp\u003eThe cells (8 \u0026times; 10⁴ cells/well) were seeded in 96-well plates, allowed to adhere, and then treated with various concentrations of cisplatin for 48 hours. IC₅₀ values were calculated via GraphPad Prism v9.5.0 on the basis of the logarithmic relationship between drug concentration and the cellular response.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry apoptosis assay\u003c/h2\u003e\u003cp\u003eApoptosis was assessed via an Annexin V-FITC/PI Apoptosis Detection Kit (Keygen Biotech, Nanjing, China). After 24 hours of cisplatin treatment, the cells were washed with PBS, resuspended in 500 \u0026micro;L of binding buffer, and stained with 5 \u0026micro;L of Annexin V-FITC and 5 \u0026micro;L of propidium iodide (PI) for 10 minutes in the dark. Apoptosis was analyzed via a CytoFLEX flow cytometer and CytExpert software (Beckman Coulter). The cells were categorized as viable (FITC⁻/PI⁻), early apoptotic (FITC⁺/PI⁻), or late apoptotic/dead (FITC⁺/PI⁺).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eFluorescence in situ hybridization (FISH)\u003c/h2\u003e\u003cp\u003eBiotin-labeled LPCRL probes (Ribobio, Guangzhou, China) were used to detect LPCRL localization with a FISH detection kit (Ribobio). Fixed cells were hybridized with probes overnight, washed, stained with DAPI, and visualized via fluorescence microscopy. The tissue samples were cryosectioned at 4 \u0026micro;m after fixation in 4% paraformaldehyde for 1 hour and processed similarly.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eRNA pull-down and RNA-binding protein immunoprecipitation (RIP) assays\u003c/h2\u003e\u003cp\u003eThe RNA pull-down assays used biotin-labeled LPCRL probes (RiboBio) and their antisense controls. SK-MES-1 cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 5 mM EDTA; 1% NP-40; 0.1% SDS; 1 mM DTT; 1\u0026times; protease inhibitor cocktail; 0.1 U/\u0026micro;L RNase inhibitor) for 15 minutes on ice. The lysates were centrifuged at 12,000 \u0026times; g for 15 minutes, and the supernatants were incubated with 100 pmol of biotinylated oligonucleotides or 2 \u0026micro;g of antibodies (MIB1, USP15 for RIP) overnight at 4\u0026deg;C. M-280 streptavidin Dynabeads (Invitrogen, 11206D, for RNA pull-down) or Protein G Dynabeads (Invitrogen, 10004D, for RIP) preblocked with 500 ng/\u0026micro;L yeast total RNA and 5% BSA were added for 2 hours at room temperature. The beads were washed with lysis buffer and high-salt lysis buffer (500 mM NaCl). The purified RNAs were analyzed via RT‒qPCR, and the proteins were analyzed via western blotting after silver staining.\u003c/p\u003e\u003cp\u003e\u003cb\u003e5\u0026prime; and 3\u0026prime; rapid amplification of cDNA ends (RACE)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRACE was performed via the SMARTer RACE 5\u0026prime;/3\u0026prime; Kit (Takara). Total RNA from SK-MES-1 cells was used to synthesize 5\u0026prime;- and 3\u0026prime;-RACE-ready cDNA, which was amplified via nested PCR with universal primers and gene-specific primers (\u003cb\u003eSupplementary Table S2\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot\u003c/h2\u003e\u003cp\u003eTotal protein was extracted via a Total Protein Extraction Kit (Bestbio, Shanghai, China) with protease and phosphatase inhibitors (Epizyme, Shanghai, China). Proteins were quantified and separated by SDS‒PAGE, transferred to PVDF membranes (Merck Millipore), blocked with 5% skim milk, and incubated with primary antibodies overnight, followed by incubation with secondary antibodies (Proteintech). Bands were detected via a Tanon multigel imaging system. The following antibodies were used: MIB1 (sc-393551, Santa Cruz, 1:500), USP15 (66310s, CST, 1:1000), c-PARP (ab32561, Abcam, 1:1000), γ-H2AX (CY6572, Abways, 1:1000), β-Actin (66009-1-Ig, Proteintech, 1:10000), GAPDH (60004-1-Ig, Proteintech, 1:10000), Ub (#20326, CST, 1:1000), Myc (60003-2-Ig, Proteintech, 1:2000), HA (51064-2-AP, Proteintech, 1:2000), DLL4 (21584-1-AP, Proteintech, 1:2000), LaminB1 (12987-1-AP, Proteintech, 1:2000), NICD (10062-2-AP, Proteintech, 1:2000), N-cadherin (22018-1-AP, Proteintech, 1:2000), PCNA (60097-1-Ig, Proteintech, 1:5000), Vimentin (10366-1-AP, Proteintech, 1:5000), and c-Myc (10828-1-AP, Proteintech, 1:5000).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eImmunoprecipitation (IP)\u003c/h2\u003e\u003cp\u003eThe cells were lysed in IP buffer supplemented with protease and phosphatase inhibitors, and the lysates were subsequently centrifuged. The supernatants were immunoprecipitated with antibodies against MIB1 (Santa Cruz), USP15 (CST), DLL4 (Proteintech), Myc (Proteintech), or HA (Proteintech) overnight at 4\u0026deg;C. Immune complexes were captured with protein A/G magnetic beads (Biolinkedin, Shanghai, China), washed with ice-cold PBS, 20 \u0026micro;L of 1\u0026times; loading buffer was added, the mixture was heated at 95\u0026deg;C for 10 minutes, and the proteins were analyzed by western blotting.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eAnimal experiments\u003c/h2\u003e\u003cp\u003eFemale NOD-SCID mice (4\u0026ndash;6 weeks old; n\u0026thinsp;=\u0026thinsp;6, 3 per group) and BALB/c nude mice (4\u0026ndash;6 weeks old; n\u0026thinsp;=\u0026thinsp;18, 9 per group) were obtained from Charles River Laboratories (Zhejiang, China). SK-MES-1 cells (5 \u0026times; 10⁶ cells/mouse) transfected with si-LPCRL or si-NC were injected subcutaneously into BALB/c nude mice. Tumor growth was monitored every 5 days. After 4 weeks, the mice were euthanized, and the tumors were fixed in 10% formalin for pathological analysis.\u003c/p\u003e\u003cp\u003eFor the metastasis assays, luciferase-labeled SK-MES-1 cells (1 \u0026times; 10⁶) transfected with si-LPCRL or si-NC were injected via the tail vein into NOD-SCID mice. Lung metastases were monitored by bioluminescence imaging (IVIS Spectrum). After 3 weeks, the mice were euthanized, and the lungs were fixed and stained with H\u0026amp;E for histological confirmation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo PDX model-based therapeutic study\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhen the PDX tumor volume reached 50\u0026ndash;100 mm\u0026sup3;, the mice were randomized into 5 groups (n\u0026thinsp;=\u0026thinsp;8/group): (a) blank control (5% glucose); (b) si-NC; (c) si-LPCRL; (d) si-NC\u0026thinsp;+\u0026thinsp;cisplatin; and (e) si-LPCRL\u0026thinsp;+\u0026thinsp;cisplatin. Si-LPCRL or si-NC (10 \u0026micro;g/tumor/dose) in 5% glucose was administered intratumorally via in vivo jetPEI\u0026reg; (Polyplus, France) every 5 days for 4 doses. Cisplatin (2.5 mg/kg) was administered intraperitoneally every 5 days for 3 doses in the combination groups. The mice were euthanized, and the tumors were excised, weighed, measured, and paraffin-embedded for IHC.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e\u003cp\u003eThe tumor sections were deparaffinized, rehydrated, and subjected to antigen retrieval with sodium citrate (Beyotime). After blocking, the sections were incubated with primary antibodies (MIB1, Proteintech, 1:200; HES1, Bioss, Beijing, 1:200; γ-H2AX, Bioss, 1:200; Ki-67, Abcam, 1:200) overnight at 4\u0026deg;C and then with HRP-labeled secondary antibodies (Boster). Staining was performed with DAB (Beyotime), and the sections were counterstained with hematoxylin. Images were captured with a Nikon microscope. Expression was quantified by the H score (staining intensity \u0026times; percentage of positive cells). Intensities: 0 (none), 1 (light brown), 2 (brown), and 3 (dark brown). Positive cells: 0 (\u0026lt;\u0026thinsp;5%), 1 (5\u0026ndash;25%), 2 (26\u0026ndash;50%), 3 (51\u0026ndash;75%), and 4 (76\u0026ndash;100%).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll the experiments were performed in triplicate. The data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SDs). Comparisons between two groups were performed via two-tailed Student\u0026rsquo;s t tests. Paired samples were analyzed with paired t tests. A \u003cem\u003ep\u003c/em\u003e value of less than 0.05 was considered statistically significant. Statistical analyses were conducted via SPSS (version 17.0).\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eLPCRL is upregulated in primary cisplatin-resistant LUSC tissues\u003c/h2\u003e\u003cp\u003eTo identify key lncRNAs associated with primary cisplatin resistance in LUSC, we established six LUSC PDX models, which were serially passaged to the third generation. After three cycles of cisplatin treatment, the models were classified into cisplatin-sensitive (tumor inhibition rate, TIR\u0026thinsp;\u0026gt;\u0026thinsp;50%; n\u0026thinsp;=\u0026thinsp;2) and cisplatin-resistant (TIR\u0026thinsp;\u0026lt;\u0026thinsp;50%; n\u0026thinsp;=\u0026thinsp;4) groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, S1B). Microarray analysis of the lncRNA expression profiles of third-generation xenograft tumors revealed a clear transcriptional distinction between the cisplatin-sensitive and cisplatin-resistant groups via principal component analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Volcano plot analysis identified 847 differentially expressed lncRNAs (|fold change| \u0026gt;1.5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Among these, uc002ktr.3, which we designated LPCRL (lung squamous cell carcinoma primary cisplatin resistance-associated long noncoding RNA), was the most significantly upregulated antisense lncRNA in cisplatin-resistant tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). RT‒qPCR analysis confirmed significantly increased LPCRL expression in cisplatin-resistant tissues (n\u0026thinsp;=\u0026thinsp;3) from eight additional PDX models, which were established and classified on the basis of cisplatin screening (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBioinformatic analysis revealed that the LPCRL is located on chromosome 18 and is transcribed from the \u003cem\u003eMIR133A1HG\u003c/em\u003e gene, which overlaps with the antisense strand of the twelfth intron of \u003cem\u003eMIB1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Kaplan‒Meier analysis revealed that high MIR133A1HG expression was correlated with poorer overall survival in LUSC patients [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Analysis of the GDC TCGA LUSC cohort revealed that 42.6% (207/486) of patients harbored MIR133A1HG copy number gains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Given the role of copy number variations (CNVs) in driving gene expression and tumorigenesis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], we hypothesized that LPCRL expression would be elevated in LUSC. Consistent with these findings, RT‒qPCR confirmed significantly higher LPCRL levels in LUSC tissues than in paired normal adjacent tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). This tumor-specific overexpression was further corroborated by fluorescence in situ hybridization (FISH), which revealed markedly stronger LPCRL signals in tumor cells than in stromal cells within the same tissue (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). In summary, these data establish LPCRL as an antisense lncRNA that not only is highly expressed in LUSC but is also further upregulated in a cisplatin-resistant context, positioning it as a key mediator of LUSC resistance to cisplatin.\u003c/p\u003e\u003cp\u003eTo molecularly characterize LPCRL, we performed 5' and 3' RACE in LUSC cells. Using a 5,912-nt reference sequence from the UCSC Genome Browser (which annotates four MIR133A1HG transcripts; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD), we identified a single 688-nt, single-exon transcript in SK-MES-1 cells. This variant was confirmed by ORF Finder to lack protein-coding potential (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE) and constitutes a partial segment of the MIR133A1HG-204 isoform (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF). RNA FISH and subcellular fractionation assays revealed that LPCRL is predominantly localized in the nucleus of both SK-MES-1 and NCI-H520 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG, S1H), two widely used cell lines in LUSC research. Given the high expression of LPCRL in LUSC, we performed siRNA-mediated knockdown in primary LUSC cells established in our laboratory (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eI). LPCRL knockdown significantly increased cisplatin sensitivity and reduced cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). Taken together, our results define LPCRL as a nuclear-enriched antisense lncRNA associated with cisplatin resistance and cell proliferation in LUSC.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003eLPCRL promotes cisplatin resistance, proliferation and migration in LUSC\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eTo further investigate the role of LPCRL in primary cisplatin resistance in LUSC, we first assessed its expression in SK-MES-1 and NCI-H520 cells. RT‒qPCR analysis revealed significantly higher LPCRL expression in SK-MES-1 cells than in NCI-H520 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Consistent with these findings, SK-MES-1 cells exhibited greater intrinsic cisplatin resistance, as indicated by a higher IC\u003csub\u003e50\u003c/sub\u003e in the MTT assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To silence the nucleus-localized LPCRL, we designed a panel of siRNAs, shRNAs, and ASOs and evaluated their silencing efficacy (Supplementary Fig. S2A-S2C). Among these, only one siRNA demonstrated high interference efficiency. This siRNA, which targets the stem region of a predicted stem loop in the LPCRL secondary structure (modeled via RNAstructure software via the minimum free energy method), was selected for subsequent experiments (Supplementary Fig. S2D). We also stably overexpressed exogenous LPCRL in NCI-H520 cells, which presented relatively low endogenous LPCRL levels (Supplementary Fig. S2E). Functionally, LPCRL silencing significantly decreased the IC\u003csub\u003e50\u003c/sub\u003e of cisplatin in SK-MES-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), whereas its overexpression increased the IC\u003csub\u003e50\u003c/sub\u003e of cisplatin in NCI-H520 cells (Supplementary Fig. S2F). Given that cisplatin exerts its antitumor effects primarily by inducing DNA damage and subsequent apoptosis, we next examined these processes. Flow cytometry analysis revealed that LPCRL silencing enhanced cisplatin-induced apoptosis in SK-MES-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), which was supported by elevated levels of γ-H2AX (a DNA damage marker) and cleaved PARP (an apoptosis marker; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Conversely, LPCRL overexpression in NCI-H520 cells attenuated cisplatin-induced apoptosis and reduced the expression of these markers (Supplementary Fig. S2G, S2H).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate the impact of LPCRL on LUSC cell proliferation and migration in vitro, we performed colony formation and Transwell migration assays. LPCRL knockdown significantly suppressed the proliferation and migration of SK-MES-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), whereas its ectopic overexpression enhanced these abilities in NCI-H520 cells (Supplementary Fig. S2I, S2J). Consistent with these phenotypic alterations, the expression of proliferation markers (PCNA) and epithelial‒mesenchymal transition (EMT)-related migration markers (N-cadherin and vimentin) was downregulated following LPCRL knockdown in SK-MES-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) but upregulated upon LPCRL overexpression in NCI-H520 cells (Supplementary Fig. S2K).\u003c/p\u003e\u003cp\u003eTo further validate the role of LPCRL in promoting proliferation in vivo, we subcutaneously injected BALB/c nude mice with SK-MES-1 cells transfected with LPCRL-targeting siRNA (si-LPCRL) or control siRNA (si-NC) (Supplementary Fig. S2L). Compared with the si-NC, si-LPCRL not only reduced the tumor incidence but also suppressed the tumor growth rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Consistently, immunohistochemical (IHC) analysis revealed that LPCRL knockdown reduced Ki-67 positivity (a marker of cell proliferation; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). To further assess the role of LPCRL in metastasis in vivo, we established an experimental metastasis model by intravenously injecting NOD/SCID mice with luciferase-labeled SK-MES-1 cells transfected with si-NC or si-LPCRL (Supplementary Fig. S2M). si-LPCRL attenuated the metastatic burden, as shown by a reduced bioluminescence signal in the lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM). Histological examination of lung tissues confirmed a reduction in both the size and number of metastatic lesions in the si-LPCRL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO). Collectively, these findings indicate that LPCRL contribute to cisplatin resistance and promote tumor proliferation and metastasis in LUSC.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eLPCRL binds to MIB1 and inhibits ubiquitin-mediated degradation\u003c/h2\u003e\u003cp\u003eAntisense lncRNAs regulate the expression of their corresponding sense genes at the transcriptional or posttranscriptional level through interactions with DNA, RNA, or proteins [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Notably, LPCRL is transcribed from an antisense intronic region within the \u003cem\u003eMIB1\u003c/em\u003e gene. We therefore hypothesized that LPCRL may promote LUSC progression by regulating MIB1, an E3 ubiquitin ligase known to be overexpressed in lung cancer and associated with poor prognosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Consistent with this hypothesis, CPTAC data and our IHC analyses confirmed significantly elevated MIB1 protein levels in LUSC tissues relative to normal tissues (Supplementary Fig. S3A, S3B). Surprisingly, neither LPCRL knockdown nor LPCRL overexpression significantly altered MIB1 mRNA expression (Supplementary Fig. S3C, S3D); likewise, modulating MIB1 expression did not affect the abundance of LPCRL transcripts (Supplementary Fig. S3E, S3F). However, LPCRL knockdown markedly reduced MIB1 protein levels, whereas LPCRL overexpression significantly increased them (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), indicating a posttranscriptional regulatory mechanism.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePrevious studies have shown that lncRNAs can stabilize their interacting proteins by inhibiting ubiquitin-proteasome-mediated degradation [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To determine if LPCRL physically interacts with MIB1, we first employed RPISeq, which predicts a high-confidence interaction (RF/SVM scores: 0.65/0.81). We next performed RNA pull-down assays in SK-MES-1 cells using a biotin-labeled LPCRL probe. Following efficient probe enrichment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), silver staining revealed a specific\u0026thinsp;~\u0026thinsp;110 kDa band (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), which was confirmed via western blot analysis as MIB1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). RNA immunoprecipitation (RIP) assays independently validated this interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). To map the binding domain, we analyzed the secondary structure of LPCRL, identifying three major loops: Loop1 (1\u0026ndash;252 nt), Loop2 (253\u0026ndash;476 nt), and Loop3 (477\u0026ndash;688 nt) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). RIP assays with full-length and truncated LPCRL constructs revealed significant enrichment only for the full-length and Loop2 fragments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), thereby mapping the core MIB1-binding region to nucleotides 253\u0026ndash;476 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003eTo assess whether LPCRL stabilizes MIB1 via the ubiquitin‒proteasome pathway, LUSC cells were treated with the proteasome inhibitor MG132 or the protein synthesis inhibitor cycloheximide (CHX). MG132 treatment significantly increased MIB1 protein levels in both SK-MES-1 and NCI-H520 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), confirming the proteasomal degradation of MIB1. Consistent with these findings, LPCRL knockdown increased MIB1 ubiquitination, whereas LPCRL overexpression reduced MIB1 ubiquitination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). In CHX chase assays, LPCRL silencing accelerated MIB1 degradation, whereas LPCRL overexpression extended its half-life (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). These results demonstrate that LPCRL stabilizes MIB1 by inhibiting its ubiquitin-mediated proteasomal degradation.\u003c/p\u003e\u003cp\u003eTo investigate whether LPCRL promotes LUSC progression via MIB1, we first examined the role of MIB1 in LUSC cells. In SK-MES-1 cells, MIB1 knockdown significantly downregulated key markers of cell proliferation and migration, including PCNA, N-cadherin, and vimentin (Supplementary Fig. S3G). Furthermore, MIB1 silencing followed by cisplatin treatment markedly increased the levels of γ-H2AX and cleaved PARP, indicating enhanced DNA damage and apoptosis (Supplementary Fig. S3H). To confirm that LPCRL functions through MIB1, we silenced MIB1 in NCI-H520 cells stably overexpressing LPCRL. MTT assays revealed that MIB1 knockdown abolished the LPCRL-induced increase in the IC\u003csub\u003e50\u003c/sub\u003e of cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). Furthermore, colony formation and Transwell assays revealed that MIB1 silencing attenuated the enhanced proliferative and migratory capacities conferred by LPCRL overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN), which was consistent with decreased PCNA and vimentin protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO). Upon cisplatin exposure, MIB1 silencing abrogated the LPCRL-mediated suppression of γ-H2AX and cleaved PARP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eP). These findings collectively support that MIB1 is a key mediator of the oncogenic effects of LPCRL in regulating proliferation, migration, and cisplatin resistance in LUSC.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eLPCRL stabilizes MIB1 via USP15-mediated deubiquitination\u003c/h2\u003e\u003cp\u003eLPCRL lacks protein-coding capacity; therefore, the precise mechanism by which it interacts with MIB1 to regulate MIB1 ubiquitination remains incompletely understood. Accumulating evidence indicates that lncRNAs function as molecular scaffolds to facilitate protein‒protein interactions [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. On the basis of these findings, we hypothesized that LPCRL may act as a scaffold to recruit a deubiquitinating enzyme that interacts with MIB1, thereby inhibiting MIB1 ubiquitination and subsequent proteasomal degradation. To identify MIB1-associated deubiquitinating enzymes, analysis of BioID proteomic datasets and BioGRID interactors identified USP9X, CYLD, and USP15 as potential MIB1-interacting candidates [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. While interactions between MIB1 and USP9X or CYLD have been reported [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], the MIB1-USP15 interaction remains uncharacterized. USP15 is a critical deubiquitinating enzyme implicated in multiple malignancies and has been reported to promote lung cancer cell proliferation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, RPISeq analysis predicted a high-probability interaction between LPCRL and USP15 (RF/SVM scores: 0.75 and 0.97). On the basis of these findings, we hypothesized that LPCRL serves as a molecular scaffold, facilitating the interaction of MIB1 with USP15 in LUSC cells. Western blot analysis confirmed the specific enrichment of USP15 by the biotin-labeled LPCRL probe (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Furthermore, RIP assays revealed significant enrichment of LPCRL in USP15-immunoprecipitated complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Similarly, using full-length and truncated LPCRL constructs transfected into SK-MES-1 cells, RIP assays revealed significant enrichment of the Loop1 region (nucleotides 1\u0026ndash;252) in USP15-immunoprecipitated complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Collectively, these results indicate that MIB1 primarily binds to Loop2 of LPCRL, whereas USP15 preferentially associates with Loop1, supporting the role of LPCRL as a molecular scaffold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese results indicate that LPCRL binds independently to MIB1 and USP15. We therefore hypothesized that LPCRL serves as a molecular scaffold to promote the MIB1-USP15 interaction. To this end, we first assessed whether MIB1 and USP15 physically interact. Molecular docking analysis predicted multiple potential binding modes between MIB1 and USP15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Co-IP assays confirmed the endogenous interaction between MIB1 and USP15 in both SK-MES-1 and NCI-H520 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, Supplementary Fig. S4A). Furthermore, immunofluorescence (IF) staining demonstrated their nuclear colocalization in these cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, Supplementary Fig. S4B), which is consistent with the established nuclear localization of LPCRL. To further validate the scaffolding role of LPCRL, we initially conducted co-IP assays to assess exogenous MIB1-USP15 interactions in HEK293T cells with or without RNase A treatment. The results showed that MIB1 interacted with USP15 in the absence of RNase A; however, this interaction was markedly diminished upon RNase A treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Additionally, LPCRL knockdown weakened the interaction between MIB1 and USP15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI), whereas LPCRL overexpression increased this interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ), thus supporting the hypothesis that LPCRL is essential for mediating this interaction. Taken together, these data indicate that LPCRL functions as a molecular scaffold to facilitate the formation of the MIB1-USP15 complex.\u003c/p\u003e\u003cp\u003eFunctionally, USP15 knockdown reduced MIB1 protein levels and increased MIB1 ubiquitination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL; Supplementary Fig. S4C, S4D). Conversely, USP15 overexpression had the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN; Supplementary Fig. S4E, S4F). Moreover, LPCRL knockdown decreased the USP15 protein level without affecting its mRNA level, whereas LPCRL overexpression increased it (Supplementary Fig. S4G-S4J). This finding suggests that LPCRL posttranslationally stabilizes both MIB1 and USP15. Collectively, these findings demonstrate that LPCRL promotes the assembly of the USP15-MIB1-LPCRL ternary complex, which enables USP15-mediated deubiquitination of MIB1, thereby shielding it from proteasomal degradation and enhancing its protein stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eThe LPCRL-MIB1-USP15 complex activates the Notch signaling pathway\u003c/h2\u003e\u003cp\u003eOur previous studies revealed that the LPCRL-MIB1-USP15 complex enhances MIB1 stability in LUSC cells. As an E3 ubiquitin ligase, MIB1 activates the Notch signaling pathway via ubiquitination and endocytosis of the intracellular domains of its ligands (Delta/Jagged). Notably, activation of the Notch signaling pathway is critical for tumorigenesis and progression and is implicated in promoting proliferation, metastasis, and chemoresistance in various cancers [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. We thus assessed whether LPCRL-driven malignant progression in LUSC cells is dependent on the Notch signaling pathway. Co-IP assays demonstrated that MIB1 interacts with DLL4, a Notch ligand, in both SK-MES-1 and NCI-H520 cells (Supplementary Fig. S5A, S5B). LPCRL knockdown significantly reduced DLL4 ubiquitination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), whereas LPCRL overexpression increased it (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Notably, this increase was substantially mitigated upon MIB1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), confirming the role of MIB1 in this process. Furthermore, LPCRL knockdown decreased the nuclear accumulation of the Notch intracellular domain (NICD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and downregulated the mRNA and protein levels of the Notch downstream effectors HES1 and c-Myc (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Conversely, LPCRL overexpression upregulated these effectors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI), indicating that LPCRL effectively activated the Notch signaling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOur experiments demonstrated that LPCRL inhibits MIB1 degradation via the USP15-mediated ubiquitin\u0026ndash;proteasome pathway in LUSC cell nuclei, thereby maintaining MIB1 homeostasis. However, MIB1 is known to activate the Notch signaling pathway by catalyzing the ubiquitination of its specific ligands in the cytoplasm [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. We thus investigated whether the LPCRL-MIB1-USP15 complex facilitates MIB1 nuclear export to enable Notch signaling pathway activation. Both nuclear-cytoplasmic fractionation and IF assays revealed that the overexpression of LPCRL or USP15 increased the cytoplasmic MIB1 level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK; Supplementary Fig. S5C, S5D). Collectively, these findings indicate that the LPCRL-MIB1-USP15 complex regulates not only MIB1 stability but also its nuclear export, providing new insights into the molecular mechanism of LPCRL in LUSC.\u003c/p\u003e\u003cp\u003eHES1 plays a pivotal role in tumorigenesis, tumor progression, and therapeutic resistance [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. To determine whether LPCRL contributes to LUSC progression by modulating HES1, we first assessed the role of HES1 in LUSC via multiple approaches. TCGA database analysis revealed significantly elevated HES1 mRNA in LUSC tissues compared with normal lung tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL), and Kaplan‒Meier survival analysis revealed that high HES1 expression was correlated with reduced overall survival in LUSC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). CPTAC database data and our IHC analysis confirmed that HES1 protein expression was increased in LUSC clinical samples compared with normal tissues (Supplementary Fig. S5E, S5F). Furthermore, HES1 silencing reduced the expression of proliferation- and migration-related proteins (PCNA, N-cadherin, and vimentin; Supplementary Fig. S5G) and elevated cisplatin-induced γ-H2AX and cleaved PARP levels (Supplementary Fig. S5H). HES1 expression was also elevated in cisplatin-resistant patient-derived xenograft (PDX) tissues (Supplementary Fig. S5I).\u003c/p\u003e\u003cp\u003eNCI-H520 cells stably overexpressing LPCRL were subsequently transfected with HES1-targeting siRNAs. MTT assays revealed that HES1 knockdown reversed the increase in the IC\u003csub\u003e50\u003c/sub\u003e of cisplatin induced by LPCRL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN) and abolished the suppressive effect of LPCRL on cisplatin-induced γ-H2AX and cleaved PARP (Supplementary Fig. S5J). Colony formation and Transwell migration assays further demonstrated that HES1 silencing mitigated the increase in cell proliferation and migration driven by LPCRL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eP). Consistent with these functional changes, HES1 knockdown also reduced the expression of PCNA and vimentin (Supplementary Fig. S5K). Notably, treatment with tangeretin, a Notch inhibitor, also inhibited LPCRL-mediated HES1 upregulation and cisplatin resistance. This was evidenced by the reversal of the effects of LPCRL on γ-H2AX and cleaved PARP (c-PARP) levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eQ, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eR). Collectively, these findings confirm that LPCRL exerts its oncogenic functions through the MIB1-USP15-Notch-HES1 axis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eLPCRL is potential therapeutic targets for the treatment of LUSC\u003c/h2\u003e\u003cp\u003eTo assess the therapeutic potential of LPCRL inhibition, we knocked down LPCRL in primary LUSC cells derived from a patient. LPCRL silencing significantly suppressed colony formation, reduced the cisplatin IC\u003csub\u003e50\u003c/sub\u003e, and enhanced apoptosis, as evidenced by elevated γ-H2AX and c-PARP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL; Supplementary Fig. S6A, S6B). MIB1 protein expression also decreased without altering its mRNA levels (Supplementary Fig. S6C, S6D). The endogenous interaction between MIB1 and USP15 was confirmed by Co-IP, and manipulation of USP15 levels altered MIB1 protein expression accordingly (Supplementary Fig. S6E, S6F). Collectively, these results corroborate our previous observations in LUSC cell lines and support the therapeutic potential of targeting LPCRL in LUSC.\u003c/p\u003e\u003cp\u003eTo test its efficacy in vivo, we further employed a PDX model established from the same LUSC patient as the primary cells. When the tumor volume reached 50\u0026ndash;100 mm\u0026sup3;, the mice were intratumorally injected with LPCRL-targeting siRNA (si-LPCRL), si-NC, or glucose solution (Blank). Cisplatin was administered intraperitoneally every five days (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Consistent with the in vitro findings, si-LPCRL significantly inhibited tumor growth and reduced tumor weight compared with those in the si-NC and blank groups. Importantly, the combination of cisplatin and si-LPCRL enhanced the inhibitory effect compared with that of cisplatin plus si-NC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). No significant body weight changes were observed across groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), indicating that si-LPCRL inhibits tumor growth and enhances cisplatin sensitivity without overt systemic toxicity in LUSC. The knockdown efficiency was confirmed by RT‒qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Compared with those in the si-NC or blank groups, IHC staining revealed markedly lower Ki67, MIB1, and HES1 expression in the si-LPCRL-treated tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG), and the si-LPCRL plus cisplatin combination notably increased DNA damage (elevated γ-H2AX) and inhibited proliferation (decreased Ki67) compared with those in the cisplatin plus si-NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). These results demonstrate that targeting LPCRL suppresses cancer cell proliferation, reduces MIB1 and its downstream HES1 expression, and enhances cisplatin sensitivity in vivo, supporting its potential as a therapeutic target.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlatinum-based chemotherapy, particularly cisplatin, is a cornerstone of LUSC treatment; however, its efficacy is severely hindered by the emergence of drug resistance [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. These challenges underscore the urgent need to elucidate the molecular mechanisms underlying cisplatin resistance and identify novel therapeutic strategies. In this study, we identified a novel antisense lncRNA, designated LPCRL, which is highly expressed in cisplatin-resistant LUSC xenograft tumors. Bioinformatic analysis revealed that LPCRL is transcribed from the MIR133A1HG locus and is located on the antisense strand within the twelfth intron of its homologous gene, MIB1, thus constituting a natural antisense transcript (NAT). While NATs typically regulate their sense counterparts through interactions with DNA or RNA [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], we uncovered a distinct mechanism: LPCRL promotes cisplatin resistance in LUSC by functioning as a molecular scaffold that directly mediates the protein interaction between MIB1 and USP15, thereby activating the Notch signaling pathway. Our data demonstrate that the LPCRL/USP15/MIB1/Notch axis is a hitherto unidentified mechanism driving cisplatin resistance.\u003c/p\u003e\u003cp\u003eThis regulatory mode is particularly intriguing given the genomic context of LPCRL. The MIR133A1HG locus is well established as a precursor for the tumor-suppressive microRNAs miR-133a and miR-1 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In addition to its role in solid tumors, MIR133A1HG has been implicated in hematological malignancies; for example, MIR133A1HG was identified among a set of autophagy-related lncRNAs that improve the prognosis of acute myeloid leukemia (AML) patients, with higher expression correlating with better outcomes [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, its potential functions in AML remain largely unexplored. In contrast, our work revealed that LPCRL, a lncRNA isoform from the same locus, functions as an oncogene in LUSC. This functional divergence underscores the remarkable transcriptional complexity of a single genomic locus and highlights the critical importance of isoform-specific investigations in cancer research.\u003c/p\u003e\u003cp\u003eWe further elucidated the spatial dynamics of this axis. Although MIB1, an E3 ubiquitin ligase, is known to increase Notch signaling in the cytoplasm, we found that LPCRL, MIB1, and USP15 are predominantly colocalized within the nucleus and activate Notch signaling. This observation prompted us to investigate their role in regulating the nucleocytoplasmic shuttling of MIB1. Nuclear‒cytoplasmic fractionation and IF analyses demonstrated that cytoplasmic MIB1 was significantly increased upon overexpression of LPCRL or USP15. This finding reveals a regulatory mechanism whereby LPCRL and USP15 modulate the nuclear stability and subsequent cytoplasmic translocation of MIB1, which is crucial for its oncogenic function. This axis is analogous to the USP7-mediated regulation of PTEN, suggesting that nuclear stabilization followed by cytoplasmic translocation may be a common strategy for regulating key signaling proteins [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFrom a translational perspective, we successfully identified a specific and efficient siRNA against LPCRL after extensive screening. The scarcity of effective inhibitors may be attributed to the nuclear localization and complex secondary structure of LPCRL. Notably, the effective siRNA targeted the stem region within LPCRL-Loop2 (Supplementary Fig. S2D), underscoring the importance of considering the RNA secondary structure in therapeutic design. Recent advancements in structural probing techniques have revealed critical structural motifs and RNA‒protein interfaces that contribute to lncRNA dysfunction. Furthermore, developing small molecules, antisense oligonucleotides, and peptidomimetic-based therapeutic agents that target these motifs and interfaces presents promising strategies for treating lncRNA-mediated diseases [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In PDX models, intratumoral administration of LPCRL-siRNA significantly suppressed tumor growth, and compared with monotherapy, its combination with cisplatin achieved superior efficacy. Moreover, with advances in drug delivery systems, such as inhalable lipid nanoparticles that enable targeted lung delivery with increased local accumulation and reduced systemic exposure [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], LPCRL-siRNA holds significant promise for clinical translation as a novel therapeutic strategy for LUSC.\u003c/p\u003e\u003cp\u003eDespite these significant findings, our study has several limitations. First, large-scale, multicenter clinical studies are necessary to validate the association between LPCRL expression and cisplatin response, as well as its prognostic value in patients with LUSC. Second, the functional role of nuclear MIB1 remains incompletely understood; it may have nuclear substrates other than Notch ligands, and proximity-labeling techniques such as BioID may help delineate the nuclear interactome of MIB1. Third, the dynamics of MIB1 nucleocytoplasmic shuttling and the precise mechanisms governing its translocation require further elucidation, potentially through live-cell imaging at the single-cell level. Together, these future directions will not only elucidate the noncanonical roles of nuclear MIB1 and the precise mechanisms of MIB1 nucleocytoplasmic shuttling but also pave the way for rationally designed LPCRL-targeted therapies.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study identified LPCRL as a novel NAT in LUSC. It functions as a molecular scaffold that facilitates the MIB1-USP15 interaction and promotes the nuclear export of MIB1, which in turn activates the Notch signaling pathway. This activation protects cells from DNA damage and cisplatin-induced cell death. Silencing LPCRL (si-LPCRL) disrupts signaling through the LPCRL/USP15/MIB1 axis and enhances the efficacy of cisplatin-based chemotherapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLUSClung squamous cell carcinoma\u003c/p\u003e\n\u003cp\u003elong\u0026nbsp;noncoding RNAs\u0026nbsp; \u0026nbsp; \u0026nbsp; lncRNAs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003enatural antisense transcripts\u0026nbsp;\u0026nbsp;NATs\u003c/p\u003e\n\u003cp\u003epatient-derived xenograft\u0026nbsp;\u0026nbsp; \u0026nbsp;PDX\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLPCRL \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; LUSC primary cisplatin resistance-associated lncRNA\u003c/p\u003e\n\u003cp\u003eCopy number variations \u0026nbsp; \u0026nbsp; CNVs\u003c/p\u003e\n\u003cp\u003eCo-IP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Coimmunoprecipitation\u003c/p\u003e\n\u003cp\u003eFISH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fluorescence in situ hybridization\u003c/p\u003e\n\u003cp\u003eRIP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;RNA immunoprecipitation\u003c/p\u003e\n\u003cp\u003eIHC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Immunohistochemistry\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the mouse experiments in this study were approved by the Institutional Animal Care and Use Committee of the University of Science and Technology of China (Approval No. 2019-N(A)-179). The collection of human samples and research were approved by the Ethics Committee of the University of Science and Technology of China (Approval No. 2019-N(H)-128), and written informed consent was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll raw lncRNA microarray data are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by funding from the National Natural Science Foundation of China (81972006) and Fundamental Research Funds for the Central Universities (WK9110000101).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBLW conceived and designed the research. PL carried out the molecular genetic studies and drafted the manuscript. DPL performed cell experiments. SZ analyzed the data and drafted the manuscript. KF performed the statistical analysis. WQD helped to draft the manuscript. SHY, BH and MXZ performed the xenograft experiments; YEW performed the immunehistochemical analyses. XLJ collected the LUSC patient samples. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the patients for making available the tumor samples that contributed to this research. The graphical abstract and schematic diagrams in this article were created via BioRender.com.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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ACS Nano. 2025;19(2):2742\u0026ndash;58.\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":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lung squamous cell carcinoma, Cisplatin resistance, Antisense long noncoding RNA, LPCRL, USP15/MIB1 complex, Molecular scaffold","lastPublishedDoi":"10.21203/rs.3.rs-8106822/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8106822/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003ePlatinum-based chemotherapy remains the first-line treatment for advanced lung squamous cell carcinoma (LUSC), but its efficacy is often hindered by the development of chemoresistance. Although long noncoding RNAs (lncRNAs) are recognized as regulators of tumor progression and drug resistance, the functional contribution of natural antisense transcripts (NATs), a major subclass of lncRNAs involved in cisplatin resistance in LUSC, remains poorly understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003ePatient-derived xenograft (PDX) models of LUSC were established and treated with cisplatin to identify cisplatin-resistant and cisplatin-sensitive tumor tissues. LncRNA microarray profiling was used to identify transcripts associated with cisplatin resistance. The functional role of a candidate lncRNA, termed LPCRL (LUSC primary cisplatin resistance-associated LncRNA), was assessed in vitro via MTT, flow cytometry, colony formation, and Transwell migration assays. Its effects on tumor growth and metastasis were further validated in vivo. Mechanistic insights were gained through RNA pull-down, silver staining, RNA immunoprecipitation (RIP), coimmunoprecipitation (Co-IP), and Western blot analyses. Finally, the therapeutic potential of LPCRL-targeting siRNA was assessed in a LUSC PDX model.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe found that LPCRL was significantly upregulated in primary cisplatin-resistant PDX tissues. Functionally, LPCRL promoted primary cisplatin resistance and enhanced the proliferation and migration of LUSC cells both in vitro and in vivo. Mechanistically, LPCRL functions as a molecular scaffold to facilitate the interaction between MIB1 and USP15. This complex enables USP15 to deubiquitinate MIB1, thereby increasing MIB1 stability and promoting its nuclear export. The subsequent cytoplasmic accumulation of MIB1 enhances the ubiquitination of DLL4, leading to Notch pathway activation and upregulation of the downstream effector HES1. Importantly, intratumoral administration of LPCRL-targeting siRNA in PDX models suppressed tumor growth and sensitized tumors to cisplatin in vivo.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur study revealed that LPCRL promotes LUSC malignancy and cisplatin resistance via the USP15/MIB1/Notch axis, highlighting LPCRL as a promising therapeutic target.\u003c/p\u003e","manuscriptTitle":"A novel antisense lncRNA, LPCRL, functions as a molecular scaffold for the USP15/MIB1 complex to promote primary cisplatin resistance and tumor progression in lung squamous cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 07:00:36","doi":"10.21203/rs.3.rs-8106822/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-25T08:56:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-23T14:46:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331597136468899324332056392745296037053","date":"2025-12-06T13:10:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"189484778849124782973430792702728306621","date":"2025-12-01T14:01:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-20T15:43:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-19T10:49:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-19T10:48:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Experimental \u0026 Clinical Cancer Research","date":"2025-11-13T14:28:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"175bea1c-4d71-466f-aa7a-9a75c9e0efe7","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T16:02:21+00:00","versionOfRecord":{"articleIdentity":"rs-8106822","link":"https://doi.org/10.1186/s13046-026-03721-7","journal":{"identity":"journal-of-experimental-and-clinical-cancer-research","isVorOnly":false,"title":"Journal of Experimental \u0026 Clinical Cancer Research"},"publishedOn":"2026-05-01 15:57:01","publishedOnDateReadable":"May 1st, 2026"},"versionCreatedAt":"2025-12-01 07:00:36","video":"","vorDoi":"10.1186/s13046-026-03721-7","vorDoiUrl":"https://doi.org/10.1186/s13046-026-03721-7","workflowStages":[]},"version":"v1","identity":"rs-8106822","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8106822","identity":"rs-8106822","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}